Thesymposiu mwa ssponsore dby :
AgriculturalUniversity ,Wageningen ,Netherland s EuroconsultB.V. ,Arnhem ,Netherland s IntervetInternationa lB.V. ,Boxmeer ,Netherland s Ministryo fAgricultur ean dFisheries ,De nHaag ,Netherland s Ministryo fForeig nAffairs ,Departmen to fInternationa lCooperation , DenHaag ,Netherland s Organization forImprovemen to fInlan dFisheries ,Nieuwegein ,Netherland s Trouw& C oNederlan dN.V. ,Putten ,Netherland s Proceedings of the International Symposium on Reproductive Physiology ofFis h Wageningen, the Netherlands 2-6 August 1982
C.J.J. Richter and H.J.Th. Goos (compilers)
A pudoc <%i*' DER 1 LANDSOUWHOGESCHOOL WAGENINGJEN 1982 Centre for Agricultural Publishing and Documentation Wageningen
f ^- Proceedings Proceedings of the International symposium on reproductive physiology of fishWageningen , theNetherland s 2-6 August 1982 /C.J.J . Richter and H.J.Th Goos (comp.). - Wageningen :Pudoc . - 111 Met index, lit.opg . ISBN 90-220-0818-5 geb. SISO 634.7UD C 636.987 Trefw. :visteel t ISBN 90 220 0818 5 © Centre forAgricultura l Publishing and Documentation (Pudoc),Wageningen , 1982 No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by anymeans ,mechanical ,photocopying , recording, or otherwise,withou t the prior written permission of the publisher Pudoc, P.O. Box 4, 6700 AA Wageningen,Netherlands . Printed in theNetherlands . CONTENTS Foreword R. Billard, Reproductive physiology and fish culture 1 GONADOTROPINS 3 David R. Idler,Som e perspectives on fish gonadotropins 4 Y. Zohar,B . Breton &A . Fostier,Gonadotropi c function during the reproductive cycle of the female rainbow trout,Salm o gairdneri,i n relation to ovarian steroid secretion: inviv o and invitr o studies 14 Elisabeth Burzawa-Gerard,Existe-t'i l plusieurs gonadotropines (GTH) chez les poissons? Données biochimiques etvitellogénès e exogène 19 POSTERS L.W.Crim , D.M. Evans,Positiv e testosterone feedback on gonadotropic hormone in the rainbow trout 23 P. Prunet, Purification and biological characterization of Salmon prolactin 24 Maura V.Val-Sella ,Antoni o Sesso,Morphometri c analysis of structural changes in Rhamdia hilarii gonadotrophic cells during the reproduc tive cycle 25 C.Weil , L.W. Crim,A study of methods of administering LHRH afnalogs: advancement of spermiation in the landlocked salmon (Salmo salar) 26 Z.Yaron ,Aliss a Bogomolnaya and V. Hilge,Applicatio n of an in vitro bioassay for fish GTH in improving harvest of carp pituitaries 27 H.A. Zenkevich, Z.M. Lace,Compariso n of purified pituitary gonado tropins of male and female pink salmon (Oncorhynchus gorbuscha Walb.) 28 HYPOTHALAMO-HYPOPHYSISAXI S 29 R.E. Peter,Nature , localization and actions of neurohormones regulating gonadotropin secretion in teleosts 30 G.V. Callard,Aromatas e in the teleost brain and pituitary: role in hormone action 40 P.G.W.J,va n Oordt,Gonadotropi c cells in the pituitary of teleosts 44 POSTERS I.A. Barannikova,O.S .Bukovskaya ,N.A . Efimova,Hormona l control of the reproductive function of sturgeons (Chondrostei) 49 R. Billard,L.W . Crim, R.E.Peter ,B .Breton ,Long-ter m changes in plasma and pituitary GTH after castration of rainbow trout at an immature stage. 50 John P. Chang and Richard E. Peter,Action s of dopamine ongonado tropin release in goldfish, Carassius auratus 51 S.Dufour ,C . Pasqualini,B . Kerdelhuê,Y.A .Fontaine ,Presenc e et repartition d'un facteur de type LHRH chez l'anguille: étude par dosage radioimmunologique 52 G.E.Fahraeus-va n Ree,C.J . Panis,J.A.F.W .Kleijne , J.Th. Gielen and P.G.W.J.va n Oordt,Gonadotropi c hormone production inenzymaticall y dispersed pituitary cells of the trout, Salmo gairdneri 53 J.Th. Gielen,H.J.Th.Goos ,Mari'êll eG.A . deMo lMoncourt-d e Bruyn and P.G.W.J.van Oordt,Direc t action of gonadal steroids on thematu ration of gonadotropic'cell s in the rainbow trout, Salmo gairdneri 54 H.J.Th. Goos, J.Th. Gielen and P.G.W.J.vanOordt ,Seasona l variations in sensitivity toGnR H of juvenile rainbow trout,Salm o gairdneri 55 0.Kah ,P . Chambolle,P . Dubourg and M.P.Dubois ,Distributio n of immunoreactive LH-RH in the brain of the goldfish 56 J.G.D.Lambert ,Petr a G. Wilms,M.L . Zandwijk,H.J.Th . Goos and P.G.W.J.va n Oordt,Localizatio n of aromatase activity in the brain of the rainbow trout (Salmo gairdneri) 57 M. Olivereau,P . Chambolle,P . Dubourg,Effec t of synthetic LH-RH on GTH cells of E2-treated eels 58 M.P. Schreibman,H .Margolis-Kazan ,L . Halpern-Sebold and H.J.Th.Goos, The functional significance of the nucleus olfactoretinalis in the platyfish,Xiphophoru s maculatus 59 H. Singh,T.P . Singh, Site of action of some agricultural pesticide in the hypothalamo-hypophysial-ovarian axis of the freshwater catfish,Heteropneuste s fossilis (Bloch) 60 SEXMANIPULATIO N 63 P. Zaborski,Expressio n of H-Y antigen in nonmammalia n vertebrates and its relation to sex differentiation 64 R. van den Hurk and J.G.D Lambert,Temperatur e and steroid effects on gonadal sex differentiation in rainbow trout 69 Terje Refstie,Practica l application of sexmanipulatio n 73 POSTERS Edward M. Donaldson, George A. Hunter,Th e ocean release and contribu tion to the fishery of all-female and sterile groups of Coho salmon (Oncorhynchus kisutch) 78 R. Johnstone and A.F.Youngson ,Th e production of genetically all-female Atlantic salmon stocks 79 H.K. Parmentier, L.P.M. Timmermans, E.Egbert s andJ.J.M . van Groningen, Monoclonal antibodies inth estud y of gonadal differentiation inth e carp (Cyprinus carpio L.) 80 C.J. Secombes, L.M.Lair d andI.G . Priede, Fish sterilisation: theauto immune approach 81 William L. Shelton, Monosex grass carp production through breedingsex - reversed broodstock 82 PHEROMONES/GONADAL STEROIDS 83 L. Colombo, P.C.Belvedere , A.Marconat o andF . Benti-vegna, Pheromones in teleost fish 84 D.E. Kime, Thecontro l of gonadal androgen biosynthesis in fish 95 J.G.D. Lambert andR .va nde nHurk , Steroidogenesis inth eovarie s of the African catfish, Clarias lazera, before andafte r anHC G induced ovulation 99 A.P. Scott, S.M. Baynes, Plasma levels of sex steroids in relation to ovulation and spermiation inrainbo w trout (Salmo gairdneri) 103 POSTERS H. Diederik andJ.G.D . Lambert, Steroids inplasm a of the female rainbow trout before and after ovulation by NCI-GCMS 107 Jeffrey Pudney, Gloria V. Callard andJaco b Canick, Topographical distribution of steroidogenic enzymes inrelatio n to spermatogenesis in the testis of Squalus acanthias 109 C. Simontacchi, D. Casciotti &C . Boiti, Plasma steroid profile during the spawning season of pike 110 P. Thomas,N.J .Brow n andC.R .Arnold , Seasonal variations of plasma androgens and gonad histology inmal e spotted seatrout, Cynoscion nebulosus (Family: Sciaenidae) 111 B. Querat, J. Leloup-Hatey, A. Hardy, Ovarian steroid metabolism in the immature european eel (Anguilla anguilla) 112 CONSERVATION OF GAMETES 113 Joachim Stoss andEdwar d M. Donaldson, Preservation of fish gametes 114 Brian Harvey, Cryobiology andth estorag e of teleost gametes 123 POSTERS S.M. Baynes, A.P. Scott, Cryopreservation of rainbow trout spermatozoa: variation inmembran e composition may influence spermatozoan survival 128 H. Bayrle, Cryopreservation of rainbow trout sperm: effect of equilibration 129 M. Morisawa, M. Okuno, S.Morisawa , Physiological study onth e initiation of sperm motility in chum salmon, Oncorhynchus keta 131 VI Nai-Haien Chao,Ne w approaches for cryopreservation of sperm of grey mullet,Mugi l cephalus 132 M.G. Plouidy,R . Billard, The chemical composition of the companion fluids of thegamete s in the common carp,(Cyprinus carpio) 134 GONADAL GROWTH 135 Murray D.Wiegand ,Vitellogenesi s in fishes 136 C.J.J.Richter,E.H . Eding, S.E.W.Leuve n and J.G.M.va n derWijst , Effects of feeding levels and temperature on the development of the gonad in the African catfish,Claria s lazera (C& V ) 147 K. Selman,R.A . Wallace,Th e inter- and intracellular passage of proteins through the ovarian follicle in teleosts 151 POSTERS N. Devauchelle,G . Brichon,F . Lamour,G . Stephan,Biochemica l composi tion of ovules and fécond eggs of sea bass (Dicentrarchus Labrax), sole (Soleavulgaris ) and turbot (Scophthalmus maximus) 155 A. Hara,H . Hirai,Structura l studies of bony fish vitellogenin 158 P. Ollevier &M . Covens,Oestradiol-induce d female specific proteins in the plasma and in the oocytes of Gasterosteus aculeatus F. trachurus.A n immunological approach 159 R.A. Wallace,K . Selman,A new procedure for the isolation of intact vitellogenin from teleosts 161 MATURATION OF GONADS 163 A.Fostier ,B . Jalabert,Physiologica l basis of practical means to induce ovulation in fish 164 I.Boëtiu s and J. Bo'étius,Experimenta l maturation of European-silve r eels 174 Edward M. Donaldson,Georg e A. Hunter,Gle n Van der Kraak and HelenM . Dye,Applicatio n of LH-RH and LH-RH analogues to the induced final maturation and ovulation of coho salmon (Oncorhynchus kisutch) 177 Ching-ming Kuo, Induced breeding of grey mullet,Mugi l cephalus L. 181 K. Yamauchi and K. Yamamoto,Experiment s on artificial maturation and fertilization on the Japanese eel (Anguilla japonica) 185 POSTERS R.Abu-Hakima , C. El-Zahr,M . Al-Shoushani, S.Akatso ,K .Abul-Ellah , Gonad development in food fishes of Kuwait 190 R. Billard, On some patterns of reproductive physiology inmal e teleost fish 192 S.Bruslë ,Ultrastructur e of testis organization and resting cells in Liza aurata Risso 1810, (teleostei,Mugilidae ) 193 E.H. Eding, J.A.L. Janssen,G.H.J .Klein e Staarman and C.J.J.Richter, Effects of human chorionic gonadotropin (HCG) onmaturatio n and ovulation of oocytes in the catfish Clarias lazera (C& V ) 195 P. Epler, K. Bieniarz,Effec t of triiodothyronine (T3) and some gonado tropin and of steroid hormones onmaturatio n of carp (Cyprinus carpio L.) oocytes invitr o 196 K.G. Forberg, Gonad maturation of female capelin (Mallotus villosus villosus) 197 F. leMenn ,Ultrastructur e of cellular and non-cellular layers surrounding the oocyte of a teleostean Gobius niger L.: preliminary observations 198 Lin Hao-ren and Lin Ding, Studies on the breeding biology of the eel (Anguilla japonica Temminck &Schlegel ) 199 R.B. Raizada, K.K. Datta and T.S.S. Dikshith,Effec t of endosulfan on biochemical changes in ovary and GSI of two species of freshwater teleost 200 S.Rothbar d and Hamutal Rothbard, Spermiation response of carp to homologous pituitary gland 201 H.J. Schoonbee, Induced spawning of the sharptooth catfish Clarias gariepinus with notes on techniques employed in the removal of egg adhesiveness 202 VII I.J. Singh, H.N. Singh and T.P. Singh, Induction of ovulation in H. fossilis-analysis of sex related differences in the potency of homologous and heterologous pituitary extracts 203 Hiroshi Urasaki, Takeyuki Abe and Sin-Iti Ohtake, Reproduction regulated by extraocular photoreceptor in a seasonally breeding fish, Oryzias latipes , 204 Glen Van Der Kraak and Edward M. Donaldson, Saturable gonadotropin binding sites in the testis and ovary of immature chum salmon 205 K.G. Waiwood, Growth history and reproduction in Atlantic cod (Gadus morhua) 206 ENVIRONMENTAL FACTORS AND REPRODUCTION 209 R.J. Wootton, Environmental factors in fish reproduction 210 A.D. Worthington, N.A.A. MacFarlane and K.W. Easton, Controlled repro duction in the roach (Rutilus rutilus L.) 220 Shelby D. Gerking, The sensitivity of reproduction in fishes to stress ful environmental conditions 224 Isao Hanyu, Kiyoshi Asahina and Akio Shimizu, The roles of light and temperature in the reproductive cycles of three bitterling species; Rhodeus ocellatus ocellatus, Acheilognatus tabira and Pseudoperilampus typus 229 N. Bromage, C. Whitehead, J. Elliott, B'.Breton , A. Matty, Investigations into the importance of daylength on the photoperiodic control of reproduction in the female rainbow trout 233 POSTERS Bertha Baggerman, Influence of temperature on gonad development in a strongly photoperiodic species, Gasterosteus aculeatus L. . 237 B. Borg, M. Reschke, Th. van Veen, Effects of photoperiod and temperature on reproduction of male three-spined stickleback during winter 238 Y. Honma, A. Chiba, Reproductive behaviour and endocrine activity of the puffer, Fugu (= Spheroides) niphobles, occurring on the coast of Sado Island in the Japan sea ' 239 Ann L. Kyle, N.E. Stacey and R.E. Peter, Sexual stimuli associated with increased gonadotropin and milt levels in goldfish 240 Duncan S. MacKenzie and N.E. Stacey, Changes in plasma levels of gonado tropin and thyroid hormones during spawning in the white sucker (Catostomus commersoni) 241 M. Pohl, R. Schmidt, W. Holtz, Manipulation of spawning activity in rainbow trout by light programs 242 0. Skarphëdinsson, A.P. Scott and V.J. Bye, Long photoperiods stimulate gonad development in rainbow trout 243 Joachim Stoss, Ulf H.M. Fagerlund, Influence of saltwater spawning and stress on quality of sex products of chum salmon (Oncorhynchus keta) 244 CONCLUSION 245 E.A. Huisman, Basic and applied research on fish reproduction: a balance ? (Final address on behalf of the Organizing Committee) 246 List of participants 248 Author index 255 VIII FOREWORD Thisboo k contains the texto fabou t 100 papers andposter spresente d atth eSecon d International Symposiumo nReproductiv e Physiology ofFish ,hel d atWageningen ,th e Netherlands,fro m2- 6Augus t1982 . Modernfis h farmingi sa highl yspecia lized,intensiv e industry,mor e andmor e requiringpossibilitie s tomanipulat ere production.Alterin g sexualcycle s andse x ratios,inductio n ofmaturatio n ofgametes , ovulation and spermation,storin gove r longerperiod s of timeo fegg san dsper m andartificia l fertilizationar e techniques commonlyuse d for several species ofcom mercial fishbu tstil lunde r development forothers .Suc h techniques canb eachieve d by trialan derro ran dsom eresult sar e impressive.However ,a bette r understanding ofth ereproductiv ephysiolog y offis hmigh t help tomor ean dfaste rprogress . Theai mo f thesymposiu mwa s tobrin g together thoseconcerne dwit h applied aspects offis h reproduction and dailyproblem so f fish farming,wit hresearcher s interested inth emor e fundamentalproblem s ofth e reproductivephysiolog y offish . Thegenerou s financial supportfro m: Agricultural University,Wageningen , Netherlands; EuroconsultBV ,Arnhem ,Netherlands ; IntervetInternationa lB.V. ,Boxmeer , Netherlands; Ministry ofAgricultur e andFisheries , theHague ,Netherlands ; Ministry ofForeig nAffairs ,Departmen to f International Cooperation,th eHague , Netherlands; Organization forImprovemen t ofInlan d Fisheries,Nieuwegein ,Netherlands ; Trouw &C oNederlan d N.V.,Putten , Netherlands wasa grea thel p toorganiz e thissymposium . Thanks are due toth estaf fo f theInter nationalAgricultura l Centre,Wageningen ,fo r housing thesymposium . Due toth eexcellen t co-operation ofth e speakers and thepublishe rPudo c thepro ceedings of thesymposiu m could appears o shortlyafte r themeeting . C.J.J.Richter, H.J.Th.Goos. IX REPRODUCTIVE PHYSIOLOGYAN D FISH CULTURE R.Billar d Laboratoire dePhysiologi ede s Poissons I.N.R.A.Campu s deBeaulie u 35042RENNE S Cedex France Agoo d knowledgeo f themechanism s regula tokno wwhe n and howthi s phase canb e indu ting the reproductive function infis h is ced and theconsequence s thatma y resultfro m requisite toth edevelopmen t ofaquaculture . agive n treatment (possibilityo f aneuploidy Onceacquired ,thi s knowledgemake s itpos or subsequent sterility).A goo d knowledge sible toexercic evaryin g degreeso fcontro l of theflu xo f energy at thesestage s (part onth edifferen t stageso f reproduction,t o used forhydratio n and remaining partfo r adapt the techniques tofi t newsituations , embryogenesis) is important and decidewha t and then toteac han d propagate thesetechni strategy should beuse d for larvaerearing . ques toextentio n officers and fishcultu - Wemus tals o study theoptima l environmental rists,Reproductiv e physiology hasprogresse d conditions forobtainin g good-quality gametes considerably during the last fewyear sowin g during gametogenesis.Th egamet e releasei s toth edevelopmen t of newan d varied techni a key point infis h culture and an improve ques; itha sprove n itsabilit y to synthesize ment of the techniqueso f induced spawning all thedat a fromman y diversified and requiresa better knowledgeo f theneuroendo complex fields sucha s endocrinology, crinemechanism s of regulationwhic har ea t cytology and molecular biology,Som eo f the least insom especie s either stimulatoryo r crucial problems isreproductiv e physiology inhibitory asshow n recently.Thi sope nno w have thusbee n studied and partly resolved. thepossibilit y (chemical orenvironmental ) Research hasusuall y been carried outo n toovercom e this inhibitionwhic h probably laboratory animals inver y artificial condi strongly linked toth eenvironment . tionsmimickin g traditional research in k) Spawning.A simplewa y toobtai nfertili animal husbandry. Butthi s ischanging ; zed eggs ist opu tmale sarr dfemale stogethe r there isno wa tren d toward studying the ina tan ko r pond and let them reproduce animal in itsnatura l environment,wher e naturally. Inthes econditions ,th ebehavio r it issubmite d toa largervariet yo f of thefis han d theenvironmenta l cues environmental factors. (physical,chemica l and social)ar eprimor dial and can,i ncertai n cases,resolv eth e problemo f induced spawning. On somebasi c problemso f physiology interesting fishculturists . 5)Gamete s physiology.Whe n artificial inse mination isused , it isessentia l tounder The control of reproductin infis hfar stand the physiology of thegametes ,thei r ming involves control of sex,th ereproduc energy requirements and their long andshort - tivecycl ean d the releaseo f gametesa swel l term storagea swel l as thecompositio no f asth emanagemen t of gametes andembryos . their companionfluids . Inorde r tomi xth e Controlling reproductio nimp lie sa knowledg e gametes ata noptima l ratedurin g artificial of thefollowin g majorproblems. . insemination,th equalit y of egg and sperm and change intim emus t first beassessed . 1)Se xdeterminis m and differentiation.W e mustdetermin e thedynamic so f theseproces 6)Fertilizatio nan d embryogenesis .Th e ses,th efactor s responsible,th eorigi n environmental conditions inwhic h thegamete s of somatic cells,th erelationshi p between areassociate d atartificia l insemination, germ cells and somatic cells and thedeve fertilization,an d theonse to f development lopment of these cells. may havedifferen t effectso n embryonic 2) Puberty. It isdesirabl e tob eabl et o development,particularl y onpregastrulatio n advance,dela yo r inhibit puberty.W emus t stages. It istherefor e imperative todefin e study the initiation of thefunctionin g at such conditions as,fo rexample ,salinity , central,pituitar y and gonadal levelsan d temperature,pH , ioniccompositio n anddilu howthes ear e related togrowth . tion rate,a saccuratel y aspossible .Th e 3)Gametogenesi san dgamete s release.W e qualityo f embryonicdevelopment ,whic hals o must acquirea knowledg eo f thedynami c dependso f theenvironmenta l conditionsma y (cycles),th eendocrin e (endocrinean d be used asa criterio n ofgamete s quality. neuroendocrine regulation as related to All thesefactor s are interdependentan d environment)an d metabolic (nutrition) mustb econsidere d alongwit hothe r functions aspectso fgametogenesi s aswel l aso f its such as nutrition,osmoregulation ,respira genetic side (ecophysiological strainsan d tion and behavior. breeds).Th eovaria n and hepatic aspectso f vitellogenesisar ecrucia l aswel l asth e Suchdetaile d data cannot beobtaine d ona final phaseo foocyt ematuratio n andsper - large number of species and therefore miationwhic hofte n has tob e induced in research has tob e limited toa fe w species numerous cultured species. It isnecessar y serving as representativemodel so f intensive rearing (aquacultureo f transformation;e.g . attractive brood fish,aeratio n of eggsan d salmonids)o r extensive rearing (aquaculture the importanceo f prédationdurin g juvenile of production;e.g . cyprinids and cichlids stages.W eshal l then knowho wt oprotec t whichofte n need ahatchery) . eggsan d fry andmanag e spawning areasan d theirannexe s (nests,eg g substrate),W e However,th e ideao f amode l species is must beabl e toestimât «broo d fish popula nota complet eanswe r toth e problemo f tionsa swel l asthei r egg and larval produc obtaining complexdat a because therear e tionan d ,i fnecessary ,extr a brood fisho r widedivergence s between specieso f thesam e juveniles. family and evenwithi n aspecies ;dynami co f oogenesis isdifferen t intenc h and-i nroac h For polyculture,i t isnecessar y todeter LH-RH ismuc hmor eactiv e inphytophagou s mineth econdition s inwhic h reproduction species than incarp ,HC G inducesovulatio n occurs inth ewil d and howthes econdition s or spermiation ingoldfis h but not incarp , change in response toa n increase inth e thesculpi n isa hatch-spawne r inDorse t diversificationo f thenumbe r ofspecies . chalk streams but laysonl ya singl ebatc h of eggs inTeesdal e streams.Dat a cannot be The reproductive physiologist should also, applied directly butmus t always beadapte d beabl e toanalys e how-and why the changes in toculture d fishwhos e reproduction ist o theenvironmen t have consequences onth e becontro lled . reproduction and the recruitement ofth e species asfo r instance thedrasti cmodifica On the necessity of newmanagin g fish species tions inth eag ecompositio n of theatlanti c inth ewil d and thenee d fororigina l data salmonfisherie s and the considerable inth e field of reproductive physiology increaseo f themal epar ran d femalegrilses . Theapproac hdescribe d above,whic h seeks Conclusion s toacquir edetaile d knowledgeo f asmal l numbero f species, issimpl y acop yo f the Ifresearc h ist ocontribut e tofis h cul approach used inanima l husbandry, i.e. ture,th e knowledgew emus t acquire inth e domestication trialswit h control of themai n field of reproductive physiology iso ftw o functions and monoculture usually ininten kinds: (i)knowledg eo f themechanism sgover sivecondition s intank so r ina relatively ning eacho f the reproductive stages,th e well controlled ecosystem.Thi s ideaha s potentialitieso f reproduction and their beenapplie d toaquacultur ewit h some manifestation inculture d fish and (ii) success butwit h many failuresowin g toth e knowledgeo f themechanism s regulating repro fact that fitness for domesticationvarie s duction inth ewild . Inth efirs tcase , a greatdea l among species,an dw ed ono t mecanismsan d potentialj tie sshoul d be haveenoug hdat a fromothe rfields . studied ina variet y of speciesan d sometimes even independentlyo f their interestt o At present there isanothe r demand in culturists;some.specie s aremor e fitted than aquacultureconcernin g themanagemen to f others toth estud yo f aparticula r problem. the natural orquasi-natura l environment; Itwil l then benecessar y toadap t the results thismean smanagin g (understanding,protec toculture d species. Inth e second case,w e ting,exploiting )comple xaquati c ecosystems must favor ecophysiological research because considered inthei r entirelywit h different thisapproac h particularly concerns species trophic levelsan d awid e rangeo f species. tob eraise d inpolycultur e and may involve Inthi scase ,monocultur e anddomesticatio n a relatively largenumbe ro f them. cannot beused ;a ne wtyp eo f knowledge is needed.W emus tdevelo p research inwhic h Inan y case,th efis h physiologist must animals,a s plants,wil l be returned totha t continually inform himselfo f newdevelop complex, interrelated system known asth e ments inmethod s and techniques.and of the trophic chain.Th emai n functions,studie d stateo f research inprogres s inother s inrelatio n toth eenvironment ,wil l include animal species.Fis h species arenumerou san d avariet y of physical-chemical,climatic , diversified andsomewel l studied species in biological and social factors.A sconcern s other classesma y servea smodels . reproduction,w eraust b eabl e toestimat e and predict thefecundity ,prolificnes san d Finally, it isno tenoug h that thecultu - reproductive periodso fdifferen t speciesi n ristunderstan d reproduction;tha t knowledge relation toth efoo d available tobroo d fish must beassociate d and integrated with know and larvaewit h special reference toclimati c ledge inothe r fields such asnutrition , conditions sucha s temperature.Th eecologi genetics and pathology.W emus t associatean d cal conditions for reproduction should be synthetize inexperimenta l fish farm labora analysed fora largenumbe ro f species.Th e tory and experimental informations comming ecology of spawning grounds needs tob e fromal l'field so f research. studied,particularl y from thepoin t ofvie w ofoxygenation ,physical-chemica l composition of theenvironment ,turbidity ,condition s I : GONADOTROPINS SOMEPERSPECTIVE S ONFIS HGONADOTROPIN S DavidR .Idle r MarineScience s ResearchLaboratory ,Memoria lUniversit y ofNewfoundland, "St . John's,New foundland Keywords : Gonadotropins,isolation ,biologica laction ,measurement ,assay . Opening remarks Yamazaki,1976 )winte r flounder (Ng& Idler , 1979)Tilapi a (Farmer &Papkoff ,1977) . Theweigh t ofevidenc e continues tofavou r Acetonepowde r of carppituitarie swa s multiple gonadotropins (GtH)i nth efe w obtained froma commercia lsourc eb yBurzawa - teleostswhic hhav ebee nexamine d indetail . Gerard (1971). Acetonepowde r frompik eee l Iti simportan t todistinguis hbetwee n pituitarieswa s similarlyprepare db yHuan g multiple formso f"similar "carbohydrate - and associates (1981). richGtH' s (e.g.isohormones )an dhormone s Solvent fractionation and salting outwa s which differ sufficiently inthei r carbohyd apurificatio n stepi n theisolatio no f rate composition orconten t tob e separated gonadotropins frompituitarie s ofcar pb y onimmobilize d lectinssuc ha swhea t germ Burzawa-Gerard (1971);chinoo k salmonb y orConcanavali nA-Sepharose . Donaldsonan d coworkers (1972);sturgeo n Forth epurpose s of thisaddres s Ishal l byBurzawa-Gerar d andassociates , (1975a); use theter mvitellogenesi s torefe r toth e chumsalmo nb yYoned a &Yamazak i (1976); synthesis ofvitellogeni nb y thelive ran d Tilapiab y Farmer &Papkoff , (1977),an d theuptak e intoth egona d"i.e .exogenou s Hydere t al., (1979);an dpik e eelb yHuang' s vitellogenesis"an d anysynthesi so r group (1981). Alcoholic percolation as assembly ofyol kprotein s thatma y take describedb yBate san d others (1959)an d place inth eovar y at this time"i.e . extractionwit h ethanolic ammonium acetate endogenousvitellogenesis" . buffer followedb yprecipitatio nwit h Let usno w turn toth eactio n offis h ethanol,tha ti sth eStockel lHartre e pro gonadotropins onfis han dparticularl y the cedure of 1966,lya semploye d inthes e evidence relating togonadotropin s thatar e studies. atypicali n thesens e that they differ from Idler andhi sgrou p (1975a,b )innovate d theclassica l gonadotropins described for theus eo faffinit y chromatography onCo n otherlif e forms,a tleas tunti lver yrecent - A-Sepharose inthei rschem e ofisolatio no f ly. Ia mreferrin g toth e gonadotropins gonadotropins. ConA-Sepharos e chromato that arerathe rpoo r incarbohydrat e andno t graphywa sadopte db yPierc e and others retained onConcanavali nA-Sepharose . The (1976)an dBreto n and associates (1978). other termsapplie d tothes ehormone sar e Chum salmonpituitar y extractyielde da ConA I GtH's todistinguis h them fromth e broadpea kunadsorbe d onCo nA-Sepharos e more conventional ConAl l typewhic har e (designated ConA I fraction)an d anadsorb adsorbed on theimmobilize d lectin. Finally, edfractio n (designated ConAl l fraction) thesehormones ,whic hhav ebee n foundi n whichwa s eluted asa shar ppea kb yinclus each teleostw ehav eexamine d todate ,hav e iono fa-methyl-D-glucosid ei nth ebuffer . been calledvitellogeni chormone ssinc e Theproteas e inhibitor Trasylolwa suse dan d theirbiologica l actionshav e onlybee n divalent cations including calcium,magnesium demonstrated during thisimportan tphas eo f andmanganes e assureefficien t adsorption. reproduction. Theimmobilize d lectin selectively adsorbs Thescop eo f thismanuscrip t isconfine d glycoproteinwit h specificstructures . toteleosts . Thelistene ri s referredt o (Goldsteinet .al. ,1965 ), leavin gbehin di n reviewsb yFontain e &Burzawa-Gerard ,1978 ; theunadsorbe d fractionprotein swit h alo w Idler &Ng ,1980 ;Crim ,1982 ;Ball ,1981 , carbohydrate contento rno thavin g thesugar s andBurzawa-Gerard , 1982 forrhythm san d requisite forbindin g toth electin . Re- other aspectsno t coveredhere . Theactio n chromatography of theCo nA I fractiono n ofmammalia nhormone so n fishwil lno tb e ConA-Sepharos e isfrequentl ynecessar y to discussed. eliminate contaminationwit h ConAl lmaterial . Idler& Hwan g (1978)foun d thatth einclusio n ofethylen e glycol(50%v/v )i nth eelutio n Isolation of gonadotropins bufferoptimize d therecovery ,fro mCo nA - Sepharose,o fmaturationa lhormon ewhic hwa s Invariou s investigations pituitary contained inth eCo nAl lfraction . glandswer e collected fresh from fishwhen everpossible ,o ra ssoo na spossibl eafte r Other immobilized lectinswit h different death,t opreven tpostmorte m degradationo f carbohydratebindin g specificities inth e hormones. Thiswa s donei nth ecas eo f isolationo fteleos t gonadotropinshav ebee n salmonids (Bretone t al., 1976;Donaldso n testedb yIdle r &N g (1979). Lentil lectin etal. , 1972;Idle re t al.,1975a ;Yoned a& Sepharoseha sa carbohydrate specificity similar totha to fCo nA-Sepharos ebu ti t was alsoactiv e inthi sassa ybu tmuc hles s has alowe r affinity according toStei n& sotha nth eTilapi ahormone . Theday-ol d associates,1971 . Wheat germlecti n cockerel testicular radiophosphate uptake Sepharosean dHeli xpomati a lectin Sepharose assaywa suse di n theisolatio no fsalmo n arespecifi c forN-acetyl-glucosamin ean dN - gonadotropinb yDonaldson ,Idler ,Pierc e & acetylgalactosamine respectively. Itwa s associates.. Thepik eee lgonadotropi nwa s found thatCo nAl l fractionwa sadsorbe d on also active inthi savia nassay . However, Lentil lectin-Sepharose,whea t germlecti n Ishii& Yamamot o (1976)foun d thata salmo n Sepharosean dHeli xpomati alectin-Sepharose . pituitary extractwa sinactiv e instimulat Bretoni n198 1showe d thatwhea t germlecti n inghypertroph y of chickSertol i cells. The adsorbed thegonadotropi nwhic hwa s adsorbed discrepancy inthei r findingsma yhav ebee n onCo nA-Sepharose . Thus,bot h groupsfoun d duet oth edosag eo rt oth eassa yparameter s thesam egonadotropi n fraction tob eadsorb employed. edo nth eimmobilize d lectins. Amphibianassay sinvolvin g spermiationan d Gelfiltratio n separates proteinsmainl y oocytematuratio nwer euse d tomonito rgon onth ebasi s ofmolecula r size. Sephadex adotropic activity during isolationo fcar p G-100o rG-7 5wa suse db ymos t investigators and sturgeon gonadotropinsb yBurzawa - although someworker s employedUltroge lAc A Gerard. 54o rAc A44 . Inth e caseo fa species , Therewa sals o avariet y ofisolatio npro sucha sflounde rwher e thepea k following ceduresutilizin g teleostassays . Yoneda& thevoi dvolum e isrelativel yhig hmolecula r Yamazaki (1976)purifie d chum salmongonad weight (62,000)the nUltroge l allowsa bette r otropinwit h the goldfish spermiation assay separation thanSephade x G-75. Thebuffe r ofYamazak i &Donaldso n (1969). Stimulat usedwa s either thevolatil eNH.HCO, ,whic h iono f testicular growthb y Schmidt et_al. , lendsitsel f tolyophilizatio n orTris-C la t 1965an dgonada lcAM Pb y Idlere t al., anea rneutra l pH. 1975ai nimmatur e trout,stimulatio no f Diethylaminoethyl (DKAE)cellulos e ora llpovitellinproductio nb yGrönlun d (1969) DEAE ionexchange r sucha sDEA EBio-Ge lA o r oocytematuratio n invitr ob yBreto n etjil ., DEAE-SephadexA 5 0wa suse d topurif y teleost 1976 andb yHirose ,198 0an d inviv ob y gonadotropins onth ebasi s of chargeprop Ng &Idle r (1978a)an dSundarara j &Nayya r erties. (1976), testicular interlobular andintra Thebuffe ruse dwa seithe rNH.HCO. ,tri s lobularhistolog yb yHyde re tal. ,1979 , buffer orsodiu m glyeinatebuffer . Acon testicualr steroidogenesisb yHuang ,an d centration gradientwa suse d toelut eth e vitellogenesisb y Sundararaj &Idler' s adsorbedmaterial . Thevirtue s ofNH.HC O grouphav ebee nused . Gonadotropin induced havebee nnote d forpurifyin gmammalia n a depletiono f freecholestero l inth e gonadotropins. Iti seas y toremov ean d matureovar yo fmurre l (C.punctatus )an d results ingoo d stability ofth epreparat thisforme d thebasi. s ofa nassa y ranging ions. However,i tha s also given trouble from1-1 0 ug,Mukherje e &Bhattacharya , withDEA EBio-Ge lA . Forexample ,fro mtime - 1981. to-timeprotei nwhic h isnormall yhel d on Iti sadvisabl e touse ,whereve rpossible , thecolum nwil l appear inth eunadsorbe d thesam eo ra closel y related speciesi n fraction fromCo nAI . This fractionmus t biologicalstudies ,t o circumvent thepro - be checked,sa yb y electrophoresis,an dru n lemo fbiologica l specificity. When againo nDEA EBio-Ge lA ifan yband s faster Fontaine et_ al., 1972compare d thepotencie s thanR f0. 2 areobserve d (Idler,1981) . ofthei rcar pgonadotropi cpreparatio nwit h Thisproble m caused us tosometime s finda salmon gonadotropin SG-G100o fDonaldso n in higherR ffo rprolacti n than0.1 4 and0.18 . stimulating adenylate cyclase activityi n Weno welut eprolacti n and thenrevers e the thegoldfish ,h e found that thecar pgonado flow toobtai n theothe rpeptide swhic har e tropinwa s 36time smor epoten t thanSG-G100 . thanisolate db ypreparativ ePAGE . Thepossibilit y has tob e takenint oaccoun t Carboxymethylio nexchange r chromatography that thecar p gonadotropic preparationan d wasuse db yPierc e &associate s (1976),b y SG-G100ma yb eo f different degreeso f Idler &N g (1979)an db yHyde r &coworker s purity,bu t iti sver y likely that thesam e (1979)employin g ammonium acetatebuffe r picturewoul d emerge afterth e differences ata nacidi cpH . Amberlite CG-50wa s inth eexten to fpurificatio nhav ebee n employedb yFarme r &Papkof f (1977). allowed for. ThusCyprinu s carpiomatura - PreparativePAG Ewa son eo fth e stepsi n tionalhormon ewa s about five timesmor e thepurificatio n protocoluse db yBurzawa - active thansalmo nmaturationa lhormon ei n Gerard (1971)wit h carpan db yHuan g& stimulating cAMP productionb y grasscar p coworkers (1981)wit hpik eeel . ovariesi nvitr oaccordin g toIdle r &Ng , Avariet y ofbioassay shav ebee nutilize d 1979. Carp gonadotropinwa smor e active tomonito r gonadotropic activitydurin g thancatfis h gonadotropin instimulatin g isolation. Testosterone outputb yra t theproductio n of cAMP inee lovar yan d Leydigcell s invitr owa semploye d asa n bothwer emuc hmor eactiv e thanwa ssturg assay forTilapi a gonadotropinb yFarme r eongonadotropi n concludeDufou r &assoc &Papkof f (1977). Pikeee lgonadotropi n iates,1979 . Itha sbee n theinteres to f many comparative endocrinologists totes t about 3week swit hou rquit e goodwalk-i n theeffec t ofmammalia n gonadotropins onfis h coldroo m facilities. This canb eacceler reproduction. Therei sa discrepanc ybetwee n atedb yus eo fsolven to rchemica lprecipi theaction s ofmammalia n and teleostgonado tationan d freeze-drying tonam etw opro tropinso nvitellogenesi si n thecatfish . cedureswhich ,w ehav e electedno t toinclud e Inductiono fvitellogeni n intoth ecirculat untilmor e isknow n about stability ofth e ionwa sachieve db ymammalia n gonadotropins fishhormones . Theoveral lyiel d through6 butn oincorporatio n ofth eyol kprecurso r majorpurificatio n stepsan d dialysisan d intoth eoocyte sresulte d (Nath &Sundararaj , concentration throughmolecula r sieves 1981). Thisphenomeno n again illustrates generally doesno t exceed 15%eve n though theprincipl e ofzoologica l specificityo f individual stepswil lrang e from50-90 %fo r gonadotropins. Inth eassa y ofmaturationa l most components of thefractio nno t adsorbed activityusin g thegermina lvesicl ebreak onConcanavali nA-Sepharose . Therelativ e downo fOryzia slatipe s oocytes invitr oa s deatho fappropriat ehomologou s bioassays theassa yparameter ,i twa s found thatovin e hasbee na bottlenec kparticularl y for LHwa sles spoten t thanSG-G10 0an dth e studieso novaria n growth. Theuptak eo f slopeso fth edose-respons e curveswer edif radioactive phosphorus and aminoacids , ferent (Hirose,1980) . Carp gonadotropin during themos t activephas eo fvitellogen andmammalia nL H differed in theirstimulat esisha sbee n studied insom edetail . The iono fee lovaria n cAMP (Fontainee tal. , interpretation ofresult s dependso nho wth e 1981). analyses areperforme d andthi srange s from Salmon gonadotropin SG-G100ha dn oeffec t countingpiece so fgonad s toprecipitatio n onth eprocesse s of luteinization andpro with trichloroacetic acid toisolatio no f gesterone secretion inculture dmonke y theglycolipophosphoprotei n fractionb y granulosa cellsaccordin g toChanning , 10 studies. Thus,th e considerableknowledg e Burzawa-Gerard,E. ,B.F .Goncharo v &Y.A . ofth estructur e and function ofgonadotro Fontaine,1975a .L'Hormon e gonadotrope pinsbase d onpituitar yhormone sma yunderg o hypophysaire d'un poisson chondrostéen, substantialmodificatio n asinformatio ni s l'esturgeon (Acipenser stellatus Pali.). gained about thecirculatin g formswhic hac t I.Purification .Gen .Comp .Endocrinol . onth etarge t tissues. Perhaps iti sapprop 27:289-295 . riate toreiterat e aple a thatA.S .Park s Bye, V.J.,B .Breto n &R .Billard ,1980 . made inopenin g a195 7Cib acoloqiu mtha t Immunological cross-reactionbetwee n we should remember "ahormon e issomethin g pituitary gonadotropin fromNort hAtlanti c thatgoe saroun d inth ebloo d toac ti n fisheries.Gen .Comp .Endocrinol .41 :130 - anotherpar t of thebody "an dh eurge dmor e 134. research on thenatur eo fhormone s inth e Campbell,CM. , 1978.I nvitr o stimulation blood. This doesnot ,o f course,preclud e ofvitellogeni n incorporation intotrou t transformation ofa hormon e enrouteto , oocyteb y salmonpituitar yextracts . ora tit starget . Ann. Biol.anim .Biochem .Biophys . 18(4): 1013-1018. Campbell,CM . &D.R . Idler,1976 .Hormonal , References controlo fvitellogenesi sb yhypophysect - omizedwinte r flounder (Pseudopleuronectes Ball,J.N. , 1981.Hypothalami c controlo f americanusWalbaum).Gen .Comp .Endo .28:14 3 thepar sdistali s infishes ,amphibian s Channing,C.P. ,P .Licht ,H .Papkof f &E.M . andreptiles .Gen .Comp .Endocrinol .44 : Donaldson,1974 .Comparativ e activities 135-170. ofmammalian ,reptilia nan dpiscin e Bates,R.W. ,R.W . Garrison &T.B .Howard , gonadotropins inmonke y granulosacel l 1959. Extraction ofthyrotropi n from culture.Gen .Comp .Endocrinol .22 :137 - pituitary glands,mous epituitar y tumors 145. andbloo d plasmab ypercolation .Endo Chappel,S.C , C Coutifaris &S.J .Jacobs , crinology 65:7-17 . 1982. Studieso n themicroheterogeneit y Bhattacharya,S .Sen ,& S .Deb ,1981 .Hor of follicle-stimulating hormonepresen t monalregulatio n ofovaria n 17g-hydro- within theanterio rpituitar y glando f xysteroid dehydrogenase inteleost . ovariectomizedhamsters .Endocrinolog y110 : Abstract ofpapers .Nint h International 846-854. Symposium onComparativ eEndocrinology . Crim,L.W. ,1982 .Environmenta l modulation HongKong .p .30 . ofannua lan d dailyrhythm s associatedwit h Bogdanov,E.M. ,& D.D .Nansel ,1978 .Biol reproduction in teleostfishes .Can .J . ogicalan d immunological distinctions Fish.Aquat .Sei . 39:17-21 . betweenpituitar yan d serumL Hi nth erat . Crim,L.W. , &D.R . Idler,1978 .Plasm a gonad- In: "Structure andFunctio n ofth e ogropin,estradio lan dvitellogeni nan d Gonadotropins" (K.W.McKerns ,ed.) ,pp . gonadPhosviti n levels inrelatio n toth e 415-430.Plenu mPress .Ne wYork . seasonalreproductiv e cycleo ffemal e Breton,B. ,1981 .A studyo fsalmo n brown trout.Ann .Biol .anim .Biochim . (Oncorhynchus tshawytscha)pituitar y Biophys. 18:1001-1005 . gonadotropin dissociation.Gen .Comp . Crim,L.W. ,E.G .Watt s &E.M .Donaldson , Endocrinol.45 :147-152 . 1975. Theplasm a gonadotropin profile Breton,B. ,B .Jalaber t &P .Relnaud ,1976 . duringsexua lmaturatio n ina variet yo f Purificationo fgonadotropi n fromrainbo w salmonid fishes.Gen .Comp .Endocrinol . trout (Salmogairdneri i Richardson) 27: 62-70. pituitary glands.Ann .Biol .anim .Biochim . Donaldson,E.M. ,F .Yamazaki ,H.M . Dye &W . Blophys. 16(1): 25-36. W.Philleo ,1972 .Preparatio n ofgonado Breton,B. ,P .Prune t &P .Reinaud ,1978 . tropin fromsalmo n (Oncorhynchus tsawy- Sexualdifference s insalmo ngonadotropin . tscha)pituitar y glands.Gen .Comp . Ann. Biol.anim .Biochem .Biophys . 18(4): Endocrinol.18 :469-481 . 759-765. Dufour,S. ,E .Burzawa-Gerar d &Y.A .Fontaine , Burton,M.P. ,D.R . Idler &T.B .Ng ,1981 . 1979 .Evolutio n deshormone sglycoprotéiq - Theimmunofluorescen tlocatio no fteleos t ueshypophysaires:Donnée sradioimmunolog gonadotropinsan d thyrotropinsi nflounde r iquessu rle ssousunité sd e l'hormone pituitary.Gen .Comp .Endocrinol .43 :135 - gonadotrope del acar p (Cyprinus carpioL) . 147. CR. Acad. Sei.Pari s289 :137-140 . Burzawa-Gerard,E. ,1974 .Etud ebiologiqu e Farmer,S.W. , &H .Papkoff ,1977 .A teleost etbiochimiqu e del'hormon e gonadotrope (Tilapiamossambica ) gonadotropin that d'unepoisso n téléostéen,l aCarp e resembles luteinizinghormone .Lif eSei . (Cyprinus carpio L.).Mem .Mus .Nat .Hist . 20:1227-1232 . Nat. Ser.Zool . 86:1-77 . Fontaine,Y.A. , &E .Burzawa-Gerard ,1978 . Burzawa-Gerard,E. ,1982 .Chemica l datao n Biochemical andbiologica lpropertie so f pituitary gonadotropins and theirimpli fishgonadotropin s and theirsubunit s: cationt oevolution .Can .J . Fish.Aquat . comparisonwit hmammalia nhormones .In : Sei. 39:80-91 . "Structure andFunctio no f theGonadotrop - 11 ins" (M.W.McKern s ed.). pp.361-380 . Fishgonadotropin(s) .III .Evidenc e for PlenumPress .Ne wYor kan dLondon . more thanon egonadotropi n inchu msalmo n Fontaine,Y.A. ,E .Lopez ,N .Delerue-Labelle , pituitary glands. Endocrine Res.Commun . E.Fontaine-Bertrand ,F .Lalie r& C .Salmon , 2(3):237-249 . 1976.Stimulatio n gonadotrope de1'ovari e Idler,D.R. , &-S.J.Hwang ,1978 .Condition s chezl'anguill e (Anguilla anguillaL. ) foroptimizin g recovery ofsalmo ngonado - hypophysectomisée.J .Physiol .72 :871-892 . gropinactivit y fromCo nA-Sepharose . Fontaine,Y.A. , C.Salmon ,E .Fontaine - Gen.Comp .Endocrinol .36 ^642-644 . Bertrand,E .Burzawa-Gerar d &E.M .Donald Idler,D.R. , S.J.Hwan g &L.S .Bazar ,1975c . son,1972 .Compariso no f theactivitie s Fish gonadotropins(s).I .Bioassa yo f of twopurifie d fishgonadotropin s on salmongonadotropin(s ) invitr owit h adenyl cyclase activity inth egoldfis h immature trout gonads.Endocrin eRes . ovary.Can .J . Zool.50:1673-1676 . Commun.2(3):199-213 . Fostier,A. ,B .Breto n &B .Jalabert ,1979 . Idler,D.R. , &T.B .Ng ,1979 .Studie so n Stimulationhypophysair e del asécrétio n twotype so f gonadotropins frombot h oestradiol-17g chezl acarp ecommune , salmonan d carppituitaries .Gen .Comp . Cyprinus carpioL .An nEndocrinol .40 : Endocrinol.38 :421-440 . 83-84. Idler,D.R . &CM . Campbell,1980 .Gonado Fostier,A. ,B .Jalabert ,C .Campbell ,M . tropinstimulatio n ofestroge nan dyol k Terqui& B .Breton ,1981 .Cinétiqu ed e precursorsynthesi s injuvenil e rainbow liberationd e17 ahydroxy-20 &dihydro - trout.Gen .Comp .Endocrinol .41 :384 - progestérone parde sfollicule s deTruit e 391. arc-en-ciel,Salm ogairdnerii .C.R .Acad . Idler,D.R. , &T.B .Ng ,1980 .Pituitar y Sei.Pari sSer .III .292 :777-780 . gonadotropins ofbon y fish.Isolatio n Goldstein,I.J. , CE. Hellerman &J.M . andbiologica l action.Nint h International Merrick,1965 .Protein-carbohydrat e inter Congresso nAnima lReproductio nan d action.I .Th einteractio n ofpolysacch ArtificialInsemination . (Madrid)2 : arideswit h ConcanavalinA .Biochim . 339-345. Biophys.Act a97 :68-76 . Ishii,S.' ,& K .Yamamoto ,1976 .Demonstra Goncharov,B.F. ,E .Burzawa-Gerar d &A.A . tiono ffollicl estimulatin ghormon e (FSH) Kuznetsov,1980 .A studyo nth ehetero activityi nhypophysea l extracts ofvariou s geneity of thepituitar y gonadotropin vertebratesb y therespons e of theSertol i from sturgeon (Acipenser stellatus Pall.). cellso f the*chick.Gen .Comp .Endocrinol . Gen.Comp .Endocrinol .4-0 :342 . 29:506-510 . Grönlund,W. , 1969.Biologica l assayan d Jalabert,B ,1976 .I nvitr o oocytematura partial characterization of thegonadotro tionan dovulatio ni nrainbo w trout pic factorso f thepituitar y glando f (Salmogairdneri )norther n pike (Esox Pacificsalmo n (Oncorhynchus)M.Sc .Thesi s lucius)an d goldfish (Carassius auratus) University ofWashington . J.Fish .Res .Boar d Can.33 :974-988 . Hirose,H. , 1980.A bioassa y forteleos t Nath,P. , &B.I .Sundararaj ,1981 .Induct gonadotropinusin g the germinalvesicl e iono fvitellogenesi s inth ehypophy - breakdown (GVDB)o fOryzia s latipes oocyte sectomized catfish,Heteropneuste s invitro .Gen .Comp .Endocrinol .41 :108 - fossilis (Bloch):Effect so fpiscin ean d 114. mammalianhormones .Gen .Comp .Endocrinol . Huang,F.-L. ,C.-J .Huang ,S.-H . Lin,T.-B . 43:191-200 . Lo andH . Papkoff,1981 .Isolatio nan d Ng,T.B. ,& D.R . Idler,1978a ."Big "an d characterization ofgonadotropi nIsohorm - "Little"form s ofplaic evitellogeni c ones from thepituitar y gland ofpik eee l andmaturationa lhormones .Gen .Comp . (Muraenesox cinereus). Int.J .Peptid e Endocrinol.34 :408-420 . ProteinRes .18 :69-78 . Ng,T.B. ,& D.R . Idler,1978b .A vitello Hyder,M. ,A.V .Sha h &A.S .Hartree ,1979 . genichormon ewit h alarg ean d asmal l Methallibure studie'so nTilapi a III. form fromsalmo npituitaries .Gen .Comp . Effectso fTilapia npartiall ypurifie d Endocrinol.35 :189-195 . pituitary gonadotropic fractions onth e Ng,T.B. ,& D.R . Idler,1979 .Studie so n testeso fmethalibure-treate d Sarothero- twotype so fgonadotropin s frombot h donspirulu s (=Tilapia nigra).Gen .Comp . Americanplaic e andwinte r flounderpit Endocrinol.39 :475-480 . uitaries.Gen .Comp .Endocrinol .38 : Idler,D.R. , 1981.Salmo nprolacti n andit s 410-420. radioimmunoassay.OR ISymposiu m onFis h Ng,T.B. ,& D.R . Idler,1980 .Gonadotropi c Migration andReproduction .Abstracts .Toky o regulationo fandroge nproductio ni n Idler,D.R. ,L.'S .Baza r &S.J .Hwang. ' flounder and salmonids.Gen .Comp .Endo 1975a.Fis h gonadotropin(s).II . Isolat crinol.42 :25-38 . iono fgonadotropin(s ) from chumsalmo n Ng,T.B. ,CM . Campbell &D.R . Idler,1980a . pituitary glandsusin gaffinit y chromato Antibody inhibition ofvitellogenesi s and graphy. Endocrine Res.Commun .2(3) :215 - oocytematuratio n insalmo nan dflounder . 235. Gen.Comp .Endocrinol .41 :233-239 . Idler,D.R. , L.S.Baza r &S.J .Hwang ,1975b . Ng,T.B. ,D.R . Idler& M.P .Burton ,1980b . 12 Effects of teleost gonadotropinsan dthei r ovarian fragments ofth eplaic e (Pleuron- antibodies ongonada lhistolog y inwinte r ectesplatessa )b yhomologou spituitar y flounder.Gen .Comp .Endocrinol .42 :355 - extract.Gen .Comp .Endocrinol .42 :151 - 464. 154. Ng,T.B. ,D.R . Idler &J.G . Eales,1982 . Yondeda,T. ,& F .Yamazaki ,1976 .Purifica Pituitaryhormone s thatstimulat e the tiono fgonadotropi n fromchu msalmo n thyroidalsyste mi n teleost fishes.(I n pituitary glands.Bull .Jap .Soc . Sei. Press). Gen.Comp .Endocrinol . Fish.42 :343-350 . Pierce,J.G. ,M.R .Fait h &E.M .Donaldson , 1976.Antibodie s toreduce d S-carbo- Acknowledgeme nts xymethylated alphasubuni t ofbovin e luteinizinghormon e and their application Iwoul d like tothan kMichell e Saunders tostud y of thepurificatio n ofgonado fortypin g themanuscript . Thisresearc h tropin from salmon (Oncorhynchustshawy - was supportedb y theNatura lScience san d tscha)pituitar y glands.Gen .Comp . EngineeringResearc hCounci l GrantNo . Endocrinol.30 :47-60 . A-6732 toDRI . ContributionNo .49 1fro m Plack,P.A. , D.J.Pritchar d &N.W .Fraser , theMarin e SciencesResearc hLaboratory . 1971 .Eg gprotein s inco d serum.Natura l occurence and inductionb y injectionso f oestradiol-3-benzoate.Biochem .J . 121: 847-856. Relnboth,R .,1972 .Hormona l controlo fth e teleostovary .Amer .Zool .12 :307-324 . Stein,M.D. ,I.K .Howar d &H.J . Sage,1971 . Studieso na phytohemagglutini n fromth e lentil.IV .Direc tbindin g studieso fLen s culinarishemagglutini nwit h simple saccharides.Arch .Biochem .Biophys .146 : 353-355 StockellHartree ,A. ,1966 .Separatio nan d partialpurificatio n of theprotei n hormones fromhuma npituitar yglands . Biochem.J . 100:754-761 . Sundararaj,B.I. ,P .Nat h &E .Burzawa - Gerard,1982 . Synthesis ofvitellogeni n andit suptak eb y theovar y inth ecatfish , Heteropneustes fossilis (Bloch)i nresp onse to carpgonadotropi n andit ssubunits . Gen.Comp .Endocrinol .46 :93-98 . Sundararaj,B.I. ,& S.K .Nayyar ,1976 . Effects of carp gonadotropin onovaria n maintenance,maturatio n and ovulationi n hypophysectomized catfish,Heteropneuste s fossilis (Bloch).Gen .Comp .Endocrinol . 30:472-476 . Upadhyay,S.N. ,B .Breto n &R .Billard , 1978.Ultrastructura lstudie so nexperi mentalinduce d vitellogenesls injuvenil e rainbow trout.Ann .Biol . anim.Biochim . Biophys.18 :1019-1025 . Vaitukaitis,J.L. , 1978.Glycoprotei nhor mones and thiersubunit s -immunologica l andbiologica l characterization.In : "Structure andFunctio no f theGonadotro pins" (K.W.McKerns ,ed.) . PlenumPress . NewYork .pp .339-360 . Wiegand,M.D. ,& D.R . Idler (InPress) . Impairment ofearl y ovariangrowt hi n landlocked Atlantic salmonb ya nantibod y to Carbohydrate-richgonadotropins . Gen.Comp .Endocrinol . Wiegand,M.D . &R.E .Peter ,1980 .Effect s ofsalmo ngonadotropi n (SG-G100)o n plasma lipidsi n thegoldfish ,Carassiu s auratus.Can .J . Zool.58 :967-972 . Yaron,Z. ,& C .Barton ,1980 .Stimulatio n ofestradiol-17f ?outpu t fromisolate d 13 GONADOTROPIC FUNCTIONDURIN GTH EREPRODUCTIV ECYCL EO FTH EFEMAL ERAINBO W TROUT,SALM OGAIRDNE - RJ_,I NRELATIO NT OOVARIA N STEROID SECRETION :I NVIV OAN D INVITR OSTUDIE S Y. Zohàr,B .Breto n& A .Fostxe r Laboratoired ePhysiologi e desPoissons ,I.N.R.A. ,Campu sd eBeaulieu ,3504 2Rennes ,Franc e Summary lard etal. , 1978;Breto n etal. , 1982b).Whi lesom e studiesreporte d amoderat e increase An inviv o studydemonstrate d considerable inplasm a GtH levelsthroughou tvitellogenesi s changes,durin googenesis ,i nth eshort-ter m (Billard etal. , 1978;Breto n etal. ,1975 , (circadianan dultradian )plasm aprofile so f 1982b), anothe r studynote d aninitia lincrea thematurationa lgonadotropin ,'GtH .Th erela sefollowe db ya continuou sdeclin e (Bromage tedprofile so fcirculatin g estradlol-17ß(E- etal. , 1982). During the sameproces s thele 17(3)an d 17a-hydroxy-20ß-dihydroprogesterone velso fplasm aE„-17 ßan d testosteroneros e (17a,206-OH-P )wer emonitored . Thefunctio considerably,the ndecline dprogressivel yfro m nal significanceo fth edifferen t plasmaGt H thefina lstage so fvitellogenesi s (Billard patternswa sfurthe r explored invitr ousin g etal. , 1978;Fostie r et al., 1978;Whitehea d anovaria nopen-perlfusio n system. Itha s etal. , 1978;Scot t etal. , 1980,1982 ;va n beenconclude d thata t earlyovaria nrecrudes Bohemen &Lambert ,1981 ;Breto n etal. , 1982b); cencehigh-amplitud eplasm aGt Hpulses,.cons thisdecreas ewa saccompanie d bymarke d ele tituting thesprin g GtHrise ,migh t berela vations inth eGt Han d the17a ,206-OH- Ple ted toth eregulatio no fE„-17 pproductio na s velswhic hinduc eoocyte s tounderg omatura wella st o early stageso fgamet edevelopment . tionan d ovulation (Billard etal. ,1978 ; During exogenousvitellogenesi s theappearan Fostier etal. , 1978,1981b ;Breto n etal. , ceo fplasm aGt Hpulse so fmoderat eamplitud e 1982b;Bromag e etal. , 1982;Scot t etal. , might regulate theincreas e incirculatin g 1982).Wherea s 17a,20B-OH- Pdecrease sregu E_-178level san d their dailycyclicity .A - larlyafte r ovulation (Fostier etal. , 1981b), round thetim eo foocyt ematuratio n andovu GtH continues toris efo r2 t o3 mor eweek s lation,continuousl y highGt H levels (and pos (Jalabert &Breton , 1980). siblythei rdail ycycles )migh t beobligator y Suchlon g termhormona lprofile smigh tre signals,adjuste d toth emodifie d ovarian ste flect onlypartiall y thegonadotropi cfunc roidogenic potential,fo r themaintenanc eo f tion (Hontela &Peter ,1978 ;Zoha r etal. , theproductio n ofmaturation-inducin g steroid 1982)an d itsrelatio nt oovaria nsteroidoge (17a,200-OH-P )an d perhapsfo r thecontinu nesis. Thepresen tpape r summarizes aserie s ousinhibitio no f enzymeactivitie s (aroma- of experiments inwhic hw e studied,a tcer tase, 17-20lyase )interferin gwit h sucha taincharacteristi c stageso f thereproduc production. tivecycl eo f thefemal erainbo w trout (1) Keywords:rainbo wtrout ,GtH ,se xsteroids , theshor tter mprofile so fplasm amaturatio dailyfluctuations ,contro lo fsteroidogenesis - nalGtH ,E -17 8an d 17a,20ß-0H- P levelsu - singfre eswimmin gfis hfitte d witha cathe Introduction ter inth edorsa laort a (Zohar,1980 )an d (2) theregulatio no fovaria nsteroidogenesi sb y Thepituitar ycontro lo fovaria ndevelopmen t salmonmaturationa lGt Husin ga n invitr oo - insalmonid s involvesmaturationa lgonadotro varianopen-perifusio n system.Wit h sucha n pin (GtH)modulatio n ofovaria nsteroidogene approach,w ehope d toobtai nadditiona ldat a sis. Exposureo f theovarie st oGt H inviv o onth egonadotropi c functionan d itsrelatio n or invitr o enhancesth esecretio no festra - toovaria n steroid secretion. diol-17ß(E-17ß )durin gvitellogenesi s (Bil lard etal. , 1978;Breto n etal. , 1982a;Na - Resultsan d discussion gahama etal. , 1982),o r earlier (Idler& Campbell, 1980),an d of 17a-hydroxy-20ß-dihy- A.Earl yovaria nrecrudescenc e (March) droprogesterone (17a,208-OH-P )befor e theon seto foocyt ematuratio n (Fostier etal. , Theovarie so f thestudie d fishcontaine d 1981a;Suzuk ie tal. , 1981;Scot t etal. , 1982) germ cellsa tdevelopmenta l stagesrangin g The studyo f seasonalvariation so fplasm a from oogonia toprimar y oocytesundergoin g GtHan d sex steroids throughout thereproduc earlyexogenou svitellogenesis .Bleedin g the tivecycl eha sdemonstrate d atemporary'in cannulated femalesa t4 o r 1-hr intervalsfo r creasei nGt Ha tearl y stageso fovaria nre 24hr sreveale d short-term (1t o2 hrs) ,hig h crudescence (Billard etal. , 1978;Breto ne t amplitude (AGtH= u p to 100ng/ml )pulse si n al., 1982a,b),whil eE„-17 ß concentrations plasma GtH levels (fig. la)i n1 2ou t of2 6 werestil llo wan d either stable (Whitehead fish;i nth eother s theselevel sremaine d low etal. , 1978;Scot t et al., 1980;va nBoheme n and stable.I nmos t cases,onl yon epuls ewa s & Lambert,1981 )o r increasing slightly (Bil observed duringth e 12-hr samplingperiods . 14 E„-17(3level sstudie d atth e sametim ewer e lowan d stable (fig. la).However ,whe n invi troperifuse d ovarianpiece swer eexpose d to GtHpulse ssimila r tothos erecorde d inviv o (amplitude:5 0o r 100ng/ml ;duration : 1.5-2 hrs),w eobserve d an immediatean d continuous amplificationo fth eE —17 ßsecretio nrate , followed by itsslo wdecrease .E -17 0releas e wasamplifie d up toa tleas t 8hr safte rGt H application. Theplasm aGt Hpulses ,whic hwer eno tsyn chronousamon g thefish ,migh t constituteth e brief spring GtHris e infemal etrou t sampled monthly or every 2week s (seeIntroduction) . Our invitr odat a suggest thestimulator yrol t ofthes epulse s inregulatin gE„-17 ßproduc tion.Th e stable inviv oE -17f 3levels ,toge therwit hth eprolonge d invitr o enhancement ofE —17 ßsecretio n inrespons et oa Gt Hpuls e might indicatemaintenanc eb y theGt Hpulse s ofconstan t circulating concentrationso fE - •17 6ove r shortperiods .Th e high-amplitude gtHpulse scoul d also playa rol e inth enume rousoogonia lmitose san d oocyteméiose sob served inth eovarie so f thestudie d fishan d thought torequir egonadotropi nsupport . B.Exogenou svitellogenesi s (Junean dSeptem ber-October) At earlier stageso f exogenousvitellogene sis (June),GtH '(1- 3ng/ml )an dE -17 ß(0.5 - 1.5 ng/ml)level swer elo wan d constant.A t moreadvance d stageso f exogenousvitellogene sis (September-October)bloo d samplestake na t intervalso f4 , 1o r0. 5 hrs showed thatGt H levelsfluctuate d again inmos t of thefemale s whilebasa l GtHlevel swer eonly;-.slightl yhigh er than thoserecorde d inJune .Thes efluc tuations included short-term (1-3hrs )rando m GtHpulse s (fig. lb)o fmoderat eamplitud e (AGtH= u p to5 ng/ml )occurrin gwit ha rela tivelyhig hfrequenc y (upt o5 pe r 12hrs) . Althoughn oregula r synchronousdail ypatter n ofGt H levelswa snoted ,mos t of theGt Hpul seswer eobserve d during thephotophaS ean d early scotophase.Th eappearanc eo fGt Hpulsa - tilitydurin gvitellogenesi swa saccompanie d bya larg e increase inplasm aE„-17 ßu p tole velsrangin g from 6t o3 0ng/ml . Individual 8 10 12 14 E-17f 3profile s revealed continuous,relative Time ofDa y(hour ) ly synchronized dailyvariation s overriding theGt Hpulse s (fig. lb).A gradual increase Figure 1.Isolate d exampleso f individualpro inth eE„-17 ßlevel swa sobserve d during the fileso fplasm a GtH andE -17 ßo r 17a,20ß - day inal l studied females,followe d bya de OH-P levels infemal erainbo w trouta tdiffe creasestartin g atlat ephotophas eo rdurin g rentcharacteristi c stageso f sexualdevelop thescotophase ,dependin g on thefish . ment:a . earlyovaria nrecrudescenc e (March) b. advanced exogenousvitellogenesi s (October) Under invitr o perifusionconditions, asin and c.periovulation :maturatio n andovula gleGt Hpuls e induced an extended amplifica tionoccurre d during a48-h r period which tiono f theovaria nE„-17 g output;thi sres included thesamplin gtimes . ponsewa sreproduce d after eacho f threesuc cessiveGt Hpulses .Whe nth efrequenc y ofth e pulses increased,mimickin g intervalsobserve d Taken together,ou r inviv oan d invitr o iüjvivo (8,4 ,3 hrs) , thesuccessiv eovaria n resultsindicat e thatdurin g exogenousvitel responsesbecam e superimposed,leadin g toa logenesis,th eshort-ter m pulses inth eplas constantly elevated (fig.2) ,o r event oa ma GtH levelsar edetecte d by theovarie s continuously increasing,rat eo fE„-17f >produc whichrespon db y amplifying thesecretio nrat e tion. l 15 8 9 10 11 12 13 14 151 6 perifusion-time/hrs Figure2 .Profile s ofE -17f >secretio n froma n invitr operifuse d rainbow trout ovaryunder going exogenousvitellogenesis ;ovaria nfragment swer e either exposed to3 successiv eGt Hpul sesa t 4-hr intervals( • •) o rno t stimulated (o o). ofE„-17( 3fo r amuc h longer time thanth edu mid-scotophase.Th eonse to f theGt Hincrea rationo f theGt Hpulse .Th efrequenc y ofth e seswa s closely related toth ebeginnin go f GtHpulse smigh tdetermin ewhethe r theE„-17 ß thephotophas ean d thescotophase ,respecti concentration inth eplasm awoul d increase vely (fig.lc) ,suggestin g theregulator yro (athighe r frequencies),remai nconstan to r leo f thephotoperiod .I nfemale sundergoin g evendecreas e (atlowe r frequencies). Inan y oocytematuratio nan d inovulate dfemales , case,th echange s inth eE_-17 3 levelswoul d 17a,20B-OH- P levelswer e increasing orhigh , begradua lrathe r thanabrupt .Therefore ,a s showing daily fluctuations thatwer eeithe r proposed for thegoldfis hb yHontel a &Pete r synchronized withth eGt Hchange so r somewhat (1978), thechange si nth eshort-ter m pattern phase-shifted inrelatio n tothe m (fig.lc) . ofGt Hlevel sdurin gvitellogenesi s inth efe Parallel toth eonse t of thechang e inth e malerainbo w trout areprobabl y essentialt o inviv o GtHprofile s (whenth egermina lvesi themodulatio no fE„-17 ßsecretio nan d ofova cleapproache d theoocyt eperiphery) ,a mar riandevelopment .Th eappearanc e ofGt Hpulsa - ked modificationwa sobserve d inovaria n ste tility could regulate theimportan t gradual roidogenic capacity and responsiveness toGtH : augmentation inplasm aE.-17 0level sthrou (1) Thecontinuou s invitr o exposureo f fol ghout exogenousvitellogenesis ,an d anunequa l liclest ophysiologica l levelso fGt H (25-50 distribution ofplasm a GtHpulse sove r the24 - ng/ml)di d notresult ,a sa tpreviou sstage s hrperio dmigh t regulate thedail y cycleo f ofoocyt edevelopment ,i nprolonge d elevation E„-17(3a tadvance d exogenousvitellogenesis . ofE -17 ßoutput ;instead ,E — 17( 3secretio n increased briefly,the ndroppe d rapidly. (2) C.Oocyt ematuratio n and ovulation (December- Androgen release,whic hwa sonl y slightly sti January) mulated atearlie r stagesprobabl y duet oef ficient aromataseactivity ,wa s stronglyen Thegermina lvesicl e inth eoocyte so fth e hanced byGtH .Androge noutpu twa smaxima l studied femaleswa s terminatingmigratio no r whileE —17 ßproductio n decreased. (3)Conti theoocyte swer eundergoin g meioticmaturation . nuousadministration ,no ta pulse ,o f GtH ini Insom ecase sovulatio nwa s completed nomor e tiated a 17a,203-OH- Preleas e 12t o 17hr s than7 2hr sprio r to thebeginnin g of sampling. after thebeginnin g of thestimulation .A t Theprofile so fgonadotropi nwer equit edif that time,androge nproductio nrat edecreased , ferent compared topreviou s stages.Gt Hlevel s reaching alo wlevel ,whil eth e17a,20ß-OH- P werehighe r andfluctuate d ina continuous output continued itsdramati c increasewhic h rather than ina nepisodi cmanne r (fig.lc ). wasfollowe d by oocytematuration . A consistent dailycycl ewa s observed inal l Thesedat a suggest that increasing GtHle sampled fish,whateve r thesamplin g frequency velsi nviv o acto nfollicle s ready tounder (1,3 o r4-h r intervals).A relativesynchro gomaturatio n (1)b y stimulating theproduc nizationappeare d among individualprofiles . tiono fandrogen swhos earomatisatio n toE - Two distinct GtHsurge soccurred ,on ea tearl y 17ß isdepressed ,possibl y duet o theGt Hris e photophase (fig.lc )an d theothe r during the and (2)b ymodulatin g theenzym e system impll- 16 cated inth esynthesi s of thematuration-in bowtrou tdurin g spermiationan d oocytema ducing steroid (17a,20ß-OH-P),perhap sinvol turation.J . Endocr.85 :371-378 . ving theactivatio no f 20ß-hydroxysteroid de Fostier,A. ,B .Jalabert ,C .Campbell ,M .Ter - hydrogenase (Suzukie tal. , 1981)an d thedi qul& B .Breton , 1981a.Cinétiqu ed elibera minutiono f 17-20lyas eactivity .Th efunctio tioni nvitr o de 17a-hydroxy-20ß-dihydropro- nal significanceo f thedail ycycle s incircu gestéronepa rde sfollicule sd etruit earc - lating GtH should bestudie d inrelatio n too - en-ciel,Salm ogairdnerii .CR . Acad.Sei . varian steroidogenesis and tooocyt ematura ParisSer .III ,293 :777-780 . tionan dovulation . Fostier,A. ,B .Breton ,B .Jalaber t &0 .Mar - Whileth e secretiono fandrogen sb yperifu - cuzzi, 1981b.Evolutio n desniveau xplasma - sed follicles drops rapidly as17a,20ß-OH- P tiquesd el agonadotropin eglycoprotéiqu e output increases,circulatin g androgenlevel s etd el a17a-hydroxy-20ß-dihydroprogestero - during oocytematuratio n are stillhigh , nea ucour sd el amaturatio n etd el'ovula thoughdecreasin g (Campbell etal. ,1980 ; tionche zl atruit earc-en-ciel ,Salm ogair - Scott et al., 1982). Themaintenanc eo fhig h denii. CR. Acad.Sei .Pari sSer .Ill ,293 : levelso fandrogen s and lowlevel so fE -17 ß 817-820. might contribute toth eestablishmen t ofa n Fostier,A. ,C .Weil ,M . Terqui,B .Breto n& adequate steroid environment forth esynthesi s B. Jalabert,1978 .Plasm a estradlol-17ßan d of 17a,20ß-OH-Pan d foroocyt ematuration .I n gonadotropin during ovulation inrainbo w fact,th eefficienc y of the invitr o induction trout (Salmogairdner i R.).Ann .Biol .anim . ofbot hprocesse s byGt H isenhance d by tes Bioch.Biophys .18 :929-936 . tosteronean d reduced byE -17( 3(Jalabert , Hontela,A .& R.E .Peter ,1978 .Dail y cycles 1975; Jalabert &Fostier ,thi s symposium). inseru mgonadotropi n levels inth egold fish :effect so fphotoperiod ,temperatur e and sexualcondition .Can .J . Zool.56 : References 2430-2442. Idler,D.R . &C.M . Campbell,1980 .Gonadotro Billard,R. ,B .Breton ,A .Fostier ,B .Jala pin stimulationo f estrogenan dyol kprecur bert& C .Weil , 1978.Endocrin econtro lo f sor synthesis injuvenil erainbo wtrout . theteleos t reproductive cyclean d itsre Gen. Comp.Endocrinol .41 :384-391 . lation to external factors:Salmoni dan d Jalabert,B. ,1975 .Modulatio npa rdifférent s Cyprinidmodels .In :P.J . Gaillard &H.H . Steroidesno nmaturant sd el'efficacit é de Boer (Ed.): ComparativeEndocrinology .Else la 17a-hydroxy-20ß-dlhydroprogesteroneo u vier/North-Holland ,Amsterdam .P . 37-48. d'un extrait gonadotrope sur lamaturatio n Bohemen,Ch .G. ,va n &J.G.D . Lambert,1981 . intrafolliculairei nvitr ode sovocyte s de Estrogen synthesis inrelatio n toestrone , la truitearc-en-cie l Salmo gairdnerii.C . estradiolan dvitellogeni n plasma levels R.Acad . Sei.Pari sSer .D ,281 :811-814 . during thereproductiv ecycl eo f thefemal e Jalabert,B .& B .Breton ,1980 .Evolutio nd e rainbow trout,Salm ogairdneri .Gen .Comp . lagonadotropin e plasmatiquet-Gt Haprè s Endocrinol. 45:105-114 . l'ovulation chezl a truitearc-en-cie l Breton,B. ,B .Jalabert ,A .Fostie r &R . Bil (Salmogairdner iR. ) et influenced el aré lard, 1975.Etud esu rl ecycl ereproducteu r tention desovules .CR . Acad. Sei.Pari s del atruit earc-en-cie l etd el atanche . Ser. D,290 :799-801 . Effet devariation s expérimentales del a Jalabert,B .& A .Fostier ,1982 .Physiologi température.J . Physiol.Paris .70 :561-564 . calbasi so fpractica lmean s toinduc eovu Breton,B. ,Y . Zohar &R .Billard , 1982a.Pho- lation infish .Proceedin g of theinterna toperiod and gonadotropin controlo f there tional symposium onreproductiv ephysiolo productive cyclei nth efemal erainbo wtrout . gyo f fish,Wageningen ,Netherlands . Proceeding of the9t hInternationa lSympo Nagahama,Y. ,H .Kagaw a &G .Young ,1982 . sium onComparativ eEndocrinology ,Hong-Kong . Cellular sourceso f sex steroids inteleos t Inpress . gonads. Can.J . Fish.Aquat .Sei .39 :56-64 . Breton,B. ,A .Fostier ,Y . Zohar,P.Y .L eBai l Scott,A.P. ,V.J .By e& S.M . Baynes,1980 . &R .Billard ,1982b .Gonadotropin eglycopro - Seasonalvariation s inse x steroids offe téiquematurant e etoestradio l 173pendan t malerainbo w trout (Salmogairdner iRichard - lecycl ereproducteu r chezl atruit efari o son). J. Fish.Biol . 17:587-592 . (Salmo trutta)femelle .Gen .Comp .Endocri Scott,A.P. ,E.L .Sheldric k &A.P.F .Flint , nol. Inpress . 1982. Measurement of 17a,20ß-dihydroxy-4 - Bromage,N.R. ,C .Whitehea d &B .Breton ,1982 . pregnen-3-one inplasm ao f trout (Salmo Relationships betweenseru mlevel so fgona gairdneriRichardson) :Seasona lchange san d dotropin,öestradio l 178an dvitellogeni n responset osalmo npituitar y extract.Gen . inth econtro lo fovaria ndevelopmen t inth e Comp.Endocrinol .46 :444-451 . rainbowtrout :II .Th e effectso falterna Suzuki,K. ,B.I .Tamaok i& K .Hirose ,1981 . tions inenvironmenta l photoperiod.Gen . Invitr ometabolis m of4-pregnene s inova Comp.Endocrinol .I npress . rieso fa freshwate r teleost,th eay u (Ple- Campbell,C.M. ,A .Fostier ,B .Jalaber t &B . coglossus altivelis):Productio no f17a , Truscott,1980 .Identificatio nan dquanti 20ß-dihydroxy-4-pregnen-3-one and its5 3 ficationo f steroids inth eseru mo frain reducedmetabolites ,an d activationo f3ß - 17 and,2018-hydroxysteroi ddihydrogenase sb y treatmentwit ha fis hgonadotropin .Gen . Comp.Endocrinol .45 :473-481 . Whitehead,C , N.R.Bromag e& J.R.M .Forster , 1978.Seasona lchange s inreproductiv efunc tiono fth erainbo w trout (Salmogairdeni ) J. Fish.Biol . 12:601-608 . Zohar,Y. ,1980 .Dorsa l aorta catheterization inrainbo w trout (Salmogairdneri )I .It s validity inth e studyo fbloo d gonadotropin patterns.Reprod .Nutr .Develop .20 :1811 - 1823. Zohar,Y. ,B .Breto n& R .Billard ,1982 . Short-term profileso fplasm a gonadotropin levelsi nth efemal erainbo w troutthrou ghout thereproductiv e cycle.Gen .Comp . Endocrinol.46 :396 . 18 EXISTE-T'IL PLUSIEURS GONADOTROPINES(GTH) CHEZ LES POISSONS ? DONNEES BIOCHIMIQUES ET VITEL- LOGENESE EXOGENE. Elisabeth BURZAWA-GERARD. Laboratoire de Physiologie générale et comparée du Muséum national d'Histoire naturelle ; Laboratoire d'Endocrinologie comparée associé au C.N.R.S., 7 rue Cuvier, 75231 Paris cédex 05, France. Summary satisfaisante. La séparation par Chromatogra Since 1963, it has been suggested that one phie d'affinité d'une gonadotropine pauvre en glycoproteic gonadotropin (cGTH) is present carbohydrate, l'hormone vitellogénèse et d' in the carp pituitary. Further biological une gonadotropine riche en carbohydrate, and biochemical data have confirmed this hy l'hormone de maturation, à partir d'hypophyses pothesis. However, if cytological data on de différents poissons téléostéens (Idler et GTH cells in the pituitary, circulating level Ng, 1979 ; Ng et Idler, 1979) suggère au con during the reproductive cycle and exogenous traire, l'existence de deux gonadotropines. vitellogenesis are considered, the hypothe L'hormone de maturation présente un spectre sis of one GTH is controversial. Affinity d'activité gonadotrope très étendu sur la ga- chromatography had led to the separation of métogénèse incluant, chez la femelle, la vi two distinct GTHs. One hormone, poor in car tellogénèse, la maturation et l'ovulation. bohydrate, acts on the vitellogenin incorpo L'hormone de vitellogénèse serait nécessaire ration in the oocyte. The second, rich in à une étape très précise de celle-ci qui est carbohydrate is able to stimulate several l'incorporation de la vitellogénine dans aspects of the gametogenesis. Such a discre l'ovocyte. Elle serait, en revanche, inactive pancy will be review relative to the bio sur la stéroidogénèse, la maturation et l'ovu chemical data of two fish GTHs and to the lation . activity of cGTH on exogenous vitellogenesis. Une disproportion importante apparaît donc Biochemically fish GTHs have shown a high entre les potentialités gonadotropes dé ces degree of homology with mammalian gonadotro deux hormones et l'on peut s'interroger sur pins especially with the lutropin. In some sa signification. Dans le présent article, hypophysectomized fish, cGTH initiates the nous nous proposons d'éclaircir le problème synthesis of vitellogenin and its incorpora de l'existence d'une ou plusieurs gonadotro tion in the oocyte by pinocytosis. In the eel, pines en posant deux questions : (1) les don a (Vg) radioimmunoassay has been developped. nées biochimiques structurales sur la GTH de Plasma level of vitellogenin can be determi carpe mettent-elles en évidence la présence ned as well as the rate of yolk incorpora d'une deuxième hormone ? (2) la cGTH possède- tion in the oocyte. Thus, cGTH is potent on t-elle intrinsèquement une activité sur la exogenous vitellogenesis. A pituitary factor, vitellogénèse exogène? able to act on ovarian growth which is not strictly speaking a gonadotropin, make up a working hypothesis. I - Données biochimiques sur les GTH de carpe (cGTH) et l'esturgeon (aci-GTH) Keywords .- number of fish GTH, biochemical characterizations, exogenous vitellogenesis, a) Structure eel vitellogenin RIA. La cGTH et l'aci GTH sont des glycoprotéi- nes de poids moléculaire de 28000 à 32000 Introduction daltons. La composition globale en acides aminés distingue ces deux hormones. Deux L'hypothèse de l'existence d'une seule sous-unités, respectivement identifiées en hormone gonadotrope chez la carpe a été pro type a et ß sont associées par des liaisons posée en 1963 par Fontaine et Gérard. Le non-covalentes pour former le dimère hormo développement des recherches biochimiques et nal biologiquement actif. Un degré d'isologie biologiques à partir de la glycoprotéine élevé ap u être mis en évidence par la compa gonadotrope hypophysaire (cGTH) a toujours raison des séquences partielles d'amino-aci- apporté des éléments en faveur de cette hy des entre la cGTHa et la sous-unité a d'ori pothèse chez la carpe (Cyprinus oarpio) et gine mammalienne d'une part, et la cGTHß et chez l'esturgeon (Aoipensev stellatus) la LHß bovine d'autre part (Jolies et al., (Burzawa-Gérard et Fontaine, 1965 ; Burzawa- 1977). L a parenté de la cGTHß avec une sous- Gérard, 1974 ; Burzawa-Gérard et al., 1975 ; unité LHß est confirmée par des recherches Fontaine et Burzawa-Gérard, 1978). Si l'on radioimmunologiques réalisées à partir d'anti considère les données cytologiques relatives sérum cGTHa et cGTHß (Burzawa-Gérard et al, aux cellules gonadotropes de l'hypophyse, la 1980). détermination des taux circulants de GTH au cours du cycle de reproduction, ainsi que 1' b) Site d'association des sous-unités étude de la vitellogénèse exogène (réf. dans (1) Des associations à partir de sous-unités Burzawa-Gérard, 1981) l'hypothèse d'une gona hétérologues ont été obtenues (Burzawa-Gérard dotropine unique ne se révèle pas toujours et Fontaine, 1976 ;Burzawa-Gérar d et Goncha- 19 rov, 1979). Entre lessous-unité s decGT He t derésume rétablissen tun erelatio nd'homolo - d'aci GTH,un edoubl e hybridation estpossi gieave cle sgonadotropine s desVertébré se t ble. Lesmême s schémas stériques surce scou plusparticulièremen t avecl agonadotropin e ples a et( 3son t donc présents pour permettre detyp eLH .Jusqu' àaujourd'hui , l'existence les liaisons hydrogènes d'une structure qua d'unetell erelatio nn' ap uêtr emontré e avec ternaire stable. Uneuniqu e hybridationes t l'hormone devitellogénès epauvr ee ncarbohy seulement possible entre lasous-unit é LHa dratedécrit epa rIdle re tse scollaborateurs . et lessous-unité s ßd ecGT H oud'ac i GTH.I l Lamis ee névidenc ed el aprésenc ed'un e est difficile dedéfini r sil'absenc e d'hy structure quaternaire decett ehormone ,ave c bridation opposée estdu eà u n empêchement uneS U "ßd evitellogénèse " seraitu nargu stérique ouà d'autres facteurs comme l'en mentmajeu rpou rprouve r l'existence dedeu x vironnement ionique dusit e d'association de gonadotropines. l'une oul'autr e sous-unité. Ilparaî t impé ratif quel astructur e dusit e d'association L'hétérogénéité vis-à-visd el acharg eo u de lasous-unit é a reste complémentaire de duconten uglycaniqu e existeauss ipou rle s celui del asous-unit é ßpou r que l'hormone gonadotropines LH etFS H (Réf.dan sWakaba - gonadotrope puisse être formée. Uneévolu yashi, 1980) ;ell esembl e êtreche zle s tion synchrone dessite s d'association sura poissonsbeaucou pplu simportante .L'étud e et 3 ad ûs eproduir e (Fontaine etBurzawa - del aséquenc ea uN-termina ld el asous - Gérard, 1978). (2 )De sinformation s relati unité a del acGT Ha montré ,qu'e nfait , ves à l'influence del'un e oul'autr e sous- cetteparti ed e l'hormone existaitsou sdeu x unité aucour s de l'association ontét é ap formes,l'un en epossédan tpa sl enonapepti - portées parl amolécul e hybride (bLH a - dea uN-termina lpa rleque lcommenc e lasé cGTHß ) . Desétude s parradioimmunologi eon t quenced e l'autre.Cett ecaractéristiqu e est suggéré quel anatur e del asous-unit é a aussi signaléepou r lessous-unité s a mamma- pourrait jouer unrôl e surl a conformation liennes (Réf.dan sJolie se tal. , 1977).Le s de lasous-unit é ßdan s ledimèr e (Dufour, GTHsson tobtenue sà parti r d'un grandnom 1980). Le sdeu x sous-unités sontpa rcontr e bred'hypophyse s dontl econten ue nGT Hpour impliquées pour déterminer lavitess e d'as raitêtr eà'de sstade svariable sd esynthèse . sociation àun etempératur e donnée (Marche- L'hétérogénéité despréparation s hormonales lidon etal. , 1979). Uneactivit é gonadotro obtenuespa r lesdifférent sauteur se nserai t pe intense del amolécul e hybride (bLHa - peut-être lereflet .L astructur ed el'hormo cGTHß ) es tobtenu e chez unamphibien . Elle nelibéré e aumomen td e lasécrétio nhypophy - est 4à 6 fois plus active quel acGT Ho u saireche z lespoisson sn' apa sét éétudiée . que laL Hbovine , mais chez un téléostéen, Ellepeu têtr edifférent e des aform ed e son activité gonadotrope estéquipotent e à stockage,de sinformation sdan sc esen spour celle decGT H (Fontaine-Bertrand et al,1981)• raientêtr eintéressantes . c) Micro-hétérogénéité del acGT H etd e II Donnéessu rl'actio n del acGT Hsu rl a l'aciGTH . vitellogénèseexogène . (1)L ecomportemen td e lacGT He nChromato L'activité de lacGT H surl avitellogénès e graphied'affinit é par laconcanavalin e met exogène aét érecherché e surdiver sreceveurs . enévidenc eun eaffinit é variabled e l'hor monepou r lalectine .Quatr e fractionspeu (1)Che z Carassius auratus hypophysectomisé, ventêtr eséparée sdon tun e (FractionI-cGTH ) le traitement parl acGT H fait apparaître un quin'es tpa s liée ;ell ereprésent e environ accroissement important del'ovaire . L'ana 5 %de sprotéines .L aparti e glycaniqued e lyse cytologique montre l'apparition d'uno u lacGT Hpeu tdon c être considérée commeva plusieurs anneaux devésicule s vitellines riable (Burzawa-Gérard, 1982). (Burzawa-Gérard, 1974). (2) Différentes préparations d'aci GTHpos (2)Che z Heteropneustes fossilis hypophysec sédant lemêm e poids moléculaire etle s tomisé, l'effet del acGT H surl a vitellogé mêmes amino-acides N-terminaux ontét éétu nèse exogène aét éplu s particulièrement diées parisoélectrofocalisation . De nom étudié. Lestau x circulants de vitellogénine, breux composants apparaissent dont les. mesurés parl edosag e duphosphor e alkali- points iso-électriques s'étendent de4, 0à labile dans lesérum , augmentent etde sovo 6,5. Ilsson t tous actifs surl etes td e cytes austad e III,caractéristiqu e del a maturation desovocyte s decrapau d invitro . présence devitellus- , apparaissent. Desdose s Il nes'agi tpa sd'u nartefac t depurifica faibles d'hormone ontic iét éutilisée s tion carl'hétérogénéit é del acharg ede s (0,08 à0,4 5pg/g ,d epoid s corporel/injec molécules hormonales estauss i observée tion) (Sundararaj etal. , 1981). dans lesextrait s hypophysaires. Cesdiffé rences neson tpa sdue s nonplu s àun edif (3)Che z Gobius niger hypophysectomisé, férence dans lenombr e derésidu s acidesia - l'action del acGTH , ainsi quecell e del a lique (Goncharov etal. , 1979; Kuznetso v Fraction I-cGTH (nonlié e surl a concanava étal., 1982). line) ontét érecherché s surl'ovair e àl a même dose de0, 4u gpa rg d epoid s corporel Lesdonnée s structuralesqu enou svenon s etpa rinjection . L'ovogénèse est fortement 20 stimuléedan s lesdeu xcas .L'activit é rela tradiol 17ß - (2) 100m gd'oestradio l 17 ß] tive àl avitellogénès eexogèn ea ét écarac sontcapable sd'inhibe r laliaiso n 125jy g. tériséepa r l'apparition de lavitellogénin e ASVg .Le ssérum sde sanimau x témoins l'in circulante (immunoprécipitationave cu nsé hibentfaiblemen tpou rle sfemelle se tpa s rumantivitellogénin e de Gobius niger) et dutou tpou r lesmâle s (Fig. 1).D etrè s parl'identificatio n denombreuse s vésicules faiblesquantité s deV gpourraien têtr epré depinocytose ,sign ed'internalisatio n deV g sentesche zle sanguille s femellesargentées . dans lesovocyte sparvenu s austad e II+dé Desquantité s très importantes sonte nrevan finiepa rL eMen n (1979).Le s traitements cheobtenue spou r lestraitement s à l'oestra par cGTHo upa r laFractio nI-cGT H induisent diol 17ß (Tableau 1). Desextrait s d'ovaires lamêm e stimulation sur lavitellogénès e (Le Menne tBurzawa-Gérard , 1982). Tableau 1. Taux circulantd eV gdan sl e sérumd'anguille s témoinse ttraitée sà (4)Che z Anguilla anguilla, lavitellogénès e l'oestradiol 178. exogènea ét érecherché epa rde s techniques biochimiques.Elle son te upou robjecti fl a misea upoin td'u ndosag e radioimmunologique Vgg équivalent/u l (RIA)d el avitellogénin e (Vg)d e l'anguille afind emettr ee névidenc el'apparitio n de <0,000 5 laV gplasmatiqu eaprè s stimulationd e l'hor témoinsO < 0,05 mone gonadotrope etd edétermine r letau x oestradiol1 7 (15mg)241,5 7 (207,97-280,60) d'incorporation dans l'ovaire par l'appari oestradiol 17ß(100mg) 688,6 (609,41-778,15) tiond evitellu s(immunoréactif) . -L avitellogénin e d'anguille aét épurifié e àparti rd e sérumsd'anguille s traitéesà d'anguille ayantde sRG Sd e 1,4 (témoin)e t l'oestradiol 173 qu ia pou r effetd estimu de 12,8o u5 0 (ayantreç ude sinjection sd ' lerl asynthès e deV gpa r lefoie .Deu xéta extraitshypophysaire s decarpe )on tét é pesd epurificatio n ontét énécessaire s (1. préparés aprèsuitraeentr^fugation .L'inhibi filtration duséru m surUltroge lA 4 ;2 . tiond el aliaiso n (125I-Vg; A SVg )es tin Chromatographie surhydroxylapatit e HAUl complète,mai sl aréactio n croisée entrel e trogele ntampo nphosphat ep H6, 8 despro vitelluse tl aV ges timportante . L'extrait téinesexclue spou rK D 0,4 à0,6) .L aposi d'ovaire témoinn'entraîn epa s d'inhibition. tiond el avitellogénin e aucour sd ece s fractionnements aét éobtenu epa r ledosag e duphosphor e alkali-labile selon latechni Eel ovarian extractGSI1- 4 quedécrit epa rWallac ee tJared , 1968.L a Vges tobtenu e aprèsapplicatio n d'undeu xième gradient (tamponphosphat ep H6, 8 de 0,2 Mà 0, 5 M). L'électrophorèseanalytiqu e (PAGE)d el afractio n obtenuequ irévèl eun e uniqueband ed eR f =0,20 -0,2 1 mete névi denceu néta td epuret é satisfaisant.C e matériela ét éutilis épou robteni r desan tiserumsche zl elapin . =U nRI Ad el avitellogénin e aét émi sa u pointaprè smarquag e del aV gà l'iod e( 125I) selon lestechnique s classiques.De ssérum s fig equivalent of /u\ of ovarian d'anguille [(1)traitemen tpa r 15m gd'oes - vitellogenin/tube extracts/tube Figure 2 - Inhibition de la liaison (125I Vg; AS Vg) par des extraits d'ovaire d'anguille. Une estimation du taux d'incorporation de la Vg dans l'ovaire est possible (Tableau 2). Tableau 2. Estimation de la quantité de vi tellogénine incorporé dans 1'ovaire en fonc tion du RGS. Extrait d'ovaire Vg (pg équivalent/g 0.4 r d'anguille pour des _ d'ovaire) /Ug equivalent of 10" 10 ' 1 10 100 RGS de : 'vitellogenin/tube fx\ of serum/tube 1,4 < 0,2 12,8 99170(90227-122785) 125 50 925900(745000-1132000) Figure 1 - Inhibition de la liaison I Vg; as Vgpar différents sérums d'anguille. 21 Desrésultat spréliminaire s ontét éobte Idler,D.R . etT.B .Ng , 1979.Gen .Comp . nusche zl'anguill e aprèsde straitement sà Endocrinol.,38 ,421-440 . lacGT Hd'un epart ,e tà l'oestradlo l 17 g Jolles,J. , E.Burzawa-Gérard ,Y.A .Fontain e d'autrepart .L amesur ed utau xd eV gplas matiqu ed el'incorporatio n deV gdan s1'ovo etP . Jolles, 1977.Biochimie ,59^ ,893-898 . cyteon tconfirm é d'unepar tl avalidit éd u Kuznetzov,A.A. ,B.F .Goncharo v etE .Burza RIA,e td'autr epar tl acompétenc ed el a wa-Gérard, 1982.Gen.-Comp .Endocrinol , cGTHsu r lavitellogénès eexogène . (souspresse ). LeMenn ,F. , 1979.Comp .Biochem .Physiol. , Conclusion 62A,495-500 . Cesdernier srésultat smontren tqu el a LeMenn ,F . etE .Burzawa-Gérard ,1982 . cGTHes tactiv e sur lavitellogénèse ,d e Soumisà Gen .Comp .Endocrinol . plusl'ensembl e desdonnée srésumée sic i sonttoute se naccor dave c l'hypothèse del a Marchelidon,J. , R.Salesse ,J . Garnier,E . présence d'unehormon e gonadotropeunique , Burzawa-Gérard etY.A . Fontaine,1979 . chezl acarpe .L'existenc e présuméed'un e Nature, 281,n °5729 ,314-315 . secondegonadotropin e ayantsu r lagonad e une actionauss ilimité equ el'es tcell ed e Ng,T.B .e tD.R . Idler, 1979.Gen .Comp . 1'incorporationdan s1'ovocyt epeu têtr ein Endocrinol., 3£,410-420 . terprétéedan sl esen sd'un eactio ncomplémen Ng,T.B. ,D.R . Idler etM.P .Burton ,1980 . taired el'hypophyse,-dan sc ecas,l'hormon ed evi Gen.Comp .Endocrinol. , A2_,355-364 . tellogénèsen eserai tpa sun e gonadotropine ausen sstric t .De srecherche s concernant Sundararaj,B.I. ,P .Nath ,E .Burzawa-Gérard , lestau xcirculant sd el'hormon e devitello 1982.Gen .Comp .Endocrinol . génèsedoiven têtr e réaliséesafi nd emieu x Wakabayashi,K. ,1980 .In. :S .Ishi ie tal . comprendre sonrôl ephysiologique . Ed.Hormones ,adaptatio n andevolution , Jap.Sei .Pres sTokyo ,271-279 . Références Wallace,.R.A .e tD.W . Jared, 1968.Can .J . Biochem., 46,953-959 . Burzàwa-Gérard,E. , 1974.Mémoire sd uMuséum , sérieA ,Zool. ,86_ ,1-77 . Burzawa-Gérard,E. , 1981.Océanis ,6_ ,655 - 676. Burzawa-Gérard, E., 1982.Can .J .Fish . Aquat.Sei. , 39,80-91 . Burzawa-Gérard, E.e tY.A .Fontaine ,1965 . Gen.Comp .Endocrinol. ,5_ ,87-95 . Burzawa-Gérard,E .e tY.A .Fontaine ,1976 . C.R .Acad .Sei. , 282,séri eD ,97-100 . Burzawa-Gérard,E .e tB.F . Goncharov,1979 . Gen.Comp .Endocrinol. ,40 ,338 . Burzawa-Gérard,E. ,S .Dufou re tY.A .Fon taine, 1980.Gen .Comp .Endocrinol. ,41 , 199-211. Burzawa-Gérard,E. ,B.F .Goncharo ve tY.A . Fontaine, 1975.Gen .Comp .Endocrinol. ,27 , 296-304. Dufour,S. , 1980.Thès ed e 3ecycle ,Paris . Fontaine,Y.A . etE .Burzawa-Gérard ,1978 . In"Structur e andfunctio no fth egonado tropins".K .Ma cKern sed. ,361-380 . Fontaine,Y.A . etE .Gérard , 1963.C .R . Acad. Sei., 256,5634-5637 . Fontaine-Bertrand, E.,J .Marchelidon ,C . Salmon etY.A .Fontaine , 1981.C .R .Acad . Sei., 292,507-510 . Goncharov,B.F. ,E .Burzawa-Gérar d etA.A . Kuznetsov, 1979.Gen .Comp .Endocrinol. ,40 , 342. 22 POSITIVETESTOSTERON E FEEDBACKO NGONADOTROPI CHORMON E INTH ERAINBO WTROU T L.W.Crim ,D.M . Evans Marine SciencesResearc hLaboratory ,Memoria lUniversit y ofNewfoundland , St.John's , Newfoundland,Canad a Summary Testosterone increases thegonadotropi c of immaturemale s receivingT-silasti ccap hormone (GtH)conten t ofpituitarie s inth e sulewa s correlatedwit h theelevate d juvenilerainbo w trout. A silastic capsule plasmaGt Hlevel ;i nfemales ,however , was themos teffectiv emetho d ofadminis ovarian stimulationwa sno tobserve d in tering testosterone comparedwit h cocoa conjunctionwit h theGt Hreleas e indicating butter or castoroil . Whenpituitar yGt H a deficiency in theendocrin emilie u reached themicrogra m level (equivalent necessary forprematur e oocytedevelopment . with thepituitar y GtH leveldurin g the spawning season)Gt Hreleas e andstimu lationo f the testesi nth eimmatur emal e was observed. Previously,Cri m& Evans ,197 9showe d thati n thejuvenil e trout intraperitoneal (IP)testosterone(T )administratio n in cocoabutte r increases thepituitar y GtH content. However,i ncompariso nwit h the GtH contento fpituitarie s obtained from precocious salmonpar r (Crim& Evans ,1978 ) we realized that thestimulatio n ofGt Hb y T in theimmatur e troutwa srelativel y small (nanograms ofGt Haccumulated) . In thepresen tinvestigatio nmethod s ofadmin istering testosteronewer e studied tode termine amor e effectivemanne r ofstimu latingGt Hproductio n inth eimmatur etrout . Threedifferen tmethod s ofadministerin g T toyearlin g troutwer eexamined : 1)multipl e IP injections ofT i n castoroil . 2)a singl e IPimplan t ofT i n cocoabutter . 3)a singl eI P implanto fT i na J J ASON DJ FMAM WEEKS MONTHS silasticcapsule . TIME AFTER TREATMENT Figure 1show s thata smal lincreas ei n fig.1 .Pituitar yGt H contenti njuvenil e pituitary GtHfollowe d Tadministratio ni n troutfollowin gT administratio n incoco a both cocoabutte r and castor oil. Ttreat butter (20u g T), castoroi l (bi-weeklyin mentvi asilasti c capsuleproduce d amuc h jections of2 0p g T), and silastic capsules greater increasei n thepituitar y GtHcon containing approximately 3m gT . Thesea tent (microgramsv snanograms) . In sonalprofil efo rpituitar yGt Hconten ti n addition toth eeffec t ofT upo npituitar y theprecociou smal e salmonpar ri sprovide d GtH,th einfluenc eo fsteroi d'administra forreference . tiono nplasm a GtHan d gonadalchange swer e alsostudied . Ifpituitar yGt Hreache d only thenanogra m leveln oincreas ei n References plasmaGt Hwa s observed and theimmatur e gonadsremaine d undeveloped. Incontrast , Crim,L.W. ,D.M .Evans ,1978 . Can.J .Zool . thedramati c increasei npituitar yGt H 56:1550-1555 . resulting from theT treatmentvi asilasti c Crim,L.W. ,D.M .Evans ,1979 . Gen.Comp . capsulewa sassociate dwit hGt Hreleas e (at Endocrinol.37 :192-196 . 16week splasm aGt Hincrease d >3-fold com paredwit h sham controlvalue s- dat ano t shown). Onset ofspermatogenesi s anda significant increase ingonadosomati c index 23 PURIFICATIONAN DBIOLOGICA L CHARACTERIZATION OFSALMO NPROLACTI N P.Prune t INRA,Laboratoir e dePhysiologi ede sPoissons ,Campu sd eBeaulieu ,3504 2Rennes ,Franc e andDepartmen t of Zoology and CancerResearc hLaboratory ,Universit y of California,Berkele y CA,US A Keywords: chinooksalmon ,prolactin ,osmore - lactogenic activity of thishormon e inth e gulation. mammaryglan d culturesyste m (Prunet etal. , 1979;Houdebin e etal. , 1981). Chinooksalmo n Introduction prolactin exhibited aclea r lactogenic acti vity,2 0time s lower thanovin eprolacti nac Theexistenc e inth eteleos t pituitary ofa tivity (performedb yDr .L.M .Houdebine ,INR A protein similar tomammalia nprolacti nha s Laboratoire dePhysiologi e del aLactation , beenwel l established.Thi shormon e isendo Jouy-en-Josas,France) . wedwit hosmoregulator y activity (seerevie w The sodium-retaining activity of chinook Elisor,1979 ;Clark e s Bern,1980 )an dlac salmonprolacti nwa sassesse d inth eFundulu s togenicactivit y inth erabbi tmammar y gland bioassaywhic hwa sdemonstrate d tob especi (Prunet etal. , 1979;Houdebin e etal. , 1981). fico fprolacti nactivit y (Grau,E.G. ;Prunet , Inthi scommunicatio nw epresen t thepurifi P.;Nlshioka ,R.S. ;Gross ,T. ;Bern ,H.A. ; cationan d thecharacterizatio n ofprolacti n submited forpublication) .Salmo nprolacti n from chinook salmon (Oncorhynchustschawyts - showed amaximu m activity for injected doses cha). aslo wa s4 ng/m lan d appeared tob e 100ti mesmor epoten t thanovin eprolactin .However , Results athigher 'dose sth erespons eobtaine dwa s muchlower ,althoug h itwa ssignificantl y Purificationan dbiochemica l characterization higher thanth econtrol . Prolactinfro m chinooksalmo n pituitaries Conclusion .» waspurifie d byaci dacetoni c extraction,fol lowedb y salineprecipitation , isoelectric Chinook salmonprolacti nwhic happeare d to focusing onio n exchangecolum n and gelfil bea basi cprotei n (pi> 9 )wa sobtaine d ina tration. highly purified form.Th eprolacti ncharacte r The purity ofth efina lproduc twa sas ofthi sprotei nwa s established by itslacto sessedb yNH.-termina laminoaci d analysisre genicactivit y inth erabbi tmammar y gland vealing thepresenc eo fonl yon eamin oaci d and itssodium-retainin g activity inFundulu s (performed byprofesso rH .Papkoff ,Hormon e heteroclitus. ResearchLaboratory ,Universit y of California SanFrancisco ,USA) ,an db y SDSelectrophore References siswhic h showed only oneband .Salmo nprolac tinappeare d tohav ea molecula rweigh to f Clarke,W.C . &H.A .Bern ,1980 .Comparativ e 22.000daltons .I nelectrophoresi sa tp H 4.5 endocrinology ofprolacti n in"Hormona lpro thishormon emigrate d asa majo rban d (R=0.5 ) teinan d peptides" (C.H.Li ,ed.) , 8:105-197. followed bya secon d band of lower intensity, Academic Press,Ne wYork . whereasa tp H9. 3 salmonprolacti ngav en o Ensor,D.M. , 1978."Comparativ e endocrinology band.Th edemonstratio no f thepurit y ofth e ofprolactin" .Chapma nan dHall ,London . salmonprolacti npreparatio nb y theabov edes Houdebine,L.M. , S.W. Farmer &P .Prunet , cribed technicsallowe du s toconside r the 1981. Inductiono frabbi t casein synthesis secondban d obtained inelectrophoresi sa t inorga ncultur eb ytilapi aprolacti nan d pH4. 5 asanothe r formo fprolacti nrathe r growthhormone .Gen .Comp .Endocrinol. ,45 : thana contaminant .Th eelectrophoresi s ofth e 61-65. rostralpar t of cohosalmo npituitarie si n Prunet,P. ,J .Djian e& B .Breton ,1977 .Ap thepresenc eo fSD S ora tp H4. 5 revealed the plicationo fa radiorecepto r assay forlac existenceo fa majo r proteinrunnin g atth e togenichormon et oth edetectio no ffis h samepositio na sth epurifie d Chinooksalmo n prolactin likehormone .J .Endocrinol. ,73 : prolactin. 391-392. Prunet.P. ,L.M .Houdebine ,C .Delouis ,& Biological characterization B.Breton ,1979 .Stimulatio n ofmil ksynthe sisi nth erabbi tb yfis hpituitar y extract. Throughout thepurification ,prolacti nbio - J. Endocrinol.,83:393-400 . activitywa sestimate d byradiorecepto r assay for lactogenichormon e (Prunet etal. , 1977). However,considerin g theheterologou snatur e ofthi sassay ,i twa snecessar y toconfir m the 24 MORPHOMETRIC ANALYSIS OF STRUCTURAL CHANGES IN Rhamdia hilarii GONADOTROPHIC CELLS DURING THE REPRODUCTIVE CYCLE Maura V.Val-Sella, Antonio Sesso Department of General Physiology, Institute of Biosciences and Laboratory of Molecular Pathology, Faculty of Medicine, University of Säo Paulo, Säo Paulo, Brazil. Hypophyseal gonadotrophic cells (GTH the RER increases. From maturing to cells) from Rhamdia hilarii in differ spent gonadal stages surface-to-vol ent phases of the gonadal cycleexhibi t ume ratio (specific density) analy conspicuous structural variations sis shows that the RER cisternae be when analysed in thin section. Such come extremely dilated while its qualitative analysis clearly shows total surface area remains unchanged. dilation of the rough endoplasmic re ticulum (RER) cisternae but does not reveal variation in the relative volumes of secretory granules (SG)an d of the remaining cytoplasmic elements. GTH cells from animals with matur ing, mature and spent gonads were studied by morphometric analyses in thick (0.25 pm) and thin araldite sec tions (Table I). In all stages, the Golgi apparatus and mitochondria occupy a very small fraction of the cytoplasmic volume. During the maturing gonadal phase the majority of GTH cells contain many SG occupying the greater part of cyto plasmic volume. The RER cisternae are moderately distended. In the mature gonadal phase, the mean number of SG Fig.1 Hypophyseal GTH cells from Rhamdia increases slightly while the RER cis hilavii with mature gonads. (*) denotes RER ternae are more dilated (Figure D.In cisternum in the perinulear region which the spent gonadal phase both the num probably coalesce with the "small", juxta ber and relative volume of the SG posed RER elements (arrows). Note polymorphic decrease while the relative volume of secretory granules. X 12,000. Table 1.Morphometri c data on gonadotrophic ells from Rhamdia hilarii (x Gonadal stages Statistical analysis maturing mature spent it (it Nuclear V (ura3) 78.3± 6.36 73.6 ± 7.76 70.2 ± 4.J4 0.40 27.8 Cytoplasmic V (um3) 262.81 20.86 264.7 ± 18.61 366.? ± 3*.«5 4.13 114.5 Vv of hyaloplasm(% ) 20.2± 1.38 17.4 ± 1.27 15.5 + 1.48 3.11 5.9 *V v of mitochondria (%) 1.3 ± 0.10 1.2 + 0.18 1.7 ± 0.53 0.85 1.4 5V v of Golgi complex(% ) 0.5 ± 0.24 0.4 ± 0.08 0.5 ± 0.03 0.13 0.6 Vv of SG(% ) 38.4± 0.10 36.8 ± 1.48 23.3 ± 3.24* 16.21 8.9 Vv of RER(% ) 36.7 t 1.16 42.4 ± A.26 57.0 ± 2.97* 32.07 à.6 Sv of SG(nn2SG/iim3cytoplasra ) 5.3 t 0.20 5.0 ± 0.10 3.5 ± 0.34* 16.85 0.4 Svo f RER (pm2RER/iim3cytoplasm) 3.1 ± 0.04 2.8 ± Ó.lé 2.2 ± 0.3>* 31.40 0.4 Üv/Vv of SG (win2/pm3SG) 13.7± 0.51 13.6 ± 0.26 15.1 ± 0.78 2.20 2.4 Sv/V„ of RER (pm2/um3RER) 8.4 ± 0.32 6.7 ± 0.39 5.9 ± 0.(0 25.06 2.0 So f SG (ill!) 1387.2± 141.15 1332.1 +• 68.88 1230.8 ± 48.35 0.68 411.è 5 of RER (um2) 807.7+ 67.40 753.9 + 82.78 824.6 + 183.91 0.09 532.8 11 of SG 3.1 + 0.01 3.2 + 0.05 2.9 + 0.03* 14.77 Ô.l RER, rough endoplasmic reticulum ; N, log number of secretory granu les; s. total surf ace ar ea;Sv, surface density; S v/Vv, surface-to-volume ratio;SG , secretoryg ranule V, volume ;v , vol une fraction, v ther 5 percentile values for these par ameterswer e 1es s than 2% and are not considered fur S5 F values for differences betwe e nmaturing , mature and spent gland s. S§S leas t significant difference ob tained with the Tuckey test( p -0 .05) * significant at p • 0.05 25 A STUDYO FMETHOD S OFADMINISTERIN GLHR HANALOGS :ADVANCEMEN TO F SPERMIATION INTH ELAND LOCKED SALMON (SALMOSALAR ) C.Weil* ,L.W . Crim Marine SciencesResearc hLaboratory ,Memoria lUniversit y ofNewfoundland ,St .John's ,Nfld . Canada,A1 C 5S7 *permanent address:INRA ,Laboratoir e dePhysiologi e desPoissons ,7835 0 Jouye nJosas ,France . Summary During anexperimenta lperio d of1 1days , miltstrippin gever y twoday san d compared LHRHanalog sadministere d by injectiono r to thatmonitore d incontrol s (groupC) . by implantaccelerate d theonse t ofspermi - Allfou rmethod s ofapplyin gLHR H analogs ationan d increased theamoun to fcollect accelerated theonse to fspermiatio n incom ablemil t comparedwit h controlfish . This parisonwit h controls. Theinitiatio no f increase incollectabl emil tvolum ei s spermiation occurred 6day searlie r thani n associatedwit h anincreas ei nspermatozo a thecontrol s afterLHR Hinjection s insalin e production. orfollowin g thesilicon e and cholesterol implants. LHRH injections inpropylen egly LHRHha sbee npreviousl y used toadvanc e colwer e lesseffective ,spermiatio nbegin thespawnin gperio d ina variet y offishe s ningjus t Aday searlie r thani nth e control (Lam,T .1982 .Canad .J .Fish .Aq . Sei.39 : group. Alltype s ofLHR H treatmentsin 111-137). However,furthe rwor k isre creased theamoun to f collectablemil tcom quired tooptimiz eLHR H orLHR Hanalo g paredwit h controlfis hdurin g theexperi treatmentschedule s especially if theai m mentalperio d (Fig.1 ). ist oprovid eLHR H stimulation forextende d periodso ftime . Thepurpos e ofth epres .' entstud ywa s toevaluat epractica lan d MILTVOLUM E effectiveway s ofadministerin g LHRHanalog s f12- XtS.E tosalmo n toreduc efrequen thandlin go f O) thefis han dmaximiz e theperio d ofbio a>10 logicaleffectivenes so f thepeptid ehor 1 1 mone. Spermiationi nprespawnin g land locked salmonwa suse d asa biologica ltest . 2» i For thispurpos eLHR Hanalog swer eadmin ^ 4. isteredb y intraperitoneal injections orb y 1 *2- intraperitoneal implanti nfis hweighin g9 0 ^- ± 5g ,maintaine d a8° Cunde r simulated n naturalphotoperiod . The experiment lasted C SaP gS iC h 11days . Intraperitoneal injections ofD-Ala 6 LHRH fig.1 .Influenc e ofLHR H analogs treatment ethylamide onth e totalamoun t ofmil t collected a)Grou p Sa- a totaldos eo f0.75 0mg / throughout theduratio no f the11-da y kgb.w .wa sdissolve d insalin ean dadmin experimentalperiod . istered onalternativ e days (totalo f6 in jections). Study ofth espermatocri t indicated thatth e b)Grou pP g- atota ldos e of0.75 0mg / increasei nmil tvolum eprovoke d byLHR H kgb.w .wa sdissolve d in40 %propylen egly analogsi sassociate dwit h anadvancemen ti n colan d administered twice,6 day sapar t spermatozoaproductio n andno tjus tdu et o (totalo f2 injections) . spermdilution . Intraperitoneal implants Inconclusion ,LHR Hanalog s canb eadmin a)Grou p Si- a singl eimplan to fsili istered invariet y ofvehicle s toaccelerat e conerubbe rcontainin g 150u go fD-Al a LH theonse t ofspermiatio n andpossibl y other RHethylamid e (1.5mg/k gb.w. )wa sadmin reproductive eventsi nsalmon . Multiplein istered atth ebeginnin g of theexperiment . jectionsi nsalin eo rsingl e siliconeo r b)Grou p Ch- asingl e implanto fcom cholesterol implantshav e comparableeffec pacted cholesterolpowde r containing 25u g tiveness. Theimplantatio nmethod shav eth e ofD-foal(2) 6LHR H (Syntex,Inc. )wa sadmin advantages ofminimizin g thecost s oflabo r istered at thebeginnin g of theexperimen t andeliminatin g thenee d for frequenthand (dose25 0ug/k g b.w.). ling offis hwhic h canb ea practica lpro After these fourLHR H treatments,th e blemi nfis heasil y stressed. volume ofsper mrelease dwa sdetermine d by 26 APPLICATION OFA N IN VITROBIOASSA YFO RFIS HGT HI NIMPROVIN GHARVES T OFCAR PPITUITARIE S Z.Yaron' 1),Aliss aBogomolnaya( 1),an dV .Hilgev 2) (1)Departmento fZoology ,G.S .Wis eFacult yo fLif eSciences ,Te lAvi vUniversity ,Israe lan d (2)lnstitut fürKüste nun d Binnenfischerei,Ahrensburg ,F.R.G . Harvest ofcar ppituitaries ,a sa spawning GTH activity in pituitaries of small and -inducing agent,i scurrentl yrestricte d in large fish Israelt oliv edono r fisho f1 k gb wo rmor e (Rothbard,198l).Th epresen t study isa nat Gonadotrophic potency measured in vitro in temptt outiliz et oa greate r extentcar p 10 glands taken from small (<300 g) but ripe pituitaries availablea ta fish-processin g fish was similar to that taken on the same plantwhil eadoptin gth etimetabl e ofth e day from larger fish (lOOO - lUoO g; Fig.2). plant. Thegonadotrophi c potencyo fth epituita rieswa sassesse db ya n in vitro bioassay recentlydevelope d inou rlaborator y (Yaron etal. ,i npress) .Aqueou s extract ofth e testedpituitarie swa sadde dt oovaria nincu bateso f Sarotherodon aureus atconcentrat ionsequivalen t to10 -6- 10 -2gland/ml .Th e resulting increasei nestradio l (E2)outpu t wascompare dt otha t elicitedb y standard fishgonadotrophin s (SG-G100,Donaldson,o r cGTH,Burzawa-Gerard) .Al ltest swer eper formed onpool so f1 0gland so rmore . Post mortem losso fgonadotrophi c potency Thegonadotrophi c potency in vitro of pituitariesharveste d fromcar phead skep t 2k h post mortem at1 * didno tdiffe rfro m 1er5 10-4 1er3 pit./ml thato fgland stake n immediately afterde capitationo fliv ecarp s (Fig.l). Fig.2 Gonadotrophic activity invitr o of pituitaries of small and large carps com The suitability ofth e post-mortem glands forspawnin g inductionwa sverifie db yinje pared with that of cGTH and SG-G100. ctinghomogenize d glands intofemal ecarp s (lgland/kg )accordin g toth eprocedur eo f Conclusions Rothbard(l98l).Non eo fth econtrols ,bu t 5/6 ofth etreate d fish spawned successfully. Theharves to fcar ppituitarie s canb e improvedb yusin ghead so ffis hon eda y afterdeat han db yutilizin g alsofis ho f smallersize . 0 -o FRESH GLANDS • • 1d POSTMORTEM References ft// Rothbard,S .1981 .Induce dreproductio ni n cultivated cyprinids- th ecommo ncar pan d il thegrou po fchines ecarps . Bamidgeh 33:103-121 . Yaron,Z. ,A .Bogomolnay aan dE.M .Donaldson , inpress .Th e stimulation ofestradio l // secretionb yth eovar yo f Sarotherodon aureus asa bioassa y forfis hgonadotrophs . *j. . , , , HongKon gUniversit yPress . 10 6 10~s KT4 10"3 l(T2 pit/mi Fig.l Gonadotrophic activity in vitro of 1 Thisresearc h is supportedb ya gran t day post-mortem, and fresh carp pituitaries. (AQ-18)fro mth eNCRD ,Israe lan dth eGKSS , F.R.G. 27 COMPARISON OP PURIFIED PITUITARY GONADOTROPINS OP MALE AND PEMALE PINK SALMON (Oncorhynchusgorbusch aWalb. ) H.A.Zenkevich,Z.M.LSc e Institute ofBiology ,Academ yo fScience s ofth eLatvia nSSR ,Riga ,USSR . Summary Purifiedgonadotropi chormon e P]_reveale d activityi nfro g (GTH)preparation shav ebee nisola oocytematuratio na tminimu mconcen tedseparatel yfro maceton edrie d trationo f1 2/tg/ml whileM igav en o pituitaries ofmal ean dfemal epin k response inthi sbiotest .Inducin g salmon.I nth ecomparativ ebioassa y mud-loach ovulationP iwa sactiv ea t usingfro gRan atemporari a oocyte theminimu mdos eo f6 0yes perfis h maturationtes tan dth eadeny lcyc whileM lgav en orespons e evena t laseactivit ymeasurement s inhomo - 180/tg/fish.Discrepanc ybetwee nP- , genates ofmud-loac hMisgurnu sfos - andM- Lwa sfoun di nadeny lcyclas e silisovarie sa pronounce ddiscre activitymeasurement s aswell . pancy inth eactivit ywa sfoun d betweenbot hGT Hpreparations .Th e e conclusionabou t dualityan dse x 0. V*V specificity ofpin ksalmo nGT Hwa s made. K > 0 Pituitarygland sfro mmal e andfe malesalmon swer e collected during - •y^ thefis hspawnin g inautum n198 0a t <^^ theSokolovs kfish-plan t (Sakhalin) oies min./mq andaceton edried .Isolatio no fth e g.« • Ml GTHwa sperforme da sdescribe db y • • 1 1 Donaldsone tal.(1972) .A tth eion - *, -4 1 LVA-1— 1 exchange chromatography onDEAE - 25 100 200 1000 2000 cellulosefou rseparat eprotei n jmqprep, /m l fractionswer eisolate dfro mth e pituitaries ofth eeithe rse xan d fig.2.Adeny l cyclaseactivit yin nodifference swer efoun d inth e ducedb y P]_an d M]_i nhomogenate s chromatographic distributionbetwee n ofmud-loac hovary . proteinfraction s extractedfro m ?2an dM 2 showeda coincidin ggo malean dfemal eglands . nadotropic activitybot ho nth e oocytes (11/^g/ml)an dth emud-loac h ovulation (80Ag/fish) . Partialantigeni cidentit y (spur) betweenM- ,an d P]_an dM 2 andP 2wa s found indouble-immunodiffusio n cross-reactions in1 %aga rge lwit h antiseraraise d inrabbit sagains t eacho fth epreparations . Soi tseem sver ylikel ytha tpi tuitaryglan d ofpin ksalmo npro ducestw o GTH-s.Th eGT Hpreparatio n 20 40 ofmal ean dfemal efis hwit hR f 0.38-0.52reveale dth emos tpronoun fig.1.Chromatograph y onDEAE - cedse xspecificit y inth euse dtes t celluloseDE-3 2Whatman .Th ecolum n systems. (1.7x9.5 cm)wa s equilibratedi n 0.02I Na-Gl ybuffe rp H9.3 .Step References wisegradien t ofNaC lwa sused . DonaldsonE.M. ,Yamazak iP. ,Dy e H.M.,& Phille oW.W .1972 . Pre Thetw oactiv efraction s offemal e parationo fgonadotropi nfro msalmo n fishpituitarie sP^ _an dP 2a swel l (Oncorhynchustshawytscha )pituitar y ascorrespondin gmal epreparation s glands.Gen .Comp .Endocrinol. ,18 , ]% andM 2 formedband swit hR f0.38 - 469-481. ~ O.52an d0.82 ,respectively ,whe n analysed on7.5 %polyacrilamid ege l (pH8.3) . 28 HYPOTHALAMO-HYPOPHYSIS AXIS 29 NATURE,LOCALIZATIONAN DACTION SO FNEUROHORMONE S REGULATING GONADOTROPIN SECRETIONI N TELEOSTS R.E.Pete r Department ofZoology ,Universit y ofAlberta ,Edmonton ,Albert a T6G 2E9,Canad a Summary Introduction Thegonadotropi n (GtH)releasin g hormone Withth eincreasin g importance offis h (GnRH)o fteleost s issimila rt o culture ona world-wid ebasis ,ther e isa n luteinizinghormone-releasing'hormon e increasingnecessit y forregulatio n of (LH-RH),perhap s differingb y aminoaci d reproduction oftraditionall y cultured fish, substitutions atposition s 7an d8 . aswel l asnewl y cultured species. To Perikarya immunoreactive for LH-RHhav e assuremaxima lproductio n fromcultur e been localized inth eventral-latera l operations, iti susuall y necessaryt o nucleus preopticusperiventriculari s (NPP) induce ovulation and spermiationfo r and theposterio rnucleu s lateraltuberi s production offry . Synthetic analogues (NLT),a swel la sothe r areas. Localization of luteinizing hormone-releasing hormone ofimmunoreactiv e fibertract s fromcell s (LH-RH)hav ebee n successfullyuse dt o inth eNP Pan dNL Tt oth epituitar y gland induce spawningo fsevera l carpspecie si n suggeststha t these areas areth eorigi no f China (Anonymous,197 7a ,b ;Jian g et al., theendogenou s releasinghormone . 1980)an dt oinduc eovulatio n ofsalmonid s Functional evidence from lesioning studies inNort hAmeric a (Donaldson et al.,1981 / has strongly implicated theNL Ti nth e 1982, 198Z). Essential forth eefficien t regulation ofGt Hsecretio nvi aGnRH ,bu t application ofneurohormone s inth e theevidenc e forinvolvemen t ofth epre regulation ofreproductio n ofculture d fish opticregio n inGnR Hsecretio n isconflict isknowledg e ofth enatur e and actionso f ing. LH-RHan d superactive analogues theneurohormones . Inthi spape r Iwil l (LRH-A)stimulat e GtHreleas e inteleosts . reviewth enature ,brai n localizationsan d There isevidenc e forself-potentiatio nan d actions ofneurohormone s regulatinggonado self-suppression ofth eactio no fLRH- Aan d tropin (GtH)secretion . Inaddition , Iwil l LH-RH. Incertai n speciesth e actiono f alsodiscus sth erol e ofthes e factors,an d LRH-A lastsmuc h longertha nLH-RH ; sex steroids,i nth eregulatio n ofcertai n analogues canb euse dt omanipulat e certain events inth ereproductiv ecycle . phases ofth ereproductiv e cycle inteleosts . AGt Hreleas e inhibitory factor (GRIF) Gonadotropin ReleasingHormone__(GnRIT ) hasbee n foundi ngoldfish . Lesioning studies indicatetha t GRIFprobabl y Nature originates fromth e anterior-ventral NPP. Dopamineha s GRIFactivit y ingoldfis ht o GnRHactivit yha sbee ndemonstrate d in block spontaneous release ofGt Han d crude extracts ofth ehypothalamu s and modulateth e actiono fLRH-A . Iti s telencephalon ofsevera l species ofteleos t difficult to induce ovulation ingoldfis h (Breton et al., 1971;Cri m et al.,1976 ; byinjectio n ofLRH-A ;however ,b yblockin g Crim §Evans ,1980) . Crim etal . (1976) theaction s ofdopamin eb y injection ofa andCri m §Evan s (1980)foun d thathypo receptor antagonist,th eGt Hrelease - thalamic andtelencephali c extractsha d responset o LRH-Aca nb epotentiate d and more GnRHactivit ype rbrai n segment,tha n ovulation inducedmor ereadily . otherregion steste d (cerebelluman d Inadditio nt oth e classicnegativ efeed medulla), suggesting that there isa back effect ofse x steroids insexuall y differential distribution ofGnR H inth e developingteleosts ,estrogens ,an dandro brain. Studieshav eno t beendon et o genstha t canb earomatize d toestrogens , determine the specific GnRHactivit yo f havea positiv e feedback effect insexuall y variousbrai nregions . immature salmonidsan d eels. Sucha King §Milla r (1979,1980 )studie dth e positive feedback effectma yb ea nimport crossreactivity ofhypothalami c extracts antpar t ofth emechanis m inducing onäeto f froma wid erang eo fvertebrates ,includin g sexualdevelopment . tilapia (Sarotherodonmossambicus) ,wit h fourantisera ,eac hreactin g with different regionso fth e LH-RHmolecule . The extracts fromra tan d amphibiawer eparalle l toeac h other andwit h LH-RH,wherea sth e extracts fromtilapia ,a nelasmobranch ,bird san d 30 reptiles wereparalle l toeac hothe rbu t ofconfirmatio n fromon einvestigatio nt o notwit h LH-RH. Whenru no ncatio n another onth esam especies ,probabl ydu e exchange,affinit y andhig hperformanc e todifference s inspecificit y ofantisera , liquid chromatography columns,th emovemen t and incorrect description ofth elocal oftilapi a immunoreactivemateria l was izationo fth ereactiv ematerial . always similart otha t ofbird san d Immunocytochemicaltechnique shav e reptiles,bu t different from LH-RHan dth e localized LH-RH-likemateria l inth eproxi immunoreactivemateria l inhypothalami c malneurohypophysis ,i nth eregio n ofth e extracts frommammal s andamphibia . GtHcells ,i nrainbo w trout (Salmo Tilapiahypothalami c extract,a swel la s gairdneri) (Dubois et al., 1979), the extracts fromothe rvertebrates ,ha d platyfish (Xiphophorusmaculatus , GnRHactivit y onculture d ovine anterior >C.helleri ,an d X. sp.) (Schreibmane t al., pituitary cells. These studies suggest 1979; Münz etal. , 1981), Japaneseee l there isconservatio n ofth ebiologicall y (Anguillajaponica )an dpuffe r (Fugu activeregio n ofGnR Hthroughou t vertebrate niphobles) (Nozaki gKobayashi , 1979). phylogeny,bu t thatther e area tleas ttw o Goos &~Murthanoglu (1977)foun dsmal l different formso fGnR H invertebrates . perikarya immunoreactive forLH-R H Brainextract s ofco d (Gadusmorhu a scattered inth eare adorsali spar smediali s morhua)wer e foundt o containthre e ofth etelencephalo n ofrainbo w trout. In different LH-RH-like immunoreactive addition,the yfoun d immunoreactive fibers moieties (Barnett et al., 1982). Basedo n scattered inth eanterio rhypothalamus ,i n theelutio npattern s ofth ematerial s from theregio n ofth ehorizonta l commissure, ionexchange ,ge l andhig hperformanc e apparently continuous with fibersendin g liquid chromatography columns,tw oo fth e dorsalt oth epituitar y stalk. Thefiber s LH-RH-likematerial s were foundt ob e couldno tb etrace d beyond thispoint . similar insiz et o LH-RH. Thethir dwa sa Other investigators haveno tbee n ablet o "big" LH-RH-likematerial ,suggeste d tob e detect immunoreactiveperikary ao r fibers aprecurso r ofth e authentic GnRHextende d inth ebrai n ofrainbo w trout (Dubois et al., atth eN-terminal . Onth ebasi s ofcross - 1979; Nozaki &Kobayashi , 1979). reactivitywit h antiserawit hknow nanti Immunoreactive fibers,bu tn operikarya , genicrequirements ,i twa s suggestedtha t were found inth eventra lpreopti cregion , codGnRH' sdiffe r from LH-RHb yamin oaci d telencephalon,infundibula rnucleu s 8 substitution atpositio n 8 (-Arg -),or , (=nucleus lateral tuberis),habenula r 7 less likely,positio n 7 (-Leu -). This nucleus,an d optictectum , aswel l asi n supports thefinding sb y King §Milla r theneurohypophysi s ofth eproxima l lobe (1979, 1980),base d oncrossreactivit y of ofth epituitar y ofth eJapanes eee l tilapiahypothalami c extract with LH-RH (Nozaki §Kobayashi , 1979). Incar p antiserawit hknow n specificity,tha t (Cyprinus carpio), Pane t al. (1979) tilapia GnRHmos t likelydiffer sfro m described LH-RH immunoreactive fibers LH-RHb y substitution atpositio n 7o r8 . amongstnucleu spreopticu s (NPO)peri Botho fthes e studies indicate thatth e karya,an d also inth e lateral forebrain C02-terminal andNH2-termina l oftilapi a bundles,ventra l toth epreopti crecess , 1 and cod GnRHar e identical toLH-R H (pGlu - theopti ctectum ,an d the lateralhypo 2 3 9 10 His -Trp -;-Pro -Gly -NH2), andtha tth e thalamic region. Theseworker sals o centralpar t ofth emolecul ema y alsob e described immunoreactiveperikary a inth e 5 6 similart oLH-R H (-Ser^-Tyr -Gly -). NPO; however,thes e cells appeared tob e Studiesb y Idler and Crim (1982), inth emeninge s located lateral toth e published inpreliminar y form, indicate preoptic region andventra l toth e thatth ewinte r flounder (Pseudopleuronectes telencephalon (R.Peter ,persona l americanus)ha stw o formso f LH-RH-like observations). immunoreactivematerial ,on etha ti s Schreibman et al. (1979)describe d similar insiz et o LH-RH,an d anothertha t reactiveperikary ai nth eare aventrali s islarger . Chromatography studiesdon eo n parsventrali s andpar s lateralis,an dth e thewinte rflounde rmateria l indicatetha t NLTo fplatyfish ;however ,th e localization thesmalle rmolecul e issimila r toLH-R H designated asbein g inth eventra l insize ,supportin g the studiesreviewe d telencephalon was actually alongth e above. lateral extensions ofth epreopti c recess inth eventral-latera lnucleu s preopticus Localization: Anatomical and Functional periventricularis (NPP) (R.Pete rS Evidence M.P. Schreibman,unpublishe dobservations ; Schreibman et al., 1982). Münze tal . (1981) The localization ofLH-R H immunoreactive alsodescribe d reactiveperikary a inth e material insection so fth ebrai nan d ventral-lateral NPP,designate d thenucleu s pituitary fromsevera l specieso fteleost s preopticus basalis lateralis,bu tno tth e hasbee n investigated. Unfortunately there NLTi nplatyfish . Schreibman et al. (1979) issom e confusion inthi sare adu et olac k andMün ze t al. (1981)describe dreactiv e 31 fibers,presumabl y fromth eNP Pperikarya , theNL Tma yb et opromot eGt Hreleas e ona dorsal toth eopti c chiasma,i nth eregio n cyclicbasi svi asecretio n ofGnRH . ofth ehorizonta l commissure,dorsa l to Injection ofgoldfis hwit h monosodium thepituitar y stalk,amongstNL Tcell san d L-glutamatecause d edemaan dhypertrophy , inth epituitar y stalk. Münze tal . (1981) upt oabou t 48hour s after injection,i n also foundreactiv eperikary a inth e theNL Tfro mth eanterio rmargi n ofth e anterior-ventral telencephalon bordering pituitary stalk throught oth eposterio r theolfactor ybulbs ,designate d thenucleu s endo fth eNLT ,an d also ina smalle rregio n olfacto-retinalis telencephali,an d inth e oftissu e inth e anterior-ventral NPP (Peter dorsalmidbrai n justposterio r ofth e et al., 1980). Thiswa s followed,fiv eday s posterior commissure. Thenucleu solfacto - afterinjection ,b ynecrosi s ofmos to fth e retinalisproject s toth e olfactorybul b perikarya inth eaffecte d region ofth eNL T viath eolfactor y tracts,th eretin avi a and at leastsom eo fth eperikary a inth e theopti c chiasm andopti cnerve ,an dt o NPP. Correlatingwit hth e initialperio d of thedorsa ltelencephalo n ipsilaterallyan d edemaan d hypertrophy,an dth e following contralaterallyvi ath e anterior commissure stageo fnecrosis ,seru m GtH levelswer e (Münze t al., 1981,1982) . Immunoreactive significantly increased foru pt o4 8hour s fiberswer e also found inmos t telencephalic afterinjection ,an dwer e similart onorma l andmesencephali c regions,wit h aprominen t levels at5 ,7 an d 8day safte r injection. projection toth eopti ctectu m (Münze t al., At 31day s after injection therewa s ados e 1981). dependent decrease ingonadosomati cinde x Münze t al. (1982)foun dtha t cichlidan d (GSI; R. Peter and C.Nahorniak , centrarchid fishpossesse d LH-RHreactiv e unpublished results). Inth e longter m cells inth enucleu s olfacto-retinalis experiments,a significan t decreasei n telencephali,simila rt oth eplatyfis h (a serumGt Hwa s found ingoldfis h injected poecilid);however ,cyprini d (including with ahig hdos e ofglutamate ,bu tno twit h goldfish),notopteri d andanabanti d fish the lowerdoses ,eve nthoug h the lowerdose s didnot . were alsoeffectiv e incausin g adecreas ei n GSI. Theseresult sprovid e confirmation Ingoldfis h (Carassius auratus), LH-RH that theNL Ti sinvolve d inregulatio n of immunoreactiveperikary awer e found along GtHsecretio n and gonadalrecrudescence . theborder s ofth eventral-latera l preoptic recess inth eNPP ,an d inth eNL Tposterio r Kahe t al. (1982b)foun dtha t injectiono f ofth epituitar y stalk (Kahe tal. ,1982a) . glutamate ingoldfis h caused degeneration Findingreactiv e cellsi nthi sparticula r ofonl ytyp e Baminergic-lik efiber s inth e part ofth eNL Tfit swel lwit h results proximalneurohypophysis . Sinceth e from lesioning experiments ongoldfis h degenerating fiberswoul dhav e originated (Peter,1970 ;Pete r §Crim , 1978;Pete r§ mainly fromth eNL Ti nth epituitar y stalk Paulencu,1980 ;R . Peter,unpublishe d region andposterio r ofth epituitar ystalk , results),tha tdestructio n ofth eNL Ti n theseresult s indicate involvement ofthi s thepituitar y stalkregio n andposterio r of particular region ofth eNL Ti nregulatio n thepituitar y stalk caused onset ofgonada l ofactivit y ofth eGt Hcell s andgrowt h regression and/orblockag e ofgonada l hormone cells inth eproxima l parsdistalis . regression. Itwa s suggested, onth ebasi s Inlesionin g studies onpaa r ofAtlanti c ofth e lesioning studies,tha tthi spar t salmon (Salmosalar ) (Dodd et al., 1978)an d ofth eNL Twa s involved inregulatio n of killifish (Fundulusheteroclitus ) (Pickford GtHsecretio nvi areleas eo fGnRH . However, et al., 1981),destructio n ofpar t ofth e nodecreas e in serum GtH levelswa sfoun d NLTcause d gonadal regression orblockag e inth e fishwit hNL Tlesion s (Peter SCrim , ofgonada lrecrudescence . 1978; Peter §Paulencu , 1980). Resolving Whileth eabov eevidenc e clearly implicates this conflict,Pete r (1982)reporte d that theNL Ti nregulatio n ofGt Hsecretio nvi a NLT lesions caused thedail y cyclei n GnRH,immunocytologica l studiesals o serum GtHt odisappear ,withou t causinga n implicateth epreopti c region,specificall y overall decrease inth emea n serum GtH theventral-latera lNPP . Lesions inth e levels. Adail y cycle inseru m GtHi s preopticregio n offemal e goldfish causeda usuallypresen t ingoldfis h that areunder significant decrease inGS Ia t4 2day s goingovaria n recrudescence ortha thav e postoperatively (Peter §Crim , 1978), completed ovarian development preparatory suggesting some involvement inth emainten tospawnin g(Hontel a SPeter,1978 , 1980), anceo fgonada l activity. However,thes e and disappearanceo fth edail y cyclei s resultshav eno tbee n confirmed (Peter,1970 ; associated with,th eonse t ofgonada l Peter 5Paulencu ,1980 ;R . Peter,unpublishe d regression (Hontela SPeter ,1980 ;Hontela , results). Perhaps sinceth e lesioning 1982). Hontela 5Pete r (1978)hypothesize d current washig h andth e lesions large inth e thata dail y cyclei nbloo d levels ofGt H studyb y Peter d, Crim (1978),th e lesionsma y iso fimportanc e forstimulatio n of haveinterfere d with aGt Hreleas e inhibitory ovariandevelopment ;thi swa s supported by factorthough tt ooriginat e from thepre subsequent findings (Hontela &Peter ,1980 ; opticregio n (Peter §Paulencu ,1980 ;se e Hontela, 1982). Onthi sbasis ,th erol eo f below), aswel l asGnR Horiginatin g fromth e 32 NPP. Itwoul db eo fgrea t interestt o LRH-A,th e interval betweeninjections , selectively lesionth e areao fth eNP Pi n temperature,an dth e GtH release-response which the LH-RHiramunoreactiv ecell sar e iscomplex . Investigators clearlynee d located todetermin eth e effects ongonada l totak e great care indefinin g the conditions activity and GtH secretion,an d compareth e underwhic h LH-RHo rit s analoguesar e resultst oth eeffect s oflesion s inth e tested. NLT. Sincether e areprobabl ytw o Aseasona lvariatio n inth ei nviv oGt H populations ofGnR Hperikary ainvolve d in release-response to LH-RHo r LRH-Aha sbee n regulation ofGt Hsecretion ,i tma yb etha t found inbrow ntrou t (Salmotrutta ) (Crim§ therear edifference s in functionbetwee n Cluett, 1974),rainbo w trout (Weile t al., thetwo;th eNL Tha sbee n implicated in 1978)an d goldfish (Line t al.,1982). In regulation ofdail ycycle so fGt Hreleas ei n each speciesth e greatest GtHrelease - goldfish. response occurred infis h atth etim eo f spawning,whe n gonadal developmentwa s Actions completeo rnearin g completionpreparator y forspawning . Iti swel l established that hypothalamic Crim et al. (1981a)demonstrate d that extracts frombot hteleost s andmammals , analogues ofLH-R Htha tbloc kth eactio no f synthetic LH-RHan d analogues ofLH-R Htha t theauthenti cmolecul e inmammal s aresuperactiv e inmammal s canstimulat e (inhibitory analogues,iLRH-a) , alsobloc k GtHreleas e fromteleos t pituitariesi n theactio no fLH-R Ho nGt Hreleas e Inviv o 'vivoan d invitr o (Ball,1981 ;Peter,~T982) . inbrow n trout. Superactive analogueswer e Until synthetic teleost GnRH isavailabl e notmor e activetha n LH-RH invivo ; invitr o itwil lb eimpossibl e todirectl y compare LRH-Awa s somewhatmor e activetha nLH-RH , itspotenc y ina teleos twit hLH-RH . but other superactive analogues hada Nevertheless,dat ab y King §Milla r (1980) similarpotenc y toLH-RH . suggeststha ttilapi a GnRHha s similar Incoh osalmo n (Oncorhynchuskisutch ) potency toLH-R Hi nstimulatin g LHreleas e LRH-Ai sclearl ymor e active than LH-RHi n fromshee ppituitar y cells invitro . Given stimulatingplasm a GtHan d inducing thatteleos t GnRH'san d LH-RHar esimila r ovulation (G.Va nde r Kraak,H.-R . Lin, instructure ,syntheti c LH-RH canb euse d E.M. Donaldson,H.M . Dyean d G.A.Hunter , instudie s on fisht o gain informationo n personal communication). A single injection theaction s ofendogenou sGnRH . ofLRH- A (20yg/kgBWt )cause d thesam e Peter (1980)foun dtha t apai r ofinject magnitude ofincreas e inplasm a GtHa s ionso fLH-RH ,o rth e superactive analogue 200u g LH-RH/kg BWt. Inaddition ,th e des-Gly10[D-Ala6] LH-RH ethylamide (LRH-A), effect LRH-Apersiste d fora t least 96hours , weremor e effective in stimulating increased whereasth e increase inplasm a GtH following serumGt H levelstha n asingl e injectiono f LH-RHinjectio n lasted foronl y about2 4 similar dosage. This indicatestha t hours. Both LH-RHan d LRH-A accelerated potentiation ofth e GtHrelease-respons e thetim e ofovulation ,althoug h LRH-Awa s canoccur . Apai ro finjection so fLRH- A muchmor e effective (Donaldson1981/1982 , ata lo wdosag e caused,i n somecases ,a 1982). Although LRH-Aca nb e ahighl y greater increase inseru m GtH levelstha n effective agent forinducin govulatio n in injections ata highe r dosage,indicatin g somespecies ,suc h asth e coho,clearl yth e that suppression ofth erelease-respons e responsiveness tothi sanalogue ,an d others can occurdu et oexces shormone . There also,ca nvar ygreatl ybetwee nspecies . weren o significant differences inth epea k A chronic increase inplasm a GtH,lastin g serum levels ofGt H induced bysimila r at least4 weeks ,wa s induced in landlocked dosages ofLH-R Han d LRH-A;however ,LRH- A Atlantic salmon implantedwit hpellet stha t caused amor eprolonge d release-response gavea continuou s release ofa superactiv e under certainconditions . analogueo fLH-R H (Crime t al., 1982). The Line t al. (1982)reporte d thatth e length pelletswer e effective in stimulating GtH ofth einterva l between apai r of injections release,an d incausin g increased pituitary andth eacclimatio n temperature ofgoldfis h GtHcontent ,i nsalmo ntha twer ei n interact toinfluenc e the GtHrelease - regressed,recrudescin g andprespawnin g response ingoldfish . At awar m temperature gonadal conditions. The effects ongonada l (20°C)injection s with an interval of3 conditionwer e complex. Insexuall y hours induced higher serum GtH levelstha n regressedmal e salmonther ewa sn o effect on whenth e interval was9 hours . Ata col d GSI,bu thistologica l examination ofth e temperature (12°C), injections ata lo w testes suggested stimulation oftesticula r dosage (O.Olug LRH-A/gbod yweight )wer e recrudescence. Inrecrudescin g females aseffectiv e asa highe rdosag e (0.1yg/g therewa s an increase inGSI ,suggestin g BWt)a t aninterva l of 3hours . With a9 stimulation ofvitellogenesis . Inrecru hourinterval ,however ,th e lowdosag ewa s descingmales ,however ,GS Iwa sdecrease d much less effectivetha nth ehig hdosage . compared tosha m controls. Theteste so f Thesepreliminar y studies demonstratetha t the analoguetreate dmale s containedpre therelationshi p between thedosag eo f dominantly spermatids and spermatozoa, 33 whereasth esha m implanted fishha d ofLRH- Awit h adopamin eblockin g agentt o spermatocytes and spermatids;th eanalogu e reduce theGt Hrelease-inhibitor y actiono f apparently induced maturation ofth etestes , dopamine (seebelow) , canb euse d toinduc e without stimulating additional growth. ovulation. Prespawning femaleswer e all inducedt o Thecellula rmechanis m ofactio n ofGnR H ovulate, insom e cases considerably earlier hasreceive d littleattentio ni nteleos t inth eseaso ntha nnormal . Prespawning fish. Following injection ofmu d carp maleswer e induced tospermiat e considerably (Cirrhrinusmolitorella )wit h 125I-LRH-A, earliertha nusual ,o rt ohav e increased Pane t al. (1981)found ,b yultrastructura l spermvolum e ifspermiatio n had already autoradiography,tha t label appeared over commenced. Inadditio n todemonstratin g thecel lmembrane ,Golg ibody ,secretor y that chronicexposur et oanalogue s of granules,mitochondri a andnucleu so fth e LH-RHca n accelerate gonadal recrudescence GtHcells . Agreate ruptak eo f labelwa s andth etim e ofovulatio n and spermiation observedwhe n themu d carpwer e injected inAtlanti c salmon,th eresult s alsosho w with apotentiatin g doseo fcol d LRH-A that there isn o down-grading ofth eGt H tenminute sbefor e administration ofth e release-response toa norma l or lesstha n label. Ingoldfis hhel d at 12°C, normal level,a swoul db eth e case ina administration ofa potentiatin g doseo f mammal. However,whethe r some suppression LRH-A 12hour s before injection of ofth eGt Hrelease-respons e occurred cannot 125I-LRH-A caused agreate ruptak e oflabe l bedetermine d fromth edat apresented . intoth epituitar y glandb y 20t o3 0 Inearl ystudies ,larg edose s ofLH-R H minutes,an da lowerconten t oflabe la t wereuse d toinduc e ovulation insevera l 24hour spostinjectio n (0.Bres ,R.E .Peter , teleost species,includin g goldfish (for H.-R. Linan d C.S.Nahorniak ,unpublishe d review,Peter ,1982 )an dcommo ncar p results). This confirms thatpotentiatio n (C.H.Pan ,persona l communication). LRH-A inducesa greate r uptake ofGnRH ,an d hasbee n foundt ob ehighl y effectivei n suggeststha t itals o causes amor erapi d inducing ovulation (>80% )i nculture d grass depletion orturnove r ofGnR Hi nth e carp (Ctenopharyngodon idellus),blac k carp pituitary gland. Although theseresult s (Mylopharyngodonpiceus),silve rcar p require confirmation,perhap s further (Hypophthalmichthysmolitrix )an d spotted investigations onteleost sma yprovid e silver carp (Aristichthys noblis) information on^thedynamic s ofGnR H metabolism inth epituitary . (Anonymous 1977a,b ;Jian g et al.,I E Grass carpovulate d within 12t o2 2hour s ofa singl e injection ofLRH- Arangin gfro m Gonadotropin Release-InhibitoryFacto r 1t o100yg/k gBWt ;tw o injections with a7 TGRÏF) to 10hou r interval reduced thetim et o ovulationt o3 t o 14hour s afterth esecon d Nature, Localization andAction s injection. Silver carp artificially spawned forth e first timewit h LRH-Awer e Thepresenc e ofa GRI F ingoldfis hwa s more sensitive (required one injection of2 suggested by Peter et al. (1978)a sth e to20ug/k g BWtt oinduc e ovulation)tha n explanation forth edramati c increasei n fishtha tha d been induced previously serum GtH levels found inmatur e female (required twoinjection stotalin g10ug/k g goldfishfollowin g large lesions inNLT - BWt). Black carpwer erelativel y less pituitary stalkregion . Theincreas ei n responsive,requirin g twoo rthre e injections serum GtH lasted fora t least 12days , with dosagesu pt o400ug/k gBWt . These although levelsha d decreased tonea r resultsindicat etha t LRH-Aca nhav ewid e normalb ythi stime . Inaddition ,nearl y application forinducin govulatio n incarp ; allth e lesioned females ovulated. however,th eresult s aredifficul tt o Peter andPaulenc u (1980)foun dtha t evaluatebecaus e they area compendiu m of destruction ofth epituitar y stalk,an dno t experiments doneunde r different conditions theNLT ,wa sth ecaus eo fth edramati c bydifferen t workers atman y different fish increase inGt Hsecretion . This indicates farms. Inm yow n experience ongoldfish , thatremova l ofth epituitar y gland from ovulation didno t occur inmor etha nhal f hypothalamic control,b y lesioningth e ofth etes t fishi nexperiment s inwhic h pituitary stalk,allow s spontaneous dosages from lOygt o lmg/kgBW twer euse d releaseo fGt Ht ooccur ,confirmin gth e (R.Peter ,unpublishe d results). However, presenceo fGRIF . Additional lesioning ahig h frequency ofovulatio n occurred when studies inmal e aswel l asfemal e goldfish goldfishwer e injected with LRH-A indicated that theorigi n ofGRI F isth e (100ug/kgBWt )an dpimozide ,a dopamin e anterior-ventral preopticregion ,i nth e antagonist (Chang §Peter , 1982a ,b ,c) . areao fth eNP Ptha t isventra l toth e Clearly, LRH-Aca nb ea highl y effective anterior commissure (Peter §Paulencu , 1980). agent forinducin g ovulation incyprinids , Thisare a ismor eanterio r thanth e location althoughther e isa wid evariabilit y in ofLH-R H immunoreactiveperikary a inth e sensitivity. However,i nthos e species lateral-ventral NPP. Tracts for GRIFar e which are lessresponsive ,th e combination locatedbilaterall y inth e lateralpreopti c 34 region,an d the lateral-anteriorhypothal significant increase. Injection ofD Aint o amicregion ,convergin gt oente rth e thethir dventricl eha dn o significant pituitary stalk. Nagahama §Pete r (1982) effects onseru m GtH,suggestin g thatth e confirmed that lesioning thepreopti cregio n actionso fD At odecreas eseru m GtHwer e andth epituitar y stalk cause elevated serum outsideth ebloo d brainbarrier . Injection GtHan dovulatio n inmatur e femalegoldfish . ofD Aan d apomorphinebot h caused amarke d Inaddition ,ultrastructur e studies ofth e decrease inth e elevated serum GtH levelso f pituitary from lesioned fishreveale d goldfish lesioned inth epreopti cregio nt o depletion ofsecretor y granules,an d abolish endogenous GRIF. Furthermore,D A extensivedevelopmen t ofth eGolg ian d andAP Oblocke d the effects ofLRH- At ocaus e dilationo fth eendoplasmi c reticulum, GtHrelease ,an dblocke d thehig h rateo f suggestingtha t secretion and synthesis GtHtha tha dpreviousl y been inducedb y werebot h occurring inGt Hcells . LRH-A. Theseresult s indicatetha t DAact s Parsdistali s transplant studies ingold atth e level ofth epituitar y tobloc k fish,summarize d ina preliminar y reportb y spontaneousreleas eo fGtH ,an d alsot o Petere t al. (1982),suppor t thepresenc e block,o rmodulate ,th eaction so fGnRH . ofGRIF . Transplanting thepar s distalis Ina preliminar y report,Cri m (1982) fromon egoldfis ht oeithe rbesid eth ebrai n indicated that DAcoul ddiminis hth eGt H ("juxta"location) ,int oth ethir d ventricle release-response toLH-R Han ddecreas eth e inth epreopti cregion ,o rth eventricula r spontaneous release ofGt Hb yrainbo w trout regionventra l toth e optictectu m resulted pituitaries invitro . This suggeststha t ina n increase inseru m GtH levels inth e the GRIFactivit y ofD Ai nteleost sma yb e recipient fish. This increase inseru m widespread. GtH inth erecipien t fish isdu et oth e Asreporte d above,injectio n ofLRH- Aint o spontaneous releaseo fGt H fromth etrans mature female goldfish resulted ina rela plantedpar sdistalis ;th eseru m GtH levels tively lowrat e ofinduce d ovulation return tonorma l levelswithi n 24hour s (<50% ) (R.Peter ,unpublishe d results). afterremova l ofth etransplante d pars Chang §Pete r (1982a,c )foun dtha t distalis fromth e"juxta "location . Fish injection ofmatur e female goldfishhel da t withth etransplan t inth e"juxta "locatio n 12°Cwit hprimozide ,a D Aagonist ,a tth e hadhighe r serum GtHlevel stha nthos e timeo fth e firsto rth esecon do ftw o withth etransplan t inth ebrai nventricles . injectionso fLRH- A 12hour sapar t This indicates there issom e factor inth e potentiated the GtHrelease-response ,an d brain,presumabl y present inth ecerebro resulted ina novulatio n rateo f87 %o r spinal fluid,tha t suppresses thespontan higher; injection ofpimozid e atth esam e eousreleas e ofGtH . timea sa singl e injection of LRH-A,o r1 2 Noattempt s havebee nmad et oextrac t and hours ahead ofth e injection ofLRH- A isolate GRIF. However,stron g evidenceha s resulted ina n ovulation rateo f35 %an d beenpresente d that dopamine (DA)act sa sa 70%respectively . Notably,thes e ovulation GRIF ingoldfis h (Change t al., 1982;Chan g rateswer eobtaine d inspit eo fth efis h and Peter,198 2a ,b ,c) . Chang et al. beinghel d at 12°C,a temperatur e at which (1982)foun dtha ttreatmen t ofgoldfis hwit h goldfish dono tnormall yunderg ospontan 6-hydroxy-dopamine, acatecholaminergi c eousovulation . At temperatures of18-20°C , neurotoxin,cause d an increasei nseru m GtH amor eusua ltemperatur efo rspawning , levels, suggesting that catecholaminergic pimozide iseve nmor eeffectiv ea t neurons inhibit GtHrelease . Treatmentwit h potentiating LRH-Aeffect so n GtHsecretio n «-methyl-p-tyrosine,whic hblock sth e and ovulation (M.Sokolowska ,R .Peter , synthesis ofdihydroxyphenylalamin e (L-DOPA) C.H. Pan,CS . Nahorniak §J.P .Chang , theprecurso r ofDA ,an dblockin gth e unpublished results). Theresult s indicate conversion ofL-DOP At oD Awit hcarbidopa , thatblockin g theGRI F activity ofD A both caused anincreas e inseru m GtHlevels . greatly facilitates theapplicatio n of Onth eothe rhand ,Clonidine ,a n»-nor LRH-A ininduce d ovulationo fgoldfish ,an d adrenergic agonist,cause d an increasei n suggeststha tth enorma lpreovulator y GtH serum GtH,an d treatment withdiethyldithio - surgei ngoldfis h isregulate db yremova l carbamate (DDC),whic hblock sth e conversion ofD Ainhibitio n onGt Hreleas ean d ofD At onorepinephrine ,ha dn o effecto n stimulation ofGt Hsecretio nb yGnRH . serum GtH levels. Theseresult s indicate Whether anyothe r factorshav eGRI Factivit y that DAha s aninhibitor y effect,wherea s and canmodulat eth e actiono fGnR H isno t norepinephrine has astimulator y effecto n known. GtHrelease . However,thes e studies dono t indicateth e level ofactio n ofD Ao r Steroid Feedback norepinephrine. Changan d Peter (1982b)foun d thatintra Anegativ e feedback effect ofse x steroids peritoneal (ip)injectio n ofD Aan d onGt Hreleas e inteleost sundergoin go r apomorphine,a D Aagonist ,cause d adecreas e ata prespawnin g stage ofgonada ldevelop inseru m GtH levels inintac t goldfish, mentha sbee ndemonstrate d (forreview , whereaspimozide ,a D Aantagonist ,cause da Peter, 1982). Bothth epituitar y andth e 35 NLTar e involved inth enegativ e feedback trout ascompare d tocontro l fish,an dtha t action ofse xsteroids ,althoug hth e LH-RHan d synthetic superactive analogues pituitary isapparentl y themos t important could stimulate GtHreleas e invitr ofro m direct feedback site (Billard §Peter , 1977). thepituitarie s ofth etestosteron e treated Sexually immature salmonidssho w aposi trout. Thisdemonstrate s that theGt H tivefeedbac k effect ofse x steroids. A accumulated in'th epituitar y ofth e marked increase inth eGt Hconten t ofth e immaturerainbo w trout-canb ereleased ; pituitary inmal ean d female Atlantic however,evidenc etha treleas e occursi n salmonpar roccurre d aftertestosteron e vivo afteraccumulatio n isno t clear. Crim implantation inth epituitar y andNLT ,bu t and Peter (1978)foun dtha t spermatogenesis not when testosterone was implanted inth e wasadvance d in immatureAtlanti c salmon preopticregio no ropti ctectu m (Crim§ witha testosteron epelle t implanted inth e Peter, 1978). Immaturerainbo w troutwer e pituitary andNLT ,althoug hplasm aGt H shownt ohav ea nincreas e inth epituitar y remained undetectable. Crim andEvan s contento fGt Hafte rinjectio no ftest (1982)reporte d thatAtlanti c salmonpar r osterone intraperitoneally ina coco a implanted with alon greleas e capsule buttervehicl et ogiv e slowreleas e (Crim containing testosteroneha d onseto f andEvans ,1979) . Recently itwa s spermatogenesis and,increase dpituitar y demonstrated thatth epositiv e feedback andplasm a GtH. Whether theappearanc eo f effect onpituitar y GtHwa s inrespons e GtHi nth eplasm a reflects GtHreleas e to estrogenic steroids (estradiol,estron e stimulatedb yendogenou s GnRHo r spontaneous and estriol)an d androgenic steroidstha t release isno tknown . Nevertheless,thes e canb earomatize d toestrogen s (testost resultssugges t that apositiv e feedbackb y eronepropionate ,17 œ-methyltestosterone, estrogens oraromatizabl e androgensma yb e androstenedione),wherea snon-aromatizabl e an importantpar t ofth emechanis m ofonse t androgens (5°=-dihydrotestoste rone , ofsexua l development orpuberty . In 11-ketotestosterone,llg-hydroxytestoster - support,th earomatas e enzyme ispresen ti n one)an dprogestin s (17Œ-hydroxyprogester- relatively high concentrations inth e one, 17œ-hydroxy-206-dihydroprogesterone) hypothalamus ofal lteleost s investigated were ineffective (Crime t al., 1981b). (Callard, 1982). However,th echange si n Thiswa s supportedb y theadditiona l aromatase activity during areproductiv e findingtha tth earomatas e inhibitor 1,4 , cycle ofan yon e specieshav eno tbee n 6-androstatrien-3, 17-dioneblocke d the investigated. actiono ftestosteron e onpituitar yGtH . vande nHur k (1982)reporte d that Concluding Remarks shortly afterhatching ,rainbo wtrou t exhibita nincreas e in immunocytochemically Therear eman ybasi cproblem sremaining ; detectable GtH inth epituitar y after the identity ofGnR Han d the function of treatment withmethy ltestosterone , thevariou sbrai nregion swher e GnRHha s progesteronean d 17œ-hydroxyprogesterone. beententativel y localized areobvious . This indicates thatth epositiv e feedback However,althoug h there seemst ob ea effect canoccu r ata nearl yage ,an di s reasonable amount ofinformatio n available, not justa phenomeno ntha t occursa to r muchmor eneed st ob eknow nabou tth e near theonse t ofsexua l development. In actions ofLH-RH ,analogue s ofLH-RH ,and , addition,thes eresult s indicatetha t ultimately,nativ e GnRH. There isn ose t progestins aswel l asaromatizabl e ofcomprehensiv e studies available foran y androgens areactiv e atthi s stage. Since onespecie st oenabl eunderstandin g the theprogestin s dono thav e apositiv efeed roleo fth ereleasin ghormon e ina back effect ata late rag e (Crim et al., reproductive cycle,o rth eeffect so n 1981b), itwoul d beo finteres t todeter pituitary and gonadal activities ofacut e mineth echange s insteroi d responsiveness and chronicexposur e torang e ofdosage s of asdevelopmen tproceeds . syntheticreleasin g hormone. Without Onth ebasi s ofligh tmicroscop estudies , development ofsuc h information for atleas t Olivereau§ Chamboll e (1978)an dOliverea u somerepresentative s ofth efamilie s within 8Oliverea u found that estradiol treatment whichther ear e commercially important ofimmatur e eels (Anguillaanguilla. )cause d species,th eattempt s attinkerin gwit h stimulation ofGt Hcells . variousphase s ofth ereproductiv e cycle Although aromatizable androgensan d with synthetic releasing hormonesma yb e estrogens caninduc e synthesis ofGt Hi n futile,o rproduc eresult swhic h could even thepituitar y ofimmatur e salmonidsan d besubjec t tomisinterpretation . Inspit e eels,i norde r forthi st ob e considereda ofthes epessimisti c cautions,ther eha s truly functionalpositiv e feedbackeffect , beenimportan t success inth eapplicatio n theremus t alsob ereleas e ofGt Hfro mth e ofLRH- Afo rinduce d ovulation ofsalmo n pituitary. Crim and Evans (1980)foun d and certain carp species. However,th e that therewa sgreate r spontaneousreleas e facttha tther ear emajo rdifference s in ofGt Hi nvitr o frompituitarie s taken therespons et o LRH-Abetwee n closely fromtestosteron e treated immature rainbow related speciesmake sth enee d forbasi c 36 research inthi s areamor epoignant . Billard,R. , §R.E . Peter,1977 . Perhapsth emos t intriguing development Gonadotropin release after implantation inthi s areaha sbee nth ediscover yo fGRIF , ofanti-estrogen s inth epituitar yan d andtha t DAha s GRIF activity ingoldfish . hypothalamus ofgoldfish ,Carassiu s The findingtha tD Aca nmodulat eth e auratus. Gen.Comp .Endocrinol .32:213 - responset oLRH-A ,an db yblockin g DA 220. activitywit h arecepto r antagonist the Breton,B. , B.Jalabert ,R . Billard§ goldfish ismor eresponsiv et o LRH-Aan d C.Weil , 1971. Stimulation invitr o more subject to induced ovulation,indi de laliberatio n d'hormone gonadotrope cates awa y ofregulatin g ovulationi n hypophysairepa ru n facteurhypo - those species that seemt ob e"resistant " thalamiqueche z laCarp e (Cyprinus toth eeffect s ofth ereleasin ghormone . carpio L.). C.R. Acad. Sei.,Pari s Thepresenc e ofGRIF ,th eaction s ofD Ao n 273D:2591-2594. GtHsecretio nthroughou t reproductive Callard,G.V. , 1982. Aromatase inth e cycles,an dwhethe r other factorshav e teleostbrai n andpituitary :rol ei n GRIF activity requires investigation in hormoneaction . In:C.J.J .Richte r(Ed) : teleosts ingeneral . The investigation of Proceedings ofth e International the interactions ofnativ eteleos t GnRHan d Symposium onReproductiv e Physiology DAo nGt Hsecretio n could leadt oimportan t ofFish . Pudoc,Wageningen . (Inpress) . developments inth eregulatio n ofvariou s Chang,J.P. ,A.F .Coo k §R.E .Peter , 1982. phases ofth ereproductiv ecycle , Influences ofcatecholamine s ongonado particularly ovulation andspermiation . tropin secretion ingoldfish ,Carassiu s The findingtha t there isa positiv e auratus. Gen.Comp .Endocrinol . (In feedback effect ofestrogen s andaromatiz - press). ableandrogen s insexuall y immature Chang,J.P. , SR.E .Peter , 1982a. Actions salmonidsan d eels changesth eperspectiv e ofdopamin eo ngonadotropi n releasei n generallyhel d onth eeffect so fse x goldfish,Carassiu s auratus. In:C.J.J . steroids onGt Hsecretion . Themos trecen t Richter (Ed.): Proceedingso fth e findingtha tth eonse t ofspermatogenesi s International Symposium/on Reproductive canb estimulate d throughthi smechanis m Physiology ofFish .Pudoc ,Wageningen . raises interestingprospect s forth e (Inpress) . manipulation ofthi sphas eo fth erepro Chang,J.P. , §R.E .Peter , 1982b. Effects ductive cycle;th epresenc e ofsuc ha ofdopamin e ongonadotropi n releasei n positive feedback effect needs tob e female goldfish,Carassiu sauratus . investigated inteleost s ingeneral . Neuroendocrinology.(In press). Chang,J.P. , SR.E . Peter, 1982c. Effects Acknowledgements ofpimozid ean ddes-Gly 10, [D-Ala6] luteinizing hormone-releasing hormone Iwis ht othan k Dr.Harr yCoo kfo r ethylamideo n serum gonadotropin reviewing themanuscript . Unpublished concentrations,germina l vesicle resultsreporte dherei nwer esupporte d in migrationan dovulatio n infemal egold partb ygrant sA63.7 1an d G0838fro mth e fish,Carassiu s auratus. Gen.Comp . Natural Sciences and Engineering Research Endocrinol. (Inpress) . Council ofCanada . Crim, L.W., 1982. Control ofgonadotropi c hormone secretion (GTH)b yth erainbo w References troutpituitar y gland. Evidence ofGT H inhibitionb y catecholamine and Anonymous, 1977a. Ane whighl yeffectiv e stimulationo fGT Hreleas eb y someothe r ovulating agent forfis hreproduction . neuroregulatory factors. In:K . Lederis Scientia Sinica20:469-474 . §D.S . Farner (Eds.):Neurosecretion . Anonymous, 1977b. A further investigation Plenum,Ne wYork .p .446 . onth e stimulatory effect ofa syntheti c Crim, L.W. SD.M . Cluett, 1974. Elevation analogueo fhypothalami c luteinizing ofplasm a gonadotropin concentration in hormonereleasin ghormon e (LRH-A)o n response tomammalia n gonadotropin spawning in"domesti c fishes". Acta releasinghormon e (GRH)treatmen t ofth e Biochem. Biophys.Sinic a 9:15-24. malebrow ntrou t asdetermine d by Ball,J.N. , 1981. Hypothalamic controlo f radioimmunoassay. Endocr.Res .Comm . thepar sdistali s infishes ,amphibian s 1:101-110. andreptiles . Gen.Comp .Endocrinol . Crim,L.W. , §D.M . Evans, 1979. Stimu 44:135-170. lation ofpituitar y gonadotropinb y Barnett,F.H. ,J . Sohn,S .Reichli n§ testosterone injuvenil erainbo wtrou t I.M.D.Jackson , 1982. Three luteinizing (Salmogairdneri) . Gen.Comp .Endo hormone-releasing hormone likesubstance s crinol.~37 :192-196 . ina teleos t fishbrain :non e identical Crim, L.W. §D.M . Evans, 1980. LH-RH- with themammalia n LH-RHdecapeptide . stimulated gonadotropin release fromth e Biochem. Biophys.Res .Commun .105:209 - rainbowtrou tpituitar y gland:a ni n 216. vitro assay fordetectio n ofteleos t 37 gonadotropin releasing factor. Gen.Comp . Goos,H.J.Th . §0 .Murthanoglu , 1977. Endocrinol.40:283-290 . Localisation ofgonadotropi n releasing Crira,L.W. ,§ D.M . Evans, 1982. Positive hormone (GRH)i nth e forebrain andneuro - steroid feedback ongonadotropi c hormone hypophsis ofth etrou t (Salmo gairdneri). injuvenil erainbo wtrout :evidenc e of Cell Tiss. Res. 181:163-168. gonadotropin synthesis and release follow Hontela,A. , 1982. Regulation andphysio ingtestosteron e administration via sil logical significance ofdail y cyclesi n asticcapsules . In:C.J.J .Richte r (Ed.): gonadotropinhormon e levels inth efemal e Proceedings ofth e International Symposium goldfish. Ph.D.Thesis ,Departmen t of onReproductiv e Physiology ofFish . Zoology,Universit y ofAlberta . Pudoc,Wageningen . (Inpress) . Hontela,A . §R.E .Peter , 1978. Daily Crim,L.W. ,D.M . Evans,D.H . Coy, 5A.V . cycles in serumgonadotropi n levelsi n Schally, 1981a. Control ofgonadotropi c thegoldfish :effect so fphotoperiod , hormonereleas e introut : influenceo f temperature,an dsexua l condition. Can. synthetic LH-RHan d LH-RHanalogue s in J. Zool.56:2430-2442 . vivo and invitro . Life Sei.28:129-135 . Hontela,A. , §R.E .Peter , 1980. Effects Crim, L.W., D'.M.Evans ,§ B.H .Vickery , ofpinealectomy ,blinding ,an d sexual 1982. Manipulation ofth e seasonal condition onseru m gonadotropin levels reproductive cycleo fth e landlocked inth egoldfish . Gen.Comp .Endocrinol . salmon (Salmosalar )wit h LHRHadmini 40:168-179. stration atvariou s stageso fgonada l Idler,D.R . §L.W . Crim, 1982. Gonado development. Can.J . Fish.Aqua .Sei . tropin releasing factor(s) (GtH-RF)fro m (Inpress) . hypothalamus ofwinte r flounder. In: Crim,L.W. ,§ R.E . Peter, 1978. The B.Loft s (Ed.): Proceedings ofth e influence oftestosteron e implantation in Ninth International Symposiumo n thebrai n andpituitar y onpituitar y Comparative Endocrinology. University gonadotropin levels inAtlanti c salmon ofHon g Kong Press,Hon g Kong. (Inpress) . parr. Ann.Biol .Anim . Biochim. Jiang,R. ,S.Huan g §W .Zhao , 1980. Biophys.18:689-694 . Changeso fseru m gonadotropin levels Crim, L.W., R.E.Pete r §R . Billard, 1976. before andbehin d induced spawning in Stimulation ofgonadotropi n secretionb y grasscar pan d silver carp. J.Fish . intraventricular injection ofhypothal China. 4:129-133. amicextract s inth e goldfish,Carassiu s Kah,0. ,P .Chambolle ,P .Dubour g § auratus. Gen.Comp .Endocrinol .30:77-82 . M.P.Dubois , 1982a. Distrubution of Crim, L.W;, R.E.Pete r §R . Billard, 1981b. immunoreactiveLH-R H inth ebrai no f Onset ofgonadotropi c hormone accumulation thegoldfish . In:C.J.J .Richte r (Ed.): inth e immaturetrou tpituitar y glandi n Proceedings ofth e International responset oestroge n oraromatizabl e Symposium onReproductiv e Physiology of androgen steroid hormones. Gen.Comp . Fish. Pudoc,Wageningen . (Inpress) . Endocrinol.44:374-381 . Kah,0.,R.E .Peter ,P . Dubourg 6H .Cook , Dodd,J.M. , P.A.C.Stuart-Kregar ,J.P . 1982b. Effectso fmonosodiu m L-glutamate Sumpter,L.W . Crim &R.E . Peter, 1978. onpituitar y innervation ingoldfish , Premature sexualmaturatio n inth eAtlan Carassius auratus. Gen.Comp .Endocrinol . ticsalmo n (Salmo salar L.). In: P.J. (Inpress) . Gaillard §H.H . Boer (Eds.): Comparative King,J.A . SR.P .Millar , 1979. Hetero Endocrinology. Elsevier/North-Holland, geneity ofvertebrat e luteinizing- Amsterdam, p.101-104 . releasing hormone. Science. 206:67-69. Donaldson,E.M. , G.A. Hunter 5H.H . Dye, King,J.A . SR.P .Millar , 1980. 1981/1982. Induced ovulation incoh o Comparative aspectso f luteinizing salmon (Oncorhynchus kisutch). II. hormone-releasing hormone structurean d Preliminary study ofth eus eo fLH-R Han d function invertebrat ephylogeny . Endo twohig hpotenc y LH-RHanalogues . Aqua- crinology. 106:707-717. culture. 26:129-141. Lin,H.-R. ,R.E .Peter ,CS . Nahorniak § Donaldson,E.M. , G.A. Hunter,H.M . Dye $ 0. Bres, 1982. Actions ofth esuper - G.Va nde rKraak , 1982. Induced active analogue ofDes-Gly 1°[D-Ala°]LRH ovulation inPacifi c salmonusin g LH-RH ethylamide (LRH-A)o n gonadotropin analogsan d salmon gonadotropin. In: secretion ingoldfish . In:B . Lofts (Ed.): B. Lofts (Ed.): Proceedings ofth eNint h Proceedings ofth eNint h International International Symposium onComparativ e Symposium onComparativ e Endocrinology. Endocrinology. University ofHon gKon g University ofHon g KongPress ,Hon gKong . Press,Hon g Kong. (Inpress) . (Inpress) . Dubois,M.P. ,R . Billard, B.Breton SR.E . Münz,H. ,B .Claas ,W.E .Stump f §L .Jennes , Peter, 1979. Comparative distribution 1982. Centrifugual innervation ofth e ofsomatostatin ,LH-RH ,neurophysin ,an d retinab y luteinizing hormone releasing «-endorphin inth erainbo w trout:a n hormone (LHRH)-immunoreactivetelen - immunocytological study. Gen.Comp . cephalicneuron s inteleostea nfishes . Endocrinol.37:220-232 . CellTiss . Res. 222:313-323. 38 Münz,H. ,W.E .Stump f $L .Jennes , 1981. Peter,R.E. ,L.W . Crim,H.J . Th.Goo s§ LHRHsystem s inth ebrai n ofplatyfis h J.W. Crim, 1978. Lesioning studieso n Brain Res. 221:1-13. thegravi d female goldfish:neuro Nagahama,Y . §R.E . Peter, 1982. Effects endocrineregulatio n ofovulation . Gen. ofbrai n lesions on gonadotropultra - Comp.Endocrinol . 35:391-401. structure and serum gonadotropin levels Peter,R.E. ,0 . Kah,'CR . Paulencu,H .Coo k ingoldfish . CellTiss .Res . (Inpress) . §A.L . Kyle, 1980. Brain lesionsan d Nozaki,M . §H . Kobayashi, 1979. short-term endocrine effectso fmono - Distribution ofLHRH-lik esubstanc ei n sodium L-glutamate ingoldfish ,Carassiu s thevertebrat ebrai n asreveale db y auratus. Cell Tiss.Res . 212:429-442. immunohistochemistry. Arch.Histol . Peter,R.E . $CR . Paulencu, 1980. Japan. 42:201-209. Involvement ofth epreopti c regioni n Olivereau,M . §P . Chambolle, 1978. gonadotropin release-inhibition ingold Ultrastructure des cellules gonadotropes fish,Carassiu s auratus. Neuro- de l'Anguillenormal e etaprè s injection endocrinology. 31:133-141. d'oestradiol. CR. Acad. Sei.Paris . Pickford,CE. ,W.R . Knight,J.N . Knight, 287D:1409-1412. R. Gallardo §B.I .Baker , 1981. Long- Olivereau,M . §J . Olivereau, 1979a. term effectso fhypothalami c lesions Effect ofestradiol-17 ßo nth ecytolog y onth epituitar y and itstarge t organs ofth e liver,gonad s andpituitary ,an d inth ekillifis h Fundulusheteroclitus . onplasm a electrolytes inth e female I. Effectso nth e gonads,thyroid ,an d freshwater eel. Cell Tiss.Res . 199:431- growth. J. Exp. Zool. 217:341-351. 454. Schreibman,M.P. ,L.R . Halpern,H.J .Th . Olivereau,M . §J . Olivereau, 1979b. Goos SH .Margolis-Kazan , 1979. Estradiol-positive feedbacko ngonado Identification of luteinizinghormone - tropic (GTH)cell s in freshwatermal e releasing hormone (LH-RH)i nth ebrai n silver eels. Gen.Comp .Endocrinol . andpituitar y glando fa fis hb y 39:247-261. immunocytochemistry. J. Exp.Zool . Pan,C.-H. ,M.-Q .Feng ,N.-C . Ling,S .Pao , 210:153.159. / W.-Q. Xu,G.-X . Xu §R.-C .Shen , 1979. Schreibman,M.P. ,H .Margolis-Kaza n§ Immunocytochemical studies ongonado L.Halpern-Sebold , 1982. Immuno- tropinreleasin g hormone (GRH)secretor y reactivegonadotropi n and luteinizing nucleus ofth e carp (Cyprinus carpio). hormonereleasin ghormon e inth e ActaBiol .Exp .Sinica .~Ï2~306-310. " pituitary glando fneonata lplatyfish . Pan, X.,D . Chen,Z . Zhao,Y . Zhu,X . Gen. Comp.Endocrinol . (Inpress) . Zhao,H . Lin,Z . Liu,Z .Li u SD .Lin , vande nHurk ,R. , 1982. Effectso f 1981. Studieso nmechanis m ofactio n of steroids ongonadotropi c (GTH)cell si n luteinizing releasinghormon e (LH-RH)i n thepituitar y ofrainbo w trout,Salm o ovulation of fish. ScientiaSinica . 24: gairdneri,shortl y afterhatching . Cell 982-988. TissT Res. 224:361-368. Peter,R.E. , 1970. Hypothalamic control Weil,C , R.Billard, B.Breto n §B . ofthyroi d gland activity and gonadal Jalabert, 1978. Pituitaryrespons et o activity inth e goldfish,Carassiu s LH-RH atdifferen t stageso fgametogenesi s auratus. Gen.Comp .Endocrinol .14:334 - inth erainbo wtrou t (Salmogairdneri) . 3S6T Ann. Biol.Anim . Bioch7~Bio"phys. 18:863- Peter,R.E. , 1980.. Serum gonadotropin 869. levels inmatur emal e goldfishi n responset o luteinizing hormone-releasing hormone (LH-RH)an d des-Gly1°-[D-Ala6]- LH-RHethylamide . Can.J . Zool.58:1100 - 1104. Peter,R.E. , 1982. Neuroendocrine control ofreproductio n inteleosts . Can J. Fish.Aquati c Sei. 39:48-55. Peter, R.E.,J.P .Chang,A.F . Cook § CS. Nahorniak, 1982. Neuroendocrine regulation ofgonadotropi n andgrowt h hormone secretion ingoldfish . In:B . Lofts (Ed.): Proceedings ofth eNint h International Symposium on Comparative Endocrinology. University ofHon g Kong Press, Hong Kong. (Inpress) . Peter,R.E . (, L.W.Crim , 1978. Hypo thalamic lesions ofgoldfish :effect so n gonadal recrudescence and gonadotropin secretion. Ann.Biol .Anim .Bioch . Biophys. 18:819-823. 39 AROMATASE INTH ETELEOS TBRAI N ANDPITUITARY :ROL EI NHORMON EACTIO N G.V.Callar d Biology Department,Bosto nUniversity ,Boston ,Massachusetts ,U.S.A . Summary (McEwene tal. ,1979 ). One concept that isno woutdate d istha tse x For certain androgen-dependent behavioralan d steroidsar e sex specific,namely ,tha tandro neuroendocrine responses,prio r conversiont o gensar emal ehormone swherea s estrogensar e estrogen isessential . Aromatization ofan femalehormones . From the earliestbehavior - drogen toestroge n occurs inth ebrai no f ists,i twa sknow n tha*tandrogen s andestro representatives ofal lmajo r vertebrate gens often share commonaction s thatar ein groups,althoug h activity isextraordinaril y dependent ofgender . To Illustrate,copula - high inteleosts . Retention ofestroge ni n torybehavio r incastrate d ratsma y beacti discretely dissected brain regions following vated eitherb y testosterone orb yestradiol . androgenperfusion ,correspond s toth eneuro 'Paradoxical'effect s of sex steroidshav e anatomy distribution ofth eenzyme . Bycon nowbee n documented for everymajo r vertebrate trast,n o freeestroge n is recovered from group. Inaddition ,th egonad so fbot h sexes brainafte rperfusio nwit h estrogen. Anac arecapabl e of synthesizing and secreting tiveconjugatin g system forestroge npresen t androgenan d estrogen. The relativeoutpu t inth egil l inactivates orexcrete scircula of these twoclasse s of steroid doesno t ting estrogen. Thus,onl y estrogensynthe necessarily conform toexpectation sbase do n sized inclos eproximit y tocentra l targetsi s sex. Infemal e rabbits,hamsters ,an dturtles , biologically active. High levels ofaromatas e tocit ethre e examples,circulatin g androgens have alsobee n identified inteleos t pituita aremuc h greater thancirculatin g estrogens ry. Ina n rnvitr o translocation system, and fluctuations arekeye d toth e ovulatory testosterone,presumabl y via conversiont o cycle. Similar examplesma yb e found inthi s 3 estradiol,compete swit h H-estradiolfo r symposium. nuclearbindin g sites. We conclude that,i n teleosts,neithe r thebrai n nor thepituitar y Androgensand^estrogen sare ,i nfact ,com iswholl y dependent oncirculatin g estrogen, ponents ofth e same steroidogenic pathway. assuming aromatizable substrate isavailable . Testosterone andandrostenedion e are theimme Keywords: brain,pituitary ,aromatase , diatean d obligatory precursors ofestradio l estrogen,teleost . and estrone,respectively . Conversiono f androgen toestroge n (aromatization) iscon trolled by aP-45 0linke d enzyme complex Introduction termed aromatase. This enzyme isnot ,however , anexclusiv epropert y ofth eprimar y estrogen A relationship between thegonad san dbreed secreting organs (placenta,gonads )bu texist s ingbehavio r inhuman s and domestic animals also ina variet y ofperiphera l non-glandular hasbee nknow n sinceancien t times. Neverthe tissues (fat,muscle ,bone) . Of particular less,identificatio no fth eactiv e principles importance toth eneuroendocrinologis t was and theirmechanis m and sites ofactio nar e the identification in197 1b yNaftolin ,Ryan , relatively recent developments. It isno w and colleagues of low levels ofaromatas eac accepted that sex steroids acting ontarge t tivity inth ebrai n (Naftolin et al., 1975). cells inth ebrai n and elsewhere inth ecen Numerousphysiologica l andbehaviora l studies tralnervou s system (CNS)trigge r sexbehavio r inth e last tenyear s support the ideatha t inadult san dpermanentl y alter the 'maleness' aromatization toestroge n inth ebrai n itself or 'femaleness'o f theCN S of fetalan dneo isessentia l for the expression of certain natal animals. Via complex positivean d central androgenactions ;however ,sinc esep negative feedbackmechanisms ,androgen san d aratebindin gmechanism s forandroge nan des estrogens controlhypophysiotrophi c centers trogenar e simultaneously present inth ebrai n inth ebrai nand ,t o someextent ,affec t the ofbot h sexes,i ti s likely that androgens pituitary directly. Through these actions havebiologica l actions inthei r ownright . they are responsible forth erhythmi c patterns Indeed,comple x behavioral responses appear ofgonada l development characteristic ofovu tob ea composit e of several androgen-an d latory and seasonal cycles. There isgoo d estrogen-dependent components. Naturalchan evidencetha t themechanis mb ywhic h steroids ges inbrai n aromatase inrelatio n toage , interactwit h neuroendocrine tissues isth e gender,an d reproductive cyclesa swel la s samea stha t inperiphera l steroidtargets , substanceswhic h regulatearomatas e inbrai n and includes,i nsequence ,bindin g to specific inviv o and rnvitr ohav ebee n reported (Naf cytosolic receptors,translocatio n ofthi s tolin etal. , 1975;Callard ,1980 ;Callar de t complex tonuclea r acceptor sites,and ,ul al., 1980b;1981a ; 1982). Bycontrast ,littl e timately,alteratio n ofgenomi c functions isknow nabou t aromatase inth epituitary . 40 The first biochemical identification of ac tivity in this gland was reported from this laboratory and was the result of investiga 117.5 RETENTION OF FORMED ESTROGEN tions in a teleost, the longhorn sculpin (Myoxocephalus octadecimspinosus) (Callard . . FOREBRAIN et al., 1981b). We review here aromatization y 10- in the neuroendocrine tissues of teleosts and speculate on the function of this enzyme in mediating androgen action. X MIDBRAIN in 7.5i CM -* ** Brain \ (3 ~ As part of a phyletic survey, aromatase ac £ HINDBRAIN tivity in the brain of certain non-mammals i 5.0 -* »- was found to be much higher than in common O S 2.5 laboratory species (Callard et al., 1978). li. In the sculpin, for example, estrogen yields were =190 fmol/mg tissue, 1000-fold greater ~n- than that reported for adult rat and rabbit -an. (0.1-1.0 fmol/mg) and higher than other spe AROMATASE ACTIVITY cies tested. To date, seven other teleost genera have been studied: Pseudopleuronectes, 80 Opsanus, Coris, Serranu s, Paracentropristis , Spicara, and Pagellus (Callard & Reinboth, in preparation). Although none have levels 60 X quite as high as the sculpin, it would appear \ that the potential for synthesizing substan o tial amounts of estrogen may be a general S 40- O characteristic of the teleost brain. Notable S / omissions from this survey, however, are u. fresh-water species and representatives of 20 less advanced orders. In contrast to tele osts, brain conversions are low in elasmo- llnnnnr, nd branchs (Squalus, Raja), only tentatively xO-«. * identified in Petromyzon, and undetectable in o** Myxine and Amphioxus (Callard et al., 1978; 1980a). These results imply that estrogen formation The idea that estrogen synthesis in neuro in situ (aromatization) may be an alternative endocrine tissues occurs in close proximity to conventional steroid receptors for achiev to receptors is supported by the observation ing high local concentrations of active hor that, in mammals, activity is restricted to mone (estrogen). Such a mechanism might be the hypothalamus, preoptic area, septum, and essential where estrogen exerts effects via amygdala, structures which have high concen non-genomic mechanisms (e.g. short-latency trations of estrogen-binding cells, and are electrophysiological changes). Even in re known by other criteria to control reproduc gions with conventional steroid receptors, tion and sex behavior. That estrogen formed responses may depend on local hormone concen locally is retained in the same brain regions trations in excess of those in plasma. In was first demonstrated directly using the the sculpin, gonadal aromatase is low or un isolated, perfused Rhesus brain, a preparation detectable. By contrast, teleost gonads are which precludes aromatization in peripheral known to synthesize large amounts of androgen tissue (Naftolin et'al., 1975). It seems from C-21 precursors. Thus, the gonad of the paradoxical in the light of these early stu fish may serve only as a source of aromatiz- dies that, in the teleost, as in other non- able pro-hormone. mammals, aromatase is found outside the 're A final consideration is whether circula productive brain', in palliai,midbrain , and ting estrogen is present in a form which has hindbrain areas which have little or no access to target sites within the brain. labelling in autoradiograms. Nevertheless, Shortly after birth in rodents,plasm a estro retention of authentic estrogen in discrete gen is extensively bound to ot-fetoprotein, brain regions is consistent with their although androgens are poor ligands for the enzyme levels (Callard et al., 1981a; Fig.1) . same binder. Coincidentally, brain aromatase activity is relatively high. Presumably, Figure 1. Comparison of aromatase activity estrogen that occupies receptor sites in the as determined by homogenate assay (lower brain at this stage of development is derived panel) and estrogen retention in the same solely from local synthesis (McEwen et al., regions after perfusion with 3H-androgen 1979). We found recently an analogous system (upper panel) (Myoxocephalus). in sculpin.- In order to test the distribution of circulating estrogen, the isolated teleost 41 headwa sperfuse dwit h 3H-estradiol (Callard proximity tocentra l targets isbiologicall y & Claiborne,i npreparation) . Incontras t active. An intriguing question iswhethe r to resultswit h androgen,n o free estrogen high brainaromatas e levels inth e sculpin was recovered frombrai n although small area caus e ora consequenc e ofestroge n amounts of labeled steroid inpola r fractions inactivating pathways inperiphera l tissues. ofth e tissue extractswer e judged tob e estrogen conjugates. A large fractiono f Pituitary •• radioactivity inth e spent perfusatewa sals o found inth e conjugate fraction. Inorde r Until recently,pituitar y tissueswer ebe to identify the site ofestroge nmetabolism , lieved tob earomatase-negativ e (Naftoline t tissues sampled from the sculpinhea dan d al., 1975;McEwe n et al., 1979). Using the trunkwer e incubatedwit h radiolabeled sub samehomogenat e assay as forbrain ,n odetec strate (Callard andManz , inpreparation) . tableestroge nwa s formedb y theglan d of rats, Activity could be ranked as follows: gill» rabbits,monkeys ,o rth ehuma n fetus. Fur liver-kidney=gut>brain=ovary>muscle. Con thermore,followin g administration of3 H-an- version ingil l filamentswa s soextensiv e drogen,n oestroge nwa s found inth ehyophy - that3 h afte radditio n of substrate only siso f ratsan dmonkey s although brain regions 40% of theadde d radioactivity was ether- known tohav earomatas ewer epositive . By extractable (Fig.2) . contrast,whe n the isolated sculpinhea dwa s perfusedwit h androgen,larg e amountso f Figure2 . Conversion of 3H-substrate( 4nM ) authentic estrogenwer e recovered fromth e topola rmetabolite s by gill filaments. pituitary (Callard et al., 1981a;Fig .1) . Minced tissues (=50mg )wer e incubateda t Wepostulate d that this estrogenwa s derived 22° forth e time indicated. Steroidswer e from thebrain ,since ,i nteleosts ,th ean separated into 'free'an d 'polar'fraction s terioran dposterio r lobesar e not readily by etherextraction . separable andbot h receive direct neuralin put from thehyopthalamus . To test thisidea , thebrain/pituitar y complexwa s cultured in tact;alternatively ,th estal kwa s sectioned and the two tissues cultured separately (Cal lard et al., 1981b). Following additiono f 3H-androgen,radiolabele d estrogenproduct s were isolated and identified. Not onlywa s thepituitar y capable ofaromatizin g androgen independently ofth ebrain ,bu t activitywa s evengreate r peruni tweigh t than inadjacen t preoptic/hypothalamic regionsknow n tohav e high aromatase (3700vs .60 0 fmol/mgtissue , respectively). Ina subsequent experiment, thepituitar ywa s sectioned transversely into cephalic and caudalhalves . Inal lcultures , the caudal half synthesized farmor e estrogen than thematche d anteriorhal f (2:1 to 8:1) (Callard et al., inpreparation) . Although pituitary cytology hasno tbee n examined in this fish,th edat a indicate thataromatas e may be restricted tocertai n secretory cell types. Using similar expiant culturetech niques,w e found that aromatase isno tuniqu e Moreover,estroge nwa smetabolize d twicea s toth e sculpinpituitar y but canals ob eiden efficiently as testosterone. Mild acidhy tified inothe r teleosts (seeabove ;Callar d drolysis (pH5.0 ,37 °x 2 4h )wit h orwithou t & Reinboth,i npreparation) ,i navia nan drep Helixpomati a digestive juice (sulfatase/glu- tilianadenohyophysis ,an d in transformed curonidase)indicate d that aportio n ofth e pituitary cell strains derived from rodents estrogenmetabolite swer e sulfoconjugates; (Callard et al., submitted). Inth e latter glucuronidase (sulfatase-free)wa s ineffec studies,activit ywa spresen t insomato/lac - tive. Inth e sculpin,therefore ,th egil l totropes butno t incorticotrope s orpresump reduces free estrogen inth e circulationand , tive gonadotropes,supportin g the idea that inthi s respect,resemble s themammalia nkid aromatasewithi n theglan d isno tubiquitous . ney. Whether orno t the gill also excretes conjugated steroids isno tknown . Ifso , There isgoo d evidence that estrogenso r conjugated steroids recently identified as aromatizable androgensactin gdirectl y onth e teleost pheromonesma y enter the environment teleost pituitary havepositiv e feedback ef via this route. From experiments inNecturu s fects. Moreover,a naromatas e inhibitor (ATD) testis,w ekno w that conjugated estrogensar e canbloc k responses toaromatizabl e androgen not acceptable ligands for receptor binding (Crime t al., 1981). Although there isn o (Mak etal. , submitted). Hence,i n thiste biochemical evidence forestroge nreceptors , leost,onl y estrogen synthesized inclos e thepituitar y isheavil y labeled inautoradio - 42 grams following treatmentwit h3 H-estradlol tary,an dman y other androgen targets,con (Davise t al., 1977). This tissue,therefore , version ofcirculatin g pro-hormone toon eo r seemed tob eadvantageou s for studyingdi more activemetabolite s at its site ofactio n rectly the relationship between estrogensyn may be required. Thisadde d step inth e thesis,bindin g and translocation of estrogen events leading tobiologica l activation pro formed in situ,an d aquantifiabl e biological videsa n opportunity for regulation andserves , response. An invitr o translocation system therefore,a sa 'fine-tuning'mechanis m for was developed (Callard &Manz , inpreparation ; adjusting responses. At the same time iti s Table1) . a siteo fpotentia ldefects . Table 1. Inhibition ofuptak e of3 H-estradiol References bypituitar y cellnucle i (Myoxocephalus) (Callard &Manz , inprep a ration ). Callard,G.V. , Z.Petro ,& K.J .Ryan ,1978 . Conversion ofandroge n toestroge nan d other steroids inth evertebrat ebrain . Radioinert competitors % inhibition Amer.Zool .18:511-523 . (200-fold excess) Callard,G.V . 1980. Aromatization iscyclic - AMP dependent inculture d braincells . Estradiol-17ß 90.5 BrainResearc h204:461-464 . Progesterone 0 Callard,G.V. , Z.Petro ,& K.J .Ryan ,1980a . Testosterone 78.7 Aromatization and 5a-reduction inbrai n 5a-dihydrotestosterone 54.4 andnon—neura l tissues ofa cyclostome , ATD 0 Petromyzonmarinus . Gen.Comp .Endocrinol . ATD +testosteron e 38.4 42:155-159. Callard,G.V. , Z.Petro ,& K.J .Ryan ,1980b . Aromatization ofandroge n toestroge nb y Thequantit y of H-estradiolboun d tocel l cultured turtle brain cells. Brain nuclei from the teleost gland closely resem Research202:117-130 . bled that reported formammalia npituitar y in Callard,G.V. , Z.Petro, /K.J.Ryan ,& J.B . vitro (4.9v s 7.8 fmol/lOmg tissue,respect Claiborne,1981a . Estrogen synthesisi n ively) (Leavitt et al., 1969). Estradiolbu t vitro and_i nviv o in thebrai n ofa marin e notprogesteron e competed fornuclea rbindin g teleost (Myoxocephalus). Gen.Comp . sites,a goo d index of receptor specificity. Endocrinol.43 :243-255 . Total displacement of radiolabeled estrogen Callard,G.V. , Z.Petro ,& K.J .Ryan ,1981b . wasals o obtained with a200-fol d excesso f Biochemical evidence foraromatizatio n of anaromatizabl e androgen;however ,th enon - androgen toestroge n inth epituitary . aromatizable androgen,DHT ,wa spartiall y Gen.Comp .Endocrinol .44:359-364 . effective. SinceDH Tan d especially itsSB - Crim,L.W. ,R.E .Peter ,& R .Billard ,1981 . reducedmetabolit e areknow n tobin d tomam Onset ofgonadotropi c hormone accumulation malian estrogen receptors,w e also tested inth e immature troutpituitar y gland in the effects of testosterone inth epresenc e response toestroge n oraromatizabl e ofa naromatas e inhibitor. Inamount s equi- androgen.Gen .Comp .Endocrinol .44:374-381 . molarwit h substrate,AT D reduces estrogen Davis,R.E. ,J.I .Morrell ,& D.U . Pfaff,1977 . yields by 50%(Callar d et al., 1980b). Al Autoradiographic localization ofsex - thoughAT D alone did notbind ,i tblocke d steroid concentrating cells inth ebrai n theabilit y of testosterone tocompet ewit h of theteleos tMacropodu s opercularis. radiolabeled ligand. Subsequent experiments Gen.Comp .Endocrinol .33:496-505. . showed thatestroge nbindin gwa s slightly Leavitt,W.W. ,J.P .Friend ,& J.A .Robinso n greater inth e cephalic half of thegland ; 1969. Estradiol-specificbindin gb y however,despit e lower levels ofaromatas e pituitary nuclear fraction invitro . inthi s region,testosteron e completely dis Science165:496-498 . 3 placed H-estrogeni nbot h anterior and pos McEwen,B.S. ,P.G .Davis ,B .Parsons ,& D.W . teriorportions . Similar methods applied to Pfaff,1979 . Thebrai na sa target for brain failed todetec t specificbinding . steroid hormone action. Ann.Rev .Neuro — Relative topituitary ,bindin g inbrai ni s sei.2:65-112 . generally low. We conclude that this isa Naftolin,F. ,K.J .Ryan ,I.J .Davies ,V.V . useful system forfurthe r examiningquanti Reddy,F .Flores ,Z .Petro ,R.J .White , tative relations between aromatizationan d Y.Takaoka ,& L .Wolin ,1975 . Theforma thebiologica l expression of steroid action. tiono f estrogens by centralneuroendo crine tissues. Rec.Prog .Horm .Res . Conclusions 31:295-319. Acknowledgements:NSF-PCM78-23214 ;HD-15594 ; These and other recent developments inendo Mount Desert Island BiologicalLaboratory ; crinology indicate clearly that Starling's and SusanKendall . definition ofa hormon e isno t strictlyvalid . Notalway s isth eactiv emolecul epresen t in theblood . Inth ecas e of thebrain ,pitui — 43 GONADOTROPIC CELLS INTH EPITUITAR YO FTELEOST S P.G.W.J,va nOord t SectionComparativ eEndocrinology ,Zoologica lLaboratory ,Universit y of.Utrecht ,th e Netherlands Summary thepar sdistalis ,ofte ni nth eventra lpar t of thePPD ,wher e theyma y forma soli dven Immunocytochemical and histophysiological tralri mo f cells,a s forexampl e incypri - evidencepoint s toglobula rbasophil s asth e nodonts. Inothe rcase s theyar e spread source ofa glycoprotei n gonadotropichor throughout therostra lpar sdistali s (RPD) mone (GTH).Thi s cell typema yb e situated and thePPD ,a sfo rexampl e insalmonid san d invariou spart s of theadenohypophysis ,bu t eels (reviews:va nOord t &Peute , 1982,1983). inman y species isconcentrate d inth even Gonadotropsbelon g toth ebasophili c cell tralregio no f theproxima lpar s distalis types.Thi smean s that thecontent so fthei r (PPD). Itha s irregular cisternaeo fth e granulesreac twit hperiodi c acidan d granular endoplasmicreticulu m (GER),secre Shiff'sreagen t (PAS).The y also stainwit h tory granuleso fvaryin g electrondensity , Alcianblu e (AB)an d otherdye s for strongly 200-500n m indiameter ,an d larger,les s acid groups.Lik eothe rbasophil s theGTH - electrondens eglobules .Amon g the contents cellshav e irregular,mor eo r lessdilate d ofgranule s and globules aremateria lre cisternaeo f theGER ,an dusuall yroun dse actingwit hanti-(E>)-teleost-GTH ,an daci d cretory granuleswit h contents ofvaryin g phosphatase.Extrusio no fGT H isaccompanie d electrondensity .Amon g suchbasophil s the bya los so fgranule s and globules anda n gonadotrops arecharacterize d by therela increase innumbe r and sizeo f theGE R cis tively largediamete r of theirgranule s ternae.Cisterna l cellsan d avariet yo f (200-500 nm), and especiallyb y thepresenc e other cell typeshav ebee ndescribe d asa ofmuc h larger,roun d or irregularly shaped separate typeo fgonadotrop .However ,i t globules,wit hmor eelectro n translucent seems thateve ni f twomorphologica l types contents. ofgonadotrop s canb edistinguished ,bot h mayb eabl e toproduc ematurationa l aswel l Identification ofGTH-cell s asvitellogeni c GTH.Th ephysiologica l sig nificance of secretoryglobule s apartfro m Amoder napproac h toth equestio no fiden granules isunknown .Th eproductio n andex tifying thegonadotrop s isimmunocytochemis - trusiono fGT H isregulate d byperiphera l try.Th efirs t toappl y thismetho d ontele - and centralfactors .Amon g thecentra l sti ostmateria lwer eM cKeow nan dva nOverbeek e muli arevariou s typeso fneurosecretor y (1971)an dBillar d etal . (1971).Applyin g fibres thati nman y teleostspenetrat eth e thedoubl eantibod y fluorescence technique adenohypophysis and directly innervateglan withanti-ovine-L H oranti-carp-GT H asfirs t dular cells,includin ggonadotrops . antibody,reaction swer efoun d intw otype s Keywords:gonadotropi c cells,immunocytoche - ofbasophil s of thepar sdistalis ,consider mistry,histophysiology ,neuroendocrin ein ed tob e thegonadotrop s and thethyrotrops . nervation. Invie wo fth echemica lrelatio nbetwee nGT H and thyrotropichormon e (TSH),a reactio no f Introduction thyrotropswit h anti-GTH isn o surprise.I n thatrespect ,Goo s etal . (1976)hav ewarne d Inmos tvertebrates ,includin g thetele thatwhe nheterologou s antibodies arebein g osts,th epituitar y gland takesa centra lpo used,on eha s tob e aware ofunspecifi cre sition inth e endocrine system (review: actions and ofimmunoreactiv edeterminants , Holmes &Ball , 1974). Itconsist s ofa neu present inmolecule s of chemically related ral component orneurohypophysi s and aglan constituants ofpituitar y cells.A s are dularpar t oradenohypophysis ,derive d from sult,othe r celltype s thangonadotrop sma y anectoderma lplacode .Th e adenohypophysis reactwit hanti-GTH . secretesmor e thansi xdifferen thormones , Agoo d example isth eresult so fPeut ee t each ofwhic h isbelieve d tob eforme d ina al. (1982)wit h theAfrica n catfish,Claria s separate celltype . lazera.Thre e cell types cross-reactedwit h The cell types canb erecognize d atth e anti-carp-GTH: large,strongl y PAS-positive lightmicroscopica l levelb y theirdistribu cellswit hgranule s andglobules ,formin g tionan d thestainabilit y of thesecretor y themai n cell typei nth ePPD ;small ,mainl y granules,an d atth eelectro nmicroscopica l AB-positive cellswit hfin e granules,als o levelb y thesiz ean d shapeo f thesecretor y inth ePPD ;an d someerythrosinophili c cells granules and thefor mo f theGER .Thus ,i n inth epar s intermedia.A nanti-salmon-GT H teleosts cells secreting GTHar esituate d in and anantibod y against the ß-subunito f 4.4 carp-GTHreacte dwit h constituants ofth e wayenhance s storageo fsecretor ymateria l globular basophils only.Obviously ,th ean inth egonadotropi c cells. (Gielen etal. , tiserumagains t carp-GTH containedmor ean d 1982).Reciprocally ,accordin g tova nPutte n lessspecifi c antigen determinants thanth e etal . (1981),remova lo f thegonad si n otherantibodies . adultrainbo w trout leads toa stron gex Other importantpoint s inimmunocytochemi - trusiono fGT H and toa los so f theglobule s calstudie s are thetechnique s ofprocessin g andmos t of thegranules .Thes esecretor y of thepituitarie s and thedilutio no fth e vesicles arereplace db y anincreasin gnum firstantibody ,applie d inth edoubl eimmu - bero fdilate d cisternaeo f theGER .I nex nocytochemical technique.Leunisse n etal . treme cases thecisterna e coalesce tofor m (1982)observe d that inplasti c embedded abi gvacuole .Simila r changeshav ebee nno blockso fglutaraldehyd e andparaformalde ticeddurin g theannua lreproductiv e cycle hydefixe d rainbow trout (Salmogairdneri ) byPeut e et al. (1978),an d strengthenth e pituitaries,dependin g on theconcentration , opinion that inth erainbo w troutglobula r anti-carp-an danti-salmon-GT Hreacte d and cisternalbasophil sbelon g toon ean d eitherwit h constituants ofal lcel ltypes , thesam e typeo fgonadotropi c cell.A secon d ordi dno treac t atall .I ncryoultramicro - typeo fpresume d gonadotrop couldno tb e tomesection so f the samemateria l onlydi found. Iti stru e that inth ePP D ofnorma l lutions lower than 1: 800 0o f the twoanti - andgonadectomize d rainbow troutva nPutte n gonadotropins and anantibod y againstth e etal . (1981)observe dbasophil s differing @-subunit of carp-GTHa ta dilutio no f 1: from theglobula r cellsb y theabsenc eo f 1000- 400 0selectivel y reactedwit hth e globules,an dfro m the cisternal cellsb y contentso f thegranule s andglobule s ofth e thepresenc eo fnumerou s granules andb y presumedgonadotrops . the smalldimension s of theGE Rcisternae . Thissomewha t intermediate form tendedt o These examplesma y suffice tosho wtha t beconcentrate d inth erostro-dorsa lPPD . immunocytochemical techniquesnee d tob e Injuvenil e troutva nPutte ne tal .(unpu perfected,an d one improvementmigh tb eth e blished results)coul d stimulate thesecell s introduction ofmonoclona l antibodies toab with goitrogens,an d observed animmunocyto solutely purehormones .Eve n so,thes etech chemicalreactio nwithi n thesecretor ygra niqueswil lneve r suffice toidentif y the nuleswit hanti-human-@-TSH .So ,i tseem s cellular sourceo fpituitar yhormones ,bu t that thesecell s are thyrotrops,no t ase willalway shav e tob e combinedwit hhisto - cond typeo fgonadotrop . physiological research. Infact ,suc ha com bination isgenera lpractice .Fo rexample , thewor kb yLeunisse ne t al. (op.cit.)form s One or two types ofGTH-cell s parto fa serie so f studies on thegonado tropic cells inth erainbo w trout,carrie d Thatdoe sno tmea n thati nth epituitar y outb y agrou p ofcomparativ eendocrinolo of therainbo w trout sucha secon d typeo f gists ofth eUtrech tUniversity . gonadotrop doesno texist .Oliverea u (1978) These studies include thewor kb yva nde n andOliverea u andNagaham a (1982)describe d Hurk (1982)o n thedevelopmen t of thegona two types inpituitarie s ofmatur etrout . dotrops. Introu t larvae of45-10 0day sold , One iscomparabl e toth eglobula r typeo f basophils canb efoun d inth edorsa lan d in gonadotrop,referre d tob y themember so f theventra l PPD.Bot h showa nimmunocytoche theUtrech t group.I ti sabundan t inth e micalreactio nwit h anti-salmon-an danti - PPD,ca nb efoun d inth eRPD ,an dha ssecre carp-GTH,bu t only thedorsa lone sreac t tory granuleswit h PAS-an dAB-positiv econ with anti-human-ß-TSH. So,i tseem stha t tents thatcross-reac twit h anti-salmon-GTH. thedorsa lbasophil s are thyrotrops andth e Theothe r onedoe sno treac twit h theanti - ventral cells gonadotrops.Thi s isi naccor GTH,ha s almost completely PAS-an dAB-nega - dancewit h thefac t that thedorsall ysitu tivecontents ,an d ismainl y restricted to ated cellswer e seen todifferentiat efirst , thedorsa lglandula r strandso f thePPD . togetherwit h the thyroid follicles,an dth e Cells ofthi ssecon d typepredominate ddu ventralbasophil s somewhat later,alon gwit h ringvitellogenesis ,appeare d activemainl y thegonads .I naddition ,th edevelopmen to f inspring ,an d showedhyperplasi a andhyper theventra l cells canb eenhance db yexoge trophy inthre eyear sol d femaleswit himma nousandrogens .Giele ne t al. (unpublished turegonads . results)hav e shown thatandrogen s acto n Thisput su s inth emiddl e of thecontro thepituitar y evenafte r itsheterotopi c versy ofon eo r two types ofgonadotrop s in transplantation,an d thatthi sdirec t action the teleostpituitary .Ther e isn oproble m leadst oth eproductio n and storageo fgra about oneo fthes e cell types,whic h inmos t nules and globules.Th ereleas eo fGT Hi s of thespecies ,studie d sofar ,seem s tore minimal.Remova l of theandrogen s results in semble theglobula rbasophil s of therainbo w alos so f secretoryvesicle s and anapparen t trout.Th edifficult y comes inwit h these dedifferentiationo f thecells . cond type,an d thereason sfo r thatar e Maturation of thegonadotrop s canals ob e firsto fal lth efac t thatman y authorsdi d obtained by injecting GTH,whic h stimulates not succeed infindin g asecon d typeo fGTH - thedevelopmen t of thegonad s and inthi s cell,an d secondly the lacko funiformit y in 45 thedescriptio n of sucha cel l type.Fo r therainbo w trout (vanOordt , 1979),sugges example: insom epublication s onth epitui ting thecleavag eo fa prohormon e andth e taryo fsalmonid s theglobula r and cisternal formationo fon eo rmor ephysiologicall yac gonadotrops areconsidere d torepresen t two tivepolypeptides .I tshould ,however ,b e functionally different typeso f gonadotropic kept inmin d thatgranule san dglobule sar e cells (e.g.Coo k &va nOverbeeke , 1972;Ued a not indispensable forgonadotropi nsecre &Hirashima , 1979;Ueda , 1980); inSarothe - tion,fo rstrongl y activated gonadotrops rodonmossambicu sbasophil s inth edorsa l continue tosecret eGT Hafte r the secretory PPDwit h relatively small secretory granules vesicleshav edisappeare d from thecytoplas m havebee n coined asth esecon d gonadotropic (e.g. vanPutte n etal. , 1981). Itseem s cell type (Berne t al., 1974); inth estick thatw ewil lhav e towai t fora bette run leback (Gasterosteus aculeatus)cell swit ha derstanding of themod eo fGT Hproductio n relatively electrondens e cytoplasm,situ beforew ewil lb eabl e toelucidat e thesig ated among theventra lbasophil s inth ePP D nificance of thepresenc e oftw odifferen t (Slijkhuis, 1978),an d inth echanne l cat types of secretoryvesicles ,i.e .granule s fish (Ictaluruspunctatus ;Massou d etal. , and globules inth eGTH-cell so f teleosts 1980)an d theplatyfis h (Xiphophorusmacula - and othervertebrates . tus; Schreibman& Margolis-Kazan , 1979) cells inth epar s intermediahav ebee ndes Innervationo fGTH-cell s cribed as thesecon d typeo f gonadotropic cell. Theproduction ,storag ean dextrusio no f Mattershav ebecom e evenmor e complicated GTHfluctuate s during theannua lreproduc by therecen tresult s ofBurto ne t al., tive cyclean d depends onstimul i ofcentra l (1981). Theauthor sdescribe d twomorphologi andperiphera lorigin .I nbon y fishes,a si n callydifferen t typeso fbasophil s inth e almost allothe rvertebrate s thesestimul i pars distaliso f thewinte r flounder (Pseu- may reach theGTH-cell svi a thecirculator y dopleuronectes americanus).Durin g thespaw system.However ,i nman y teleostsneurose ning seasonbot h types showed immunofluores cretory stimulima y alsopas s alongnerv e cencewhe n treatedwit h antibodies against fibrespiercin g thelamina e thatseparat e floundermaturationa lGT Han d floundergly - theneuro -fro m theadenohypophysis ,an dpe coprotein-TSH.However ,on e typereacte d netrating into theendocrin eparenchym ao f with anti-floundervitellogeni c GTHdurin g the parsdistalis . (Reviews:Ball ,1981 ; thevitellogeni c period only,wherea s the vanOord t &Peute , 1983).Thes efibre sdi otherreacte dwit h thisantibod y throughout rectly contact thehormon eproducin gcells , thereproductiv e cycle.Thus ,fo r thebaso including thegonadotrops ,an dma yeve nter phils inth epar sdistali s of thewinte r minatewit h synapseso nthes ecells .Th ema flounder the traditional axiom of "onemor jorityo ffibre snea r thegonadotrop s and phological cell type,on ehormone "i sn o making synaptoid contactwit h the cellshav e longerapplicable ,an d thephysiologica l so-called largegranulate dvesicle s of60 - differencesbetwee n thetw o types seemst o 100n m (type B).Others ,seldo mmakin gsy changewit h thetim e ofyear .I tma yb egoo d naptoid contact,hav e elimentarygranule so f tokee p this inmin dwhe n trying toelici t 100-200n m indiamete r (typeA) .Thi sdoubl e thenumbe r ofgonadotropi c cell typesi n innervation,however ,i sno tnecessaril y the other teleosts,an d itma ywel lb e thatth e samefo r allteleost s and foral l cellsa t present controversy is largely due todif all times ofth ereproductiv e cycle.I neel s ferences inth edefinitio no f theconcep t (Knowles& Vollrath , 1966), the tench (Tinea "celltype" . tinea;Vollrath , 1967)an d insalmonid s (Fridberg &Ekengren , 1977)al lneurosecre tory fibresen d inth eneurohypophysis ;i n Functiono fgranule s andglobule s theroac h (Rutilusrutilus ;Ekengre n et al., 1978)neuroendocrin e typeB fibre s innervate Itha sbee n argued that if teleostgonado gonadotrops adjacent toth eneurohypophysis , tropsproduc e twohormone s atth e sametime , butd ono tpenetrat e deeper layers ofth e theon ema yb e stored inth egranule san d parsdistali s (Bâgee t al., 1974), leaving theothe r inth eglobule s (Boddingius,1975 ; themajorit y of theGTH-cell swithou tneuro Chinesescientists , 1978). However,fo rseve glandular connections.Moreover ,synaptoi d ral species,includin g therainbo w trout contactswer e observedwit hvesicular ,no t (vanPutte ne t al., 1981), intermediatesta withglobula r gonadotrops.Ver y significant gesbetwee ngranule s andglobule shav ebee n ly,Abraha m (1974)suggeste d thatwhe ni n described,an d thepresenc e of glycoprotein themulle t (Mugilcephalus )typ eA fibres gonadotropinha sbeen\demonstrate d inbot h wereno t observedbetwee n thegonadotrops , typeso fsecretor yvesicle s (Leunissene t thismigh tb edu e toth efac ttha t thefis h al., 1982).Thi sdoe sno tpreclud e thepre wereno t caught during the spawningseason . senceo fa secon dGT H inth egranules ,th e Inlik emanner ,Kau lan dVollrat h (1974) globuleso rboth .A t any rate,th eproteoly described agradua l decrease inth eamoun t ticenzym e acidphosphatas eha sbee ndemon ofgranule s intyp eA andB fibre sinnerva strated inth egranule s and toa lesse rex tingGTH-cell s inth egoldfis h (Carassius tenti nth eglobule s of thegonadotrop s in 46 auratus)durin g and shortly after thenup forsch.130:338-350 . tialperiod ,a swel l asa nincreas e inth e Ekengren,B. ,K .Lindah l &G .Fridberg ,1978 . amount of granules inth e typeB fibre s that Immunocytology and innervation of thego innervate thegonadotrops ,followin g an nadotropic cells inth e teleostfis hRuti oestradioltreatment . lusrutilus .Act a Zool. (Stockh.)59:125 - Thus itseem s thata direc t innervationi s 133. not indispensable for theneuroendocrin ere Fridberg,G . &B .Ekengren , 1977.Th evascu gulationo f thegonadotropi c cells,an dtha t larizationan d theneuroendocrin epathway s when sucha direc t innervation ispresent , of thepituitar y gland of theAtlanti c itchange swit h thephysiologica l condition salmon,Salm o salar.Can .J . Zool.55:1284 - of thefish .Thi s isno t theplac e todis 1296. cuss theorigi no f thevariou s typeso fneu Gielen,J.Th. ,H.J.Th . Goos,J . Peute,R.A . rosecretory fibres innervating theGTH-cell s vande nBosc h &P.G.W.J ,va nOordt ,1982 . and thenatur eo f thereleasin g orinhibi Thebrain-pituitary-gona d axis inth erain ting substances thateac ho f themproduce s bow trout,Salm o gairdneri:gonada lhor (review:Peter ,thi svolume) ,bu t itma yb e mones and thematuratio no f gonadotropic good topoin t toth edirec t innervationa s cells.Cel lTissu eRes. ,i npress . providing anextr amorphologica l tooli nth e Goos,H.J.Th. ,R . Seldenrijk &J. .Peute , study of the central controlo f theGTH - 1976.Th egonadotropi c cells inth epitui cells inman yteleosts . taryo f theblac kmolly ,Mollienisi a lati- pinna,an d other teleosts,identifie db y References theimmunofluorescenc e technique innorma l and androgen-treated animals.Cel lTissu e Abraham,M. , 1974.Ultrastructur e ofth e Res. 167:211-219. cell types and theneurosecretor yinnerva Holmes,R.L .& J.N . Ball, 1974.Th epituita tioni nth epituitar y ofMugi lcephalu sL . rygland .A comparative account.Cambridg e from freshwater ,th ese aan dhypersalin e Univ.Press ,London . / lagoon. II.Th eproxima lpar sdistalis . vande nHurk ,R. , 1982.Effect so f steroids Gen.Comp .Endocrinol .24:121-132 . ongonadotropi c (GTH)cells inth epituita Bage,G. ,B .Ekengren ,B .Fernhol m &G . ryo frainbo w trout,Salm ogairdneri , Fridberg, 1974.The 'pituitar y gland ofth e shortly afterhatching . CellTissu eRes . roach,Leuciscu s rutilus.II .Th eproxima l 224:361-368. pars distalis and its innervation.Act a Kaul,S .& L .Vollrath , 1974.Th egoldfis h Zool. (Stockh.).55:191-204 . pituitary. II.Innervation .Cel lTissu e Ball,J.N. , 1981.Hypothalami c controlo f Res. 154:231-249. thepar s distalis infishes ,amphibian s Knowles,F . &L .Vollrath , 1966.Neurosecre and reptiles.Gen .Comp .Endocrinol .44 : tory innervations of thepituitar y ofth e 135-170. eelsAnguill a and Conger. II.Th estruc Bern,H.A. ,R.S .Nishiok a &Y .Nagahama , turean d innervation of thepar sdistali s 1974.Th erelationshi p betweennerv efi atdifferen t stages of thelife-cycle . bres and adenohypophysial cell typesi n Phil. Trans.Roy . Soc.Londo nB250:329-342 . thecichli d teleostTilapi amossambica . Leunissen,J.L.M. ,A.M .d eLeeuw ,J . Peute& Recherch.Biol . Contemp.Manfre d GabeMe H.J.Th. Goos, 1982.Immunocytochemistr y of morialVol .p.179-194 . gonadotropic cellsan d identificationo f Billard,R. ,B .Breto n &M.P .Dubois ,1971 . cell types inultrathi n cryosectionso f Immunocytologie ethistochimi ede s cel thepituitar y of therainbo w trout,Salm o lulesgonadotrope s etthyréotrope shypo gairdneri.Cel lTissu eRes. ,i npress . physäres chez lacarpe ,Cyprinu scarpio . Massoud,A.A. ,B.A . Simio &K.B .Davis ,1980 . C.R .Acad . Se.Pari s SérieD 272:981-983 . Twogonadotropi c cell-types inth epitui Boddingius,J. , 1975.Th e cell typeso fth e taryo f the channel catfish.Amer .Zool . adenohypophysis inth erainbo w trout(Sal - 20:729. mo irideus).A histologica l study.Holy McKeown,B.A . &A.P .va nOverbeeke , 1971.Im - wellPres sLtd. ,Oxford . munohistochemicalidentificatio no fpitui Burton,M.P. ,D.R . Idler &T.B .Ng ,1981 . taryhormon eproducin g cells inth esokey e The immunofluorescent localization ofte salmon (Oncorhynchusnerka ,Walbaum) .Z . leostgonadotropin s and thyrotropins in Zeilforsch. 112:350-362. flounderpituitary . Gen.Comp .Endocrinol . Olivereau,M. , 1978.Le s cellulesgonadotro 43:135-147. pes chez lessalmonidés .Ann .Biol .anim . Chinese scientists, 1978.Th erol eo fLH-R H Bioch.Biophys .18:793-798 . ininductio no f spawning inth egras scar p Olivereau,M . &Y .Nagahama , 1982.Immunocy - (Ctenopharyngodon idellus). II.Ultra - tological study ofee lan d troutgonado structural studyo fpituitar ygonadotrops . trophs.Gen .Comp .Endocrinol .46:365-366 . Scientia sinica21:397-400 . vanOordt ,P.G.W.J. , 1979.A typology ofth e Cook,H . &A.P .va nOverbeeke , 1972.Ultra - gnathostome adenohypophysiswit h someem structure ofth epituitar y gland (pars phasiso nit sgonadotropi c function.Basi c distalis) insocke y salmon (Oncorhynchus Appl. Histochem.23:187-202 . nerka)durin g gonadmaturation .Z .Zell - vanOordt ,P.G.W.J .& J . Peute, 1982.Gonado - 47 tropic cells inth evertebrat epituitary . Proc.Nint h Intern.Symp .Comp .Endocri nol.Hon gKong ,i npress . vanOordt ,P.G.W.J .& J . Peute, 1983.Th e cellular origino fpituitar y gonadotropins inteleosts .In :W.S .Hoar ,D.J . Randall & E.M.Donaldso n (Ed.):Fis hPhysiology , vol. IX.Reproduction .Academi cPress , Inc.,Ne wYork ,i npress . Peute,J. , H.J.Th. Goos,M.G.A .d eBruy n& P.G.W.J,va nOordt , 1978.Gonadotropi c cellso f therainbo w troutpituitar ydu ring theannua l cycle.Ultrastructur e and hormone content.Ann .Biol .anim .Bioch . Biophys. 18:905-910. Peute,J. , C.d eZoeten-Kam p &P.G.W.J ,va n Oordt, 1982.Immunocytochemica lidentifi cationo fGTH -an dTSH-cell s inth epitui taryo fth eAfrica n catfish,Claria slaze - ra.Gen .Comp .Endocrinol .46:366 . vanPutten ,L.J.A. ,J .Peute ,P.G.W.J ,va n Oordt,H.J.Th . Goos &B .Breton ,1981 . Glycoprotein gonadotropin inth eplasm a and its cellular origin in theadenohypo - physis of sham-operated andovariectomize d rainbow trout,Salm ogairdneri .Cel lTis sueRes .218:439-448 . Schreibman,M.P . &H .Margolis-Kazan ,1979 . The immunocytochemical localization ofgo nadotropin,it s subunits,an d thyrotropin inth e teleost,Xiphophoru smaculatus . Gen. Comp.Endocrinol .39:467-474 . Slijkhuis,H. , 1978.Ultrastructura l evi dencefo r two types ofgonadotropi c cells inth epituitar y gland of themal ethree - spined stickleback,Gasterosteu saculea - tus. Gen.Comp .Endocrinol .36:639-641 . Ueda,H. , 1980.Change so f two types ofpi tuitary gonadotrophs inwhite-spotte d char,Salvelinu s leucomaenis,durin ggona daldevelopment .Bull .Fac .Fish. ,Hokkai doUniv . 31:1-15. Ueda,H . &T .Hirashima , 1979.O n twodiffe rent typeso fputativ egonadotroph s inth e pituitary gland of themas u salmon,Onco - rhynchusmasou .Anot .Zool .Jap .52:114 - 124. Vollrath,L. , 1967.übe rdi eneurosekretb - rische Innervation derAdenohypophys evo n Teleostiern,insbesonder eHippocampu s cuda undTine a tinea.Z .Zellforsch .78:234 - 260. 48 HORMONAL CONTROLO FTH EREPRODUCTIV EFUNCTIO NO FSTURGEON S (CHONDROSTEI) I.A. Barannikova,O.S .Bukovskaya ,N.A .Efimov a Physiological Institute,Leningra d University,Leningrad ,USS R Summary Peculiarities of the hormonal regulation Intramuscular administration ofsyntheti c ofgonad s functiono fChondroste ia tdiffer LH-RHwa s shownt oactivat e GTHcell san dt o entstage so fth ereproductiv e cycle innat increaseGT Hconcentratio n inbloo d offemal e urean d atfis hfarm swer estudied . stellate sturgeon3 0minute s afterinjection . Radioimmunoassay of gonadotropin (GTH), In one hour a drop of blood GTHoccurred , sexsteroid san d Cortisoli nbloo d seruman d whichwa s followedb ya mor e significantris e histological andultrastructura l analysiso f lasting 4-15 hours.Ovulatio nwa s obtained, pituitary and gonadswer eused . whichi sretaine d 5-6 hours incompariso nt o Inth e seaperio d atth ebeginnin g ofvitel - ovulation inducedb yGTH . logenesis theactivatio no fgonadotropi c (GTH) cells,th eincreas e ofpituitar y GTHa swel l as elevation of 17ß-estradiol and Cortisol levels inth ebloo d ofstellat e sturgeonwer e observed.Th e contento fGT Han d sexsteroid s atth ebeginnin g ofth eanadromou smigratio n from seat orive rha dmea nvalues ,GT Hcell s rich in secretory granulations predominated inth epituitary . Closer to the spawning period thenumbe r of devasted GTHcell sincreased .A tth ebe - gining of spawning GTHi srelease d fromth e pituitary,it sconcentratio ni nbloo dsharp lyrising .Afte r spawning exhausted cisternal cells predominated in the pituitary,man y cells were destroyed,th econten to fGT Hi n the pituitary and inbloo d as well asse x steroids and Cortisol levels were lowbot h inmal ean dfemale . Injections ofeithe rdifferen t hypophyseal preparations orAci-GT H atfis hfarm scause d the riseo fbloo d GTH.Th ehighes t levelwa s observed 4-8 hoursafte r administration,de pending on the preparation being used.A t the time of ovulation this level slightly decreased,bu twa s still significantly higher thani nth e samefis hbefor einjection . GTH 100_ E3cf ng/mi IV VI Ind.ov. Fig. 1.GT Hlevel s inth ebloo d serum ofAci - penser stellatus Pallas onvariou s stageso f thesexua lcycle . IY-before spawning,Y-spawning ,YI-afte r spawning,Ind.ov.-induce d ovulation. 49 LONG-TEEMCHANGE S INPLASM AAN DPITUITAR YGT HAFTE R CASTRATIONO FRAINBO WTROU TA TA N IMMATURE STAGE R.Billard* ,L.W . Crim**, R.E.Peter*** ,B .Breton * * Laboratoire dePhysiologi e desPoisson s I.N.R.A.Campu s deBeaulieu ,3504 2Renne sFranc e **Memoria lUniversit y ofNewfoundland ,Marin e SciencesResearc hLab .S tJohn' sNewfdl .Canad a ***Departmento f Zoology TheUniversit y ofAlberta ,Edmonto nAlberta ,T6 G2E 9Canad a Inorde r tostud y hypothalamus-pituitary- Theresult s show that i)plasm aGT H gonad relationships during the establishment increases atth enorma l timeo fpubert y in ofpubert y inrainbo w trout,juvenil e trout fishcastrate d ata nimmatur e stage.GT H were castrated ata nimmatur e stage,an dth e leveli shighe r than inth esha mcastrate d GTH inth eplasm aan d thepituitar ywa s inful lgametogenesi s suggesting anegativ e examined oneyea r laterwhe n the controls feed-back from thegonads ;ii )th epituitar y were inful lgametogenesis . GTH issimila r inth e castrates as insham s at least forth emale s indicating that Material andmethod s pituitary canbuil d GTHi nth e absenceo f gonads.Th epituitar y gland canthu s function Six-month oldmal e and femalerainbo w normally (synthesis and release ofGTH ) trout,weighin g about 20g ,wer e castrated without thegonads .Thi swoul d suggestthat , or sham-operated inJune .W ecastrate d the asi nth ehighe rvertebrates ,pubert y is fishunde rbinocular ,totall y castrating controlledb y thecentra lnervou s system somean dpartiall y castratingothers , (CNS).However ,th eplasm a andpituitar yGT H leavinga ver y smallpar t of the anterior levelan d therespons e tocastratio n areno t parto fth e gonad.Th e fishwer e thenstore d ashig ha si nolde r fish suggesting that ina racewa y atth eGourna y experimental thiscontro l of theCN So nth epituitar y is trout farm.The ywer ekille d oneyea r later. notye t fully established.Finall y thesefis h After thebloo dwa s sampled theplasma , exhibited aconsiderabl e compensatoryhyper obtainedb y centrifugation at4 CC,an dth e trophic activity inthei r gonadaldevelopment . pituitaries,wer e frozena t-20 eCunti l This demonstrates theabilit y of afe wger m GTH (maturational)determinatio nb y RIA.Th e cells todivid eactivel y and reinitiatea body and thegonad swer eweighe d and apar t normalgametogenesis . ofth egona d (whenpresent )wa s fixedfo r histological examination. Results (seeTable ) Table.GT Hrespons et oful lo rpartia lcastratio ni njuvenil erainbo w trout.Value s+ • SD Only onemal e inth esham-operate d group was immature;al l theother swer e inful l GSI PlasmaGT H spermatogenetic activity,showin g some SexTreatmen t Body Pitui.GT H » weight % ng/ml Dtg/pit. spermatozoa inth e lobules.Al l thepartiall y castratedmale swer e inful lspermatogenesis , Shamcastratio n 10 6731160 1.1411,1 3.79*2.61 18.86+11.6 thetesti shavin g reached the samesiz ea s j Fullcastratio n 6 574*112 16.46*** 20.45±11.61 in the sham-operated group (theGS Iwa s »5.11 Partialcastratio n 13 682±143 1.39*0.89 5.93* similar),althoug h its initial sizeha dbee n ±2.34 strongly reduced by surgery.Ther ewa sn o regeneration ofth e spermduc t and thetesti s Shamcastratio n 11 594±70 0.51±0.15 6.96±3.25 20.82±6.12 Fullcastratio n 6 570*79 13.65*** 12.35 wasentirel y located inth eanterio rpar to f ±2.16 ±6.28* thebod y cavity.Bod yweight ,althoug h + Partialcastratio n slightly loweri nth ecastrate dmales ,wa s immatureo 5 659+107 0.02*** 9.67±4.69 15.15±6.54 *0.01 not significantly different.Pituitar y GTH Q inexog.vitell . 4 639*96 0.26* 10.87*3.01 wasno tsignificantl y differentbetwee n ±0.12 castrated and shamsbu tplasm aGTH ,muc h *P< 0.05 ; ***P< 0.001 comparedt osha mcastrate sb yvarianc eanalysi s higher (P< 0.001 )i nth ecompletel y castra tedmale s thani nth esham-operate d group, was alsohighe r inth epartiall y castrated fish (P<0.05). Inal lsham-operatê d females vitellogenesis had started. Someo fth e partially castrated oneswer e still immature whileother s showed signs ofvitellogenesis . As compared toth e sham-operated fish,th e castratedha d ahighe rplasm aGT Hleve l (P<0.001)bu t thepartia l castrates didnot . Therewa s asignifican t difference (P<0.05 ) inth epituitar y GTH contentbetwee nsham s andcastrates . 50 ACTIONS OFDOPAMIN E ONGONADOTROPI N RELEASE IN GOLDFISH,CARASSIUS AURATUS. JohnP . Chang andRichar d E. Peter Department ofZoology, University of Alberta, Edmonton, Alberta, CANADA, T6G 2E9. Intraperitoneal injection (ip)o fdrug s capable of blocking specific enzymes ofcatecholamin e synthesis LATE RECRUDESCENCE suggest that in goldfish, dopamine (DA) inhibits gonadotropin (GtH) release (Changet al., 1982). Further experiments demonstrated that ip injections ofD A or its agonist, apomorphine (APO),decrease d and pimozide (PIM),a D A antagonist, increased serum GtH levels inintac t female goldfish. Inth e present experiments, the mechanismo fD A inhibitiono fGt H release infemal e goldfish,acclimate d to 16L8D and 10-12°C,wa s further studied.I nthes e experiments, DA, APO,an d PIM,wa s dissolvedo rsuspende d in a vehicle of0.7 % NaCIwit h 0.1%l\la 2S205. A GtHrelease-inhibitor y factor (GRIF) is present in the goldfish, and its influence canb eabolishe d by lesions inth e preoptic region, resulting ina prolonged massive spontaneous releaseo fGt H(Pete r &Paulencu , 1980; Peter, 1982). Serum GtHconcentration s in preoptic lesioned goldfish remained elevated (200t o 400 ng/ml) for several days post-lesion comparedt o levels insha moperate d animals (5t o 15 ng/ml).Com pared to vehicle injected controls, ip injections ofD A LHRH-A LHRHA LHRH-A (10 & 100 ug/g) inpreopti c lesioned fish on day2 vehicle AP0 vehicle veh.cl post-lesion significantly depressed the elevated serum PS LHRH-A LHRHA LHRH GtH levels at2 hour s (h)post-injection . Both doseso f AP0 vehicl DA reduced the elevated serum GtHlevel s by 40t o 50% (P<0.02). Similarly, ip injections ofAP O (20 ug/g) on day 1post-lesio n significantly reduced the lesion induced increase inseru m GtH levels by 24 h post- Fig. 1.Effect s ofAPO , injected with the 1st or the 2nd injection. These results indicate that DA canac t as a LHRH-A injection, on serum GtHlevel s in female GRIFt o inhibit spontaneous GtHrelease . However, goldfish. intracranial (3rd ventricle) injections ofD A did not alter serum GtH levels.Thi s suggests that DA acts directly on the gonadotrophs to block the spontaneous GtH Table I.Effect s ofPI M on serum GtH levels and secretion. ovulation at times (h)afte r the 2nd LHRH-A injection. Values that are similar (P>0.05) are identified by similar Des Gly10,[D-Ala 6] LHRHethylamid e (LHRH-A), superscripts. dissolved in fish saline (PS),was effective in stimulating treatment GtH, ng/ml # ovulated GtHreleas e ingoldfis h when injected astw oi p (24 h) (72 h) injections (0.1 ug/g), 12 hapart .Th e 1s t LHRH-A PS+vehicle 1±V injection isthough t to potentiate the actiono fth e 2nd LHRH-A+PIM 294±512 injection (Peter, 1982). Ip injections ofD A (10 & 100 LHRH-A+vehicle 66±193 ug/g) at4 h afte r the 2nd LHRH-A injection significantly reduced the elevated serum GtHlevel s by 2h afte r DA injection. TheLHRH- A induced increase in serumGt H Insummary , DA has GRIFactivit y inth e goldfish by levels was also abolished when APO (1 0 & 20 ug/g) actions directly on the gonadotrophs to inhibit the was injected simultaneously with thetw o LHRH-A spontaneous releaseo fGtH ,an db y blocking the injections.Administratio n ofAP O(2 0 ug/g, ip)wit h the actions ofGnRH .Th e removal ofth e DA inhibition on 1st , but not the 2nd, LHRH-A injection was effectivei n GtHreleas e may be anintegra l part ofth e mechanism abolishingth e increase inseru m GtHmeasure d at 24h regulating the preovulatory GtHsurg e andovulation . after the 2nd LHRH-A injection (fig. 1). However, injectiono fAP O with the 1st LHRH-A injection did not alter the LHRH-A induced increase inseru m GtH levels References measured at the time ofth e 2nd LHRH-A injection (fig. 1).Thes e results indicate that DA canbloc k the Chang, J.P.,Cook , A.F.,an d Peter, R.E. 1982. Influences potentiating effects ofmultipl e doseso fth e GtH of catecholamines on gonadotropin secretion in releasing hormone (GnRH)o n GtHreleas e ingoldfish ,a s goldfish, Carassiusauratus. Gen. Comp. Endocrinol. well asdecreas e the GtH release-response. (inpress) . Peter, R.E. 1982. Neuroendocrine controlo f Two injections ofLHRH-A , 12 hapart , stimulated reproduction inteleosts . Can.J. Fish. Aquat. Sei. GtHreleas e but did not significantly induce ovulation in 39: 48-55. gravid (sexually mature) female goldfish. Ip injectionso f Peter, R.E.,an dPaulencu ,CR . 1980. Involvement ofth e PIM(1 0 ug/g) at the time ofth e 2nd LHRH-A injection preoptic region ingonadotropi n release- potentiated the effects ofLHRH- A on GtHreleas e and inhibition ingoldfish , Carassius auratus. increasedth e occurrence ofinduce d ovulation (tableI) . Neuroendocrinology. 31: 133-141. 51 PRESENCE ET REPARTITION D'UN FACTEUR DE TYPE LHRH CHEZ L'ANGUILLE ETUDEPA RDOSAG E RADIOIM- MUNOLOGIQUE. (*) S.Dufour ,C .Pasqualin Pasqualini1 ~',iB .Kerdelhué, Kerdelhu1 ',Y.A é. Fontaine , avec lacollaboratio n techniqued eN .Delerue-L eBell ee tV . Lenoir (*) Laboratoired ePhysiologi e généralee tcomparée ,Muséu m ;L A 90,C.N.R.S ;>,7 ru eCuvier , 75231Pari scéde x05 ,e t (£)Laboratoir e desHormone sPolypeptidiques ,C.N.R.S. ,9119 0Gif - sur-Yvette,France . Summary : Eel brain contains a factor which présenced'I RLHR H n'apa sét édétecté edan s produced a complete cross-reaction in a RIA lesa cvasculaire .L'hypophys e contientun e for mammalian LHRH and which exhibited on Se- grandequantit éd'I R LHRH (304+ 6 9pg )à un e phadex G25a behavior similar to that of mam concentration (112+ _2 6pg/mg )dépassan tmêm e malian LHRH. This LHRH-like immunoreactive celled ulob emédia nd el'hypothalamus . factor (IR LHRH) had a wider distribution in the eel brain than LHRH in mammals. Pituita Dans lesautre stissu sétudiés ,u ndébu t ry also contained a large amount of IR LHRH, deréactio n croiséees tparfoi sobservée .L a at a concentration which was higher than in concentration enI RLHR Hy es t< 0,0 2 pg/mg median hypothalamic lobe. Finally, plasma (moelledistale ,foie ,ovaire ,muscle )o ues t samples gave important cross reactions in the proched ecett evaleu r (tubedigestif ,peau) . RIA. This RIA makes possible further physio Enfin,l aprésenc ed'I R LHRH ap uêtr edé logical studies in the eel ; it can already tectéedan sl eplasm aà de s concentrations be concluded that the lack of sexual develop importantes (de4 0à plusieur s centainespg / ment and the weak gonadotropic function of ml). the eel are not due to a lack of IR LHRH. Unecaractérisatio npartiell e de l'IRLHR H cérébrale aét éréalisé epa r filtration surr « Lecycl ebiologiqu ed el'anguill e euro SéphadexG2 5d'u nextrai td'u npoo ld edien péenne, Anguilla anguilla, se singularise céphalese tmésencéphales .L'activit é IRLHR H par l'absence dedéveloppemen t sexuel touta u estélué e enu npi cd eK D -0,76 , semblableà longd es avi edan sle scour sd'eau ,cec i celuidétermin é surl amêm ecolonn epou rl a étantd ûà l afaibl e activité del a fonction LHRHmammalienne . gonadotrope (Dufoure tal. ,sou spresse) . Nousabordon s icil'étud ed u contrôlehypo - Enconclusion * lecervea ud'anguill e con thalamiqued el afonctio n gonadotropepa r tientu nfacteu rI RLHR Hplu slargemen tré l'étuded el arépartitio n d'un facteurimmu - partiqu eche zle sMammifères ,e naccor dave c noréactifd etyp eLHR H (IRLHRH) , aumoye n lesdonnée simmuno-histologique s obtenues d'un système dedosag e radioimmunologique chezle spoissons ,résultat ssuggéran tqu ec e (RIA)préalablemen tmi sa upoin tpou r laLHR H facteurpeu tavoi rd'autre s fonctionsqu'hy - mammalienne (Kerdelhuée tal. , 1973, 1976). pophysiotrope (cfpa r ex.Mün ze t al.,1982 , Kahe t al., 1982). Dans l'hypophyse, l'IR Leséchantillon s sontprélevé s surde s LHRH estprésent e enquantit éimportante ,c e anguilles femellesd e 200à 35 0g ,aussitô t quies tà rapproche rd u faitque ,che zle s aprèsdécapitation ,e tcongelés .Le s tissus Téléostéens,1'eminenc emédian e estincorpo sontextrait s dansHC l 1N etle ssurnageants , réeà l'hypophys e (revued eBail , 1981).L e aprèscentrifugation ,son tneutralisé se t RIAmi se noeuvr eperme tdon cd edose ru n congelés jusqu'audosage . facteur detyp eLHR H chezl'anguill e etouvr e Lesextrait sd ecervea uo ud'hypophys ed ' lavoi e àde sétude sphysiologiques .I lpeu t anguillemontren tun eréactio n croisée com déjàêtr econcl uqu e lemanqu ed edéveloppe plètedan sl eRIA .L e cerveauentie r contient mentsexue le tl afaibl e fonction gonadotrope 920+ 135p gI RLHR Hdon tl amajorit é (75% ) de l'anguille nepeuven tpa sêtr erelié sà estprésent e dans l'ensemble dudiencéphal e uneabsenc ed'I RLHRH . etd umésencéphale , tandisqu el etélencépha - lee ncontien t 21% e tl ecervea upostérieu r Références 4 %,à l aconcentratio n respective de25, 0+ _ Ball,J. , 1981.Gen .Comp .Endocrinol. ,44 , 0,2,7, 8 + 1,6 et 1,1^0, 3 pg/mg tissufrais . 135-170. L'ensemble diencéphale etmésencéphal e aét é Dufour,S. ,N .Delerue-L eBelle ,Y.A .Fontain e ensuitedisséqu ée ntroi sparties ,le slobe s Gen. Comp.Endocrinol. ,sou spresse . hypothalamiques (trèsdéveloppé sche zle s Kah, O.,P . Chambolle,P .Dubourg ,M.P .Du poissons), les lobesoptique se tl aparti e bois, 1982.Abstrac t fromthi sSymposium . intermédiaire (àl afoi sdiencéphaliqu e et Kerdelhué,B. ,M .Jutisz ,D . Gillessen,R . mésencéphalique)qu icontiennen trespective Studer, 1973.Biochem .Biophys .Acta ,297 , ment 147+ 10,12 8+ 5 8e t35 0+ 2 3p gI R 540-548. LHRH auxconcentration s de 19+ 1,2 5+ 2 e t Kerdelhué,B. ,S .Catin ,C .Kordon ,M .Jutisz , 30j ^1 pg/mg .Quan tau xlobe shypothalamiques , 1976. Endocrinology, 98_,1539-1549 . lamajeur eparti ed el'I RLHR Hes tcontenu e Münz,H. ,C .Class ,W.E .Stumpf ,L .Jennes , dans lelob emédia n (128+ _2 0pg )à un econ 1982. Cell.Tissu eRes. ,222 ,313-323 . centration de3 4+ 1pg/mg ,bie n supérieure àcell ede s lobes latéraux (<7 pg/mg) . La 52 GONADOTROPICHORMON EPRODUCTIO N INENZYMATICALL YDISPERSE D PITUITARY CELLSO FTH ETROUT , SALMOGAIRDNER I G.E.Fâhraeus-va nRee ,C.J . Panis,J.A.F.W .Kleijne ,J.Th . Gielenan dP.G.W.J ,va nOord t Zoological Laboratory,Universit y ofUtrecht ,Th eNetherland s Inorde r tostud y invitr o controlo fth e activity ofgonadotropi c (GTH)cell si n rainbow trout,pituitarie s of juvenile fishes (ca. 1yr. )ar eenzymaticall ydisso ciated according toth emetho d ofFähraeus - vanRe ee tal. .Th e dispersed cells arekep t ina suspensio n system for 72hrs . ori n primarymonolaye r culturesunde r different culturecondition s forperiod s rangingu pt o 21 days.Th ecell s arecultivate d incontro l medium or inmediu mwit h either 17a-methyl- testosterone (MT)o restradiol-17 g (E2)o r withM Tan d dopamine (DA)i nconcentration s of8. 5 x 10~'Man d subsequently incubated for3 hrs . incontro lmediu m or inmediu m with 8.5 x 10_7MM Tand/o r syntheticLH-R H orwit hM Tand/o r 8.5 x 10"8Mdes-Gly 10-|D- Fig. 1.Phas econtras tmicrograp h of 1wee k Ala6I-LH-RH ethylamide (LH-RHa).GT Hcell s oldprimar ymonolaye r culture inMT-enriche d areidentifie db yAlcia nBlue-Periodi cAci d medium.X224 .Fig .2 .Ligh tmicrograp h of3 Schiff-OrangeG staining and doubleantibod y weekol d culture inMT-enriche dmediu m immuno-enzyme cytochemical techniqueusin g showing strong immunostainingo fGT Hcell s anti-carpBGTHa sfirs tantibody .Percentage s withanti-carpgGTH .X560 . of immunoreactive cells among theisolate d and cultivated cells arecalculated .Secre strongerwit h longer culture time.Primar y tory activity isestimate db ymeasurin g the monolayer culture givesa bette rresul t than GTHconten t incel l extracts and culture thesuspensio n system.Cell s cultivated in mediab y radioimmunoassay. steroid-enriched medium givea bette rmono Inbot h culture systemsGT Hcell s showa n layer (fig. 1)tha nthos ecultivate d incon accumulation ofGT H (seetable )an d anin trolmedium . crease innumbe r of immunoreactive cells Subsequent treatment of thecell swit hM T (fig.2 )afte r treatmentwit hMT ,indicatin g for 3hrs .cause s stimulationo fGT Hsynthe a stimulation ofGT H synthesisb y thisste sisan d inhibitiono fGT Hrelease .Subse roid.E 2seem s tohav e thesam eeffect .A quent treatment of thecell s 18hrs .afte r synergetic stimulation ofGT H synthesisi s dissociationwit hLH-R Ho rLH-RH a for3 hrs . causedb yM T andD Ao rb yM Tan d ametabo doesno t affectGT Hproduction ,bu tstimu liteo fDA .Autonomou s GTHreleas e israise d latesGT Hreleas e intomediu m afterprimin g above thato f thecontro lmediu m levelb y of thecell swit h steroids for 72hrs . thesesteroids .Th eeffect s of steroidsar e GTH synthesis and release arebot h stimulated by simultaneous treatment of thecell swit h MT andhypophysiotropi c substances after Treatment 72hrs .suspensio n culture priming of thecell swit h steroids for8 days . cell extracts medium These observations clearly show thatth e cells shortly afterdissociatio n retainthei r Untreated 0.20+0.04 0.31+0.02 ability torespon d tosteroids ,bu tno tt o Control 0.22+0.03 0.16+0.02 hypophysiotropic substances.Afte r 72hrs . MT 0.68+0.03 0.36+0.02 thecell s apparently recoveran d startt o MT+ D A 1.09+0.16 0.48+0.03 respond toLH-R Han d itsanalogue . hrs.monolaye r culture Reference Untreated 0.56+0.03 0.69+0.12 Control 0.73+0.05 0.77+0.11 Fahraeus-vanRee ,G.E. ,S.E.F.Guldenaa r& MT 3.03+0.64 1.30+0.23 J.Th.Gielen.Fin e structure and function of isolated cells as revealed frompitui taries of immature rainbow trout,Salm o Tableo fGT Hconten t (AU)+ _S Di nextract s gairdneri,b ymean s ofa ne wenzymati c ofuntreate d and treated cells (0,25x 106) dispersion technique.Cel lTissu eRes. , and culturemedi a (0.5 ml). inpress . 53 DIRECTACTIO NO FGONADA LSTEROID SO NTH EMATURATIO NO FGONADOTROPI C CELLSI NTH ERAINBO W TROUT,SALM OGAIRDNER I J.Th.Gielen ,H.J.Th .Goos ,Mariëll eG.A .d eMo lMoncourt-d eBruy nan dP.G.W.J .va nOord t ZoologicalLaboratory ,Res .Grou pfo rComparativ eEndocrinology , StateUniversit yo fUtrecht , Padualaan 8,350 8T B Utrecht,Th eNetherland s Injuvenil e trouti tha sbee n demonstrated treated host animals.Testosteron e (1ygr/g r thatadministratio no fgonada l steroids,e.g . b.w.)an dcoco abutte r treatment started2 4 testosterone leadst oa naccelerate dmatura hoursprio rt oth eimplantatio no fth epi - tiono fth egonadotropi c (GTH) cells.Thes e tuitaries.GT Hmeasuremen tb yRI Aan da nE M maturational effectscompris e synthesisan d studywer ecarrie dou to nth ei nsit uan d storageo fGTH ,accompanie db yultrastruc graftedpituitarie so fth ehos tanimals . tural changeso fth eGTH-cell s sucha s en PlasmaGT Hlevel swer eals o analysed. largemento fth ecell ,nucleu san dsecretor y Testosterone treatment caused GTH-cellma granules,developmen to fth eGolg iapparatu s turationo fth ei nsit upituitarie sa swel l andth eappearanc eo fglobules .Simila r asi nth egrafts .Accordingly ,th eGT Hcon signso fGTH-cel lactivatio ncoul d alsob e tento fth ei nsit uan dth eimplante dpitui achievedb yinjectin g gonadotropin,an di t tariesha dincrease d inth etestosteron e hasbee nprove ntha tthi seffec ti scause d treatedanimals . by endogenous gonadalhormone sbein gsynthe Itca nb econclude d thattestosteron eact s sizeda sa resul to fth egonadotropi c acti directlyo nth ematuratio no fth eGTH-cell s vityo fth eadministere dhormon e (Gielene t and thatth ebrai ni sno tnecessaril yin - n-1? n-13 1.4- 1 1.2- 1*11 r>8 n-18 —m 0.8- 0.6 0.4 0.2 GTU(assay units/ml plasma) GTH(assay units/pituitary) •control treatment in situ •in situ testosteronetreated •graft testosteronetreated •control treatment graft •untreatedin situ al., Cell Tissue Res., 1982, 225, 45-57). volved in this process. Admittedly, one Do steroid hormones act on the GTH-cells could argue that circulating GnRH was in directly or indirectly via affecting gonado volved. However, in vitro studies using iso tropin releasing hormone (GnRH)-centres in lated pituitary cells confirm our conclusions the brain ? To study the direct effect of (Fâhraeus-van Ree et al., this symposium). steroids on the gonadotrops, the influence of testosterone on GTH-cells of ectopic im planted pituitary homografts was investiga ted. Pituitaries of male and female donor animals were transplanted into the caudal musculature of testosterone and cocoa butter 54 SEASONALVARIATION S INSENSITIVIT Y TOGnR HO FJUVENIL ERAINBO WTROUT ,SALM OGAIRDNER I H.J.Th.Goos ,J.Th .Gielen ,J .Janse nan dP.G.W.J .va nOord t ZoologicalLaboratory ,Res .Grou p forComparativ eEndocrinology ,Stat eUniversit y ofUtrecht , Padualaan8 ,350 8T B Utrecht,Th eNetherland s Injuvenil e trout thematuratio no fgona .responsewa sno talway sidentica l (Fig. 2). dotropic (GTH)-cells canb e stimulatedb y Fluctuations inrespons e toLH-RH a andi n gonadal steroids sucha s testosterone,bot h basicGT Hplasm a levels (Fig.3 )migh tre exogenous aswel la sendogenou s secretedb y flecta seasona lvariatio ni nth esensitivi thegonad s asa resul to fGT H treatment tyt oGnR H injuvenil etrout . (Gielene tal. ,1982a) .Testosteron eact sdi rectlyo nth egonadotrop s (Gielene tal. , 1982b). Asa consequenc e of thesteroi dhormon e inducedmaturatio n of theGTH-cells ,synthe siso fgonadotropi n isenhanced ,bu t thisi s notaccompanie db y anincrease d releaseo f GTH.Possibl e explanation for thisobserva tionmigh tb e that i)th eGTH-cell sar e stillno treceptiv e forgonadotropi nre leasinghormon e (GnRH)o r ii)th eGnRH-cell s inth ebrai nd ono t secrete theirhormon eo r iii)th ereleas e isinhibite db y theste roid. To investigate thereceptivit y of theGTH - Fig.2.Increase of plasma GTH after injection cells toGnR H testosteronepre-treate d and of LH-RHa at different times of the year. à 3 pl»»ma before LH-RHa injection D plaint! after LH-RHa injection mui _fi mQm Qmm 2.S >ig r LH-RHa Fig.1.GTH level in plasma (open columns) and Fig.3..Basic plasma level of GTH in juvenile pituitary extracts after LH-RHa injection trout during part of the year (assay units/ (assay units/ml plasma; a.u./pituitary). ml plasma). nonpre-treate d juvenile,sexuall y immature References troutwer e injected i.p.wit h aLH-R Hana 1 6 logue (des-Gly 0-|D-Ala |-LH-RHethylamide) . GielenJ.Th. ,H.J.Th .Goos ,J .Peute ,R.A . Testosteronewa s administered bya coco a vande nBosch ,P.G.W.J ,va nOordt ,1982a . butter implantation (1ugr/g rb.w. ) 2week s Thebrain-pituitary-gonada laxi s inth e prior toth eLH-RH a treatment.GT Hlevel s rainbow trout,Salm ogairnderi :Gonada l inth eplasm aan d inpituitar yextract swer e hormones and thematuratio no fgonadotro analysedb yRIA . piccells .Cel lTissu eRes. ,225 :45-57 . Twohour s aftera singl e injectiono fLH - GielenJ.Th. ,H.J.Th .Goos ,Mariëll eG.A .d e RHa (50ngr/g rb.w. ) intono npre-treate d MolMoncourt-d eBruyn ,P.G.W.J .va nOordt , and testosteronepre-treate d animals the 1982b.Direc t actiono f gonadal steroids plasmaGT H levelswer e increased inbot h on thematuratio no fgonadotropi c cells in groups (Fig. 1).Despit e thereleas eo fgo therainbo w trout,Salm o gairdneri.Thes e nadotropin thepituitar y GTHconten twa sno t proceedings. decreased,indicatin g thatLH-RH ano tonl y induced thereleas eo fGT Hbu tals oit ssyn thesis. Surprisingly,whe nLH-RH a (10ngr/g rb.w. ) was givena tdifferen t timeso f theyea r the 55 DISTRIBUTION OFIMMUNOREACTIV ELH-R HI NTH EBRAI NO FTH EGOLDFIS H O.KAH ,P . CHAMBOLLE,P .DUBOUR Gan dM.P .DUBOIS * Centred eMorphologi e Expérimentale CNRS,Av . des Facultés,3340 5Talenc e Cedex FRANCE *INRA 37380Nouzill y FRANCE Summary Using anindirec t immunofluorescencetech medialis of thetelencephalo n andalon g the nique,LH-R Hwa s detected in twohypothala wall ofth eventricle .A n important tract mic centers :th eventrolatera l NPPan d the presumably originating from thecel lbodie s posteriorNLT .Immunoreactlv e fiberswer e ofth eNP P travelled caudally to theNL Tan d observed inman y brainarea s and inth epro possibly thepituitar ywher e fiberswer eob ximal pars dlstalis.o f thepituitary . served inth eproxima l pars distalis .Nume rous fiberswer e foundalon g thewall so f Experiments basedupo n pituitarytrans theventricl e in thediencephalon . Thehabe - plantations,brai n lesioning studiesan d nularnucleu s receiveda massiv e innervation hypothalamic extracts injections havesug aswel l as theopti c tectum inth estratu m gested that,i nteleosts ,th egonadotropi c albumcentral e and toa lesse r extentth e function of thepituitar y isunde rhypotha stratumgriseu mcentral ean d thestratu mmar lamic stimulatory control.Howeve r thenatu ginale.Immunoreactiv e fiberswer eals onume rean d the localization ofth e teleostean rousa t the level ofth eposterio r commissure gonadotropinhormone-releasin g hormone(Gn and in thelatera lpart s of thedorsa lmid RH)ar eno twel l established. Severalau brain tegmentum.Mor e posteriorly,o nlongi thors attempted tolocaliz eLH-R Hi nth e tudinalsections ,fiber swer e located inth e brainan d pituitary ofdifferen tspecies , nucleus diffusus lobiinferioris ,th efasci however informations concerning thegold culus longitudinalis medialis and themedul fish,whic h ison eo fth emos t studiedte - laoblongata .A few fiberswer e observed in leostwit h regards toneuroendocrin e control thecorpus,o f thecerebellu mbu tnon e inth e ofreproduction ,ar e lacking.W e reporther e valvula. ourobservation s on the immunocytochemical localization ofLH-R Hi n thebrai no fth e Ourresult s demonstrate thata t leasttw o goldfish (Carassiusauratus) . LH-RHcenter s arelocate d inth eNP Pan dth e posteriorNLT .thes e findings arepartl y in Males and females goldfish of thecommo n agrémentwit h previous reports onothe rte varietywer euse d inthi s study. Several leosts (seereview s by Ball,198 1 ;Peter , fishwer egive ncolchicin e intraperitoneally 1982). TheNL Ti sknow na sbein g involved in (lOmg/lOOgbod yweight )4 8hour s beforede theregulatio n ofth egonadotropi c function capitation. Paraffin orcryosta t sections ofth epituitar y :lesionin g of theNL Tpar s wereprocesse d forth eindirec t immunofluo posterior results ina blockag eo fgonada l rescence techniqueusin g aprimar y antibody recrudescence (Peter,1982) .Mor e recentlya directed towards synthetic LH-RH.Th especi gonadotropin inhibiting factorha s beendes ficity of thereactio nwa s checked byommis - cribed inth eNP P (Peter,1982) . Furthermore, sion ofon e stepo f thereaction ,us eo f bothNP Pan dNL Tar ecapabl eo fconcentratin g other primary antisera orabsorptio no fth e labelled steroids (Kime tal . 1978). antiserumwit h syntheticLH-RH . Norelationshi pwa s observed betweenth e References distribution ofLH-R Han d these xo r there Ball,J.N. , 1981.Hypothalami c control ofth e productive cycle.Immunoreactiv emateria l pars distali si nfishes ,amphibian s andrep was observed inconstantly in twogroup so f tiles. Gen.Comp .Endocr .44 :135-170 . cellbodie s of thehypothalamus .Th e first Kim,J.A. ,W.E .Stump fan dM .Sar ,1978 .To onewa s located in theventrolatera l parts pography ofestroge n target cells in thefo - of thenucleu s preopticus periventricularis rebraino fgoldfish ,Carassiu sauratus . (NPP ;Pete ran d Gill,1975 )an dconsiste d J. Comp.Neurol .18 2 :611-620 . ofbipola rneuron s directed rostrocaudally. Peter,R.E. ,1982 .Neuroendocrin e controlo f An othergrou po fimmunoreactiv ecel lbodie s reproduction in teleosts,Can .J . FishAquat . was found in theposterio rpar to f thenu Sei. 39 :48-55 . cleus lateralis tuberis (NLT ;Pete ran d Gill, 1975). These perikaryawer e lessnume Peter,R.E .an dV.E .Gill ,1975 .A Stereota rous andwer eobserve d only intw ofishes . xic atlas and technique forforebrai nnucle i Immunoreactive fiberswer ewidel ydistribu ofth egoldfish ,Carassiu s auratus.J .Comp . ted inth ewhol ebrai nbu twer e particularly Neurol.,15 9 :69-102 . numerous insevera lareas .O n longitudinal sections,fiber s directed rostrocaudally were observed in theventra l telencephalonan d theolfactor y tract.Whethe r they originated in theNP Pi sno t sure.Fiber swer eals o frequently observed inth eare a dorsalisan d 56 LOCALIZATIONO FAROMATAS EACTIVIT YI NTH EBRAI NO FTH ERAINBO WTROU T (SALMO GAIRDNERI) J.G.D. Lambert,Petr aG .Wilms ,M.L .Zandwijk ,H.J.Th . Goosan dP.G.W.J .va nOordt . ZoologicalLaboratory , sectionfo rComparativ eEndocrinology ,Stat eUniversit yo fUtrecht , TheNetherlands . Aromatization (conversiono fandrogen s BIOCHEMICAL PROCEDURE intooestrogens )coul db edemonstrate di n severalpart so fth ebrai n (Lamberte tal . PARTS of brain sections [0.5-3 mg) 1982) .I nthi sstud ya metho d ispresente d incubation H an drostene dio ne 0.1 AiCi/mg tissue fora mor epreciz e localization ofth e 3 hours 20 °C NAD/NADP/NADPH 2 mM total volume: sucrose 125 mM aromatase activity.I tcomprise sa 50pl/m g tissue propylenegtycol 5 %(v/v) combinationo famicroanatomica l anda phosphate buffer (pH 7.U) 100 mM biochemicalappraoch . INCUBATION MEDIUM Brainso fadul t troutwer e quickly frozen with CO2.Transvers e sectionso f40 0 fimwer e cuta ta temperatur eo f-10°C .Th e lefthal f of thesewer eprocesse d forhistologica l extraction with dichloromethane studyt oidentif y thedifferen tbrai nareas . ORGANIC FRACTION Therigh thalf swer e cutint o2 o r3pieces , TLC: diisopropylether:chloroform:hexane 7:2:1 HISTOLOGICAL PROCEDURE scanning of plates UNKNOWN TESTOSTERONE ANDROSTENEDIONE OESTRADIOL OESTRONE determination of enzyme activity by percentage distribution method ENZYME ACTIVITY PRELIMINARY RESULTS 3 area dorsalispar s mediate ["") n.preopliou s H n 'aletalis (uberis Q3n . recessus tolerate 6 5 4 3 '2.' '1' Saccus dorsaks TRANSVERSE FROZEN SECTIONS (400/jm) area dorsahspar s mectats ructeus ereooncus Cfüasma oplKun LEFT HALF RIGHT HALF processedfo r processedfo r histological study biochemical study ( fixed inBouin's fluid divided into parts I 5ju m sections £3 stained with Haematoxylin-Eosine \ n rec lot* laterals \ hypophyse identification of different each part incubated brain areas to determine aromatase activity OPTIC TECTUM AND CAUDAL HYPOTHALAMUS correspondingt odistinc tbrai n structures (i.g.hypothalamus ,tectu mopticu m etc.). Thearomatizin g capacitywa s studiebd y Reference: incubating thesepiece swit h-^H-androstene - J.G.D. Lambert andP.G.W.J ,va nOordt : dionan d expresseda sth eamoun to foestron e Cathechol-oestrogensi nth ebrai no f Salmo +oestradiolforme d (fmol/mg tissue/min). gairdneri.Gen .comp .Endocrinol.(46), p401 , Someo fth epreliminar y results areshow n (1982) as thefigure si nth e Q's 57 EFFECT OF SYNTHETIC LH-RH ONGT H CELLS OF E2-TREATED EELS M. Olivereau, P. Charabolle,P . Dubourg Institut Océanographique, 75005 Paris,Centr e deMorphologie , Talence,Franc e 5ummar y Estradiol treatment induces thesyn or dilated cisternae were more abun thesis of gonadotropin (GTH)i n the dant at the periphery of thecells . pituitary ofAnguill a anguilla L.In Large globules were lessnumerous . jection of LHRH appears able torelea Some globules showed an excentric seGTH : exocytotic figures and lysis dense core of avariabl e size,an d a of some large globules and granules peripheral area of a low orver y low are observed. However, this response electron density and afin e granular does not occur in allGT H cells which structure.A muriform structure was remain granulated. occasionally observed. Inmos t cases, themembran e was no longer discerni Freshwater male silver eels have ble. This process does not occur in smallGT H cells poorly differentiated allGT H cells after LHRH injections; containing abundant mitochondria, and it israrel y observed inE2-treate d rather few granules with a moderate eels. In addition, several exocytotic electron density and an average diame figures were observed in LHRH-injec- ter of 125 nm (80-200nm ) (Olivereau ted eels. The figures were located 6 Chambolle, 1978). Estradiol-17ß near the basal lamina, often facing a (E2) treatment induces development capillary. They occurred rarely in and hyperplasia ofGT H cells although eels treated for one day, and were a mitotic activity was not detected. more abundant in those receiving 4 A well developed Golgi complex,nume and 6 injections, whereas lytic pro rous secretory granules (200-500 nm cesses in-th e globules were observed diameter), dilated cisternae and some after a single injection. The Golgi large globules (1.2-2.2 urn)ar e obsep area remained highly developed, and ved (Olivereau & Chambolle, 1979)- As Golgi vesicles were abundant. These no macroscopic effect is discernible ultrastructural data suggest that on the gonad, the release of thesyn some GTH is released after injection thesized hormone remains questionable. of synthetic LHRH although degranula- Despite a careful study, exocytotic ted cells areno t observed. These da figures were not observed.Suppres ta agree with those of Crim & Evans sion of E2 treatment induces agra reporting that LHRH increases plasma dual regression ofGT H cells (Olive GTH levels in immature trouts previou reau &Olivereau , 1979)- sly treated with gonadal steroids. Twenty-two male silver eels were References treated with E2 (10 or 13 injections, Crim, L.W.,& D.M . Evans, 1980.LHR H I25 ug/100g , within2 0 or2 6 days stimulated gonadotropin release from respectively. Eight eels were subse therainbo w trout pituitary gland: quently injected withNaC l 0.6 'foplu s an invitr o assay for detection of gelatin 1% in the body cavity.Four teleost gonadotropin-releasing fac- teen eels received LHRH in the same tor(s). Gen.Comp . Endocrinol. 40: solvent (l injection, or2 injections 283-290. 90mi n apart, or4 or 6 injections within2 or 3 days respectively,2 5 Olivereau, M. & P. Chambolle, I978. tagpe r injection). Th e eels were kil Ultrastructure des cellules gonado- led 1h after the last injection.Pi - tropes de l'Anguille normale etaprè s injection d'oestradiol. C.R . Acad. tuitaries were fixed in 6.5 i° gluta- raldehyde solution inSörense n buffer Sei. Paris,28 7 D: l409-l4l2. (pH7.2 ) and post-fixed in 1$ osmium Olivereau, M. &P. Chambolle, 1979- tetroxide.The y were embedded in Epon Ultrastructure of gonadotrophs inth e and stained with uranyle acet.3; ean d eelfollowin g estradiol treatment. lead citrate. Proc. Indian Natl Sei. Acad. B 45: 478-482. Olivereau, M. &J . Olivereau, 1979. Solvent injections had noe.'fec t Estradiol-positive feedback ongona on the developed GTH cells.Th e res dotropic (GTH)cell s in freshwater ponse.o fthes e cells to LHRH isno t male silver eels. Gen.Comp .Endocri uniform,Som e areas of the p:oxima l nol. 39=247-261 . pars distalis were not clear;y affec ted. Other islets ofGT H cell:,appea red less granulated. Large vacuoles 58 THEFUNCTIONA L SIGNIFICANCE OFTH ENUCLEU S OLFACTORETINALIS INTH EPLATYFISH ,XIPHOPHORU S MACULATUS M.P. Schreibman,H .Margolis-Kazan ,L .Halpern-Sebol dan dH.J.Th .Goos-' - Department ofBiology ,Brookly nCollege ,Brooklyn ,Ne wYork ,11210 ,U.S.A . and ^Zoology Laboratory,Utrech tUniversity ,Utrecht ,Th eNetherland s Thenucleu s olfactoretinalis (NOR)ha s inth ebrai n thatar e involved inth edevel beenidentifie d asa nimmunoreactiv elutein opmentan dmaintenanc e ofth ereproductiv e izinghormon e releasinghormon e (ir-LHRH) system (see figure). Thesequentia lappear containing center inth ebrain . Becausei t anceo f ir-LHRH inbrai nnucle ibeginnin g islocate d at theboundar ybetwee n theven with theNO Ran dprogressin g ina cauda l traltelencephalo nan d theolfactor y lobe direction toward thepituitar y gland,lead s and sendsprojection s tobot h theopti can d ust osuspec t that theNO R serves toiniti olfactory nerves,i tha sbee n termedth e ate theactivit y inth eothe rnucle iessen nucleusolfactoretinali stelencephal i (Münz tialfo r theproces so fpubert y tooccur . etal. ,1981) . Inou r immunocytochemical Wesuspect ,too ,tha t theNO R ismos tac (ICC)studie so f sexuallymatur eplatyfish , tivewhe n gonadotropin levelsar elow ,a s ir-LHRHha sals obee n localized intract s forexampl ei nth e immature fishan d inth e betweenth eNO Ran d thepinea lcomple x hypophysectomized adultstha tw ehav estu (habenularnucleus )an d between theNO Ran d died. (Supported byNIH-NI A (AG01938)an d their-LHR H containing neurons of thenucle theCit yUniversit y ofNe wYork . uspreopticu speriventriculari s (NPP)an d thenucleu slaterali s tuberispar sposteri - oris (NLT). THE! NOR i : INTEGRATOR OFINTERNA LAN DEXTERNA L SIGNALS FORTH EREGULATIO NO FREPRODUCTIV ESYSTE MMATURATIO NAN D MAINTENANCE. Wehav eals o found thatdurin g platyfish development theNO R isth efirs tregio ni n PinealComple x thebrai ni nwhic hir-LHR H canb eidenti fied. Itappear s insexuall y immaturefis h OlfactorySyste m (stage 1)a t4 week so fag ei nearl ymatur inggenotype s and at 9week s ofag ei nlat e VisualSyste m maturers. Ir-LHRH canfirs tb edemonstrate d HormonalFeedbac k inneuron s of theNP P asth eproces so f sexualmaturatio n isinitiate d (stage2 ) and inth eNL Ton et otw oweek slate ra s pubertal development continues. Thesam e sequence ofdevelopmen t occurs inlat e maturersbu twit ha mor eprotracte d time scale. PITUITARY GLAND Ina nIC Cstud yo fhypophysectomize d platyfishw efoun d that there isa marke d increasei nth eir-LHR H content inperi - GONADS karyaan dprocesse s of theNO Rbu t adeple tiono f ir-LHRH inth eNP P andNLT . If hypophysectomized fishreceiv e fivelOu g injections of salmon gonadotropin onalter References natedays ,ther e isa nincreas ei nth e Münz,H. ,W.E .Stumpf ,an dL .Jennes ,1981 . number of ir-LHRHcontainin g fiberso fth e LHRH-systemsi nth ebrai no fplatyfish . NPPan dNL Tbut ,althoug h stillabov econ BrainRes. ,221:1-13 . trollevels ,a significan t decrease ofir - Schreibman,M.P. ,L .Halpern-Sebold , LHRH inth eNOR . RIAanalyse s ofwhol e M. Ferin,H .Margolis-Kaza nan d brains confirm thedecreas e inLHR Hcon H.J.Th.Goos , 1982. Theeffec to f tentfollowin ghypophysectom y (mean+ S.E.: hypophysectomy and gonadotropinadminis 649+ 28p gpe rbrai ncompare d to44 6+ 3 1 tration onth edistributio nan dquantit y pgpe rbrain ) (Schreibmanet .al ., 1982 ). ofLHR H inth ebrain so fplatyfish :A combined immunocytochemistryan dradio Based onou robservation s andbecaus eo f immunoassay study. Submitted toBrai n itsanatomica lposition ,th eNO Rma ypla y Res. animportan t role intransmittin g (transla ting)environmenta l cues tothos ecenter s 59 SITE OF ACTION OF SOME AGRICULTURAL PESTICIDE IN THE HYPOTHALAMO-HYPOPHY- SIAL-OVARIAN AXIS OF THE FRESHWATER CATFISH. HETEROPNEUSTES FOSSILIS(BLOCH ) H- SINGH, T. P. SINGH Fish Endocrinology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi -221006 , India. Deleterious effect of some agricultural pesticides on of the pituitary gland and the blood serum of the fish reproductive physiology of Heteronopustes fossilis exposed to pesticides was substantially reduced is now well established ( Singh & Singh, 1980a, b, (P^O.001) as reflected'b y decreased ovarian 32p uptake 1981 ). These pesticides decrease gonadotrophin secretion and inhibit ovarian activity. The present study was undertaken to explore the action of four pesticides - Cythion, Paramar M50 (organophosphate), Aldrin and Hexadrin (organochlorine) at sublethal PITUITARY EXTRACT concentration in 3 sets of experiments on hypothalamo- A hypophyseal-ovarian axis in H. fossilis. Fish were exposed to sublethal concentration of in À pesticides for four weeks. In the first set of experiment effect of the above pesticides on ovarian 32p uptake in sham-hypophysectomized and hypophysectomized fish was studied. In the second experiment ovarian 32p uptake was measured in response to the injections of pituitary extract or blood serum collected from fish exposed to pesticides. And the third experiment was conducted to assess ovarian 32p uptake response after the intraperitoneal administration of hypothalamic extract pooled from pesticides exposed fish. in hypophysectomiïed recipients in comparison to the control ( Fig. 2 ). Hypothalamic extract of pesticide exposed fish also showed significantly decreased amount of GnRH-like substance ( Fig. 3 ). Thus these pesticides apparently inhibited the secretion of »p 400CLO-. HYPOTHALAMIC EXTRACT A* I i X Fig. 1 All the four pesticides Cythion, Paramar M50, Aldrin and Hexadrin significantly reduced (P<0.05 to 0.001) the ovarian incorporation in sham-hypophysectomized fish. In hypophysectomized fish Cythion and Paramar M50 had no effect on 32p uptake of ovary but Aldrin and Hexadrin were potent enough to further reduce the 32p uptake by ovary ( P^D.OO1 , Fig. 1 ). This clearly indicate that these organochlorine compounds directly Fig. 3 affect the ovary in H. fossilis. Gonadotrophic potency 60 Reference : Singh, H. and T, P. Singh, 1980a. Effect of two pesti cides on ovarian 32p uptake and gonadotropin concentration during different phases of annual reproductive cycle in the freshwater catfish, Hete- ropneustes fossilis ( Bloch ). Environmental Res, 22 : 190-200. Singh, H. and T. P. Singh, 1980b. Effects of two pesticides on testicular 32p uptake, gonadotropic potency, lipid and cholesterol content of testis, liver and blood serum during spawning phase in in H. fossilis (Bloch). Endokrinologie, 76: 288-296. Singh, H. and T. P. Singh, 1981. Effect of Parathion and Aldrin on survival, ovarian 32p uptake and gonadotrophic potency in a freshwater catfish, Heteropneustes fossilis ( Bloch ). Endokrinolo gie, 77 : 173-178. Acknowledgements : Financial assistance from DST ( No. HCS/DST/928/ 30 ) and CSIR (No. 7/13 ( 1422 )/80-81 Govt, of India, New Delhi is gratefully acknowledged. 61 SEX MANIPULATION 63 EXPRESSION OF H-Y ANTIGEN IN NON MAMMALIAN VERTEBRATES AND ITS RELATION TO SEX DIFFERENTIATION P. Zaborski Centre de Cytologie Expérimenta le , C.N.R.S., 67 rue M. Glinsbourg,9420 0 1VRY-SUR-SEINE,FRANC E Summary cell. However H-Y serology is far from being easy to handle, and numerous technical dif Among the factors involved in primary ficulties may led to conflicting results gonadal differentiation, a cell surface com of H-Y serotyping. Because H-Y is a minor ponent, the H-Y antigen, has been proposed antigen, the main technical problem is the as the principal determining factor of mam weakness of the H-Y antiserum. A solution malian testicular organogenesis. Since the to this problem is to use monoclonal H-Y H-Y antigen has been found in the heteroga- antibodies. metic sex of species from all the classes In spite of these difficulties, since 1975 of vertebrates, it has thus been assumed the H-Y antigen was suspected as being the to play an important role in XY testicular mammalian testis determining factor, and, or ZW ovarian differentiation. However in due to its phylogenetic conservation, to nonmammalian vertebrates, experimental fin be involved in the differentiation of the dings suggest a role for both sex hormones heterogametic gonad in vertebrates (Wachtel and H-Y antigen in gonadal organogenesis, et al., 1975a). In mammals at least, there since the gonadal H-Y expression depends is a strong body of evidence to suggest that on a sex steroid control. H-Y might play a role in primary sex deter Keywords: H-Y antigen, gonadal sex differen mination. Although this will not be discus tiation, sex hormones, sex manipulation. sed here in detail, it is important to know that, inmammals : Introduction - testicular tissue is generally associated with the expression of H-Y antigen, whatever H-Y antigen was first considered as ami the sex genotype or the sex phenotype may nor histocompatibility antigen specific of be the male in mammals. It was originally dis - there is a gonad^specific receptor for covered in the mouse, since male skin grafts H-Y antigen (in gonadal tissue of both sexes) on females were rejected while other graft - H-Y antigen is presumed to control testis combinations were tolerated in the highly differentiation via its receptor inbred (syngeneic) C57BL/6 mouse strain (Ei- - dissociated ovarian cells reorganize into chwald & Sflmser, 1955). This male-specific testicular-like structures in the presence antigen was then termed 'H-Y' (Billingham of exogenous H-Y antigen (for review on mam & Silvers, 1960), 'H' as 'histocompatibi mals see: Haseltine & Ohno, 1981;0hno,1979 ; lity' and 'Y' as 'Y-chromosome', because Wachtel & Koo, 1981). the expression of this surface antigen was attributed to the Y-chromosome, being the I Phylogenetic conservation of H-Y antigen main genomic difference between males and and of its gonad-specific receptor females of the same syngeneic strain. Studies on the involvement of H-Y antigen The homology of mouse H-Y antigen with in transplantation are numerous (review: a cross-reactive or identical plasma membra Billingham and Hings, 1981). However, graft ne component in other vertebrates has been experiments are not the only way to study demonstrated. Since direct cytotoxicity or the H-Y antigen and a serological approach binding assays cannot be used in nonmamma has been developed. H-Y antiserum is usually lian vertebrates, H-Y typings are performed obtained by hyperimmunisation of female mice by serological absorption experiments using or rats with spleen cells from males of the mouse or rat H-Y antiserum. Cells to be tes same syngeneic strain. Even though the H-Y ted are incubated with H-Y antiserum. The antigen is present on all male cells, only residual anti-H-Y activity is then estimated a few cell types are good targets for H-Y on mammalian H-Y positive target-cells. A antibodies. Among them, the mouse epididymal decrease of anti-H-Y activity indicates that spermatozoon is the most widely used in nu the +tested cells are H-Y antigen positive merous serological assays (review: Koo,1981). (H-Y ),wherea s no (or a few) loss of anti- H-Y typing is usually determined by serolo H-Y activity indicates that the_teste d cells gical absorption. This procedure uses the are H-Y antigen negative (H-Y"). The sero ability of the cells to be tested to remove logical assays that have been used for non- H-Y antibodies from H-Y antiserum. The resi mammalian vertebrates are the following: dual anti-H-Y activity of the recovered H-Y cytotoxicity test on mouse sperm (Goldberg antiserum is then estimated in a direct se et al., 1971); cytotoxicity on rat epidermal rological assay using an H-Y positive target cells (Scheid et al., 1972); protein A ro- 64 Table 1.H- Yantige n expression infishes . References Species H-Y antigen in Tested tissues Shaleve tal . (1978) Salmo gairdneri femal e blood Muller& Wol f (1979) Salmo gairdneri ? gonads Salvelinus alpinus ? gonads Rutil us rutilus ? gonads Carassius auratus ? gonads Barbus tetrazona ? gonads Lebistes reticulatus male gonads Xiphophorus helleri male gonads Pechan et al. (1979) Xiphophorus maculatus male (XY or YY) brain Haplochromis burtoni male brain Orizias latipes male (XY) brain,liver Ti lapi a male Shalev & Huebner(1980; Lebistes reticulatus male 1i ver, heart,spleen,gonads Wiberg (1982) Anguilla anguilla femal e (ZW) gonads Zaborski & Bruslé Serranus cabri 11a testicular part blood,spleen,ovotestis Park &Grimm ,198 1 sette assay on mouse sperm (Koo& Goldberg, cells from normal Xenopusjnale s (inXenopus , 1978); cytotoxicity on human lymphoblastoid normal ZZ testes areH-Y/an d normal ZWova cells (Fellous et al., 1978); immunobacte- ries are H-Y+). As inmammals , thehomoga rian rosette assay onmous e sperm (Zaborski, metic gonad gossess H-Y receptors, although 1979a). normally H-Y. In a more functional pers Using such techniques, antigenic determi pective, Zenzes et al. (1980) cultivated nants similar to H-Y have been found in dissociated testes from chickens inth epre birds (Wachtel etal. , 1975a; Müller etal. , sence of mammalian H-Yantige n andthe yob 1980), reptiles (Zaborski et al.,1979 ; served that thetesticula r cells reorganized Engel et al., 1981), amphibians (Wachtel into ovarian-like structures (normal avian et al.,1975a ; Zaborski, 1979b; Engel & ovaries areH- Y ). Schmid, 1981), and fishes (see Table1) . The widespread phylogenetic representation Moreover, a strong correlation between the of H-Y antigen as well asth e phylogenetic H-Y sexan dth eheterogameti c sexha sbee n conservatism ofit sgonads-specifi c receptor established (XY-male= H-Y; ZW-female=H-Y ) suggest that H-Y antigen could have anim at least within thebird s andth eamphibi portant sex-associated function in nonmam- ans. However, there are some intra-class malian vertebrates. exceptions, mostly in mammals, dependant on genetic anomalies. Theus eo f detection II Manipulation ofse xan do fH- Yexpressio n of H-Y antigen asa too l for identification of an unknown heterogametic sexi sno t al Sex as well asH- Ymanipulatio n byepige - ways possible because oftechnica l difficul netic factors is possible in nonmammalian ties in H-Y serology. It should be noted vertebrates butno ti nmammals . All the ob that, forreason s probably involving defects servations that have been made so fari n in cross-reactivity, the greatest problems this respect are reviewed in this section. are encountered in fishes. However, since the studies infishe s were generally perfor Change ofse xwit h change ofH- Yexpressio n med ona preliminary way, further more con clusive studies are+ required. A strict homo Sex hormone treatments inbird s andamphi logy between theH- Yse xan dth eheterogame bians. In female birds, sexdissymmetr y of tic sex seems to be exagerated. However, gonads isa specific feature. Only thelef t the identification ofth eH- Yphenotyp emi gonad differentiates into an ovary while ght be used as a substitute which reflects the right one remains rudimentary. Sexdis differences inse xallele s between malean d symmetry occurs aswel l infeminize d males, female individuals, when sex chromosomes since male embryos treated with estradiol orth eheterogameti c sexar eunknown . benzoate develop a left ovotestis whileth e Further studies have demonstrated thecon right gonad remains testicular. Muller et servatism ofth egonad-specifi c H-Yrecepto r al. (1979) have shown that such anovotesti s Wachtel et al. (1980) shown that soluble becomes H-Ylik e a control ovary (normal mammalian H-Y antigen and soluble avianH- Y ZZ testes areH- Y) . Similar results were antigen bound specifically to testicular obtained in quails: H-Y antigen waspresen t 65 in ovotestes of male embryos feminized fol antigen (Reyss-Brion et al., 1982). lowing diethylstilbestrol treatment, but H-Y was not induced in non-gonadal_tissue s Thus, sex hormone treatment or ovariectomy such as spleen that remained H-Y (Müller (which also influence the hormonal balance) et al., 1980). Moreover, the quail ovotestis both induce an'inversio n of H-Y phenotype is only transiently feminized (as also in on the gonadal cells.-Thes e experiments de the chick) and reverts to a testis a few monstrate that the H-Y structural gene(s) weeks after hatching. The H-Y study of the is present in both the heterogametic and post-hatching period in treated males shown the homogametic sex. The correlation between an evolution of H-Y expression: H-Y antigen the presence of H-Y antigen and the presence disappeared from the reverting left ovotes of gonadal structures of the heterogametic tis while,th e unfeminized fight testis re sex is confirmed. However, these experiments mains H-Y (Zaborski et al., 1981). In am do not permit one to discern whether the phibians, complete and functional sex inver H-Y expression is the result of the treat sion can be achieved by steroid treatment ment and the cause of the gonadal inversion of larvae. Wachtel et al. (1980) in the frog or the result of the gonadal inversion. Xenopus laevis and Zaborski & Andrieux(1980) in the salamander Pleurodeles waltlii (both Change of H-Y expression without change of amphibians displaying a ZZ-male/ZW-female sex sex-determining mechanism) demonstrated that ZZ male larvae treated with estrogens were As sex steroids are apparently involved sex-inversed and subsequent adult 'neo-fema- .in the sexual differentiation of gonadal les' typed H-Y in ovarian cells but remai and H-Y phenotype, the question arose whe ned H-Y in non-gonadal cells, according to ther H-Y antigen expression might also be their homogametic sex genotype. hormonally controlled in already differen tiated gonads. The following experiments Temperature treatments in reptiles. In has been undertaken in adult amphibians be many species of turtle the sexual differen longing to either the ZZ-male/ZW-female or tiation of the gonads is temperature depen the XX-fema'le/XY-male sex-determining system. dant and complete sex-inversion can occur. In Pleurodeles, when adult ZZ males were In Emys orbicularis, egg incubation below treated with estradiol-17beta, the testes 27.5°C results in 100% phenotypic males, were not feminized but became H-Y , and a while egg incubation above 29.5°C results drop of piasma"testosteron e was observed. in 100% phenotypic females. H-Y typing of Thus, the appearance of H-Y antigenis the blood cells in animals of each lot shown direct consequence of the estrogen treatment that some individuals were H-Y and the and does not necessarily depend on the fe - others v^ere H-Y~. Since wild adult females minization of the gonads (Zaborski & And- are H-Y (no heteromorphic sex chromosomes) rieux, 1980b). Though the estradiol treated H-Y individuals were considered as geno- testes were not inversed; the spermatogene typic females (and as sex-inversed in the sis was inhibited and the glandular tissue phenotypic male lot), and H-Y individuals was atrophied. It is well known that the were considered as genotypic males (and as very same effects on testes are obtained sex-inversed in the phenotypic female lot). when adult males are hypophysectomized. H-Y The H-Y typing of gonadal cells has shown typing in such males in Pleurodeles shown that, whatever the blood H-Y phenotype, ova that testicular cells became H-Y . Moreover, rian cells were H-Y and testicular cells both plasma testosterone and estradiol le were H-Y~(Zaborski et al., 1982). Tempe vels were very low, suggesting that estra rature dependant changes in sex hormone diol per se was not required to induce H-Y concentrations in the embryonic gonads could expression. This conclusion was confirmed be involved. in hypophysectomized female Pleurodeles: ovarian cells remained H-Y as in normal Ovariectomy in birds. In the female chick, ovaries although estradiol level as well a left ovariectomy induces the transforma as testosterone level were very low (Zabor tion of the rudimentary right gonad into ski & Andrieux, 1980a). a testis. Studies of the H-Y antigen in such Thus a repressor control exerted by andro a model shown that it progressively disap gens was suspected. However testosterone peared in the initially H-Y right gonad treatment of adult females failed to show during the course of its masculinization, any modification of the ovarian H-Y pheno to reach a levej of H-Y expression similar type which remained H-Y , suggesting a con to that of H-Y~control testes. Non-gonadal stitutive expression of H-Y in ZW females, cells of operated animals were not affected while it was inducible in ZZ males. To elu and remained H-Y (Zaborski et al., 1980). cidate this specific problem, more precise Although the gonadal transformation occured and contrôlable in vitro experiments were without any added exogenous hormones, an undertaken with Pleurodeles ovaries. They important increase of testosterone and a shown that a three-week culture with dihy- decrease of estrogen production occured du drotestosterone led to the inhibition of ring the period of disappearance of H-Y H-Y expression in ovarian tissues which be- 66 came H-Y~(Penrad-Mobayed & Zaborski,1982). tles; Differentiation 20:152-156. Thus it is the absence (or a low level) of Engel, W. & M. Schmid, 1981. H-Y antigen androgens rather than the presence of estro as a tool for the determination of the gens that controls the gonadal H-Y expres heterogametic sex in amphibia. Cytogenet. sion in aZZ/Z W system. Cell Genet. 30:130-136. Complementary experiments in the frog Rana Fellous, M., E. Günther, R. Kemler, J. Wiels ridibunda shown that estradiol treatment R. Berger, J.L. Guenet, H. Jakob & F. Ja of XY adult males induced an inhibition of cob, 1978. Association of the H-Y male H-Y expression on initially H-Y testicular antigen with beta?-microglobulin on human cells. It appears that the H-Y expression lymphoid and differentiated mouse terato- is repressed in ZW PIeurode le s ovary by an carcinoma cell lines. J. Exp. Med. 148: androgen, whereas the very same effect is 58-70. induced by an estrogen in XY Rana testis. Goldberg, E.H., E.A. Boyse, D. Bennett, M. Scheid & E.A. Carswell, 1971. Serological demonstration of H-Y (male) antigen on Conclusion mouse sperm. Nature 232:478-480. Haseltine, F.P. & S. Ohno, 1981. Mechanisms Once the technical difficulties have been of gonadal differentiation. Science 211: overcome, H-Y serology may be used as atoo l 1272-1278. in sex identification, since a stronger re Koo, G.C., 1981. Serology of H-Y antigen. activity of the H-Y antiserum is observed Hum. Genet. 58:18-20. in the heterogametic sex rather than in the Koo, G.C. & C.L. Goldberg, 1978. A simpli homogametic sex. Associated with classical fied technique for H-Y typing. J. Immunol. experiments, it also permits us to enhance Methods 23:197-201. our knowledge on the conditions of expres sion of H-Y antigen. Maurel, M.C. & P. Zaborski, 1982. Inhibition of H-Y antigen expression in the frog tes In nonmammalian vertebrates, the sex hor tis following estradiol treatment. Biol. mones appear to be the primary inducers of Cell. 45:86. / H-Y antigen. An inhibitory or repressor con Müller, U., A. Guichard, M. Reyss-Brion & trol by androgens in ZZ/ZW vertebrates and D. Scheib, 1980. Induction of H-Y antigen by estrogens in XX/XY vertebrates may also in the gonads of male quail embryos by be proposed. However, since each sex pro diethylstilbestrol. Differentiation 16: duces both kind of sex hormones, the control 129-133. involves a quantitative rather than aquali tative hormonal change. In all these res MUller, U. & U. Wolf, 1979.Cross-reactivit y pects, H-Y might play an intermediary role to mammalian anti-H-Y antiserum in tele- as a part of a chain of factors leading to ostean fish. Differentiation 14:185-187. morphogenetic events. Müller, U., M.T. Zenzes, U. Wolf, W. Engel & J.P. Weniger, 1979. Appearance of H-W Indeed, the fact that the H-Y antigen mi (H-Y) antigen in the gonads of oestradiol ght be involved in sex differentiation is sex-reversed male chicken embryos. Nature an exciting suggestion. In this connection, 280:142-144. what remains is to design convenient experi Ohno, S., 1979. Major sex-determining genes. ments of the 'cause and effect relationship' Springer-Verlag, Berlin Heidelberg pp.140. type using exogenous H-Y antigen and undif ferentiated embryonic gonadal cells. Park, E.H. & H. Grimm, 1981. Distribution of C-band heterochromatin in the ZW sex The role of H-Y antigen in the process chromosomes of european and american eels of sex differentiation is far from being (Anguillidae, Teleostomi). Cytogenet. Cell clear and it seems premature to adopt a Genet. 31:167-174. definitive position regarding the present Pechan, P., S.S. Wachtel & R. Reinboth,1979. state of our knowledge on immunologic, ge H-Y antigen in the teleost. Differentia netic, hormonal and environmental sex tion 14:189-192. factors. Penrad-Mobayed, M. & P. Zaborski, 1982. Control of H-Y antigen expression in am phibian gonads: possible inhibitory role References of androgens in the heterogametic ovary. Proc. XV Internat. Embryol. Conf. Stras Billingham, R.E. & I.M. Hings, 1981. The bourg France p.89 . H-Y antigen and its role in natural trans Reyss-Brion, M., Th.M. Mignot & A. Guichard, plantation. Hum. Genet. 58:9-17. 1982. Development of steroidogenesis in Billingham, R.E. & W.K. Silvers, 1960. Stu the right gonad of domestic fowl masculi dies on tolerance of the Y chromosome an nized by left ovariectomy. Gen. Comp. En tigen inmice . J. Immunol. 85:14-26. docrinol. 46:68-74. Eichwald, E.J. & CR. Silmser, 1955. Unti Scheid, M., E.A. Boyse, E.A. Carswell & L.J. tled communication. Transp. Bull. 2:148- Old, 1972. Serologically demonstrable 149. alloantigens of mouse epidermal cells. Engel, W., Klemme, B. & M. Schmid, 1981. J. Exp. Med. 135:938-955. H-Y antigen and sex-determination in tur Shalev, A., I. Berczi & J.L. Hamerton,1978 . Detection and cross-reaction of H-Y anti Zaborski, P. &B . Andrieux, 1980a. H-Y anti gen by haemagglutination. J. Immunogenet. gen in sexual organogenesis of amphibians: 5:303-312. recent studies on its expression in some Shalev, A. & E. Huebner, 1980. Expression experimental conditions. Proc. XIV Inter of H-Y antigen in the guppy (Lebistes re- nat. Embryol. Conf. Patras Greece, p.114. ticulatus). Diefferentiation 16:81-83. Zaborski, P. &B . Andrieux, 1980b.Hypothè Wachtel, S.S., P. Bresler & S.S.Koide,1980 . se sur l'action inhibitrice de latesto Does H-Y antigen induce the heterogametic sterone dans l'expression de l'antigène ovary? Cell 20:859-864. H-Y chez un Triton.Arch .Anat .Microsc . Wachtel, S.S. & G.C. Koo, 1981. H-Y antigen Morphol. Exp.69:333 . in gonadal differentiation. In CR. Aus Zaborski, P. & S. Bruslé. H-Y antigen in tin & R.G. Edwards (Ed.): Mechanisms of a synchronous hermaphrodite teleost fish: sex differentiation in animals and man. restriction of its expression to the tes Academic Press,London , p.255-299 . ticular part of the ovotestis of Serranus Wachtel, S.S., G.C. Koo & E.A. Boyse, 1975. cabrilla L. Reprod. Nutr. Develop, (sub Evolutionary conservation of H-Y (male) mitted). antigen. Nature 254:270-272. Zaborski, P., M. Dorizzi & C. Pieau,1979 . Wachtel, S.S., S. Ohno, G.C. Koo & E.A.Boyse, Sur l'utilisation de sérum anti-H-Y de 1975. Possible role for H-Y antigen in Souris pour la détermination du sexegéné the primary determination of sex. tique chez Emys orbicularis L. (Testidines Nature 257:235-236. Emydidae). CR. Acad. Sei. Paris, D., Wiberg, U., 1982. Serological cross-reacti 288:351-354. vity to rat anti-H-Y antiserum in the fe Zaborski, P., M. Dorizzi & C. Pieau,1982 . male european eel (Anguilla anguilla). H-Y antigen expression in temperature sex- Differentiation 21:206-208. reversed turtles (Emysorbicularis) . Zaborski, P., 1979a. Detection of H-Y anti Differentiation (to bepublished) . gen on mouse sperm by the use of Staphylo Zaborski, P.,A. Guichard &M . Reyss-Brion, coccus aureus. Transplantation 27:348-350. 1980. H-Y.antige n in ovariectomized chicks Zaborski, P., 1979b. Sur la constance de : disappearance of its expression during l'expression de l'antigène H-Y chez le the transformation of the right gonad into sexe hétérogamétique de quelques Amphi- a testis. Biol. Cell.39:291-294 . biens et sur la mise en évidence d'un di- Zaborski, P., Aw Guichard & D. Scheib,1981 . morphisme sexuel de l'expression de cet Transient expression of H-Y antigen in antigène chez 1'Amphibien Anoure Pelodytes quail ovotestis following early diethyl- punctatus D. CR. Acad. Sei. Paris, D, stilbestrol (DES)treatment . Biol.Cell . 289:1153-1156. 41:113-122. Zenzes, M.T., E. Urban & U.Wolf , 1980.Mam malian cross-reactive H-Y antigen induces sex reversal in vitro in the avian testis. Differentiation 17:121-126. 68 TEMPERATUREAN D STEROIDEFFECT S ONGONADA L SEXDIFFERENTIATIO N INRAINBO WTROU T R.va nde nHur k andJ.G.D . Lambert ZoologicalLaboratory ,Universit y ofUtrecht ,Th eNetherland s Summary after fertilization,a perio d thatcoincide s with onseto fgonada l sexdifferentiation . Testicularhomogenate s ofrainbo w trout Anearlie r starto f treatmentwit h steroid, fromD(ay ) 100afte r fertilizationincubate d i.e.fro mhatchin g onward,inhibit snorma l invitr o inth epresenc e of C]4-labelled development ofgonads ,ofte nresultin g in 11ß-hydroxyandrostenedion e (llßAdione)ar e sterilization.Treatmen t fromD5 7onwar d able toconver t this steroid into 11-keto doesno t affect thenorma l sexratio .8 androstenedione (11-keto Adione),whic hi s weeks treatmentwit hmethyltestosteron e and indicative of thepresenc e of11ß-hydrox y llßAdioneadde d toth efoo d from thefirs t steroid dehydrogenase (llß-HSD).Ovarie s day of feedingwer ecarrie d outi norde rt o fromD10 0 convertH3-labelle d 17a-hydroxy investigatewhethe r such long treatments progesterone (17aP4)int oandrostenedione , willresul t inal lmal ebroods ,an dwit hP 4 which indicates thepresenc e of 17a,20 - tostud ywhethe r thissteroi d excertsa fe desmolase.Thes ean dpreviou s observations minizing effectwhe nadde d toth efood . (vande nHur ke tal. ,1982a )sho wtha tjus t Experiments inwhic hgonada lhomogenate s developing testes and ovariesposses s the areincubate d invitr owit h labelled steroid sameenzym e systems,an d testes inadditio n precursors demonstrate thatundifferentiate d have those tofor m steroidswit ha functio gonads and justdevelopin g ovaries inrain nal group at the 11-position.Bot hovarie s bow troutposses s enzyme systemswhic hsyn and testes fromD10 0d ono t show 170-HSDan d thesizeP 4an d other C21-steroids,wherea s aromatase activity and thusar eunabl et o justdifferentiatin g testes inadditio nca n synthesize testosterone andoestrogens . synthesize theandrogen s androstenedionean d Administration of llßAdione (60ug/g )fo r llßAdione (vande nHur ke tal. ,1982a) . 8week s from first feeding results ina nal l Theseresult s and thosefro m experiments in male troutpopulation .Administratio n ofa which fishwer e treatedwit h steroidspossi lowerdos e (6ug/g )an do fmethyltestoste - bly indicate that thepresenc eo rabsenc eo f rone (0.6an d 6ug/g )als oresul t inhig h androgens at thestar t ofgonada l sexdiffe proportions ofmale s (94-99%). Iti sconclu rentiationdetermine s gonadal sex.O n ded that 11ßAdionei sa nendogenou s androgen account of itshig handrogeni cpotenc y in inrainbo wtrout ,whic h initiates thede fish,w ewondere d whether 11-ketotestoste velopment oftestes . rone issynthesize d injus tdifferentiatin g Contrary to itsfeminizin g effectwhe n testes.Therefor e testicular tissuefro m suspended inaquariumwater ,progesteron e D100ha sbee n incubated invitr o inth epre (P4)doe sno t influence sexrati owhe nadde d senceo f 11@Adione-C14i norde r todetermin e toth efood .Neithe r does exposureo f3 1t o apossibl e synthesis ofothe r 11-steroids. 57da yol d trout tohig h temperaturessig Furthermore,homogenate s ofovari a fromD10 0 nificantly alter these xratio . were incubated invitr o inth epresenc eo f Keywords:gonads ,se xdifferentiation ,ste 17aP4-H3,sinc e itwa sno tobviou s frompre roids,temperature ,Salm ogairdneri . vious studies,whethe r justdifferentiatin g ovarieshav e 17a,20-desmolasefo r thesyn thesiso fandrostenedione . Introduction Commercialproductio n ofmonose xpopula Inbrood s ofrainbo w trout,Salm ogaird tions isver y important infis hfarming .In neri,th ese xrati o canb einfluence db y creasing theproportio n offemale sresult s treatmentwit h steroids (Johnstone etal. , ina numbe r ofbenifit si ntrou tfarming . 1978;Okad a etal. , 1979;va nde nHur k& Feminization canb eachieve db y steroidhor Slof, 1981;va nde nHur k et al., 1982b). moneadministration ,however ,huma ncon This effect isdependen t ontyp ean d doseo f sumptiono f steroid treated fish isno tper added steroid,tim eo f starto f treatment mitted.Cros sfertilizatio no fmasculinize d andmetho d of steroid administration.I nou r animals of femalegenotyp ewit hnorma lfe experiments the sexrati owa s shifted toward malesproduce s all-female offsprings,bu t amal edirectio nb y treatment for4 week s this isa tim econsumin g and expensive withmethyltestosteron e or llßAdione.Th e method.I nthi srespect ,feminizatio nwit ha sexrati owa s shifted toward afemal edi biotic factorwoul d bemor eattractive .Tem rectionb y treatmentwit hP 4 suspended in peraturema ypla y an importantrol e inde aquariumwater .Th ebes tresults ,64-84 % terminationo fgonada l sexi nteleost s dominanceo fon ese xwer eobtaine dwhe n (Bruslé& Bruslé , 1982).Possibl e influences treatmentwa s started betweenD4 3an dD5 7 ofhig h temperature ongonada l sexdifferen - 69 tiation inrainbo w troutwer e investigated. 28ovarie sfro mDl0 0trou twer epoole dan d homogenized in 1m l 0.1M phosphatebuffe r Materialsan dmethod s (pH7.4 )containin g0.25 Msucrose ,0.2 5m l homogenatewa s thenincubate dwit h 37KB q Animals 17aP4-H3 (spec,act. :'39 6GBq/mmol )dis solved in2 5u lpropyleneglycol ,2m Meac ho f Fertilized eggso frainbo w trout,Salm o NADan dNADP Han d0.1 5m lphosphat ebuffe r gairdneri,wer e incubated inaquari a at 11C for3 ha t20°C .Befor eextraction , 100u g and 12h l:12h d.Hatchin g occurred atD28 - eacho f 17aP4,androstenedione ,testosterone , 29.Befor ean dafte r treatment,fr ywer e oestradiol-17ßan doestron ewer e addeda s rearedunde r thesam econdition s asth e carrier steroids.Separatio no f steroidswa s eggs.Fr ywer efe d (Trouvit)4 %o fthei r carried outwit hTL Ci nbenzene-ethylacetat e bodyweigh tdaily . (3:1).Androstenedion ewa s furtherpurifie d by crystallizationt oa constan t specific Steroid and temperature treatments activity. 200fis hwer e treated for 8week s from Results first feeding (=D47 )unti lD10 5wit h ste roid added toth efoo do rwit h controlfood . Steroid and temperature treatments Preparations of theexperimenta l dietha s been describedb yva nde nHur k (1982).Me - Highpercentage s ofmale swer eobserve d thyltestosteronewa sadde d toth efoo d in aftertreatin g troutfro mD4 7t oD10 5wit h concentrations of0. 6 and 6ug/g ,11SAdion e HBAdione andmethyltestosteron e (Table1) . in6 an d 60ug/g ,an dP 4 in6 0an d 600ug/g . Ineac hexperimen t thegonad s of 100random Table 1.Effect s ofandrogen s onse xratio . ly sampled fishwer e sexeda tD12 0o rD150 . Number of fish sexed= 100.Perio d oftreat Gonadswer e examinedhistologicall y (vande n ment= D47-105 .a = p <0.0001 . Hurk &Slof , 1981). Statistical analysiso f Treatment Dose %o %o sexratio' swa sdon ewit h thechi-squar e Ug/g test;Student' s t-test formea nbod yweight , a meangona dweigh t andmea ngonadosomati cin 11fSAdion e 6 94 6 dex (GSI)wa sused .GS Iwa s expressed in 60 100a °/oo. Methyltestosterone 0.6 99 1 High temperature shockswer egive na tD31 , 6 98 2 Control D44an dD5 7afte r fertilization,respective - 47 53 ly.Fis hwer ebrough t atth edesire dtempe ratureleve lwithi n2 0min .Subsequently , Thehighes tdos eo f llfSAdioneused ,eve nre they remained at25° C for 3ho ra t28-29° C sultsi na 100%mal epopulation .Excep tfo r for 10min .Anothe r groupo f fishwa skep t malefis htreate dwit h 6ug/ g llfSAdione, fromD4 3t oD5 7a t 22-23°C.Afte r eachexpe meantesti sweigh tan dGS Iwer e stronglyde riment,temperatur ewa sgraduall y lowered creased inandroge ntreate d trout (Table2) . again to 11°C. Treatmentwit hP4 adde d toth efoo ddi d notchang ea norma l 1:1 distributiono f Invitr o experiments males andfemales .Female s treatedwit h60 0 ug/gP 4showe da significantl ydecrease d 34teste s fromD10 0fis hwer epooled ,an d ovariumweigh t (0.63+0.36mg ;p = 0.0003 ) homogenized in 1m l 0.1M phosphatebuffe r andmea nGS I (0.36+0.20;p <0.0001 )a tD12 0 (pH7.4 )containin g 0.25Msucros ea t0°C . ascompare d tocontro lvalue s (1.41+0.42m g 0.25m lhomogenat ewa s incubatedwit h 1KB q and 1.09+0.25,respectively) .Female s llgAdione-C14 (spec,act. :2 GBq/mmol )dis treatedwit h 60ug/ gP 4showe d asignificant solved in2 4u lpropyleneglycol ,2m Meac ho f ly (p= 0.01 ) decreasedmea nGS I (0.76+ NADan dNADP Han d0.1 5m lphosphat ebuffer . 0.25).Mortalitie s amonggroup s offish ,ex Incubationswer e carried outa t20° Cunde r posed tosteroi d or controltreatments , continuous automatic shaking.Afte r 3hen varied from2-10% . zymereaction swer e terminatedb y adding Neitherhig h temperature shocksno r 2 1m l dichloromethane. 100u geac ho f 11gAdi- weekso fenhance d temperature fromD4 3t o one, 11-ketoAdione ,11-ket o testosterone D57 significantlychange d these xratio .A and llg-hydroxy testosteronewer e addedt o shocko f 25°Cfo r3 ha tD4 4an dD5 7result s theincubatio nmixtur ebefor e extraction ina tendenc y towardsmor efemale s (59%)an d withdichloromethan e (3x 10ml) . Theex males (61%),respectively .Mortalit ypercen tractwa s evaporated invacu o and theresi tagesvarie d from 10-25%.Mea ngona dweigh t duewa s subjected toTL C indichloromethane - (1.59+0.62mg ;p <0.0001 )an dmea nGS I methanol (97:3).Afte rformylatio nan dre (0.66+0.47;p < 0.005 )wer esignificantl y peatedTL C indichloromethane-methanol ,th e decreased atD15 0i nfemale skep t for2 11-ketoAdion efractio nwa sfurthe rpurifie d weeks at 22-23°Ca scompare d tocontro l by recrystallization (seeva nde nHur ke t values (3.71+1.07mg ; 1.14+0.40). al., 1982). 70 Invitr o experiments ministrationo fmethyltestosteron e (0.6an d 6ug/g )result s ina decreas eo ftesti s After incubationo fD10 0teste swit h lip weight andGSI . Sterilegonad swer eno t Adione-C14, 11-ketoAdion ewa s identied found inandroge ntreate d fish.Thi si s asth eonl y formed product.Thes eyoun g incontras t toth efinding s ofJohnston ee t testes thuscontai n llß-HSD,bu t lack 17ß- al. (1978).Althoug h theseauthor s claimed HSD tosynthesiz e 11ß-hydroxy testosterone allmal ebrood safte r treating troutfo r3 or 11-ketotestosterone . monthswit h 3Ug/ gmethyltestosterone , 17% D100ovarie sposses s ]7a,20-desmolaseac appeared tohav esteril egonads .Thi smigh t tivity,sinc e 17aP4appeare d tob econver bedu et oth elonge randroge n treatment ted intoandrostenedione .D10 0ovaries -ar e used.Okad ae tal . (1979)foun d 60,8 0an d notabl e tosynthesiz e testosteronean d 88%male s afterfeedin g troutwit h 1,5 o r oestrogens from 17aP4 10ug/ gmethyltestosteron e for 2month s from swim-up,respectively .Th edifferenc e Table 2.Effect s ofandrogen s onbod y betweenth epresen tresult san d thoseo f weight,gona dweigh t and GSI.Perio do f Okadae tal . isprobabl y duet oa differenc e treatmentD47-105 .Value s areestimate d at infoo dpreparation ,sinc ew euse dth e D150. method ofJohnston e etal . (1978)i nwhic h steroidwa s incorporated intoportion so f Compound Dose Meanbod y Meangona d MeanGS I defatted food. (tig/g)weigh t(g ) weight(mg ) (/oo ) Frompresen t andpreviou s (vande nHur ke t Females al., 1982a)i nvitr o experiments itappear s thatovarie s fromD10 0ar eunabl e tofor m llßAdione 6 2.91+0.79 0.66+0.59 0.25+0.27 oestrogens.Thi s excludesoestrogen s froma Control - 3.39+0.76 3.71+1.07 1.14+0.40 rolea sa "gyno-inducer "a ssuggeste db yYa - Males mamoto (1969).Possibl y iti sth eabsenc eo f 11ßAdione 6 4.29+1.94 1.86+1.00 0.42+0.09 enzymes tosynthesiz e steroidswit h afunc 60 3.53+0.92 0.69+0.29 0.21+0.10a tional group atth e 11-positiontha tenable s MT 0.6 3.60+1.03 0.98+0.58 0.27+0.14 a ovariumdevelopment .P 4appeare d tohav ea 6 3.60+1.56 0.78+0.70°0.20+0.11 ' feminizing effecto nth egonads ,whe nadde d Control - 2.79+0.82 1.46+0.69 0.50+0.18 toaquariu mwate r (vande nHur k &Slof , 1981).Th erelativ ehig h doseo fP 4admi a= p <0.0001 ,b = p <0.005,- c p<0.0 3 MT= methyltestosterone . nistered probably affectsgonada l sexdiffe rentiationb y ablockag eo fenzyme s involved Discussion inth esynthesi s ofandrogens .Presen tin vestigations dono t confirm afeminizin gef Frompreviou s (vande nHur k etal. ,1982a ) fecto fP 4whe nadde d toth efood .I tdoe s andpresen tobservation s itappear s thatth e not appear tob ean yreaso n tous ehighe r maindifferenc e insteroidogenesi s between doseso fP 4 infutur eexperiments ,sinc eth e justdevelopin g trout testes andovarie s highest dose tested significantlydecrease d fromD10 0i sth epotenc y of theteste st o ovariumweigh t andGSI .P 4take ni nb yth e synthesize 11ßAdionean d 11-ketoAdion e alimentary tract ispossibl ymor eeasil y through 11ß-hydroxylasean d llß-HSD (Fig. subjected todegradatio nb y thelive rtha n 1). Thisma y indicate thata t thetim e when takenu pb y thegills . theseenzyme sar epresen t troutgonad sdif Inteleosts ,temperatur ema ypla y anim ferentiate into testes.11-ket o testosterone portant role indeterminatio no fgonada l sex and 11ß-hydroxytestosteron ear eno t formed (Bruslé& Bruslé , 1982).Fro m thefe winves inD10 0testes ,whic h indicates thatthes e tigations reported sofa ri tappear s thata androgens areno t involved ingonada l sex period ofrelativel yhig h temperaturema y differentiationi ntrout .Synthesi s ofthes e increase theamoun to fmale s orfemales ,de 11-steroidsha spreviousl ybee n demonstrated pending onth especie s investigated,wherea s inteste so fon eyea rol d trout (Arai& Ta - insom especie s aperio d of lowtemperatur e maoki, 1967). enhances thepercentag eo fmales .Recently , Thepresen tobservatio n thatadministra itha sbee nfoun d thata temporar y risei n tiono f6 0ug/ g 11ßAdionefo r2 month sfro m temperaturefro m3 0 to39° Cwithi n3 ha tD 9 first feeding results ina nal lmal e trout ofdevelopmen t results ina nal lmal ebroo d population shows that this 11-steroidex - from theAfrica ncatfish ,Claria slazer a certsa masculinizin g effect ongona ddiffe (vande nHur k &Richter ,unpubl.) . Inth e rentiation.Thi sfinding ,an d results from present study,hig h temperature,eithe r invitr o studies,poin t to llßAdionea sa n givena sshock s or ina 2 week sperio ddi d endogenous gonadal androgen inrainbo w not affect these xrati o introut .Furthe r trout,whic h induces theonse t oftesti s studieshav e toh ecarrie d outt olear n differentiation.A similarmasculinizin g ef whether iti spossibl e toinfluenc e sexi n fectwa sobserve d after additiono f thear troutb ykeepin g fisha thig htemperatures . tificial androgenmethyltestosterone .Lik e thehig h dose (60ug/g )o f llßAdionegiven , but contrary toth e lowdos e (60ug/g) ,ad 71 Testesfrom D100* Ovariesfrom D100 11-KETO TESTOSTERONE 11/Î-0H TESTOSTERONE TESTOSTERONE 0ESTRADI0L-17/i Fig. 1. Steroidbiosynthesi s introu tgonad s fromD100 . Enzymeabbreviations :O H= hydroxylase ,D = desmolase ,HS D= hydrox y steroiddehy drogenase. References phological and experimental studyo fgona dal sexdifferentiatio ni nth erainbo w Arai,R . &B .Tamaoki , 1967.Biosynthesi so f trout.Cel lTiss .Res .218:487-497 . llg-hydroxytestosteron e and 11-ketotes Hurk,R .va nden ,G.A . Slof,J.G.D .Lambert , tosterone inth etesti so frainbo wtrout . H.J.Th.Goo s &P.G.W.J .va nOordt ,1982b . Can.J .Biochem .45:1191-1195 . Effectso f androgens onth edevelopmen to f Bruslé,J . &S .Bruslé , 1982.L agonadogene - thepituitary-gonada l axis injuvenil e sede spoissons .Repr .Nutr .Dev .22 :i n rainbow trout.Gen .Comp .End . 46:p .394 . press. Johnstone,R. ,T.H . Simpson &A.F .Youngson , Hurk,R .va nden ,1982 .Effect s of steroids 1978.Se xreversa l insalmoni d culture. ongonadotropi c (GTH)cell si nth epitui Aquaculture 13:115-134. taryo frainbo w trout,SaIm pgairdneri , Okada,H. ,H .Matumot o &F .Yamazaki ,1979 . shortly afterhatching . CellTiss .Res . Functionalmasculinizatio n of geneticfe 224:361-368. males inrainbo w trout.Bull .Jpn .Soc . Hurk,R .va nden ,J.G.D .Lamber t &J .Peute , Sei.Fish .45 :413-419 . (1982a). Steroidogenesis inth egonad so f Yamamoto,T. , 1969.Se xdifferentiation , rainbow trout fry,Salm ogairdneri ,befor e 117-175.In :Fis hphysiology ,Vol .III . and after theonse to fgonada l sexdiffer Acad.Press ,He wYor k &London . entiation.Repr .Nutr .Dev .22:413-425 . Hurk,R .va nde n& G.A . Slof, 1981.A mor 72 PRACTICAL APPLICATION OP SEX MANIPULATION TERJE REFSTIE Department of Animal Genetics and Breeding Agricultural University of Norway, 1432Âs-NLH ,Norwa y Abstract Practical application of sex mani further investigated before the value pulation involves,induce d maturation of triploids in commercial fish farm and ovulation, off season-spawning, ing can be evaluated. mono sex culture,an d production of sterile fish. Induced breeding To induce final maturation and ovulation purified gonadotrophins Induced breeding is essential for seem to be effective alone orto species which do not breed incapti getherwit h other hormones.Gonado vity. It involves induction of final trophins also synchronize spawning oocyte maturation and ovulation in time. females and induction of spermatoge nesis inmale s (ifnecessary) . Even Off season spawning can be induced when fish can breed in captivity and by photo or photothermal regulation, artificial incubation can be con and in salmonids spawning have been trolled, it is practiced to achieve obtained 90 days prior to and 220 better control of spawning time and days after normal spawning peak. This fry production. For some species shows the possibilities to obtain prespawning mortality can become evi fertilized eggs during most of the dent. This is spesially aproble m for year, which allow the aquaculturist salmonids during the holding period todevelo p an integrated processing when brood stocks are taken from wild marketing-program for fresh fish. stocks. Induced maturation and ovula Mono sex culture are recognised as tion can than reduce the prespawning a solution to over-population caused mortality and thereby increase the by high fecundity. Mono sex culture egg takes. (Hunter et_al. 1979). also have advantage when one of the sexes have superior production The use of hormones to induce spaw traits. Mono sex culture canb e pro ning has been widely practiced spe duced by admimistration of sexhor sially in carps.Recen t reviews in mone during the time gonads are not clude Shaudhuri (1976), Fontaine differentiated. A disadvantage with (1976) and Lam (1982). Today purified this method is that fish distined gonadotrophins are commercially for human consumption have been fed available (Syndel Laboratories Ltd) hormones. This problem can beover and its effect to induce ovulation come by sex reversing the homogame when given alone or together with tic sex and then produce mono sex other hormones are clearly shown in offspring. Crosses between related pasific salmon (Hunter et_al. 1978, Tilapia species can in some cases 1979, 1981,Donaldso n et_aIT 1981, produce all male population. Induced Donaldson et_ali 1981 and'jalabert diploid gynogenesis can produce all 1978,Atlanti c salmon (Gjerdeunpu female population. blished), rainbo w trout (Gjerdeun published) and Walleye (Lessman 1978) Sterility can be produced by sur gical castration, autoimmune castra Hormone injections also synchronize tion, administration of high doses ovulationi nlarg e groups of animals of sex steroids,hybridizatio n bet which often are important when carry ween species and chromosomal mani ing out a selection program. pulation. Disadvantages with hormone injec Production of triploid fish by tion is extra laboure and stress chromosomal manipulation appear to caused by handling of fish.Th e be a suitable technique inman y commercially produced gonadotrophins species. However, the production is also relatively expensive,an di t traits in triploids have to be seems appropriated to look for 73 cheaper methods to induce ovulation nized as the best solution to over and spawning. population caused by high fecundity, which is amai n problem in Tilapia Off season spawning under almost.any pond condition (Hickling, 1963). Mono sex culture Inman y species off-season produc also have advantages when,on e of the tion of fertilized eggs would prove sexes have abette r growth rate or beneficial to aquaculturist and a higher marketing value. fisheries research. When producing In fish,a s in other animals,se x pen-size fish schedual spawning would is genetically determinded,however , allow the fish farmer to develope an many species are not clearly differen integrated processing marketing pro tiated into either sex at hatching. gram for fresh fish.Fo r research Sex reverse can be induced by admini purpose, off-season production of stration of sex hormone at early fertilized eggs would give fish of stage of life.Thi s has been reported standard weight or age several times for rainbow trout,Atlanti c salmon, during the year. Coho salmon,Tilapi a and goldfish Off season-spawning can be induced (forrevie w see Yamamoto, 1969, inman y species by regulation of Schreck, 1974,Lam , 1982,Donaldso n photoperiod, photothermal treatment and Hunter, 1982). A disadvantage of or photothermal treatment together technique istha t all fish require with hormon administration. In sal- treatment and fish destined forhu monids regulation of photoperiod man consumption will have been fed have shown to be effective for brood hormons. When females are.homogameti c trout(Allison , 1951,Hazar d and Eddy, sex this problem can be overcome by 1951, Corson, 1955.Henderson , 1963, producing fenotypic males from gene Carlson and Hale, 1973), coho salmon tic females and mate them with nor (MacQuarrie et_al. 1978) pink salmon mal females.Thi s will give all fe (MacQuarrie et_al. 1979) and rainbow male population without hormone treat trout (Whitehead et_ali 1978). ment of the slaughtering fishes.Th e In coho salmon spawning has been problem might be to distinguish bet obtained asmuc h as 90 days prior to ween sex reversed genetic females normal spawning (MacQuarrie et_al. (fenotypic males) and normalmales . 1978), and inpin k salmon spawning In Tilapia mono sex male culture has been obtained 220 days after nor have been produced by crossing T. mal spawning (MacQuarrie el_al. 1979). nilotica,T . aurea. (Pruginin et_al. This shows the possibilities to 1975). obtain fertilized eggs during most of Artificial gynogenesis have been the year in salmonids simply by regu induced inman y species (Purdom, lation of photoperiod. However, the 1969, Stanly, 1976,Chourrout ,1980 , broodstock have tob e kept in build Refstie et al_. 1982) and will pro ingswer e normal light can be ex duce mono sex female populations cluded for one year minimum. when the female are the homogametic Photothermal treatment haveproduce d sex. However, the gynogenetic fish precociously gravid catfish as early are higly inbreed, and will probably as April,an d spawning was induced by suffer from significant inbreeding administration of luteinizing hormone depression (Aulstad and Kittelsen, (LH), (Sundaraj,-andVasa l 1976). The 1981, Kincaid, 1976). normal spawning season was July- August. The process was repeated and Sterile fish the same set of fish spawned four times between April and July the same Sterile fish have many advantages year and gave 2-6 times the number in fish farming. Problems with over of eggs contained from untreated population caused by high fecundity spawners. Thismean s that higher and loss of growth and quality dur numbers of eggs can be colledted from ingmaturatio n and spawning, can be selected females,an d will improve avoided. For salmonidsmaturit y also the selection intensity in a breeding set an upper limit for size and scheme. slaughtering time. Sterile fish can be produced by Mono sex culture irradiation,chemosterilization ,sur gical techniques, immunological tech- Mono sex culture has been recog 74 niques, hormone administration,hy Channel Catfish (Walters et_al. 1981) bridization and chromosome manipula and Carp (Ojima and Makino, 1978), tion (for review see Stanly, 1978, and today techniques for production andDonaldso n and Hunter, 1982). of high numbers of triploid salmonids Irradiation claim placement of by heat shock are available. fish inwate r containing an isotope An alternaltive method to heat or injection of fish with an isotope shock for production of triploid sal and would not be acceptable unless monids is productions of a tetraploid the isotope had aver y short half- strain (Thorgaard et_ali 1981, life and could be used under con Refstie, 1981,Standl y and Allen, trolled conditions. Chemosteriliza- 1979). Tetraploid fishes are ex tion also posess problems when the pected to be fertile and will give fish are used for human consumption triploid fish when back crossed to and the effects are often temporary. diploids. These methods aretherefor e not like Lincoln (1981) reports that fully ly to be practical procedures in motile spermatoza were produced from fish farming. several triploid plaice. Thorgaard Surgical castration is labor inten and Gall (1979) report that six tri sive and costly, and the fish can not ploid rainbow trout found in a full- be operated on until the gonads are sib family were normal in size and of sufficient size.Thi s ruling out external appearance,an d haveunde its use inocea n ranching, and it is veloped gonads. probably also too labor intensive More research is needed to study for use inpractica l fish farming. the degree of sterility, and produc Little work has been conducted tion traits of triploids have to be with immunological techniques in investigated before their value in fish, and although some promising fish farming can be evaluated. How results are reported (Laird et_al. ever,th e results so'fa rar epromis 1978, 1981), more research is needed ing and production of sterile fish before this technique can be used in by chromosome manipulation have not practical fish farming. the disadvantages as administration Hybridization between species often of sex hormones have,whe n the fish results in sterility in fish (Buss are destined for human consumption. and Wright, 1957,Suzuki ,1974 , MacCrimmon et_al. 1974). Hybrids bet ween salmonid species ordinary have References low hatchability (for review see Allison,L.N . 1951.Dela y of spawning Chevassus, 1979). Refstie (1981) in eastern brook trout bymean s of reared hybrids between four salmonid artificial prolonged light inter species together with their parent vals. Prog. Fish-Cult.,13 : species in seawater and concluded 111-116. that this hybrids was of no advan Aulstad,D , and Kittelsen, A.,1971. tage in fish farming in seawater.To Abnormal body curvatures of rain day it does not look as if production bow trout (Salmo gairdneri)inbred of sterile species hybrids have any fry.J.Fis h Res.Bd . Canada 28: advantages inpractica l fish farming. 1918-1920. Administration of relatively high Buss, K.W. and Wright,J.E . Jr.,1957. doses of sex hormones at early stages Appearance and fertility of trout of life,hav e produced sterile fish hybrids. Trans.Am . Fish Soc. 87: inman y species and can easely be 172-181. applied to fish farming conditions. Carlsson,A.R . and Hale,J.G . 1973. However,whe n fish are destined for Early maturation of brook trout human consumptions,thi smetho d has in the laboratory. Prog. Fish-Cult. the same disadvantages as using sex 35: 150-153. hormones to induce sex reverse. Chaudhuri, H., 1976.Us e of hormones Triploid fishes may be totally or in induced spawning of carps. partially sterile. Tripoloidy have J. Fish.Res. Bd. Canada 33:940-947. been induced by heat shock in flat Chevassus, B. 1979.Hybridizatio n in fish (Purdom, 1972) Salmonids salmonids: results and perspec (Chorrout, 1980,Thorgar d and Jazwin, tives. Aquacultur e 17:113-128 . 1981, Holmefjord et_ali 1982), Chourrout. D., 1980.Therma l induction of diploid gynogenesis and triplo- idy inth e eggs of the rainbow trout (Salmo gairdneri Richardson). Reprod. Nutr. Dev. 20:727-733. 75 Corson, B.W., 1955. Four years pro (Oncorhynchus kisutch). J.Fish gress in the use of artificially Res. Bd. Canada 35:1423-1429 . controlled light to induce early Kincaid, H.L., 1976. Inbreeding in spawning of brook trout. Prog. rainbow trout (Salmo gairdneri) Fish-Cult. 17:99-102. J. Fish. Res.Bd . Canada 33: 2420- Donaldson, E.M., Hunter, G.A. and 2426. Dye,H.M. , 1981.Induce d ovula Laird,L.M ., Ellis;'M.A. ,Wilson , tion in coho salmon (Oncorhynchus A.R. and Holliday,F.G.T. , 1978. kisutch). II.Preliminar y study Thedevelopmen t of gonadal salmon of the use of LH-RH and two high (Salmo salar L.) and a consider potency LH-RH analogues.Aquacul ation of thepossibilit y of in ture 26:129-141. ducing autoimmune destruction of Donaldson,E.M . and Hunter, G.A., thetesis .Ann .Biol .Anim .Bio - 1982. Sex control in fish with chem. Biophys. 18:1101-1106. particular reference to salmonids. Laird,L.M. , Wilson,A.R . and Can. J.FishAquat . Sei. 39:99- Holliday,F.G.T. , 1981.Fiel d 110. trials of ametho d of induction Fontaine,M. , 1976.Hormone s and the of autoimmune gonad rejection in control of reproduction in aqua- Atlantic salmon (Salmon salar L.) culture. J.Fish Res.Bd . Canada Reprod. Nutr. Dev. 20:1781-1 788 . 33:922.939. Lam,T.J., 1982.Application s of endo Hazard, T.P. and Eddy,R.E. ,1951 . crinology to fish culture. Can. J. Modification of the sexual cycle Fish Aquat. Sei. 39:111-137 . in brook trout (Salvelinus fonti- Lessman,C.A.,1978 . Effects of gonado nalis)b y control of light.Trans . tropin mixtures and two steroids Am. Fish Soc. 80:158-162. on inducing ovulation in the Henderson,N.E. ,1963 . Influence of Walleye. Prog. Fish-Cult.40:3-5 . light and temperature on there Lincoln,R.F. , 1981.Sexua lmatura productive cycle of the eastern tion in triploid male plaice brook trout,(Salvelinus fontinal- (Pleuronectes platessa) and plaice is) (Mitchill). J.Fish Res.Bd . x flounder (Platichthys flesus) Canada 20:859-897 . hybrids. J. Fish. Biol. 19:415- Hickling,CF. , 1963.Th e cultiva 426. tion of Tilapia. Sei. Am.208 : MacCrimmon,H.R. , Stewart,J.E. and 143-152. Brett,J.R . 1974.Aquacultur e in Holmefjord, J., Refstie,T . and Canada. The practice and promise. H0stmark,J. , 1982. Preliminary Bull. Fish.Res .Bd . Canada 188: results of induction of triploidy 1-84. in salmonids using heat shocks. MacQuarrie,D.W. , Markert,J.R . and 33rd Annual Meeting of the EAAP, Vanstone,W.E. ,1978 . Photoperiod Commission of Animal Genetics, induced off-season spawning of Leningrad 16-19 August 1982. coho salmon (Oncorhynchus kisutch). Hunter,G.A. , Donaldson, E.M., Ann. Biol.Anim . Biochem.Biophys . Stone, E.T. and Dye,H.M. , 1978. 18: 1051-1058. Induced ovulation of femalechin MacQuarrie,D.W. , Vanstone,W.E .an d ook salmon (Oncorhynchus tshawyt- Markert,J.R. , 1979. Photoperiod scha) at aproductio n hatchery. induced off-season spawning of Aquaculture 15:99-112 . pink salmon (Oncorhynchus gorbuscha) Hunter, G.A., Donaldson,E.M. , Dyé, Aquaculture 18:289-302 . H.M. and Peterson, K., 1979. Oijma, Y. and Makino,S . 1978. Triplo A preliminary study of induced idy induced by cold shock inferti ovulation in coho salmon (Oncor lized eggs of the carp. Proc. Japan hynchus kisutch) at Robertson Acad. 54.Ser .B :359-362 . Creek Salmon Hatchery. FishMar . Pruginin,Y . Rothbard,S . Wohlfarth, Serv.,Technica l Report 899; G., Halevy,A. ,Moav ,R . and Hulata, 15p . G., 1975 .All-mal e broods of Tilapia Hunter, G.A., Donaldson, E.M. and nilotica xT . aurea hybrids.Aqua - Dye, H.M., 1981.Induce d ovula culture 6:11-2 1. tion in coho salmon (Oncorhynchus Purdom,C.E . 1969. Radiation -induce d kisutch). I. Further studies on gynogenesis in fish. Heredity, the useo f salmopituitar y prepa London 24:431-444 . rations. Aquaculture 26: 117-127. Purdom,C.E . 1972.Induce d pöliploidy Jalabert,B. ,Goetz ,F.W. , Bretan,B . in plaice (Pleuronectes platessa) Foster,A . and Donaldson, E.M., and its hybrid with the flounder 1978. Precocious induction of (Platichthys flesus). Heredity, oocyte maturation on coho salmon London 29:11-24. 76 Refstie,T . 1981.Tetraploi d rainbow Yamamoto,T. , 1969. Sexdifferenti trout produced by cytochalasin B. ation. InW.S .Hoa r and D.J. Aquaculture 25:51.58. Randall (eds.). Fish Physiology, Restie, T., Stoss,J . and Donaldson, Vol 3: 117-175. Academic Press, E.M., 1982.Productio n ofall - New York,N.Y . female coho aalmon (Oncorhyn- chus kisutch) by diploid gyno- genesis using irradiated sperm and cold shock. Aquaculture (in press). Shreck,C.B. , 1974.Hormona l treat ment and sexmanipulatio n in fishes. P. 84-106. In C.B. Schreck (ed.)Contro l of sex in fishes. Extension Division,Virgini a Polytechnic Inst, and State Univ. Blacksburg,V.A . Stanley, J.G. 1976. Production of hybrid, androgenetic and gynogene- tic grass carp and carp.Trans . Am. Fish Soc. 105: 10-16. Stanley, J.G., 1978.Contro l of sex in fishes with special references to checking reproduction in grass carp. Proceedings of the Inter national Grass Carp Conference, Gainesville,Florida , January 9-12, 1978:4 2pp . Stanley, J.G. and Allen,S.K . jr. 1979. Polyploid mosaics induced by cytochalasin B in landlocked Atlantic salmon (Salmo salar). Trans. Am. Fish. Soc.108 : 462-466. Sundararaj, B.I.an d Vasal,S. , 1976. Photoperiod and temperature con trol inth e regulation of repro duction in the female catfish, (Heteropneustes fosilis)J.Fis h Res. Bd. Canada, 33:959-973 . Suzuki, R. 1974. Inter-crossings and backcrossing of F-|hybrid s among salmonid fishes.Bull . Freshw..-.Fish . Res.Lab .24:11-32 . Thorgaard, G.H. and Gall,G.A.E . 1979. Adult triploids in arain bow trout family. Genetics 93: 961-973. Thorgaard, G.H., Jazwin,M.E . and Stier,A.R. , 1981. Polyploidy induced by heat shock in rainbow trout. Trans.Am . Fish Soc. 110: 546-550. Walters,W.R. , Libey, G.S. and Chrisman,C.L. , 1981. Induction of triploidy in channel catfish. Trans. Am. Fish Soc. 110-312. Whitehead, C., Bromage, N.R. and Forster,J.R.M. , 1978. Seasonal changes in reproductive function of the rainbow trout (Salmo gairdneri). J. Fish Biol.12:601- 608. 77 THEOCEA N RELEASEAN D CONTRIBUTION TOTH EFISHER Y OFALL-FEMAL E AND STERILE GROUPS OFCOH O SALMON(ONCORHYNCHU SKISUTCH ) EdwardM .Donaldson ,Georg eA .Hunte r WestVancouve r Laboratory,Fisherie s ResearchBranch ,Dept . ofFisherie s andOceans ,416 0 MarineDrive ,Wes tVancouver ,B.C .V7 V 1N6 Canada Introduction contributed toth ecommercia l fisheriesa t Theapplicatio n of sexcontro l similar rates,however ,th esteril e group techniques has thepotentia l tomaximiz e contributed ata lowe r rate toth e economic returns from the searanchin go f recreationalfishery . Inth e fallo f 1981, Pacific salmon. Theproductio no f theall-femal e group returned toth e all-female groupswoul d increase the hatchery asexpected . Returning females landed value of thecatc h bymaximizin g from thetreate d groupwer esimila ri nsiz e theproportio n offis hcontainin groe . and gonadosomaticinde x tountreate d Sterilization provides amean so f females. No sterile fishreturne d toth e inhibiting thenorma l propensity for hatchery,however ,a smal lnumbe r offis h anadromousmigration ,thereb y increasing which contained maturing single orpartia l theaverag e size ofth efis h inth ecatc h testes didreturn . and extending theperio d that thefis har e available toth efishery .Bot h sexcontro l Discussion techniques eliminateprecociou smales . Theresult s indicate thati ti sfeasibl e This study presents the results fromth e ona production basis to produce essentially firstocea n release of sterilean d 100%femal e and sterile groupso fcoh o all-female groups ofsalmon . salmonb y steroid treatmenti nth eearl y life stages. Further,fis h treated inthi s Materialsan dMethod s manner maintain their altered gonadal Twogroup s ofapproximatel y40,00 0coh o phenotypes. Thesomewha t slowergrowt hsee n salmonwer e treated witheithe r insteril e fishma y becompensate d forb y estradiolo rmethyltestosteron ea seye d continued growtffan dmaintenanc e ofhig h eggs,alevin s and frya t the Capilano fleshqualit y inth esteril e fish duringan d SalmonHatchery . Treatment ofth eeye d beyond theperio do fnorma lsexua l eggswa s conducted byimmersio n inwate r maturation. containing thesteroid s at20 0yg/1 . Two The tag datashow s thatbot hgroup s treatments of2 h r durationwer e applied7 survived afterreleas e intoth eocea nan d daysapart . Alevins were treated contributed significantly to thefishery . similarily. Frywer e fed for thefirs t 90 Interpretation ofth e differential days adie t containing either 5mg/k g contribution toth erecreationa l fishery is estradiolo r1 0mg/k gmethyltestosterone . notpossibl ebase do n present data. The Both groupswer enos e tagged and fin return toth ehatcher y of substantial clipped at 6month s ofage . Before numbers ofphenotypicall ynormal , release,a sampl e of 1,000 from each group exclusively female fish from theestradio l were transferred toa seawater net pent o group demonstrates the feasibility ofthi s monitor gonadalmorphology , survivalan d treatment forenhancin g theproportio no f growth. Contribution toth efisherie swa s femalesi nth ecommercia l catch. To determined fromnos eta g recoveries andt o producea nall-femal e group suitable for thehatcher y byfi nclips . broodstock purposesi twoul d benecessar yt o use thealternat e techniques described by Results Donaldsonan dHunte r (1982). Perhapsth e The estrogenan d androgen treated groups most significant biological finding from contained> 97%femal ean d sterile fish, this studyi sth e demonstration that respectively. Bothgroup s survived at sterilizationeliminate s thepropensit y of similar ratesunti lmaturatio n ofth e thecoh o salmont ounderg oit sanadromou s estradiolgroup . Sterile fishgre wa ta migration. Theevaluatio no f thetota l slower rate thanfemal e fish butunlik e contribution ofth esteril efis h toth e thefemales ,continue d togro w through the fishery willawai t tag returns insubsequen t period of sexualmaturation . Asa result , years. at femalematurit y thecarcas sweight s were similar inth e twogroups . Reference Furthermore,th esteril efis hdi dno t Donaldson,E.M .an dG.A . Hunter,1982 . Sex display thefles h deterioration associated controli nfis hwit h particular reference with sexualmaturation . to salmonids. W.S.Hoa rSymposium ,Fis h To date,a totalo f 1007tag shav e been Reproduction:Behavio ran dEndocrinology . recovered from thefishery . Bothgroup s Can.J .Fish .Aquat .Sei. ,39 :99-110 . 78 THEPRODUCTIO NO FGENETICALL YALL-FEMAL EATLANTI C SALMONSTOCK S Johnstone,R. ,an dA.F .Youngso nDept .o fAgricultur e &Fisherie s forScotland , MarineLaboratory ,Aberdee n Theattainmen t of sexualmaturit y isa majo r or precociously mature (20%) individuals constraint on Atlantic salmon cultivation. (characterised by theirdarke r colouration, Inth e freshwate r rearingphase ,precociou s more obviouspar rmark s and distended genital sexualmaturit y inmale s entering theirsec papillae).Matur e fishwer e furthercategor ondwinte r isassociate d with increased dis ised as thosewhic h gavemil tb ymanua lex ease andmortalities .I nth ese awate rrear pression (14%)o r as those whichfaile dt o ing phase,maturit y ofadul t fishmark sth e dos o (6%).Upo n sacrificean dvisua linspec end ofth eperio d inwhic hthe yma yb euseful tiono fth egonads ,th elatte rgrou pwa s seen ly reared and often occurs atbod yweight s to containanimal swit ha rang eo ftesticula r which are undesirably or unacceptably low. typesvaryin g fromlobe dteste s lackingduct s Insalmonid s generally,mal e fishbecom emat tomor enorma lteste sblocke d atth egenita l ure at ayounge r age thand ofemales .A sa papilla. consequence ofthis ,advantage smigh tb eex pected to follow from growing only female Lobed testes from five individualanimal s stocks. were dissected out,choppe d and placed in modified Cortland extender (Ihra t10°C )an d Bytreatin g fishshortl y afterfirs tfeed theresultin gmil tsolution swer euse dsepar ingwit hdietar y 17ß-oestradiol,i tha sprov atelyt ofertilis e theov a fromsevera lnor ed possible toover-rid e thenorma lexpres mal salmon females.Fertilisatio n rateswer e sion of genetic sex in genotypically male excellent and subsequent survivals to the fish inbot h salmonan d rainbowtrou t(John eyed-egg stagewer e good/(>95%).Firs tfeed stonee t al., 1978). Groupso f fishsuccess ing mortalities were satisfactory (<5%)i n fullytreate d inthi swa yprov e tob eentire threeou to fth e fivegroups ,bu twer ehighe r lyfemale ,wit happarentl ynorma l gonads,an d (20%an d41% )i nth etw oothe rgroups . have thedeferre d maturation characteristics ofth e female type (Johnstonee t al.,unpub After eight weekso f feeding (July 1982), lished results). thegonad so f5 0fis hi neac heg gbatc hwer e inspected microscopically (x25 ) following Oestrogen treatment is not always fully fixation inBouin s fluid.Al lanimal sexamin effective however, andnorma lmal e fishan d ed were female. The sex ratio ina contro l fishwit hvariabl ehermaphroditi c gonadsma y group of 42 fish was 1.1 °*: i.oç .Thes e occur.Suc hanimal s tend to retainth etempor results demonstrate that,a si nrainbo wtrout , al pattern ofsexua ldevelopmen t typicalo f theAtlanti c salmon female ishomozygou s for males (Johnstone etal. ,unpublishe d results). sex(XX) . Analternativ emetho d ofproducin gall-fe The performance ofth eall-femal eprogen y male stocks exists.Th enorma l geneticexpres ofprecociousl ymatur emale swil lb e compared, sion of sex in female rainbowtrou tma yb e infutur eyears ,wit hth eperformanc e ofall - overridden by exposure to dietary androgen females generated from sex-reversed malepar resulting inth e fishhavin g gonads inwhic h entsmaturin g relatively lateri nth erearin g testicular elements predominate.I ntreate d cycle. mixed-sex stocks, genetically female fish canb e identified atmaturit yb ythei rherma phroditism orb y theabsenc e ofpaten tsper m ducts. The genetic constitution of rainbow References trout is suchtha twhe n crossed withnorma l female fish, they yield all-female progeny Johnstone,R . et al., 1978.Aquaculture ,13 , (Johnstone, et al.,1979a ;By ean dLincoln , 115-134. 1981).Thi s techniqueha sno wbee nvalidate d Johnstone, R. et al., 1979a.Aquaculture , forAtlanti c salmon. 18, 13-19. Bye,V .& Lincoln ,R . 1981.Fis hFarme r4(8) , InMa y 1979a grou po fAtlanti c salmonfr y 22-24. were fed adie t containing 17ot-methyl-testos- terone at3 mg/k g diet forth efirs t6 0day s offeeding .Thereafte r theywer e fednormally . Aton eyea r ofage ,a proportio n ofth estoc k (10%)becam e smolts andwer e transferred to sea water. Asth e remaining fishapproache d theirsecon d winter infres hwate r (November 1981), theywer e classified asimmatur e (80%) 79 MONOCLONAL ANTIBODIES INTH ESTUD Y OFGONADA L DIFFERENTIATION INTH ECAR P (CYPRINUS carpio L.] H.K. Parmentier,L.P.M .Timmermans ,E .Egbert s andJ.J.M .va nGroninge n Department ofExperimenta l Animal Morphology andCel l Biology,Agricultura l University, Wageningen,Th eNetherland s Summary Table 1. Characteristics of the monoclonal antibodies specific for carp spermatozoa*) Monoclonal antibodies (MA) have been raised against spermatozoa of carp, in order to study MONO ELISA IMMUNOFLUORESCENCE HEAVY AGGLU COMPLEMENT the appearance of specific antigens during CLONAL on smears frozen CHAIN TINATION CYTOTOXICITY gonado- and spermatogenesis in fish. After ANTI SPERM of sections TYPE TITER LYSIS TITER characterizing the antisera obtained, using BODIES SPERM TESTIS 3) •) 1) immunohistochemical and immunological methods, WCS 1 0"' Y 6 4 eleven MA appeared to define cell membrane 2a determinants specific for spermatogenic cells. WCS 3 0 *3 8 4 These MA are expected to form a handsome tool WCS 7 0 V 7 4 WCS 11 in the study of cytodifferentiation during 0 y'Y2a 8 4 spermatogenesis in fish, and might be used WCS 12 0 M2a 6 4 to induce male sterility in carp. WCS1 4 0 Y2b 6 4 WCS 16 - - 6 4 Introduction WCS1 7 0 V 5 2 WCS2 7 0 v,y2b 5 5 Studies on mammalian spermatogenic cells WCS2 8 0 Y Y 11 3 have shown that the membranes of spermatozoa, 3' 2a WCS2 9 0 V 11 3 spermatids and spermatocytes contain deter minants normally not present on somatic cells, 1)befor e subcloningo fth ehybridoma s whereas spermatogonia do not (Millette, 1979). 2) precursor cells 3) determined with anti-mouse Ig heavy chain antisera in an In these studies it was concluded that the Ouchterlony double diffusion test blood testis barrier formed by Sertoli cells 4) 2-log dilution provides an environment in which specific spermatogenic antigens may appear. These Eleven MA reacted with spermatozoa, and antigens are probably involved in cellular except for MA WCS 16, also with spermatogenic interactions and the process of cytodiffer cells. However, none of the MA reacted with entiation in the mammalian testis. somatic tissue, i.e. gut, liver, brain, spleen MA derived from individual hybrid cell lines and kidney as determined on frozen sections. provide highly specific reagents, which can All MA belong to the mouse IgG- or IgM- be used in the study of the membrane composi class, and are able to agglutinate and, in tion of individual cell types. Recently, the presence of complement, lyse mature sperm Bechtol et al. (1979) and Gaunt (1982) suc cells. This latter property suggests that the ceeded in defining differentiation antigens MA might be used to kill spermatogenic cells of spermatogenesis in the mouse with MA. of carp in vivo, and so to induce male In fish, little is known about the process sterility. of cytodifferentiation in the testis. However, it is likely that, here too, specific sperma- References togenic antigens are present, because Sertoli cells have been demonstrated in several spe Bechtol, K.B., S.C. Brown & P.H.. Kennett, 1979. cies (Grier, 1981). Recognition of differentiation antigens of To obtain evidence of such antigens, MA spermatogenesis in the mouse by using anti have been raised against mature spermatozoa bodies from spleen cell-myeloma hybrids of carp. after syngeneic immunization. Proc. Natl. Acad. Sei. USA 76: 363-367. Results and conclusions Gaunt, S.J., 1982. A 28-K Dalton cell surfaca auto antigen of spermatogenesis: characteri Hybridomas were obtained from a fusion be zation using a monoclonal antibody. Dev. tween cells of the mouse myeloma NS1 and Biol. 89: 92-101. splenocytes from Balb/c mice hyperimmunized Grier, H.J., 1981. Cellular organisation of with carp spermatozoa. The MA obtained were the testis and spermatogenesis in fishes. characterized in several tests as shown in Amer. Zool. 21: 345-357. table 1. Millette, CF., 1979. Cell surface antigens during mammalian spermatogenesis. Curr. Top. Dev. Biol. 13: 1-29. 80 FISHSTERILISATION :TH EAUTOIMMUN EAPPROAC H C.J. Secombes,L.M . Laird and1.6 .Pried e Department ofZoology ,Universit y ofAberdeen , Scotland U.K. Recent work at Aberdeen University has showntha ti ti spossibl e toinduc e anauto immune reaction inAtlanti c salmon (Salmo salar)leadin g todegeneratio n ofeithe rtes tiso rovary .Thi s ispropose d asa possibl e method forsterilisin g largebatche s offis h onfarms .Th eprocesse s involved inthi s res ponsear eno wbein g investigated usingrain bowtrou t (Salmogairdneri )an dAtlanti c sal mona slaborator ymodels .Clearl y acellula r response isinvolved ,a ssee ni nhistologica l studies (Laird et al., 1978, 1980),an di t isimportan t tokno w ifa humora l (antibody) response also occurs.T othi sen d spermag Fig. 1. Sperm agglutination seen afterin glutinationtest shav ebee nperforme d inth e cubationwit hseru m froma trou timmunise d present study on serum from gonad injected 42day spreviousl ywit htesti splu sFCA . trout. Bar= 2 0|jm . Methods This detectiono fsper m agglutinins isth e Rainbow trout,weighin g 35-40g ,wer ein firstobservatio n ofpossibl e auto-antibodies jected intraperitoneallywit hallogenei cgon in fish. Agglutination occurred along the ad material (testis or ovary plus testis) whole length ofth esper m (Fig. 1).Indica emulsified in Freund's complete adjuvant tions that ahumora l immune response isin (FCA). Each fish received 0.1 ml of a50 % volved come frompreliminar y immunofluores gonad solutionmixe dwit ha nequa lvolum eo f cence studies using rabbit anti- troutim - adjuvant.Fis hwer e kepta tth eambien ttem munoglubulin antiserum,showin g theobserve d perature, 12-17°C,durin gth ecours eo fth e sperm agglutinins tob e immunoglobulin sperm experiment. Blood samples were taken 4,6 agglutinins. The detectiono fsper magglut and 8 weeks after immunisation. Sera were inins in theser a fromsom eo fth e fishin decomplemented at 50°C for 30min . before jected only FCA isi nagreemen twit hprevi use. Sera were serially diluted inBaker' s oushistologica l studieswher ea noccasiona l buffer pH8. 1 (166m M glucose, 34m M NaCl, fish was found to have asurfac e granuloma 17m MN aHP0 , and 1m MK HP0, )i nmicrotitr e onth e gonad.I tno w remains tob eelucidate d plates.T oeac hwel lwa « added5 0u lo ffres h ifth e reason forthi s response isth erout e trout spermatozoa,adjuste d to4 x 107cell / of injection,possibl yallowin gFC At ocom e ml Baker's buffer. After 3 hours a sample into contactwit hth eanimal s owngonad . was taken from each well and examined for sperm agglutination. Amajo rprogramm e isno wunderwa y toeval uate this technique as a tool forth efis h Results and discussion farming industry. Titres of 1:32 and 1:64 were reached as early as day 28i nth emixe d gonad injected References group,an d themea ntitre s (±standar d devi ation) were 3.63 ± 1.92, 0.75 ± 0.50 and Laird,L.M. ,A.E .Ellis ,A.R .Wilso n& 3.25 ± 1.89 onday s28 ,4 2an d5 6respective F.G.T.Hollida y (1978).Th edevelopmen to f ly. The meantitre s forth etesti s injected the gonadal and immune systems inth eAt groupwer e 2.25 ± 1.16,2.2 5 ± 1.26 and 2.25 lantic salmon (Salmo salarL. )an d acon ± 1.50.Abou t 50%o fth e controlFC A injected sideration of the possiblity of inducing fish responded, compared to almost 100%o f autoimmune destruction ofth etestis .Ann . gonad injected fish.Mea n titreswer e 1.50± Biol. anim.Bioch .Biophys . 18,1101-1106 . 1.31 on day2 8an d 3.00 +3.0 0 onda y56 .A Laird, L.M., A.R. Wilson & F.G.T.Hollida y phosphate buffer injected groupha dsignif (1980).Fiel d trialso fa metho d ofinduc icantly lower aglutination titres than all tion of autoimmune gonad rejection inAt othergroup s atal ltimes . lantic salmon (Salmo salar L.). Reprod. Nutr.Develop .20 ,1781-1788 . MONOSEX GRASSCAR PPRODUCTIO NTHROUG HBREEDIN G SEX-REVERSED BROODSTOCK WilliamL . Shelton Alabama CooperativeFisher y ResearchUnit ,Aubur nUniversity ,Alabama ,US A Summary Monosexgras s carpprovid ea viabl e During 1981,1 2mor e sex-reversedmale s alternative aquaticplan t control. Usinga fromth e 1978an d 1979yea r classesha d monosexpopulatio n isno t an infallible motile spermatozoa,an d severalwer euse d meanso fpreventin g reproduction asindiv ina secon d spawning test. Thisyear , iduals arefertile ,bu t itca nb euse di n 650,000egg swer e fertilizedwit hmil tfro m areaswher egras s carphav eno tbee n sex-reversed males. A totalo fabou t stocked. Further,thi sprotoco l shouldb e 250,000fr ywer estocke d inpond s forlate r applicable toothe r exotic specieswit h examination. similar reproductivebiology . Fish from the 1980an d 1981yea r classes Monosex grass carp canb eobtaine d byin (presumed all-female)wer e treatedwit hM T duced gynogenetic fertilization (Stanley implants;late r theywil lb eexamine d as 1976). Femalegras s carpar epresume d to potentialbroodstock . Sex-reversed males be homogametic forse xdeterminatio n(XX) ; mayb eavailabl e from the 1980yea r classi n therefore,al lgynogeneti c grasscar p 1982. shouldb e females. But theyiel d ofviabl e Themechanis m ofse xdeterminatio n in offspring isinsufficien t tob epractical . fishes isunresolved ,thu si ti spossibl e Todevelo p aprogra m ofmonosexin g that that somemale sma yb eproduce d inthi s couldmee t largedemands ,w ehav estudie d breeding program. With thiscaveat ,i fn o functionalse xreversa lwit hmethyltestos - malesar efoun d inth epresume d all-female terone (MT). The sex-reversed fishar e populations',applicatio n of theapproac h can phenotypicmale sbu t genotypic females (XX) be considered. Relatively fewmale sar e andwhe nbre d tonorma lfemale s (XX),shoul d required asth eful lreproductiv e potential produceonl yXX-progeny . ofnorma l females isused . Wehav edescribe d thegonada ldifferentia tion forgras scar pan dhav e developed an References extended release capsule (Shelton& Jense n 1979). This capsulewa s implanted inth e Shelton,W .L. , 1982. Production ofrepro - body cavity of grass carp ofappropriat e ductively limited grass carpfo rbiologica l sizean d thetreate d fishwer e thenstocke d control ofaquati cweeds — Phase II.Off . inpond sdurin g theperio d ofhormon e WaterRes .& Tech. ,USDI ,Wate rResour . release. Only female (XX)wer e treated,s o Res. Inst.Bull . 45,Aubur nUniv . atmaturity ,an ymale s inth epopulatio n would beconsidere d tohav ebee nfunc Shelton,W .1 , &G .L .Jensen ,1979 . Produc tionally sexreversed . tiono freproductivel y limited grasscar p Twosize so fimplan t (5& 1 2mg )hav e forbiologica l controlo faquati cweeds . been tested,bot hwer eeffectiv ebu tth e Off. WaterRes .& Tech. ,USDI ,Wate r smaller onepermitte d treating fishearlie r Resour.Res .Inst .Bull . 39,Aubur nUniv . ingonada ldifferentiation . Wehav eha d thegreates t successusin g 80-110mm , 10,t o Stanley,J . G.,1976. Production ofhybrid , 16-week-old fish,the nrapidl y growing them androgenic,an d gynogenetic grasscar p toabou t 180-200mm . and carp.Trans .Am .Fish .Soc .105:10-16 . Fishwer e treatedb y thismetho d in1977 , 1978, and 1979. During 1980,th efirs tsex - Stanley,J . G.& A .E .Thomas ,1978 . Absence reversedmal emature d andwa suse d inth e of sexreversa l inunise xgras s carpfe d initial spawning test (Shelton 1982). methyltestosterone. In:R .0 .Smitherman , About 160,000egg swer e fertilizedwit h W.L . Shelton,& J .H . Grover (Eds): milt from thesex-reverse dmal e (XX-o ) ; Culture ofexoti cfishe s symposium 380,000egg s from thesam efemal ewer e proceedings.Fis h Cult.Sect. ,Am .Fish . fertilizedwit hmil t froma norma lmal e Soc, AuburnAL .Page s194-199 . (XY). Therewa sn odifferenc ei ndevelop mento f the twogroups ;abou t 85,000fr y hatched inth eexperimenta l group;o fabou t 500tha thav ebee n sexed;al lwer efemales . Anadditiona l 8,000-10,000 presumed mono sexfis har ebein ggrown . 82 PHEROMONES/GONADAL STEROIDS PHEROMONES INTELEOS TFIS H L.Colombo ,P .C .Belvedere ,A .Plarconat oan d F.Bentivegn a Institute ofAnima l Biology, University ofPadua ,Padua ,an d ZoologicalStation ,Naples ,Ital y Summary terproximit y ordirec t contact isneede d for bioelectric (Hopkins,1981) , hydrodynamic (Ta Pheromonescontro l anumbe r of socialan d Volga, 1971)an d tactileinteractions . sexualinteraction s inteleos t fish and their Theorgan swhich ,i nfish ,generat e orde interindividual integrative function canb e tect physical signals areofte n highlysophi regarded asa n extension ofth e intercellular sticated and specialized and theirrapid ,cl o communication mode.Actually , ectocrineo r se-rangemod eo factio n contrasts withtha t pheromone-releasing cellsrepresen t thecen characterizing theinterchang e ofchemica l trifugal component ofth echemorecepto-signa l messengers.I nparticular ,odorou s signals ling systemwhic h processes the chemicalin propagate slowly inth ewater ,accordin g to formation flux through theorganism .I nthi s their speed ofmolecula r diffusion andtrans paper,th erecen t literature onfis hpheromo port bycurrents ,bu t their source can betr a nesi ssystematicall y reviewed and available eed byfis hfro m very longdistances ,a sdu data about their origin and chemistry arecr i ring homing (Johnsen & Hasler, 1980),i f tically discussed.Integumentar y peptidesar e they areassociate d with adifferentia l rate suggested tomediat epheromonall y conspecific ofwate rflo wallowin g orientation byrheota - recognition and aggregation whereastesticu xis (Hara, 1971).I n theabsenc e ofth edire laro rovaria n androgens in conjugated form ctional clueinheren t ina current ,fis hca n might play therol eo f sex attractantsi n still search along aconcentratio n gradient differentspecies . by exploratory strategies thatar emor ecom Key words:teleost ,pheromone ,behaviour ,re plex than trueosmotropotaxi s (Timms &Klee - production,growth . rekoper, 1972). Taste too canguid e somefis h tofoo d by sensing chemical cues,eve n ifre Introduction leased in stillwate r (Hara,-1971).Th eus eo f gustatory receptors to reveal semiochemicals Teleost fish rely upon acomple xmultisen - from conspecifics hasbee n advocated forth e sory communication system forthei r social response ofth emal e ofcave-dwelle rpoecili - and sexual interactions.Th e balance between idst oth efemal ese xattractan t (Parzefall, physical (visual,auditory , hydrodynamic, 1973)an d inth e caseo fth emouthin g ofth e tactilean d bioelectric]vs .chemica l signals male'sgenita l papilla byth efemal eo fTila - (olfactory andgustatory ) involved inintra - piamacrochi rdurin g courtship (Crapond eCa specific communicationma y vary indifferen t prona, 1976). speciesan d even between sexeso fth esam e From an evolutionary viewpoint,chemorece - species (Crapon deCaprona , 1982). Thisrela ption,wit h its extreme sensitivity andra test obot hth etransmissio n characteristics ther conservative traits,wa s probablyper (speed,directionalit y and attenuation)o fa fected earliertha n theperceptio n ofphysi given signalling output and thediscriminato cal stimuli,bein g thedominan t sensei nth e ry potential ofth ecorrespondin g sensoryap lessorganize d aquatic invertebrates and uni paratus inth efis h natural habitat. cellularorganisms .It s rolei n intraspecific Forinstance ,althoug h anumbe r ofteleost s communication ofmetazo a can beregarde d as can emit and perceive sounds of lowfrequen either aspecialize d adjunct toit sfunctio n cy, predominantly below 1KHz ,thei rsoni c in environmental scanning andinterspecifi c exchangesma yb ehampere d by high levelso f recognition inprey-predato r and host-parasi ambient noisei nth e samefrequenc y range te/symbion t relationships (Kittredge, 1974) originating from wavemotion ,frictio n bywa or, rather,a sth econsequenc e ofa ninterin ter currents,waterfalls ,win d orfro m other dividual extension ofth e intercellular com soniferous speciesincludin gma nwit h his municationmod ewhic h is chemically-mediated. traffic and industry (dyrberg, 1978). More Thisproces smus t haveoccurre d repeatedly over,despit e thefas t and efficientpropaga and independently indifferen t taxa when the tion ofacousti c energy inwater ,fishe sca n rewa sa selectiv eadvantag efo ra populatio n locatea soun d sourceonl y inth enea rfield , toimplemen t social integration or sexual co presumably bymean so fth e lateral line(Ta - ordination and itrequire d thedifferentia volga, 1971). tiono fenvironmentall y diffusible chemical Visual stimuli haveth eadvantag e ofbein g signals (pheromones)matche d totha t ofspe immediate,directiona l and highly informative cificreceptors . but their effectiverang e ismuc h shorteri n water than inai rdependin g upon turbidity Thepheromon e concept infis h and lightquenchin g with depth.A n evengrea 84 Karlsonan d Luschsr(1959)propose d theter m "pheromones" for "substanceswhic h aresecre ted toth eoutsid e by an individual andrece ived by asecon d individual ofth esam e spe cies,i nwhic h they releasea specifi creac tion,fo r example,a definit e behaviouro ra developmental process".The y added alsotha t pheromones,a schemica lmessengers ,shoul d be active inminut e amountsan d berelativel y species-specific,onl y limited molecularo - verlapping between closely-related species being tolerated. Therequisit e ofa definite ,stereotype d behavioural ordevelopmenta l response inth e recipient organism, asofte n observed inin sects,i sactuall y too restrictive to bea - dopted in thedefinitio n ofpheromone si n Fig.1.Th echemorecepto-signallin g system of vertebrates.A s argued byMykytowyc z (1979) afish .Th efis hbod y isportraye d asa toro in thecas eo fmammals ,thei r superior level idal-like figurewhil echemorecepto-secretor y ofcerebra l information processingmake sth e cellsar erepresente d bytw o concentricrings - reaction toa give n odour signal conditionalo n Signallingmodes :A = centripetal ;B =centri 'ase t ofmanifol d variables,suc ha spreviou s fugal; C ' internal. experience,physiologica l condition,socia l status, ecological context etc.T oa mino r tract,th e luminao fth egonoduct s and ofci degree,thi s lacko fstric t automatism inth e ther secretory or excretory ducts,th eepen - responset oa nolfactor y stimulus islikel y dimal canal,th eencephali c ventriclesetc . tooccu rals oi nteleosts ,a sdiscusse d by Both interfaces can bethough ta s selectively Myrberg (1975)abou t parentdiscriminatio n of permeable totw obidirectiona l fluxes:on eo f young inCichlasom a nigrofasciatum. energy-matter (tob echannelle d essentially Therevers epossibilit y thata give nrespo n intometabolism ,growth ,reproductio n andre sei striggere d by amulti-componen t odorant action to "stress")an d theothe ro fchemica l mixture,a s iti swel l documented ininsect s signals (tocontro l theinterna l balancea - (Ritter, 1979), should also be kept inmin d mong thefou renergy-matte r channels).T csi m when hunting forfis h pheromones.I nthi sca plify,physica l signals areno t considered se,th especies-specificit y ofth e signalma y here. beentruste d notonl y tomolecule swit hdif Thetransfe r ofchemica l signalsacros san d ferent chemical structures (symbolic coding) between thetw o interfaces isno t bydiffu butals ot oodorou sblend swit h differentra siono rpassiv etranspor t alone,bu t iti s tioso fth e sameingredient s (syntacticco also relayed by cellso fa specialize d type, ding), asreporte d in lepidoptera (Ritter, thechemorecepto-secretor y cells.Thes ecell s 1979). are characterized by thefacult y of secreting Forth eabov e criticism,w efee l thatth e orstoppin g to secrete achemica lmessenge r term "pheromones"i sadequat e todesignat e after interacting with an incoming signalling theintraspecifi csemiochemical so ffis honl y molecule through asurfac e or cytoplasmicre ifi ti sgive n an acceptationmor ecomprehen ceptor.Thi sfunctio n isrequeste d forbot h sivethe n inth eorigina ldefinition . information processing and transduction,i.e . tochang eth echemica lvector . Thechemorecepto-signallin g system According towherefro m thefirs t signal is received andwheret o thesecon d isdirected , Itmus t bepointe d outtha t thepheromone - cellular relaysca n operatei nthre eways : releasing or ectocrine cells (tob edistin centripetally, internally and centrifugally guished from esocrine cellswhos e secretions (Fig.1). areessentiall y ofmetaboli c significance) Inth e centripetalmode ,th e cellaccept s belong toth echemorecepto-signallin g system the signal from theexterna l orenclose dspa which transduces and processes thechemica l cesan d directs itsow nmessenge r intoth ein information flux through theorganism . ternal space.Th echemosensor y cells sucha s By adrasticall y reductive approach,th e olfactory,gustatory ,vomero-nasa lan dpara - body ofa fis h (and ofan y otheranima la s neuronal cells ofth eope n type (Fujita et al. well)ca nb eassimilate d toa toroidal-lik e 1980)wor k inthi smanner . figurewhos einterna l spacei sseparate d from Inth einterna lmode ,cell sreceiv ean d theenclose d spaceb y aninne rinterfac ean d transmit signals only within theinterna l from theexterna l spaceb ya noute rinterface , space.I ti stypica l ofnervous ,neurosecre asshow n inFig.1 . tory,endocrin ean d paraneuronal cellso fth e Theenclose d spacecorrespond st oal lthos e closed type (Fujitae tal. ,1960) . spaces "inside"th ebod y ofa fis hwhic ho - In thecentrifuga lmod e cellsreceiv esig pen,o ra t leastopene d once,int oth eoute r nalsfro m theinterna l spacean d secretese space, such asth ecavitie s ofth edigestiv e miochemicals into the external and enclosed 85 spaces.I ti sexhibite d by ectocrinecells . (1977)an dSolomo n (1977)an d referencei s However, asigna l released intoa n enclosed made tothe mfo ra mor e extensive coverageo f spacewhic h communicates no longerwit h the previous literature.Ai m ofthi spape r ist o outside [asi nth ecas eo fth eCSF-contaotin g update thesurve y with adiscussio n onphero neuron systemaroun d theencephali cventri mone sourcesan d chemistry and topresen tso cles (Franzoni &Fasolo ,1982) ]canno t bere me unpublished datafro m our laboratoryo n garded aspheromona lb ydefinition .Thi ssi thepossibl erol eo fandroge n conjugatesi n tuation resembles that ofa nanima l smelling fishsexua lcommunication . itsow nodou ran d iti sclassifiabl ea sauto - ectocrinecommunication . Pheromonal control of social behaviour It should beemphasize d thatth eabov emo delrefer s tosignallin gmode s and nott oac Chemical communication hasbee n documented tual celltypes .I nth eintricac y offeed orclaime d toshap edifferen tfis h socialbe backcircuits ,a give n cellmay ,i nfact , haviourswhic hma y bebroadl y classified as function inmor etha n oneway .Fo rinstance , hierarchical,associative (schooling),aggre a chemosensory cell isswitchin g toth ein gative (homing)an d alarmed (fright reaction). ternalmod ewhe n itmodulate s itstransmis sion inrespons et o hormones.I nmal egold Hierarchical behaviour fish,i tha sbee n showntha t the sensitivity toodour so fth eolfactor y epithelium isan - Researcho nth eweigh to folfactio n inso drogen-dependent (Yamazaki,1979 ). cial hierarchy is limited tocatfishe s ofth e Further,whe n an endocrineo rneurosecreto genusIctaluru swhic har ebasicall yterrito ry cellsucceed st o send its secretion into rial and nocturnal animalswit h a keen sense the external spacet oac ta sa pheromone ,it s ofsmell . role becomes ectocrine.Interestingly ,th e Yellow bullheads,I.natalis , have beenfo fishpheromone sfo rwhic hw e havemor e hints undt ous echemica l cuesno tonl y torecogni about theirchemica l identity seemt ob epro zeindividua l conspeoifles,bu tals ot oas duced just by endocrine (Colombo etal.,1980 ) sessthei rrelativ edominan to r submissive and neurosecretory cells (Richards,1974 )a s attitudei norde rt o avoid undue challenges discussed below. ofsocia l status (reviewed byBardac han d Todd,1970 ;Solomon , 1977).However ,whe na Discrimination bymean s of pheromones dominantfis hwa sreturne d toit sterritor y after experiencing alosin g encounter,i twa s An impressive numbero fintraspecifi cinter immediately attacked by itsformerl y submis actionshav ebee n showno r suggested tob e siveneighbour ,suggestin g that stressha d underpheromona l regulation infish .Firs to f altered itsodour . all,teleost s usechemica l cuest omak eim Ast oth epossibl e sourceo fth epheromone - portantdiscrimination s sucha sth erecogni (s), itwa sfoun d that theintegumentar y sli tion ofone' s species (Qshima etal. ,1969 ; me, presumably uncontaminated from urineo r Höglund 8Astrand , 1973;Bloo m SPerlmutter , fecal residues,wa s sufficient fordono r1 - 1977),rac e orpopulatio n (Nordeng,1971,1977 ; dentification,thoug h lesseffectivel y than Deivinge t al., 1974), brood (îlyrberg,1B75 ; donorambien twate r (Todd et al., 1967). McKayeS Barlow , 1976), individualconspeci - Ina ninterestin g study,Richard s (1974) fics (Tod d etal. , 1967;Richards ,1974) , hasshow n that bothurin ean d extractso fth e their social status (Todd et al., 1987), life urophysis provideinformatio n fordiscrimina stage (Miles,1968) , sex (Tavolga, 1956;Gan - tiono fconspeclfic s in I.nebulosus. dolfi, 1969;Crapo n deCaprona ,1982 )an dse Several and evenunusua l neuropeptides have xual condition (Partridge etal. , 1976;Cro w been isolated from thefis h urophysis (Bern, & Liley,1979 ;Honda ,1979 ,19B0a,b) . 1982). Nevertheless,i tseem s unrealistict o In somecases ,thi sabilit y isdependen t postulate thatth europhysi s containsa uni upona previou s exposureo fth efis h toth e quecompoun d foreac hindividua l fisha ssug pheromonedurin g acritica l period (imprin gested byBarnet t (1981), especially ifon e ting effect) (Myrberg,1975 ;Crapo n deCapro considers therathe r conservative natureo f na, 1982). Recognitionma y trigger aquic k neuropeptidemolecula r evolution.A syntactic change inbehaviou r (releaseeffect ). Alter coding based onfe w compounds ismor e likely. natively,a pheromon ema y cause aslo wphy Receptors forneuropeptide s arewidesprea d siologicalmodificatio n affecting laterbeha inth ebod y ofvertebrate san d thus itwoul d viouro reve n survival (priming effect).O n notb esurprisin g tofin d themals o inth e thewhole ,pheromone s havebee nreporte d to dendritic terminalso folfactor y cells.Sign i influence fish socialbehaviou r (mostlythro ficantly peptideswher ereporte d tostimulat e ughreleas e effects),bod ygrowt han dviabi quiteeffectivel y theolfactor y epithelium of lity (mainl y through priming effects)an d thewhit e catfish,I.catu s (Suzuki &Tucker , reproduction (through both). 1971). An externalization forpheromona lcom Valuablereview so nth etopi c offis hpher o munication ofth einterna l neuropeptidesig mones havebee nprepare d byBardac h andTod d nalling system is,therefore ,conceivable . (1970),Har a (1971),Pfeiffe r (1974),Crapo n However, ifth e hypothetical pheromonal deCapron a (1976),Tavolg a (1976),Barnet t neuropeptides aresecrete d by theurophysis , they should overcomedilutio n inth evas tex nals (Hemmings,19663 . ternal spacewithou t flooding receptorsi n Wrede (1932)an d Goz (1941)demonstrate d theinterna l body volume.T o comply withthi s thatminnows ,Phoxinu s phoxinus,maintai nco restriction,th epeptide smigh t beeithe rho r hesion becausethe y areattracte d byodour s monally inactiveo rquickl y removed from the released from the skinmucu s of schoolmem circulation intoth eurine . bers.A simila r observation wasmad e byKino - On theothe r hand,ther e areals o hintst o sita (1975)i nth emarin e catfish eel,Ploto - a completely different alternative:tha tth e susanguillaris ,i nwhic h adializabl ean d peptides areactuall y released from cellsi n heat-stable attractant is secreted from the theintegument .I nrainbo w trout,typica lol body surface of schooling fish. factory bulbar responses were induced byski n Considerable evidence exists that odour mucus collected from conspecifics,a swel la s trailsar eutilize d byanadromou ssalmonid s otherteleosts ,i nsuc h awa y ast oavoi d con for homestream selection (Johnsen &Hasler , taminationb yalar m substances (HaraS PlacDo - 1980). Nordeng (1971, 1977)wa sth efirs tt o nald, 1976). Activit y wasfoun d tob easso propose thatorientatio n for homewardmigra ciated with heat-stable,non-volatil ecompo tion isactuall y provided bypopulation-spe nentso fmolecula rweigh t between 500an d cific pheromonesrelease d from the skinmucu s 1000an d was pH-dependent.Thoug h notremark ofresiden t juveniles and descending smolt. ed by theauthors ,thes epropertie s seemt o Recordings from singlecell s in theolfac parallel thoseo f smallpeptides . tory bulb ofth eArti echar ,Salm oalpinus , Moreover,th econclusio n by Richards (1974] disclosed thatfis h could discriminatecha r 'thaturin ean d urophysiswer emor eeffectiv e populationsfro m different areaso n thebasi s forinterindividua l recognition than aninte ofodorant s contained inth e integumentary gumentary extract prepared by homogenizing slime (Deiving etal. , 1974).Sinc e thisi s 1 cm section oftissu e in 12m lo fpon dwa copiously secreted by thefish ,i tma y bea teri sope n to criticism. Infact ,catfis h majorvehicl efo r impregnation ofwate rwit h were conditioned tomanifes t recognitionb y charodours .Differentia l responses ofth e approach orfligh t reactions but,sinc ea ca t olfactory bulbt o homo-an d heterospecific fish skin homogenatemus t contain alarm sub skinmucu swer eobserve d alsoi nth erainbo w stances (Pfeiffer, 1974], therewa s astron g trout,Salm o'gairdner i (Hara &McDonald,1976) , biastoward sfligh t responses,thu sinvalida asdiscusse d above. ting theassessmen t ofrecognitio n bymean s Recently,Stabel l and Selset (1980)hav e ofintegumentar y signals. warned toavoi d carefully contaminationb y Theabilit y offis ht o smell peptides (and intestinal juice inth ecollectio n ofski n aminoacidsa swel l (Suzuki STucker ,1971) ) mucusfo rolfactio n studies infish .I nfact , isunderstandabl e ifw e consider that,i na n intestinal residues contain bileaci dderiva aqueousmediu m odours correspond towater-so tiveswhic h arepowerfu l odorants inteleost s luble (i.e.polar )rathe r thanvolatil e (of (Selset, 1980),thoug h they are not likelyt o ten unpolar)compounds . contain peptides oraminoacids . Whetherfis h contain pheromonalpeptide si n Iti so finteres t thatelver s ofth ecata - theski nremain s tob edemonstrate d experimen dromous species,Anguill a rostrata,ar edraw n tally,bu t it isinspirin g thefac t thata upstream bya heat-stable ,non-volatil e sub numbero finterestin g peptides havebee nre stance emitted by theadult s (Miles,1968) , vealed inth e skino famphibian s (Erspamere t and not byth eyoun g asi n salmonids.Atem a• al., 1977)where ,b yth eway ,the ymigh tper et al.(1973 )reporte d thatalewive s (Alosa form a similarfunction . pseudoharengus)wer e attracted tothei rhome - An advantage (forth efish )o fth eexterna - stream by compounds ofmolecula rweigh t less lization notonl y ofth ereceptor s butals o than 1000Dalto nwhic hwer eno tfre eaminoa ofth eeventua l peptide-releasing cellsi s cids. thatther ei sn onee d for special hormonally Thepossibilit y thatfis har eguide d to inactivepeptide s because,contrar y toth e their homestream also by non-pheromonal che urophysialmodel ,ther ei sn odange r ofhor mical cueswashe d outfro m soil (enviromones) monalunbalances . orplant s (allomones)canno t bepresentl ydi smissed.However ,th edemonstratio n thatcoh o Schooling and homing salmon imprinted tomorpholin ea tth etim eo f smolting wereabl e toretur n asadult st oth e Itshoul d beemphasize d that theinforma sites scented with it,i nth eabsenc eo fcoh o tion conveyed by integumentary water-soluble _salmon upstream, (Johnsen &Hasler ,1980 )i s odoursfro m conspeclficsma y beuse d notonl y weakenedb yth efac t thatmorpholin e canac t tostabiliz e hierarchical systemsbu tals ot o asa non-specifi c attractant for salmonids support associativean d aggregative behavio (Mazeaud, 1981). urssuc h asschoolin g and homing,respective - ly. Alarm substances Afis h school isa n integrated association ofisospecifi c individualswhic h isstabili Aclas sof,integumentar y pheromones,dist zed cybernetically through visual,olfactor y inctfro m thosepresumabl y involved inhiera r (especially at night)an d hydrodynamicsig chical,associativ e and aggregativebehaviours , 87 isrepresente d by theso-calle d alarm substan together innatur eten d tob eo f similarsiz e esso fOstariophys ian d Gonorhynchiformes(cf . (Keenleyside, 1955)thu s equalizing theadver Pfeiffer, 1974). seeffect s ofcrowding . Thesepheromone s are stored In specialized Besides,i ti sconceivabl e that,i nnature , epidermal elements,th eclu b cells,whic hre themai n function ofcrowdin g factors isjus t leasethe m not byactiv e secretion butupo n topreven t crowding byfavourin g fishdisper breakagecause d bymechanica l skininjuries . sal, theiractio n becoming moredramati c only Their intimidatory effect helps nearbycon - under abnormally high populationdensities . specifics to escape further attacks bya pre Forthi sreason ,researc h oncrowdin g factors dator buti t isscarcel y activated incas eo f is likely tob eo fgrea t importance forth e cannibalism (Verheijen 8Reuter , 1969). practice offis hcultur ewher eproductivit yin Testosterone hasbee n shown toreduc eth e creaseswit h theinorsTs ao f stockdensities . numbero fclu b cells inmal e fatheadminnows , Preliminary fractionation studiesd ono t Pimephalespromelas , sothe y canru b their support thesuggestio n that acrowdin g factor back toclea n aspawnin g sitewithou t freeing and anaggregatin g pheromonemigh t beactual alarm substancetha twoul d frighten awayin lyth e same substance acting inopposit eway s terested females [Smith, 1973). Malyukin ae t atdifferen t concentrations (Solomon, 1977). al. (1977)hav eals oreporte d adecreas eo f Thecrowdin g factorso fdifferen t fisheswer e alarm pheromonei nth eskin s ofspawnin gma extracted withorgani c solventsfro m thewa lean d femaleminnows ,P.phoxinus , and ade terwher ethe y had been secreted and thusap pression ofth efrigh t reaction in starved or peart ob elipid s (Yu8 Perlmutter , 1970jPfu - unhealthy recipients. derer etal. , 1974),wherea s aggregatingphe - Alarm substances areapparentl y heat-labile •romoneswer efoun d to bewater-solubl e (see and water-soluble and havebee n claimed tob e above). Moreover,populatio n densitywoul d be either pterinoic compoundswit h UVautofluo optimized more effectively bya feed-bac kme rescence (Pfeiffer &Lemke ,1973 )o raminosu - chanism based ontw odifferen t signalsfo r garswithou t UVabsorptio n (Kasumyan &Lebe - fishdispersa l and aggregation then byon eba deva, 1979).Whateve r their nature,the yre sed onth e level ofa singl e signal only. present afascinatin g example ofth eevolu tion ofa naltruisti c character,a sdiscusse d Pheromonal control ofreproductio n bySolomo n (1977). Teleosts,a sothe ranima lgroups ,hav eampl y Pheromonalcontro l ofgrowt h and viability exploited chemical communication forbot hth e control offertilit y and thecoordinatio n of Convincing evidence exists thatfis hca n sexual and epigamicbehaviours . respond to homospecific semiochemicalsno t only behaviourally but also physiologically Pheromonal control offertilit y (priming effects).I nthi s kind ofcommunica tion,th epheromona l influence penetratesde Sincereproductio n increases populationden eply intoth echemorecepto-signallin g system sity,it sdepressio n bycrowdin g factorsappe ofth erecipient . arst ob ea mor e radical and lesswastefu lme An interesting case istha t inwhic hchemi chanism ofautoregulatio n than thestuntin g calcue sfacilitate s (beneficialconditio or killing ofalread y bornrelatives . ning)o rinhibi t (crowding factors)th egrowt h Swingle (1953)foun d that,i funcrowde dfish , and eventh eviabilit y ofconspecific s (cf. about tospaw nwer e exposed toth e holdingwa Solomon, 1977). It hasbee n reported that tero fa dens egroup ,thei r spawning waspre fish livebette r inwate rwher eothe rfis h vented.Y uan dPerlmutte r (1970)quot eth e had lived and that sucha conditionin g can thesiswor k by Greene (1964)wh o extracteda makethe mmor eresistan t to stress (McCauley , species-specific spawning inhibiting factor 1968). fromgoldfis hwate rwit h chloroform.Ros e Conversely,whe n crowded,fis h liberatei n (1959)discovere d that,i nth e live-bearing themediu m species-specific inhibitorswhic h guppy,Lebiste sreticulatus ,th enumbe ro f stunt orshorte n the life-span of smallero r young perfemal ewa s inversely proportional weakermember s ofth egrou p (Yu& Perlmutter , toth enumbe ro ffemale spe rtan k butwa sin 1970). Crowding hasbee n shown topromot eth e creased byreplacin g mosto fth ewate rdaily . releaseo f heart-rate depressants inth egol d It isreall y unfortunate thatthes e original fish (Francise tal ., 1974 )an d ofimmuno-sup - findingsdi d not arouse sufficient interest pressivefactor s inth eblu egourami,Tricho - tostimulat efurthe r investigation.Density - gaster tricopterus,thu sincreasin g vulnera dependent inhibitorso ffecundit y would be bility by infective agents (Perlmutter etal. , obviously detrimental inth erearin g offis h 1973). forbreeding ,especiall y ifth e latterar ege Sincelarge ranimal sproduc emor e crowding netically selected. factor,wherea s smaller onesar emor esensi Itshoul d benote d that fertility may bede tivet oit ,i n anovercrowde d group ofyoung , pressed not only by crowding butals o byiso 'fishthos ewhic h are initially smallerwil l lation.Female so fPterophyllu m scalare,whic h stopt ogro w andwoul d eventually die (Rose had been isolated chemically from themal efo r &Rose , 1965). Significantly,fis h schooling twomonths ,wer efoun d to spawna t lowerrate s with respectt onon-deprive d controls (Chien, ceseem st ob eessentia l indeterminin g the 1974). subsequent preference forth eodou ro fth e isospecificgravi d female (Crapond eCaprona , Sexual behaviour 1982), justa sth efirs t broodcare experien cei sdecisiv efo rth e subsequent olfactory Muchmor e information isavailabl e onth e discrimination ofyoun gb y cichlid parents roleo fchemica l signals infis h sexualbeha (Myrberg, 1975). viour.Th efollowin g interactions havebee n reported tob emediate d bypheromones : Sources of sexpheromone s a)attractio n ofth emal eb y thefemal ei n Poecilia reticulata (Gandolfi,1969) ,Ictalu - Several authors,wh o havedemonstrate d the ruspunctatu s (Timms8 Kleerekoper , 1972)an d occurrence of sexattractant s inteleost s Brachydanio rerio (Bloom &Perlmutter , 1978; haveals o tried to point outthei r source vande nHur k etal. , 1982); and togiv e hintsabou t their chemistry.A b)attractio n ofth emal eb y theovulate d or general consensus existsabou t thegonada l responsive femaleonly ,wit h stimulation of origin offis h sexpheromones ,wit hth epos male courtship inBathygoblu s soporator CTa- sible exception ofBlenniu s pavo inwhic h volga, 1956), Colisa lalia,C.labios a (Rossi, themal eattractan t isproduce d byspeciali 1969),Astyana x mexicanus (Wilkens, 1972), zed appendices ofth eana l fin spines (Lau Carassius auratus (Partridge et al., 1976), men et al., 1974). SignificantlyMalyukin a Poeciliamexican a (Parzefall, 1973),Poecili a etal . (1974)reporte d that evenmicrosmati c reticulata (Crow &Liley , 1979),Poecili a chi fish react togonada lodours . ca (Hilton Brett SGrosse ,1982 )Hypomesu so - Inth efemale so fgob y (Tavolga, 1956), lidus (Gkada et al., 1979J,Plecoglossu s alti goldfish (Partridge et al., 1976), ayu (Hon velis (Honda, 1979), Salmogairdner i (Honda, da, 1979), trout (Honda,1980a )an d loach 1980a), Misgurnusanguillicaudatu s (Honda, (Honda, 1980b)a novaria n pheromoneelicitin g 198Gb)an d Haplochromisburton i (Crapond e malecourtshi p hasbee n shownt o accumulate Caprona, 1980); inth eovaria n fluid chiefly orexclusivel y c)increas e ofmal et omal einteraction sin just afterovulatio n and to berelease d with duced byth efemal ei nColis a lalia,C.labio iti nth e surrounding medium through theovi - sa (Rossi,1969) , Mollienesia latipinna (Thi- pore.Sexua l attractants and stimulantswer e essen SSturdivant ,1977 )an dPoecili a chica revealed inth e ovarieso f pond smelt (Okada (HiltonBret t 8Grosse , 1982); et al., 1979),gupp y (CrowS Liley ,1979 )an d d)attractio n ofth efemal eb yth emal ei n zebrafish (vande nHur k etal. , 1982).A te Blennius pavo (Laumen etal. , 1974), Brachida sticular sexpheromon e hasbee n postulated nioreri o (Bloom andPerlmutter , 1977)an d also inth eblac kgob y (Colombo et al., 1980). Gobiusjoz o (Colombo etal.,1980) ; There areindications ,however ,tha tfemal e e)intrasexua l attraction inHypsoblenniu sr o fishma y utilizeals o urine,beside sovaria n bustus,H.jenkinsi ,H.gentili s (Losey, 1979), fluid,a sa pheromona lvehicle .Althoug hfe Salmogairdner i (NewcombeS Hartman , 1973), males ofPoecili a reticulata becomemaximall y Hacropodusoperculari s (Davis8 Pilotte ,1975 ) attractivet omale s during theperio d ofrec e and Brachydanio rerio (Bloom &Perlmutter , ptivity which follows parturition,whe n the 1977). proximal end ofthei roviduc t isope n (Crow 8 Liley, 1979), the holding water ofadul t Parental behaviour virgin females,whos eoviduc t isclosed ,stil l attractsmale s (Gandolfi,1969 ;Cro w8 Liley , Chemical information isimportan t alsoi n 1979; ourow nunpublishe d observations). epigamicbehaviou ro ffis h as exemplified by Similarly, even thoughmal egoldfis h prefer theparent-youn g interactions ofcichlids .I n the smell ofrecentl y ovulated overtha to f Hemichromisbimaculatus ,broodin g behaviour unovulated females (Partridge et al., 1976), and selective recognition ofyoun g by thepa yet they are easily attracted byth eholdin g rents areinitiate d by chemical emanations water offemale s in latevitellogeni ccondi from thewigglin g fry (Plyrberg,1966) .Als o tionwhe n theovar y containsver y littleflu inCichlasom anigrofasciatu m (Myrberg, 1975) id (unpublished). and C.citrinellum (HcKaye& Barlow , 1976), Amor edecisiv e experiment wasrecentl y parents learn todistinguis h their broodb y made byu si nGobiu sjozo .Urine ,fre efro m itsodour . ovarian contaminants,wa sobtaine d from both Conversely,youn g cichlids canrecogniz e gravid (i.e.ovulate d but unspawned)an dpost - conspecific adults,bu tno tthei rparents , vitellogenic females bytyin g upth eurogeni through urinary cues (Barnett, 1981),thei r talpapill aan d collecting theurin e (usually detection ofparentalnes s being essentially 0.1ml )wit h a1-m lsyring edirectl y from the visual (Myrberg, 1975). swollen urinary bladder 10-12h rafterwards . Studieso ncichlid s haveprovide d consistent Itwa sthe ndilute d 1:50wit h saline.Ovaria n evidence ofth eimportanc e ofearl y experien fluid,freefro m urinary contaminants,wa sob ce (imprinting)i nth edevelopmen t ofappro tained byremovin g anovary ,slittin g itopen , priateresponse s topheromones .I nmal eHaplo shaking iti na watc hglas s containing 5m l chromis burtoni,th efirs t breeding experien offis h salinefo r 5mi nan d pipetting off 89 thesupernatan t (cf.Tavolga , 1956). terol esters (Algranati,1979 )an d steroid Behavioural testswer e performed onsexual glucuronides (vande nHur k etal. , 1982)i n lyactiv emale sisolate d in 50-1aquari awhic h Brachydanio rerio. wereprovide d withgrave l on thebottom ,a The latterfindin g isi n keeping withprevi halved plastic tubea sa shelte ran d running ousreport sfrom ,ou r laboratory abouta testi seawater.Behaviou rwa smonitore d for 10mi n cular steroid pheromone inGobiu sjoz o (Colom afterth eadditio n toth eaquariu m watero f boe tal. , 1979,1980)."I n this species,th e 1m l ofsalin e alonean d for 10mi nafte rth e male bearsa prominen tmesorchia l glandcompo addition of 1m l of either urine solutiono r sed cf Leydig cellswhic h synthesize mainly ovarianwashing . 5ß-reduced androgen conjugates,particularl y Urinewa smuc hmor e effectivetha n ovarian etiocholanolone glucuronide (E-G), together washing intriggerin g courtship and fluids with small amountso fnon-conjugatabl e11-oxy obtained from gravid femaleswer emor eeffec genated androgens,namel y 11(3-hydroxyandroste- tivetha nthos eo fpostvitellogeni c ones.Ma nedionean d adrenosterone.Whil eth e latter leurin ewa s uneffective (unpublished).Thi s compounds haveprobabl y ahormona lfunction , result contrastswit h theobservatio n byTa E-Gwa sfoun d toattrac tgravi d femaleswhe n volga (1956)tha t urinewa s inactive inano tested at lowconcentratio n (2"10~ Ma tth e thergobii d fish. stimulus source)i na four-choic e system.Un - Thisvariabilit y imposest oassa y onlyova ovulatedfemale swer e unresponsive (Colombo rian fluid uncontaminated from possibleuri et al., 1960). nary attractantswhe n trying to locateth e Gravid femaleswer eals oattracte d bydilut e sourceo fa se xpheromone .Th eusua lprocedu male urine (1:600dilutio n atth e stimulusso reso f stripping,squeezin g or evenrunnin ga urce)delivere d into onear m ofa glas sY-ma - finger lightly along thefis h sidesd ono t zei na constan tflo wo fseawate rwhic hwa s seem tob esaf eenough . equal totha to fth econtralatera l arm (76% Thedifferenc e between ovarian fluid andu - correctresponses ,n = 17 ,agains t 50 %i n rinea spheromona lvehicle s isno ta trivia l control runs, n= 42) . one. Firstly,owin g to peripheralmetabolis m Thepheromona l system ofG.joz o isver yin and binding toplasm a proteins,a novaria n teresting because it ischaracterize d by: hormonei sno t likely tob eals o aurinar y a)undertakin g ofth ecentrifuga l signalling pheromonewherea s this possibility existsi n mode by endocrine elements associated with thecas eo fth eovaria nfluid .Moreove r since theiroverdevelopmen t to copewit h signalat a systemic catabolite ofa novaria n hormone tenuation inthe*externa lspace ; isjus t afractio n of it,onl y aninactiv eo - b)pheromona l androgens prevailing overbu t varian product,sinthesize d inparalle lt o different from hormonal androgens topreven t hormones and scarcely transformedperipherally , flooding of hormonal androgenreceptors ; isexpecte d toreac ha urinar y concentration c)pheromona lmolecula rfor m suitablefo rbot h adequatefo r long-rangetransmission .However , urinary excretion and environmental diffusion a simple extra-ovarianmolecula rfinishin g as steroid conjugates arewater-soluble ; (e.g.conjugation )ma y berequire d ifth eo - d)femal e responsivity toth emal e pheromone varian product isactuall y apropheromone . induced byovulation . Secondly,a urinar y pheromonemus t beo f Most striking thepheromona l system ofth e lowmolecula rweight ,wherea s sucha restric boardisplay s justth e samefeatures ,th eon tiondoe sno tappl y inth e caseo fth eovari lydifferenc e being that thetesticula r5a - anfluid . androst-16-enes,whic h induceth ematin g stan Thirdly,intraovaria n secretion shouldal cei nth e estrous sow,ar evolatil ean drelea lowth eaccumulatio n ofa greate r pheromonal sed inth eai rwit h thebreat h (Genningse t charge than urinary excretion owing tolowe r al., 1977;cf .Colomb o etal. , 1980 fordis clearance rate (especially infreshwater )an d cussion). greaterflui d storage capacity.Presently ,i t We haveals o extended ourstud y tospecies , isunclea r howfis h exploit thesevehicle st o sucha sCarassiu s auratusan dPoecili areticu deliver sex pheromones,especiall y becausea - lata,i nwhic h iti sth emal etha t looksfo r vailable information about thechemistr yo f thefemal efo r spawning (and notth eothe r the latteri sstil l scanty and contradictory. way round asi nG.jozo )an d should thenplay s therol eo fth ereceive r inth epheromona lin Chemistry of sexpheromone s teraction. Malegoldfis hwer eteste d ina two-choic e Disparatemolecula r identities havebee n system inpair sbecaus e singlemale sar eat proposed forfemal e sex pheromones infis h tracted notonl y byth eodou ro ffemale sbu t sucha sestroge n in Lebistes (= Poecilia ) also bytha to fmales .Pellet so ffis hfoo d reticulatus (Amouriq, 1965), a ether-soluble weregive n tofis h immediately beforeth e substancei nCarassiu s auratus (Partridgee t test tocal m them down and toimpregnat eth e al., 1976),water-ethe r solublebasi csubsta n water ofth esyste m withaspecifi c odours.A s cesi nPlecoglossu saltiveli s (Honda,1979) , shown in Fig.2,mal egoldfis hwer eattracte d Salmogairdner i (Honda,1980a )an d Misgurnus byE- Gwherea s femalesdi d not react.Male s anguillicaudatus (Honda,1980b) ,a protei ni n responded alsot oovaria n fluid (possiblycon Hypomesusolidu s (Okada etal. , 1979), choies taminated with urine)bu t not tocharcoal-tre - 90 100 tion.Significantly ,hig hyield so ftestoste n= n = 8 roneglucuronid ewer eals oobtaine d afterin ? PO.001 cubation ofpregnenolon ean d progesteronewit h ovarian tissuefro m theIndia n catfish.Heter o | 50H pneustesfossili s (Colombo andSundararaj ,un published).Als o thefindin g byKim e (this symposium)tha t thetesticula rglucuronide / free steroid ratio ishighes t introu t atsper - miation,whe n environmental temperature isa t o itsminimum ,ca n berationalize d interm so f pheromoneproduction . Wilw-H 0 E-G w-H 0 E-lG 2 2 T t t ft Theconclusio n that notonl y theinterna l Fig.2. %Tim e spent byon eo ftw omal e (A)o r spacebu tals o theexterna l spacearoun d fish female (B)goldfis h inth etw o compartments may besexualize d bygonada l steroids supports ofa 150- 1tan k (halved bya pane l endinga t theconcep t that theevolutio n ofth ebioregu - 6.5 cmfro m thebottom )fo r3 0mi n beforean y latoryfunctio n of sex steroidsi nvertebrate s input CT)an d during addition toon ecompart isaime d atth eprogressiv e elongation ofth e ment (arrow)o f5 ml/mi nfo r3 0mi no fwar m communication channel connecting thesteroid - water alone (W-H2O;4 C abov eth etan Kwate r secreting with thesteroid-responsiv e sites, t°C)o retiocholanolon e glucuronide (E-G)a t asdiscusse d byColomb o et al. (1980).Accor aconcentratio n of1ug/m l inwar m water.Stu ding tothi smodel ,th eectocrin erol eo fgo dent's t-test onmatche d pairs (withrespec t nadal steroidsoriginate d from theirendocri to W-H2O). nefunctio n which,i nit sturn ,emerge d from theirparacrin e orintragonada l activity.I n 51 A B thefemal efo rinstance ,w ema y supposetha t x: _ n=10 P'0?01 n.10 itwa sth e estrogen in excesst otha tneede d 4 forgranulos a mitogenesisan ddifferentiatio n (Richards,1975 )tha twas^utilize d forth een docrinecontro l ofextra-ovaria n targets,jus t asth e hormonal progestogenswer ederive d from theovaria n overflow ofth ematurationa lste roidsinvolve d inoocyt edifferentiatio n (i.e. resumption ofmeiosis ) (cf.Colomb o etal. , III III 1980).A tthi spoint ,w ewoul d liket osugges t that pheromonal androgen conjugates represents T t t T t t WHO E-G w-HaO A-G analternativ e utilization ofth eandroge nin termediates left overb yth edeclin e of a- Fig.3.Numbe r offis h (c hecked every 30sec ) romatization (vanBoheme n Lambert ,1981 )and , among fivemal eguppie s visiting thetw ocom - & possibly, 11(3-hydroxylationi n somemal efish , partmentso fa 35- 1tan k (halved bya pane l occurring atth ecompletio n ofgamet ematura endinga t11. 5c mfro mt hebottom )fo r3 0mi n tion. before any input (T)an d during additiont o onecompartmen t (arrow) of4. 2ml/mi n for3 0 min ofwar mwate ralon e (w-hteO),etiocholano - loneglucuronid e (E-G)( A)o randrosteron e References glucuronide (A-G) (B)a t a concentration of 0.6 yg/ml inwar mwater , Student's t-test as Algranati,F.D. , 1979.Rol eo f sexattractan t above. pheromone(s)o n spawning aggregation inth e zebrafish ,Brachydani o rerio,an d theiso ated ovarian fluid,testosterone ,estradiol - lationan d partial purification ofth eat - 173an d its3-glucuronide . tractants (abstract). 2nd Conf.Ethol .Be - Ina simila rtest ,als omal eguppie sdispla hav.Eool .Fishes ,Octobe r 19-22,Illinoi s yed apreferenc e forE- G but notfo r itsepi - StateUniv .Bloomington . mer,androsteron e glucuronide (Fig.3),whil e Amouriq,L. ,1965 .Origin ed e lasubstanc edy femalesdi d notrespon d toboth .Thes eexpe namogène émisepa r Lebistesreticulatu sfe riments,whic hwil lb ereporte d indetai lel melle (PoissonPoeciilidae ,Cyprinodontifor sewhere demonstrate that therespons et oE- G me). C .R .Acad .Se .Paris ,260:2334-2335 . is specificwit h respect to sexan d chirality Atema,J. , S.Jacobson ,J . Todd & D. Boylan, but nott ospecies . 1973.Th eimportanc e ofchemica l signalsi n Presently,w e haveno tye t established whe stimulating behaviour ofmarin eorganisms , ther 5ß-androgen conjugates aresecrete d by effectso faltere d environmental chemistry theovar y orexcrete d withth eurin ei nth e onanima l communication.In :G .E .Glas s femaleso fgoldfis h and guppy.However ,Lam (Ed.):Bioassa y Techniques and Environmental bert and vande nHur k (this symposium)hav e Chemistry.An n ArborScienc ePublishers ,Mi revealed ahig h conversion rateo fandroste - chigan,p .177-197 . nedionet o steroid glucuronides,mainl ytesto Bardach,J . E. &J . H.Todd ,1970 .Chemica l steroneglucuronide ,b yth eovar y ofth eAfri communication infish .In :J .W .Johnston , cancatfish ,Clari a lazera,jus tafte rovula 0.G .Moulto n &A .Tur k (Ed.): Communication 91 by Ehsmical signals.Appleton-Century-Crofts , nari,1977 .Occurrenc e oftachykinin s (Phy- NewYork .p .205 . salemino rSubstanc eP-lik epeptides ) inth e Barnett,C, 1977.Aspect s ofchemica lcommu amphibians skinan d theiractio n onsmoot h nication with special reference tofish . musclepreparations .In :U.S .vo nEule r& Biosci. Commun.,3:331-3.92 . B. Pernow (Ed.): "SubstanceP" . RavenPress , Barnett,C, 1981.Th erol eo furin ei nparent - NewYork . offspring communication ina cichli d fish. Francis,A.A. ,F .Smith" &P .Pfuderer ,1974 . Z.Tierpsychol. ,55:173-182 . A heart-rate bioassay forcrowdin g factors Bern,H.A.,1982 .Th ecauda l neurosecretory ingoldfish .Prog .Fish .Cult. ,36:196-200 . system offishes :th ebeginnin g of"urophy - Franzoni,M.F .S A .Fasolo ,1982 .Ultrastruc siology".Gen .Comp .Endocrinol. ,46:349 - turalanalysi s ofth eposterio r hypotalamus 350. ofsalamander .J . Submicrosc.Cytol. ,14 : Bloom,H. &A .Perlmutter , 1977.A sexualag 355-368. gregating pheromonesyste m inth ezebrafish , Fujita,T. ,S .Kobayashi ,R .Yu i &T .Iwanaga , Brachydanio rerio [Hamilton-Buchanan).J . 1980. Evolution ofneuron san d paraneurons. Exp. Zool.,199:215-226 . In: S.Ishil ,T .Hiran o &M .Wad a (Ed.): Bloom,H. D. &A .Perlmutter , 1978.Possibl e Hormones,Adaptatio n and Evolution.Sprin pheromonemediate d isolation intw o species ger-Verlag,Berlin ,p .35-43 . ofcyprini d fisho fth egenus ,Brachydanio . Gandolfi,G. , 1969.A chemica l sex attractant J. FishBiol. ,13:47-50 . inth egupp yPoecili a reticulata Peters(Pi Bohemen,van Ch.G . &J . G. D. Lambert,1981 . sces,Poeciliidae) .Monitor eZool .Ital. , Estrogen synthesis inrelatio n toestrone , 3:89-98. estradiol and vitellogenin plasma levels •Gennings,J.N. , D.B.Gowe r &L.H .Bannister , during thereproductiv ecycl eo fth efemal e 1977. Studieso nth ereceptor s to5a-androst - rainbow trout,Salm ogairdneri .Gen .Comp . 16-en-3-onean d 5a-androst-16-en-3a-oli n Endocrinol.,45:105-114 . sownasa lmucosa .Biochim .Biophys .Acta , Chien,A .K. , 1974.Reproductio n behaviouro f o496:547-556. theangelfish ,Pterophyllu m scalare (Pisces: Goz, H.,1941 .Übe rde nArt-un d Individualge- Cichlidae):Influenc eo fmal e stimuliupo n ruch beiFischen .Z .vergl .Physiol. ,29:1 - the spawning rate offemales .Anim .Behav. , 45. 21:457-463. Hara,T.J. , 1971.Chemoreception .In :W.S . Colombo,L. ,P .C .Belveder e &A .Marconato , Hoar &D.J . Randall (Ed.): FishPhysiology . 1979. Biochemical and functional aspectso f AcademicPress, "Ne wYork .p .79-120 . gonadal biosynthesis of steroid hormonesi n Hara,T.J . 8S .MacDdnald , 1976.Olfactor yre teleost fish.Proc .India n Natl.Sei .Acad. , sponsest oskin -mucou s substances inrain B45:226-234. bowtrou tSalm ogairdneri .Comp .Biochem . Colombo,L. ,A .Marconato ,p .C .Belveder e& Physiol.,54A:41-44 . C.Friso ,1980 .Endocrinolog y ofteleos t Hemmings,C.C. , 1966.Olfactio n and visioni n reproduction: atesticula r steroidpheromo fish schooling.J . Exp.Zool. ,45:449-464 . nei nth eblac kgoby ,Gobiu sjoz o L..Boll . HiltonBrett ,B.L .S D.J . Grosse,1982 .A re Zool.,47:355-364 . productivepheromon e inth eMexica npoecii - Crapond eCaprona ,M-D. ,1976 .Chemica lstimu lidfish ,Poecili a chica.Copeia ,1982:219 - lian d reproduction infish .Conclusion . ..223. Experientia,32:1098-1100 . Hoglund,L.B . & ». Ästrand, 1973.Preference s Crapond eCaprona ,M-D. ,1980 .Olfactor y com among juvenile char (Salvelinus alpinus L.) munication ina cichli d fish,Haplochromi s to intraspecificodour san dwate r currents burtoni.Z .Tierpsychol. ,52:113-134 . studied with thefluviariu m technique.Inst . Crapond eCaprona ,M-D. ,1982 .Th e influence Freshwater Res.Drottningholm..Repor t No53 : of early experience onpreference s foropti 21-30. calan d chemical cuesproduce d by bothsexe s Honda,H. , 1979.Femal e sexpheromon e ofth e inth ecichli d fishHaplochromi s burtoni ayu, Plecoglossus altivelis,involve d in (Astatotilapia burtoni,Greenwood , 1979). courtship behaviour.Bull .Jap .Soc .Seien . Z.Tierpsychol. ,58:329-361 . Fish.,45:1375-1380 . Crow,R .T . &N .R .Liley ,1979 .A sexualphe Honda,H. , 1980a.Femal e sexpheromon e ofrain romone inth eguppy ,Poecili a reticulata bow trout,Salm ogairdneri ,involve d inco (Peters).Can .J . Zool.,57:184-188 . urtship behaviour.Bull .Jap .Soc .Seien . Davis,R .E .S N .J . Pilotte,1975 .Attractio n Fish.,46:1109-1112 . toconspecifi can d nonconspecificchemica l Honda,H. , 1980b.Femal e sexpheromon e ofth e stimuli inmal ean d femaleMacropodu soper loach,Misgurnu sanguillicaudatus,involve d culars (Anabantoidei).Behav .Biol. ,13 : incourtshi p behaviour.Bull .Jap .Soc . 191-196. Seien.Fish. ,46:1223-1225 . Driving,K.B. ,H . Nordeng 8B .Oakley ,1974 . Hopkins,CD. , 1981.O n thediversit y ofelec Singleuni tdiscriminatio n offis hodour s tric signals ina communit yo fmormyri delec released bycha r (Salmoalpinu s L.)popula tricfis h inWes t Africa.Amer .Zool. ,21 : tions. Comp.Bioohem .Physiol. ,47A:1051 - 211-222. 1063. Hurk,va nde n R.,L.A.' tHart ,J.G.D .Lamber t Erspamer,V. ,G . Falconieri Erspamerâ G .Li - &P.G.W.J .va n Oordt,1982 .O nth eregula - 92 tiono f sexual behaviour ofmal szebrafish , visual stimuli inth epreferentia ldiscrimi Brachydanio rerio.Gen .Comp .Endocrinol. , nation ofyoun g byth ecichli d fishCichla 46:403. soma nigrofasciatum (Günther). Z.Tierpsy - Johnsen,P.B . 8A.D .Hasler ,1980 .Th eus eo f chol.,37:274-279 . chemical cuesi nth eupstrea mmigratio no f Myrberg,A.A. , 1978.Ocea n noisean d thebeh a coho salmon,Oncorhynchu s kisutchWalbaum . viour ofmarin eanimals :relationship s and J. FishBiol. ,17:67-73 . implications.In :J.L .Fletche r 8R.G .Bus - Karlson,P . 8M . Luscher,1959 . "Pheromones": nel (Ed.): Effects ofNois e onWildlife . a new termfo ra clas s ofbiologicall yac Academic Press,Ne wYork .p .169-208 . tive substances.Nature ,163:55-56 . Newcombe,C .8 G .Hartman ,1973 .Som echemi Kasumyan,A.0. 8N.E .Lebedeva ,1979 .Ne wdat a cal signals inth e spawning behaviouro f onth enatur eo fth ealar m pheromone incy - rainbowtrou t (Salmogairdneri) .J .Fish . prinids (Phoxinus phoxinus). Vopr .Ikhtiol. , Res. Bd.Canada ,30:995-997 . 19:890-895 (inRussian) . Nordeng,H. ,1971 .I sth e localorientatio n Keenleyside,M.H.A. ,1955 .Som easpect so f ofanadromou sfishe sdetermine d bypheromo the schooling behaviouro ffish .Behaviour , nes?. Nature ,233:411-413 . 8:183-248. Nordeng,H. ,1977 .A pheromon e hypothesis for Kinosita,H. , 1975.Schoolin g behaviouro f homewardmigratio n inanadromou ssalmonids . marine catfish eel (Plotosu sanguillaris) . Oikos,28:155-159 . InModer nBiologica lSciences ,vol.9 ,Iwa - Okada,H. ,D.K .Saka i 8K- iSugiwaka ,1979 . nami,Tokyo . Chemical stimuluso n thereproductiv ebe Kittredge,J.S. , 1974.Comparativ ebiochemi haviouro fth epon d smelt.Sc .Report sHok stry:marin ebiochemistry .In :R.N .Maris - kaido FishHatch. ,33:89-99 . cal (Ed.): Experimental MarineBiology . Dshima,K. ,W.E .Hah n SA .Gorbman , 1969.Ol AcademicPress ,Ne wYork .p .225-267 . factory discrimination ofnatura lwater sb y Laumen,J. , U.Per n 8V .Blüm ,1974 .Investi salmon.J .Fish .Res .Bd .Canada ,26:2111 - gations on thefunctio n and hormonalregu 2121. lation ofth eana l appendices inBlenniu s Partridge,B.L. ,N.R .Lile y 8N.E .Stacey , pavo (Risso).J . Exp.Zool. ,190:47-56 . 1976. Therol eo fpherdmone si nth esexua l Losey,G.S. ,1969 .Sexua l pheromones insom e behaviouro fth egoldfish .Anim .Behav. ,24 : fish ofth egenu sHypsoblenniu s Gill.Scien 291-299. ce, 163:181-183. Parzefall,J. , 1973.Attractio n and sexualcy Malyukina,G.A. , G.V. Devitsyna 8E.A .Maru - cleo fpoeciliids .In :J.H .Schröde r (Ed.): sov, 1974.Communicatio n on thebasi so f Genetics andMutagenesi s ofFish .Springer - chemoreception infishes .Zh .Obshch .Biol. , Verlag,Berlin ,p.357-406 . 35:70-79 (inRussian ). Perlmutter,A. ,D.A .Sarot ,M- LYu ,R.J .Fi - Malyukina,G.A. , A.D.Kasumyan ,E.A .Naruso v lazzola 8R.J .Seeley , 1973.Th e effecto f & N.I.Pashchenko ,1977 .Th ealar mpheromo crowding on theimmun erespons eo fth eblu e nean d its significance infis hbehaviour . gourami,Trichogaste r trichopterus,t oin Zh. Obshch.Biol. ,38:123-13 2 (in Russian). fectious pancreatic necrosis (IPN)virus . Mazeaud,F. , 1981.Morpholine ,a nonspecifi c LifeSei. , 13:363-375. attractant for salmonids.Aquaculture ,26 : Pfeiffer,W. ,1974 .Pheromone s infis h andam 189-191. phibia.In :M..C .Birc h (Ed.):Pheromones . flcCauley,R.W. ,1968 .Suggeste d physiological NorthHolland/America n Elsevier,Amsterdam , interaction among rainbow troutfingerling s p.269-296 . undergoing thermal stress.J . Fish.Res , Pfeiffer,W .8 J . Lemke,1973 .Untersuchunge n Bd.Canada ,25:1983-1986 . zurIsolierun g und Identifizierungde sSchr e McKaye ,K.R .8 G.W . Barlow, 1976.Chemica l ckstoffes ausde rHau tde rElritze ,Phoxinu s recognition ofyoun g by theMida scichlid , phoxinus (L.) (Cyprinidae,Ostariophysi ,Pi Cichlasomacitrinellum .Copeia ,1976:276 - sces). J.Comp .Physiol. ,82:407-410 . 282. Pfuderer,P. ,P .William s &A.A .Francis , 1974. Miles,S.G. ,1968 .Rheotaxi s of elverso fth e Partial purification ofth e crowding factor American eel (Anguilla rostrata)i nth ela from Carassius auratusan d Cyprinus carpio. boratory towate rfro m different streamsi n J. Exp.Zool. ,187:375-382 . NovaScotia .J . Fish.Res .Bd .Canada ,25 : Richards,I.S. , 1974.Cauda l neurosecretory 1591-1602. system:possibl erol e inpheromon eproduc Mykytowycz,R. , 1975.Som edifficultie si n tion.J . Exp.Zool. ,187:405-408 . the study ofth efunctio n and composition Richards,J.S. ,1975 .Estradio l receptorcon ofsemiochemical si nmammals ,particularl y tent inra tgranulos a cellsdurin g follicu wild rabbit,Oryctolagu s cuniculus.In : F.J. lardevelopment:modificatio nb y estradiol Ritter (Ed.): OdourCommunicatio n inAnimals . andgonadotropins .Endocrinology ,97:1174 - Elsevier/NorthHollan d BiomedicalPress , 1184. Amsterdam,p .105-115 . Ritter,F.J. ,1979 .Chemica l Ecology:odou r Myrberg,A.A. , 1966.Parenta l recognition of communication inanimals :genera lintroduc young incichli d fishes.Anim .Behav. ,14 : tion and overview.In :F.J . Ritter (Ed.): 565-571. Chemical Ecology: OdourCommunicatio ni n Myrberg,A.A. , 1975.Th erol eo fchemica lan d Animals.Elsevier/Nort h Holland Biomedical 93 Press,p .1-15 . 519. Rose,S.U. ,1959 .Failur eo f survival ofslo Yamazaki,F .â K .Watanabe ,1979 .Th erol e wlygrowin g members ofa population .Scien ofsteroi d hormones inse x recognitiondu ce,129:1026 . ring spawning behavior ofth egoldfis hCa - Rose,S.M . SF.C .Rose ,1965 .Th econtro lo f rassiusauratus .Proc .India n Natl.Sei . growth and reproduction infreshwate rorga Acad.Par tB Biol .Sei. ,45:505-511 . nismsb y specific products.Mitt .int .Ve Yu,M- L SA .Perlmutter- ,1970 .Growt hinhibi rein,theor .angew . Limnol.,13:21-35 . tingfactor s inth ezebrafish ,Brachydani o Rossi,A.C. ,1969 .Chemica l signals andnest - rerio and theblu egourami ,Trichogaste r building intw o specieso f Colisa (Pisces, trichopterus.Growth ,34:153-175 . Anabantidae].Monitor eZool .Ital. ,3:225 - 237. Selset,R. , 1980.Chemica lmethod s forfrac tionation ofodorant s produced by charsmo - ltsan d tentative suggestionß forpheromo - neorigins .Act aPhysiol .Scand. ,108:97 - 104. Smith,R.I.F. ,1973 .Testosteron e eliminates alarm substance inmal efathea dminnows . Can.J . Zool.,51:675-876 . Solomon,D.J. ,1977 .A reviw ofchemica lcom munication infreshwate r fish.J . FishBiol . 11:363-376. Stabell,O.B . &R.Selset ,1980 . Comparison ofmucu s collectingmethod s infis holfac tion.Act aPhysiol .Scand. , 108:91-96. Suzuki,N . &D .Tucker ,1971 .Amin o acidsa s olfactory stimuli infreshwate rcatfish , Ictalurus catus (Linn.). Comp.Biochem .Ph y sioi.,40A:399-404 . Swingle,H.S. , 1953.A repressivefacto rcon trolling reproduction offishes .Proc .Eigh t Pacif.Sei .Congr.,IIIA:865-870 . Tavolga,W.N. , 1956.Visual ,chemica lan d sound stimulia scue s inth e sexdiscrimi natory behavior ofth egobii d fishBathy - gobius soporator.Zoologic a N.Y.,41:49-65 . Tavolga,W.N. ,1971 .Soun d production andde tection.In :W.S .Hoa rS D.J . Randall (Ed.h FishPhysiology .Academi cPress ,Ne wYork . Vol.V:135-205. Tavolga,W.N. , 1976.Chemica l stimuli inre productive behaviouri nfish:communication . Experientia,32:1093-1095 . Thiessen,D.D . SS.K .Sturdivant ,1977 .Fema lepheromon ei nth eblac kmoll y fish (Mol- lienesia latipinna): apossibl emetaboli c correlate.J . Chem.Ecol. , 3:207-218. Timms,A.M . &H .Kleerekoper ,1972 .Th elo'co - motory responses ofmal eIctaluru spuncta - tus,th echanne l fish,t oa pheromon ere leased byth erip efemal e ofth especies . Trans.Am .Fish .Soc , 101:302-310. Todd,J.H. , J.Atem aS J.E .Bardach ,1967 . Chemical communication in social behaviour ofa fish ,th eyello wbullhea d (Ictalurus natalis).Science ,158:672-673 . Verheijen,F.J . &J.H . Reuter,1969 .Th eef fecto falar m substanceo nprédatio namon g cyprinids.Anim .Behav. ,17:551-554 .• Wilkens,H. , 1972.Übe rpreadaptatio n furda s Hohlenleben,untersuch t am Laiverhalten o- ber-und unterrirdischer Population desAst - yanaxmexicanu s (Pisces).Zool .Anz. ,188 : 1-11. Wrede,W.L. ,1932 .Versuch eübe rde rArtduf t derElritzen .Z .vergl .Physiol. ,17:510 - 94 THECONTRO LO FGONADA LANDROGE NBIOSYNTHESI S INFIS H D.E.Kim e Departmento f Zoology,Th eUniversity ,Sheffield ,S1 02TN ,Unite dKingdo m Summary ing.Smal lpiece so ftesti s (50-100mg ) wereincubate d in1 m lKrebs-Ringe rbicarb Theeffec to ftemperatur eo nreproductio n onatemediu m inth eabsenc eo fcofactors . infou r specieso f fish,Salm ogairdneri , Forincubation swit hradioactiv eprecursor s Carassiusauratus ,Sarotherodo nmossambicu s 1-2 uCio ftritiate d steroidwa sadde dt o andScyliorhinu s caniculus isdiscussed . themedium .Whe nendogenou sprecursor swer e Incubationso fteste swit hendogenou san d used,a preincubatio nwa scarrie dou tfo r exogenousprecursor s indicate that1 ) threehours ,afte rwhic hth emediu mwa sre utilisation ofexogenou s substrate placedb y freshmediu m containing salmon increaseswit h temperature,2 )competitiv e pituitary extract.Afte rthre ehour sth e formationo f 'inactive'steroid s athig h incubationmediu mwa sextracted ,separate d temperature limits 'active'steroi dforma intofre ean dconjugate d fractionsan d tiont oth enorma lreproductiv e temperature, chromatographedt oseparat e individual and 3)gonadotropi n stimulation ofsteroid steroids.Radioactiv eproduct swer eidenti ogenesis istemperatur edependent .Seasona l fiedan dquantitate db y scintillation studiesindicat etha t factorsothe rtha n counting;non-radioactiv eproduct swer e temperaturema y alsoaffec tglucuronid e quantitatedb yradioimmunoassay .Ful l synthesisan dthei rpossibl enatur ei s detailso fthes eprocedure sar edescribe d discussed. inothe rpublication s fromou rlaboratory . Keywords:reproduction ,fish ,temperature , androgens,glucuronides ,testis . Resultsan ddiscussio n Introduction Investigationshav e sofa rbee ncarrie d outo nfou rspecie so ffis h:th erainbo w Oneo fth emajo r factors influencing trout (Salmogairdneri) ,th egoldfis h reproductive developmenti nman y species (Carassiusauratus) ,th etilapi a (Sarother offis hi stemperature ,an dthi sha softe n odonmossambicus )an dth edogfis h (Scylio been considered toac tvi aa hypothalami c rhinus caniculus),an di neac hcas emajo r thermoreceptor andthenc ereleas eo fpitui changesi nth e steroidprofil ewer e found tarygonadotropi n to stimulate gonadal tooccu rove rth etemperatur e range steroidproduction .Recentl yw emad eth e examined. suggestion (Kime,1979 )tha ttemperatur e Inth etrou tan dgoldfish ,tw odistinc t could alsoac tdirectl yo nth e steroidogenic patternswer e apparentwhe n testistissu e enzymeso fth etesti st ogiv emaxima lstim wasincubate dwit hradioactiv e testosterone ulationo fsteroi dproductio n attempera overa rang eo ftemperatures . Firstly, tureswhic hwer eenvironmentall ymos tsuite d thetw ocompound swhic hmigh tb e considered forgonada ldevelopment .T otes tthi shyp asth echaracteristi c teleostandrogens , othesisw e incubated testes froma numbe r 11-ketotestosteronean dllß-hydroxytestos - of speciesa ta rang eo ftemperature san d teroneros e fromver y lowlevel sa t1 toa examinedth eeffec to ftemperatur e onth e plateaucorrespondin g verybroadl yt oth e naturean dth eyiel do fsteroid sproduce d temperature rangeove rwhic h reproductive frombot h endogenousan dexogenou spre developmentoccur san dthe na ttemperature s cursors.Som eo fthes e resultshav ebee n approaching theletha l limit forth e reported indetai lelsewher e (Kime,1979 ; speciesthe ydecline d againt olo wvalue s 1980;Kim e &Hews ,1982 ;Kim e &Hyder ,1982) , (Figurela) .Utilisatio n ofsubstrate ,how butw e consideri ti sno wopportun e tocom everincrease d steadilywit h temperature pareth eresult sobtaine d insevera l (Figurelb )an dthi s increase isaccounte d different speciesan dt odiscus swhethe r forb yth e second distinctpatter n found there isa genera lmechanis mb ywhic h forformatio no fsteroi d glucuronides environmental temperaturema yac tdirectl y (Figure lc). Inbot h speciesther e isa onth egona d toregulat e reproductive steadyincreas e informatio no ftestoster development infish . oneglucuronid e with increasei ntempera turewhic hi si ncontras tt oth eplatea u Materials andMethod s foundwit hth e11-oxygenate dandrogens . Since formationo fth etw oproducts , glucuronidesan d11-oxygenate d androgens Fish.wereobtaine d from commercial are incompetitio nw ehav e suggestedtha t sources,an da tth etim eo f incubation thepreferentia l formationo fglucuronid e were sexuallymatur ean dusuall yspermiat - 95 a) 19- 60 60- ; -> ' / \ \ '•S 8 Il H- -—^ 40 // \ ' / / / 20 20 / / / 20 40 0 20 40 20 40 Incubation temperature Figure 1.Th e effecto ftemperatur eo nth emetabolis m oftritiate dprecursor si nvitr ofo r four specieso ffish . (-- - trout , • •goldfish , dogfish, ....tilapia ) athighe rtemperature sma yb e aprotectiv e predominantly ofth eglucuronide so fth e mechanism toinhibi tandroge n synthesisan d 50-diolan dtestosterone .Th e formationo f hencereproductiv e development attempera 5ß-androstane glucuronides iso fparticula r tureswhic hma yb eto ohig h for successful interest sinceColomb oe tal . (1982)hav e reproduction. shownthes ecompound st ohav epheromona l Ina nelasmobranc h fish,th espotte d activity insome 'species .Sinc epheromone s dogfishScyliorhinu s caniculus,a ver y mustb e speciesspecifi c iti slikel ytha t similarmechanis m appearst ob eoperative . sensitivity tothes e compoundsha sbee n Whentesti stissu eo fthi sspecie swa s developed insom especie st oindicat eth e incubatedwit hradioactiv epregnenolone , presenceo fa potentia lmat ebu ttha tth e yieldso fth eelasmobranc h androgen,test primarypurpos eo fthes e 'inactive'metabo osterone,showe da broa dpea k inyiel d litesi si nsom ewa yconnecte dwit hregula around 11-16 .Agai nther ewa sa stead y tiono freproductiv ecycles . increase inutilisatio n ofsubstrat ewhic h Inmammals ,bot hglucuronide s andSB - wasaccounte d forb yth eincreasin gyiel d reduced steroidsar econsidere d tob e ofa nunidentifiabl ebu textremel ypola r primarilyhepati cmetabolite s servingn o steroid (Fig.1).Th ebalanc ebetwee nth e functionothe rtha ndeactivatio n and activeandroge n andthi sunusual ,an d excretion.I ti stherefor ever y surprising probablyinactive ,steroi dbear sa remark tofin dthe mproduce d insuc hlarg equan ableresemblanc e toth epatter n foundi n titiesb y fishtestes .Thi sfac tan dth e thetrou tan dgoldfish . differential effecto ftemperatur eo nth e The fourth specieswhic hw ehav eexamined , rateo fsynthesi so fth eactiv e andinactiv e thetilapi a Sarotherodonmossambicus ,agai n steroids (Figure2 )suggest stha tthes e fitted thisgenera lpatter n (Figure 1), but metabolitesma ypla y someprotectiv erol e witha rathe rdifferen tvariation . inth ereproductiv e endocrinologyo f species Utilisation oftestosteron e substrate again sucha sfis hi nwhic henvironmenta l andbod y increasedwit htemperature ,bu tth eyield s temperature varyove ra considerabl erange . ofth e11-oxygenate dandrogen swer e fairly Weca nthu s summarise the findingso fou r constantove rth etemperatur e rangechose n invitr oexperiment swit hradioactiv epre (15-40) althoug h 11-ketotestosteronedi d cursorsi na genera l formapplicabl e to showa smal lbu tsignifican tdro pi nyiel d allfou rspecies :1 )th emetabolis mo f from 30-40 .Onceagai nw e found thatth e precursor toproduct s increaseswit hin glucuronides increasedwit htemperature , crease intemperature ,2 )optimu m formation but inadditio nw e foundformatio no fsub ofactiv eandroge n correspondsver ybroadl y stantialquantitie so f5ß-androstane-3a , toth etemperatur e forreproductiv e develop 17ß-diolwhic h followeda simila rbu tles s mento fth e speciesan ddecrease s abovean d steeppatter nt otha to fth eglucuronides . belowthi srange ,an d 3)formatio n ofa n Althoughw eha dmeasure dtota lglucuronide , inactivemetabolit e increases steadilywit h wehav e showntha tthi s fractionconsist s increase intemperatur e anda thig h 96 /dogfis h portionso fsteroid swit htemperatur ean d theincreas ei nglucuronid ea thighe rtemp erature,i tdiffer sfro mth eexperiment s with exogenous steroid inth emuc hmor e definedpea k intota lsteroi dformation . / ' goldfish Thisindicate stha ttemperatur ema ypla ya n htrou t evenmor e important rolei non eo fth eearl y stageso f steroidogenesis,possibl ya tth e stageo fgonadotropi n stimulated cleavage ofth echolestero l sidechain . Figure3 clearly showstha tfo rth e sameamoun to f gonadotropin stimulation,yield so ftota l / //' ytilapia unconjugated androgensar e fourtime s greatera t22 °tha na t40 ° (9.6time si f \ only testosterone isconsidered) .I nvitr o incubations,eve nwit hth eus eo fendogenou s AV precursors,ma yno tb ea tru e representation ,<>•"/ / ofth e situationi nviv obu tpreliminar y resultswit hthre e species,Salm ogairdneri , Cyprinus carpioan dSarotherodo nmossambicu s indicatetha tth erelativ eproportio no f glucuronide isgreate r infis hhel da t \ - ' highertemperature s thani nthos ea tth e lowertemperature ,thu sconfirmin g thei n vitroresult s (Kime& Manning ,Unpublishe d Temperature observations). Ourstudie shav ethu sshow ntha tth e directeffec to ftemperatur e onth etesti s Figure 2.Th eeffec to ftemperatur eo nth e mayaffec tbot hth erelativ eproportion so f ratioo finactive :activ e steroidi nvitro . thesteroid s formed andth etota lamoun to f temperatures isth epredominan tmetabolite , steroidproduce dpe runi to fgonadotropin . theseresult sar econsisten twit h1 )Comp Itals o followstha tth erelativ eproportion s etitive inhibitiono fformatio no fth e ofgonadotropi n andandroge n inplasm awil l biologically active steroids atto ohig ha varyconsiderabl ywit htemperature . Temp- temperature forsuccessfu lreproduction , and2 )tha ti nth eabsenc eo fthi scompet itivesteroid ,androgen swoul db eforme d optimally ataroun d theletha ltemperatur e forth especies . Theexperiment stha tw ehav e sofa r describedhav eIn-rolve dth eus eo fradio activeprecursors ,bu t formationo f steroids fromendogenou sprecursor smigh tb eexpecte d tosho wth eeffec to ftemperatur e atearlie r stageso fsteroidogenesi s andmigh tb e nearert oth e situationi nvivo .Preliminar y experimentswit hradioisotope s are,however , essential forth e identificationo fth e steroids formed sotha t suitableassay sca n be devisedwhe nw eexamin eendogenou sprod ucts.W ehav e sofa ronl yexamine d formation ofendogenou s steroids inon especies , Sarotherodonmossambicus .Th eeffec to f temperaturewa s foundt ob erathe rdifferen t totha tobserve dwit hexogenou sprecursor s andal lsteroid s showed amaximu m yielda t 22 witha stead ydro pt o4 0 (Figure3) . Glucuronides alsopeake d atthi stemperatur e butthei rrelativ eyield sincrease d at highertemperature s atwhic hthe ywer e Temperature comparable tofre e steroid formation.Un fortunatelyn oantiseru m isavailabl e for measuremento fth e5ß-dio lan d itsglucer - onidewhic hw ewoul dhav eexpecte d tofollo w Figure 3.Th eeffec to ftemperatur eo nfre e a similarris ewit htemperature .Althoug h andconjugate d steroid formation fromen thisexperimen tconfirm s the change inpro dogenousprecursor si nteste so fSarothero donmossambicu s (- free, glucuronide). 97 eraturema yals oaffec tothe rendocrin epro ofhabitat san dbreedin g conditions infis h cessessuc ha shepati c catabolism (Kime& iti slikel ytha tth erelativ e importance Saksena,1980) , feedback ofgonada l steroids ofthes edifferen tfactor swil lvar ycon onth epituitary ,steroi d actiono ntarge t siderablybetwee nspecies . tissue (Dasmahapatrae tal.. ,1981 ), recep toran dprotei nbinding ,an dhormon esynth Acknowledgement esisb yth ehypothalamus ,pituitary ,adrenal * thyroidan dpineal .Wha tha softe nbee nre Parto fthi swor kwa s supportedb ya ferred toa sth e 'hypothalamic thermorecep grant,GR/A/94843 ,fro mth e Sciencean d tor'ma yi nfac tb ea temperatur e dependent EngineeringResearc hCouncil . enzyme system.Th e effecto ftemperatur eo n reproduction infis hwil lb e areflectio n References ofal lo fthes eindividua lfactors . Colombo,L. ,A .Marconato ,P .Colomb o Todetermin ewhethe rglucuronide splaye d arol ei nseasona l changesi nreproductiv e Belvedere &S .Raineri ,1982 .Pheromone s activityw ehav emeasure dplasm a levels inteleos t fish.Thi svolume . ofbot h freean dconjugate d androgens Dasmahapatra,A.K. ,A.K .Ra y &A.K .Medda , duringth e seasonal cycleo fth ebrow n 1981.Temperatur e dependentactio no f troutSalm otrutt a (Kime& Manning ,1982 ). estrogeni nSing ifis h (Heteropneustes We foundtha tplasm a levelso fglucuronide s fossilisBloch) .Endokrinologi e 78:107 - increased rapidly asspawnin gapproache d 110. and,afte r spermiation,level so fthes e Dutton,G.J. , 1980.Glucuronidatio no f conjugatescoul d exceedthos eo fth efre e drugsan dothe rcompounds .CR CPress , steroid.Sinc eglucuronide s areexcrete d BocaRaton ,Florida . approximately tentime smor e rapidlytha n Kellie,A.E .& E.R . Smith,1957 .Rena l thefre esteroi d (Kellie &Smith , 1957), clearanceo f17-ox osteroi d conjugates thisindicate sgonada l secretion tob eo f formed inhuma nperiphera lplasma . majorimportance .Thi sincreas ei nglucur - Biochem.J.66 :490-495 . onide secretion,howeve rcorrespond st oa Kime, D.E.,1979 .Th e effecto ftempera seasonal falli nenvironmenta l temperature tureo ntesticula r steroidogenic enzymes andthu sappear st ocontradic t thefinding s ofth erainbo wtrout ,Salm ogairdneri • both invitr oan di nfis hhel d atdifferen t Gen.Comp.Endocrinol .39 :290-296 . temperatures.I twoul d therefore appear Kime,D.E. ,198t> .Androge nbiosynthesi sb y likely thatothe r factorsapar tfro m testeso fth egoldfis h (Carassiusauratus ) temperature,ma ywel lals oregulat eglucur - invitr o- th eeffec to ftemperatur eo n onide synthesisan dtha tglucuronide sma y theformatio no f steroidglucuronides . play somerol e inth enorma l reproductive Gen.Comp.Endocrinol.41 :164-172 . functioning infish .I na recen textensiv e Kime.E., &E.A.Hews ,1982 .Th eeffec to f review,Dutto n (1980)ha sdescribe d alarg e temperature onsteroi dbiosynthesi sb y numbero ffactor swhic hhav ebee n shownt o testeso fth edogfish ,Scyliorhinu s actdirectl yo nth eglucurony l transferase caniculum.Comp.Biochem.Physiol.71B : complex,bu tunfortunatel y themajorit yo f 675-679. thiswor k isconcerne dwit hmammalia n liver Kime,D.E.,& M.Hyder ,1982 .Th eeffec to f tissue andit svalidit y toteleos ttesti s temperature andgonadotropi n ontesticula r mayb equestioned . Someo fthes e factors steroidogenesis inSärotherodo n (Tilapia) whichma yb ewort h considering,however , mossambicusi nvitro .Gen.Comp.Endocrinol . are theeffec to fse xan dadrena l steroids, InPress . phospholipids,unsaturate d fattyacids ,an d Kime,D.E.,S N.J .Manning ,1982 .Seasona l somemeta lions .Sinc econcentration so f changesi n freean dconjugate d androgens someo fthes e factorshav ebee n shownt oin inth ebrow ntrout ,Salm otrutta .Gen.Comp . creasei nteleos tplasm adurin g sexual Endocrinol.46 :I nPres s maturation,i ti spossibl e thati nsom e Kime,D.E.,& D.N . Saksena,1980 .Th eeffec t speciesthe yma ypla y arol e inregulatin g oftemperatur e onth ehepati c catabolismo f thebalanc eo fgonada l steroids,eithe rb y testosterone inth erainbo wtrou t (Salmo affecting theenzym eactivit yo rit ssyn gairdneri)an dth egoldfis h (Carassius thesis.Th eincreas e inglucuronid e forma auratus).Gen.Comp.Endocrinol .42 :228-234 . tionwit h sexualmaturatio n iso fparticula r interestwit hrespec tt oit spossibl ephero - monalrol ei nsom especie s (Colomboe t al., 1982). A fullerunderstandin g ofho wthes efac torsma y affectgonada lsteroidogenesi si n fishan dthei rrelationshi p toth ereprod uctivedevelopmen tan dmaturatio n ofth e speciesma ylea dt omethod s forth earti ficialmanipulatio no freproductiv e cycles inaquaculture .Wit h thewid e diversity 98 STEROIDOGENESIS INTH EOVARIE S OFTH EAFRICA N CATFISH,CLARIA SLAZERA ,BEFOR EAN DAFTE RA N HCG INDUCED OVULATION J.G.D.Lamber t andR .va nde nHur k ZoologicalLaboratory ,Sectio nfo rComparativ eEndocrinology ,Stat eUniversit y ofUtrecht , Padualaan 8,350 8T B Utrecht,Th eNetherland s Theafrica n catfish,Çlaria s lazeralend s givenegativ eresult s forthi senzyme .Thi s itselfadmirabl y tofis hcultur eo naccoun t indicates that,precedin g oocytematuratio n of itshardiness ,it somnivorou s character theovaria n steroidogenic potency isres and itsabilit y toliv ei npoorl y oxygenated tricted toth e special thecacells . water (Richter, 1976).Unde rnatura lcondi 6Hour safte rHCG .Th e first signo fmatu tions thisfis hshow s anannua l reproductive rationbecome svisible .Th enucleu s orger cycle,whic h culminates ina spawnin g oncea minalvesicl eha smigrate d toth emicropyle , year.I nfis hculture ,i ti sdesire d tohav e which issituate d near thecente ro f thefu youngbroo d throughout theyear .A t thede tureanima lpole .A fe whour s latergermina l partment ofFis h Culture and InlandFishe vesiclebreakdow ntake splac ean d thecylin rieso f theAgricultur eUniversity ,Wage drical cellso f thegranulos abegi n tose ningen,Th eNetherlands ,catfis hwer e cul cretea nattachmen tdisk ,whil ea tth esam e tured,whic har eabl e todevelo pvitellogeni c timeth egranulos a cellsbecom esmaller .N o oocytes atan ydesire d time.Unde r thesela changei n35-HS Dactivit y canb eobserved . boratory conditions,maturatio nan dovula 10Hour s afterHCG .Th eformatio no fth e tionhav e tob e inducedb y atreatmen twit h attachment diskha sbee ncomplete d andth e gonadotropins,suc ha shuma n chorionicgona granulosano wconsist so fNearl y flatcell s dotropin (HCG)o rcar ppituitar y suspensions (fig. la).Fro m thismomen t onwards achang e (Eding et al., 1982).Wha thappen s inth e inlocalizatio n of 38-HSDca nb eobserved . ovaryafte r sucha treatmen t isunknown . Besides thespecia l thecacells ,th egranu Therefore,w e studiedwit henzymecytochemi - losacell s ofth ematurin g follicles showa calan dbiochemica lmethod s thesteroidoge positive3g-HS Dactivit y (fig.lb) . nesis inth eovarie sbefor ean d aftera nin 16Hour s afterHCG .Man ymatur e oocytes ducedovulatio nb yHCG . havebee nevacuate d from their follicular envelopes towards thelume no f theovary , To induceovulatio nadul t femalecatfis h butovipositio nha sno t takenplace .S oa withrip eovaries ,age d 10-12month swer e masso fovulate d ovai sfoun d inth eovaria n treated oncewit h 4I.U .o fHC Gpe r gramo f cavities,an dnumerou s postovulatoryfolli fish.Sixtee nhour safte rHC G injectional l clesca nb eobserve d inth eovaria nwall . theanimal shav eovulated .A t 0,6 , 10,16 , The33-HS Dactivit y remains inth egranulos a 28, 36,4 8an d 72hour s afterHC G injections cellsbu t these cellsno wbelon g toth e respectively twoanimal swer e anaesthetized postovulatory follicles. and theovarie swer eremove d andprepare d 28,36 ,4 8an d 72Hour s afterHCG .Durin g forhistologica l (Richter &va nde nHurk , theseperiod s thepostovulator y follicles 1982),enzymecytochemica l (vande nHur k& are stillpresen t inth eovar ybu t theyd o Richter, 1980)an dbiochemica l (Lambert& not showa constan t 36-HSDactivity .Fro m vanBohemen ,1979 )studies . ovulationonward s the36-HS D activityde creases slowlyan d 48hour s afterHCG ,abou t Morphological and enzymecytochemical results 30hour safte rovulation ,activit y cann o longerb eobserve d inth egranulos acell so f Theovarie so f control animals contain thepostovulator y follicles.Th especia l largepostvitellogeni c follicles.Thes efol thecacells ,however ,ar estil lactive . licleshav e completed theirvitellogenesi s andhav ereache d adiamete ro f 1000-1200urn . Invitr o incubationswit h H-pregnenolone Thenucleu s islocate d centrally.Th egranu losacell s of thesefollicle s constitutea The steroid synthesizing capacity ofth e thickened ringo f cylindricalcell saroun d ovarieso f catfishbefor ean d afterovula theregio no fth efutur eanima lpole ,whil e tionha sbee nstudie db y incubating ovarian thecell s at theopposit e sidear e smallan d homogenateswit h tritiated pregnenolonet o cubic.Fo r tracing thesit eo fsteroi dsyn determine A5 and A4 pathway steroids andi n thesis thecytochemica l localization ofth e particular theso-calle d oocytematuratio n enzyme36-hydroxysteroi d dehydrogenasei s inducing steroids:deoxycorticosteron e and used.3g-HS Dactivit y islocate donl yi nspe 17a-hydroxy,20ß-dihydroprogesterone.Th e cial thecacell s (stromacells )aroun dvi bioconversion towate r soluble compounds i.e. tellogenic andpostvitellogeni c oocytes.Th e testosteroneglucuronide has alsobee nstu granulosa cells,althoug hwell-developed , died,an d itappeare d thatfro movulatio n 99 X _. *^wrr^ •f Fig. 1. 3&-HSD activity in the ovary of Ciarias lazera. a. Ovary 10 hours after HCG injec tion; maturating follicles with a well-developed attachment disk (AD). (56x). h. Ovary 10 hours after HCG injection; 38-HSDactivity in granulosa cells (GR) and special theca cells (STC). (56x). c. Ovary 16 hours after HCGinjection; postovulatoty follicles (POF). (185x). d. Ovary 16 hours after HCGinjection; 3&-HSD activity in granulosa cells of the postovula- tory follicles and in special theca cells. (18Sx). onwardsth eradioactivit ywa srathe rhig hi n HCG treatment effectsa shif ti nsynthesis . theremainin gwate r fractions afterorgani c Incontro lanimal s theyiel do fdehydroepian - extraction. drosteronei shigh ,whil eth eyiel do f17a - After incubationth esteroid swer eisola hydroxyprogesterone israthe rlo wa ttha tmo tedan dpurified ,usin g thinlaye rtech ment.Bu tb y6 hour s afterHC Gtreatmen ta niques,derivativ e formationan drecrystal - complete shiftca nb eobserved .Thi s indi lizationt oconstan t specific activity.Th e cates thatgonadotropi n stimulates the17a - quantitative data,th epercentage so fyield , hydroxypregnenolonespecifi c 3E3-HSDand/o r wereobtaine da )b ycalculatio no nth ebasi s inhibits the17a-hydroxypregnenolon especifi c ofprecurso r initial radioactivityan dcor 17a,20-C21desmolase . rectedfo rprocedura l lossesb ya G Crecove Deoxycorticosterone,a hormon e thatca nin rydeterminatio no fth eadde d carriers,an d ducematuratio ni ncatfis hi nvivo ,i sno t b)b ycalculatio no fth epercentag edistri formed.Therefor eth ematuratio n effecto f butiono fth eradioactivit ywhic hi srepre deoxycorticosterone ispharmacologica lan d sentedb yth earea so fth epeak so nth era notphysiological .However ,i tseem st ob e dioscanso fth ethi nlaye rplates . that 17a-hydroxy,20ß-dihydroprogesteronei s Theresult so fth epregnenolon e incubation thematurationa l factora sa nincreas ei n are summarized infig .2 .I nal lth eincuba synthesiso fthi s steroidca nb eobserve d tions carriedou tpregnenolon eha sbee n justbefor eovulation . largelymetabolized ,a sonl ya littl eo f Theovulatio nproces sca nals ob ecorrela thisprecurso ri sleft .Th econversio nt o tedwit ha chang ei nandroge nmetabolism . progesterone isnearl y zero.Thi sma yB edu e Thedecreas eo ftestosteron ema yb ecause db y toa rapi d conversiont oothe rproduct so r adecreas ei n173-HS Dactivit ya sandrostene - toa lac ko fpregnenolon e specific 3B-HSD.A dioneaccumulates ,an db ya conversio no f lacko f3B-HS Di sth emos tplausibl eexpla testosteronet oit sglucuronide . nationa sincubation s forshorte r timepe riodsneve r showa large ryiel do fprogeste Acompariso no fth eenzymecytochemica lre rone.Th ebiosyntheti c pathwaymus tthere sultswit hth ebiochemica l dataoffer sth e foreg ovi a17a-hydroxypregnenolone .Thi si s possibility toge tmor e information aboutth e confirmedb yth eresult so fincubation swit h steroid synthesizing potencyo feac ho fth e shorter timeperiods . 3J5-HSDpositiv e structures.Th especia l theca 100 30 ,* 20 10 PREGNENOLONE PROGESTERONE DEOXYCORTICOSTERONE t*»-\ Tto(OH) PROGESTERONE 17ff(0H) ,20/SD!HYDROPROGESTERONE i# *V*VV / DEHYDROEPIANDROSTERON E ANDROSTENEDIONE TESTOSTERONE TESTOSTERONE - GLUC. Fig. 2. Bioconversions in ovaries of Claviae lazera 0, 6, 10, 16, 28, 36, 48 and 72 hours after HCG injection. Percentages of yield of steroids isolated from incubations of ovarian homogenates with pregnenolone-?'a-3'H. • before ovulation; •after ovulation. cellsar epresen t incontro l animalsa swel l Identification of 17a-hydroxy,20ß-dihydro- asi nanimal s treatedwit hgonadotropins , progesterone inplasm ab y GC-MS. and they arealway s 3B-HSDpositive .Thi s may indicate that thesecell s arerespon 17a-Hydroxy,20(3-dihydroprogesteron eca nb e siblefo r thesynthesi s of thosesteroid s synthesized by theovar y justbefor eovula which aresynthesize d throughout theannua l tion.Wit hpreliminar y experimentsw ehav e cycle (androgens).However,"th egranulos a demonstrated thepresenc eo f this steroid in cells around theoocytes ,a swel l asi nth e theplasm ao f theafrica ncatfish .Thes eex postovulatory follicles areinvolve d in perimentswer e carried outwit h agaschroma - steroid synthesis only during specialphy tographical-mass spectrometricalmethod . siological processes for instance during 17a-Hydroxy,20ß-dihydroprogesteronei sderi - maturationwit h 17a-hydroxy,20ß-dihydropro- vatized toit smethoxime-d iTM S compound. gesterone formation,an d duringvitelloge - From theelectro n impact (70eV )fragmenta nesiswit ha nestroge n synthesis (vanBohe tionspectru m itappeare d that themolecula r men &Lambert , 1981). The 3ß-HSDactivit y in ionpea k issmal lbu t that theion s 388an d thegranulos a cells of thepostovulator y 298ar echaracteristic .Io n38 8i sth etota l follicles shouldb econsidere d asa residua l moleculewithou t C20-C21 sidechai n (fig.3) . activity of thegranulos a cellsbefor eovu Following both these ionsb y selected ion lation.Th e formation of testosteroneglucu - monitoring after capillarygaschromatograph y ronideafte rovulatio n canb ecorrelate dwit h of theextracte d steroids from theplasm ao f thepresenc eo fpostovulator y folliclesbu t catfish 28hour s afterHC G injectioni t notwit h the 3(5-HSDactivit y inthes efolli appeared thatbot h ionswer epresen tan d cles. Soi tmigh tb epossibl e that theenzym e coincidewit h theretentio n timeo f thestan glucuronosyl transferase is located inth e dard 17a-hydroxy,20(3-dihydroprogesterone(M0 - granulosa cells of thesepostovulator yfolli diTMS ) (fig.4) .Thes e ionscoul dno tb e cles.Th e significance of theseglucuronide s detected inplasm ao f controlanimals . isunknown .The yma yb eo f importance ina biological inactivation of steroids assug gestedb yKim e (1980)o r theyma yb efunc Acknowledgement tioning as sexpheromone s (vande nHur ke t al., 1982;Colomb o etal. , 1982). Theauthor s thankMrs.J.CM .Granneman ,Mr . G.M.H.Engel s andMr.V.C.M.M .d eNij sfo r 101 17atOH ) ,20/JOlHYDROPROGESTERONE - ra (C.& V.) . This symposium. MO— dITMS vande nHurk ,R. ,L.A . 'tHart ,J.G.D .Lam 298 bert &P.G.W.J ,va nOordt , 1982.O nth e regulationo f sexualbehaviou r ofmal eze - brafish,Brachydanio ,rerio .Gen .Comp .En Ml IIIM L I Ui lulu Illn . docrinol.46 :403 . 288 vande nHurk ,R . &C.J.J .Richter , 1980.His - tochemical evidence forgranulos a steroids infollicl ematuratio n inth eafrica ncat fish,Claria s lazera.Cel lTissu eRes . 211:345-348 . Kime,D.E. , 1980.Androge nbiosynthesi sb y testis of thegoldfish ,Carassiu sauratus , invitro :Th e effecto f temperatureo nth e formationo f steroid glucuronides.Gen . Comp.Endocrinol .41 :164-172 . Fig. 3. Mass spectrum (EI) of 17a-hydroxy, Lambert,J.G.D . &Ch.G .va nBohemen ,1979 . 20^-dihydropvogesterone-methoxime-di 'TMS. Steroidogenesis inth eovar yo f therain bow trout,Salm ogairdneri ,durin g there productive cycle.Proc .Indian ,natn .Sei . Acad.B45 :414-420 . I OH = 298.45 Richter,C.J.J. ,1976 .Th eafrica ncatfish , F.S,= '36 Clarias lazera (C.& V.) , ane wpossibili tyfo rfis hcultur e intropica l regions? 1 In:Aspect so f fishcultur ean d fishbree ding.Ed .E.H .Huisman .Miscellaneou spa i i pers 13,Landbouwhogeschool ,Wageningen , TheNetherlands . I UN = 388.45 Richter,C.J.J .& R .va nde nHurk , 1982.Ef F.S.= 99 fectso f 11-desoxycorticosterone-acetate and carppituitar y suspensiono nfollicl e maturationi nth eovarie so f theafrica n catfish,Claria s lazera (C. &V.) . Aqua- culture 28,i npress . 20 25 30 Fig. 4. Selected ion monitoring •profiles at m/e 388 and 298 as recorded with a HP S992B. GC-MS during SIM analysis of the organic fraction of plasma (Clarias lazera) after methoxime and TMS derivatization. theircompeten t technical assistence,Dr . C.J.J.Richte r (Department ofFis h Culture and InlandFisheries ,Agricultur eUniversi ty,Wageningen ,Th eNetherlands )fo rth e supply of catfish.HC Gwa s agif tfro mIn terpretInternationa l B.V.,Boxmeer ,Th e Netherlands. References vanBohemen ,Ch.G .& J.G.D . Lambert,1981 . Estrogen synthesis inrelatio n toestrone , estradiol,an dvitellogeni nplasm alevel s during thereproductiv e cycleo f thefe malerainbo wtrout ,Salm o gairdneri.Gen . Comp.Endocrinol .45 :105-114 . Colombo,L. ,A .Marconato ,P .Colombo-Belve dere &S ,Raineri ,1982 .Pheromone s inte - leostfish .Thi s symposium. Eding,E.H. ,J.A.L . Janssen,G.H.J .Klein e Staarman& C.J.J .Richter ,1982 .Effect so f humanchorioni c gonadotropin (HCG)o nma turationan d ovulationo foocytes -i nth e ovaryo f theafrica ncatfis- hClarias -laze - 102 PLASMALEVEL S OFSE XSTEROID S INRELATIO NT OOVULATIO NAN D SPERMIATION INRAINBO WTROU T (SALMOGAIRDNERI ) A.P .Scott ,S .M .Bayne s MAFF,Directorat e ofFisherie sResearch ,Fisherie s Laboratory, PakefieldRoad ,Lowestoft ,Suffol kNR3 3OHT ,U.K . Summary (3)C2 1 -*C1 9desmolase ,(4 )aromatase , (5)17ß-hydroxysteroi ddehydrogenase ,(6 ) Thephysiologica l eventsassociate d with 11ß-hydroxylase,(7 )11ß-hydroxysteroi d bothovulatio nan d spermiatlon inrainbo w dehydrogenase. Iti sassume d inthi sstud y trout involve afal li nplasm alevel so f that therelativ eactivit y of theseenzyme s androgensan da concomitan t risei nplasm a isth emajo rinfluenc eo nplasm alevel so f levels ofprogestagens . Onepossibl e sexsteroids . Gonadal formationo fsteroi d explanation istha tgonadotrpphi nblock s glucuronides (Kime,thi svolume )an d pos androgenbiosynthesi s inth egonad sb y sibledifferentia l rateso freleas ean d inhibiting C21 •*C1 9desmolase ,thereb y metabolism ofsteroid shav eno tbee n causing thebuild-u p of 17a-hydroxyprogeste- investigated. ronean d its 20ß-hydroxylatedderivative , Oneobservatio nmad ei nthi sstud ywa s 17a,20ß-dihydroxy-4-pregnen-3-one . Inth e that spermiatingmal efis hha d substantially male rainbowtrout ,th ehighes t levelso f elevated levels of 17a,20ß-dihydroxy-4 - 17a,20ß-dihydroxy-4-pregnen-3-on ehav ebee n pregnen-3-one. Thissteroi dha sbee nimpli foundi nspermiatin g fish. Wepropos etha t cated inth econtro l ofoocyt ematuratio n in amajo r function ofthi ssteroi d inth emal e femalerainbo wtrout . Wesugges ta functio n isth econtro lo f theK compositiono f for iti nth emale . seminalfluid . Methods Keywords:Salm ogairdneri ;spermiation ; ovulation;se xsteroids ;17a ,20ß-dihydroxy - Females 4-pregnen-3-one. Detailso fth eexperimenta lwor ko nfemal e rainbow trouthav e beenreporte d previously Introduction (Scotte t al., 1982),o rar eawaitin gpubli cation (Scotte t al.,i npress) . Thispape r describes thechange s inplasm a levelso fse xsteroid s that takeplac e Males around the timeo fovulatio n and spermiation Agrou po f 16mal erainbo wtrou t inrainbo wtrout . Thesteroid sw ehav e (18-monthsold )wer ekep t incircula r tanks investigated (shownunderlined )for ma par t suppliedwit hrive rwater . Allmil twa s of the A1*biosyntheti c pathway: stripped outan d collected attwo-wee k intervals. Blood sampleswer e takenever y Progesterone fourweeks . Measurementswer emad eo f (D| (2) volumeo fmilt ,spermatocri tvalue ,an d sodium andpotassiu m ionconcentration s in 17a-hydroxyprogesterone—»-1 7q ,20ß-dihydrox y seminalfluid . Spermdensit y and total 4-pregnen-3-one sperm countwer eestimate d from the (3) spermatocrit. Androstenedione ••Oestron e (?) Experimental (5)J (4) Threegroup so fspermiatin gmal e rainbow Testosterone- 17ß-oestradiol (?) trout (c.100 0g weight )wer e injected (6)| intraperitoneally with 3.0,0. 3 or0 m go f 11 ß-hydroxytestosterone(< 0 17a,20ß-dihydroxy-4-pregnen-3-on edissolve d (7)\ in50 0 |ilo fa polypropylen eglycol ,0.9 % 11-ketotestosterone ( 103 Results Ovulation (a) Ovulationan dth eevent simmediatel y preceding it (i.e.oocyt ematuration )wer e associated witha fal li ntestosterone , androstenedione and 17ß-oestradiollevels , anda nabrup tris ei n17a-hydroxyprogester - onean d 17a,20ß-dihydroxy-4-pregnen-3-on e levels (Fig.1) . (b) Injectiono fchu msalmo npituitar y powder extracts intomatur e femalerainbo w troutraise d plasmalevel so f 17a,2 0ß - dihydroxy-4-pregnen-3-one and lowered plasma levelso ftestosteron e (Fig.2) . Spermiation (a) Spermiation (theperio dwhe nsper m couldb eexpresse dmanuall y frommal e fish) Days lasted for6- 7 months inth eparticula r straino frainbo w troutw e studied (Fig.3) . Fig.1 . Changesi nplasm asteroi d levelso f Thehighes tandroge nlevel scoincide dwit h femalerainbo w trout around the timeo f thebeginnin g of thisperio d (November). ovulation. 17a,20ß-dihydroxy-4-pregnen-3-on elevels , however,peake d severalmonth slate r (March) whensper mreleas ewa sa tit smaximum . 800 INDUCED OVULATION 17a-hydroxyprogesteronean d17ß-oestradio l levels (notshown )di dno tvar ysignifi cantly. Androgenlevels ,whic hstarte d to riseagai ni nJune/Jul yan d accelerated sharply inSeptembe rwer e correlated with thegonadosomati c index,an dwit h thepro- ' portion of spermatocytes inth etestis , butwer eno t correlatedwit han yo fth emil t parameters. 17a,20ß-dihydroxy-4-pregnen-3 - onelevel s correlatedwit hvolum eo fmil t (r= 0.45),tota lsper m count (r= 0.43) ,K + content (r= 0.48 )an dK +/Na+rati o (r=0.52 )i nsemina l fluid (allP < 0.001 , n =67 ;steroi d levelslog . transformed) (b) Injections of 17a,20ß-dihydroxy-4 - pregnen-3-one intospermiatin g fishsignifi cantlyraise d theK +/Na+rati oo fth e seminalflui d (Fig.4 )bu tdi dno taffec t thevolum eo fmil t ortota lsper mcount . A linearrelationshi p existsbetwee nth e concentrations ofK + and Na+ insemina l fluid. Theregressio nequatio nfo rcalcula tingpotassiu m ionlevel s (calculated from thedat acollecte d onday s0 ,2 ,4 an d 6o f theexperiment )wa s (0.240x sodiumio n concentration)- 0.973 . Thecorrelatio n coefficientwa s 0.83 (n= 84), andth e interceptwa sno t significantly different from zero. Thislatte rfac tmean s that observed-expected K+ concentrations (as showni nFig .4 )an dK +/Na+ratio s (referred toabove )ar eequivalen tmeasurements . Fig.2 . Responseo fplasm alevel s of 17a, 20ß-dihydroxy-4-pregnen-3-onean dtesto sterone tointraperitonea l injectionso f salmonpituitar yextrac ti nthre efish . Ovulationoccurre d betweenday s7 an d10 . Arrowsindicat einjectio ndays . 1stinjec tion,1. 5 mgpowde rkg -1; 2ndinjection , 6m gpowde rkg -1 bodyweight . 104 Discussion Ourdiscussio nwil lb elimite d toth etw o topicswhich ,w efeel ,ar eo fmos tinterest : (1)th efal li nplasm a androgen levelsa t spermiationan dovulation ,an d (2)th érol e of 17 105 havefoun dlevel s ashig ha s6 0n gml - In References somerainbo wtrou tmales . Wehav eals o clearly demonstrated thatelevate d levelso f Arai,R .& B .Tamaoki , 1967.Steroi dbiosyn 17a,20ß-dihydroxy-4-pregnen-3-on ear easso thesisi nvitr ob yteste so frainbo w ciatedwit hspermiation . Since,b ythi s trout,Salm ogairdneri .Gen .Comp . stage,al lmeioti c andmitoti c divisions Endocrinol.8:305-31,3 . havebee ncomplete d andmos t ofth esperma Baynes,S .M. ,A .P . Scott& A .P .Dawson , tozoaar efull yformed ,a rol efo rthi s 1981.Rainbo wtrout ,Salm ogairdneri , steroid inth econtro lo fsom easpec to f spermatozoa:effect so f cations andp Ho n spermatogenesis (assuggeste d byDepêch e& motility.J .Fis hBiol .19:259-267 . Sire,1982 )woul d seemunlikely .Ou rinitia l Campbell,C .M. ,A .Fostier ,B .Jalaber t& thoughtswer e that 17a,20ß-dihydroxy-4 - B.Truscott ,1980 .Identificatio nan d pregnen-3-onemigh t controltesticula r quantification ofsteroid s inth eseru mo f hydration (cf.th ehydratio no foocyte s that rainbowtrou tdurin gspermiatio nan d takesplac e concurrentlywit hmeioti c oocytematuration .J .Endocrinol .85 : maturation),an d that injectiono fthi s 371-378. steroidwoul d increase thetota lvolum eo f Depêche,J .& 0 .Sire ,1982 . Invitr ometa - themilt . However,althoug h therewa sa bolismo fprogesteron e and 17a-hydroxy - significant correlationbetwee nplasm a progesterone inth etesti so fth erainbo w steroid levels and spermvolume ,injection s trout,Salm o gairdneri,a tdifferen t of 17a,20ß-dihydroxy-4-pregnen-3-on eha dn o stageso fspermatogenesis .Reprod .Nutr . influenceo nthi sparticula rparameter . Develop.22:427-438 . Ourinteres t infactor s controlling the Inano,H. ,H .Nakano ,M . Shikita& activationo fspermatozo a (Baynese t al., B.Tamaoki ,1967 .Th einfluenc e ofvariou s 1981),however ,le du st oth ediscover yo f factorsupo n testicularenzyme srelate d to another,muc hcloser ,associatio n- between steroidogenesis. Biochim.Biophys .Act a 17a,20ß-dihydroxy-4-pregnen-3-on elevel s 137:540-548. and thepotassiu mconcentratio no fsemina l Schmidt,P .J .& D .R .Idler ,1962 .Steroi d fluid. hormoneë inth eplasm ao fsalmo na t Seminalfluid so fman yvertebrates ,amon g variousstate so fmaturation .Gen .Comp . them therainbo wtrout ,hav ea nunusuall y Endocrinol.2:204-214 . highpotassiu mconten t (5-10time s thato f Scott,A .P. ,E .L .Sheldric k& blood plasma). Iti sknow n that,i nrainbo w A.P .F .Flirft ,1982 .Measuremen t of17a , trouttestes ,potassiu mion spla ya nimpor 20ß-dihydroxy-4-pregnen-3-onei nplasm ao f tantrol e inth einhibitio n ofspermatozoa n trout (Salmogairdneri) : seasonal changes motility (seeBayne se tal. , 1981). It and responset osalmo npituitar yextract . would notb eunreasonable ,i nvie wo fwha t Gen.Comp .Endocrinol .46:444-451 . isknow no fsteroid-mediate d ionictranspor t Scott,A .P. ,J .P .Sumpter ,an d systemsi nothe rtissues ,t oexpec t acatio n P.A .Hardiman .Hormon e changesdurin g withsuc ha nimportan t function tob eunde r ovulationi nth erainbo wtrout . Gen. thecontro l ofa testicula r steroid. Corti comp.Endocrinol ,(i n press). sol,desoxycorticosteron e and aldosterone Sire,0 .& J .Depêche ,1981 .I nvitr oeffec t + + directly control thetranspor t ofK and Na ofa fis hgonadotrophi n onaromatas ean d acrossmembrane s oforgan s sucha sth e 17ß-hydroxysteroid dehydrogenaseactivi kidney and gills. Theyar eall ,lik e17a , tiesi nth eovar yo f therainbo wtrou t 20ß-dihydroxy-4-pregnen-3-one,C2 1steroids . (Salmogairdneri) . Reprod.Nutr .Develop . If themechanis mw e suggest exists inth e 21:715-726. testis,an d isi nan ywa ycomparable ,the n we should expect tofin d specific receptors for 17a,20ß-dihydroxy-4-pregnen-3-on ean d alsoa Na +/K+-activatedATPas ewithi nth e wallso fth eva sdeferentia . Thisremain s tob eestablished . Abbreviations used onfigure s: 17-P: 17a-hydroxyprogesterone 17,20-P:(17 a20ß-P) : 17a,20ß-dihydroxy-4 - pregnen-3-one A: Androstenedione T: (Testo): Testosterone Ed: Oestradiol 11-T:11-ketotestosteron e 106 STEROIDS INPLASM AO FTH EFEMAL ERAINBO WTROU TBEFOR EAN DAFTE ROVULATIO N BYNCI-GCM S H.Diederi k and J.G.D. Lambert ZoologicalLaboratory ,Sectio n forComparativ eEndocrinology , StateUniversit y ofUtrecht , Padualaan8 ,350 8T B Utrecht,Th eNetherland s Inconnectio nwit h the studyo f thehormo Drying by filtration nal regulationo freproductio n inteleosts , Evaporation under N„(0,-free) 50 C. researchha sbee n started toidentif yan d RESIDUE II ' quantify steroids inplasm ao ffemal erain TOLUENE-FRACTION (Non-phenolic steroids) bowtrout ,abou t threeday sbefore ,an don e Washing with 0.2 N HCl (0.5 ml) and fourweek s after ovulation.A ne wad Drying by filtration vancedmetho d isuse d todetermin e steroids Evaporation under N„(0„-free) 50°C. RESIDUE III i inplasm a ata lo wleve l and for isolation andpurificatio n ion-pair systemsar eintro Derivatization duced. Identification andquantificatio n takesplac eb y capillary gaschromatography- KETO-FUNCTIONS to oxims by FLOROX-Reagent negative chemical ionization-mass-spectro- (O-Pentafluoroben zylhydroxylamin e HCl/Pyridin e ) metry.T oobtai n full expressiono f thene gative chemical ionizationprocess ,steroid s HYDROXYL/PHENOLIC-FUNCTIONS to HFB-esters by Heptafluorobutirylimidazole or to TMS-ethers by withhydroxy l groups arederivitize d to N-Trimethylsilylimidazole theirheptafluorobutiry l (HFB)derivative s and steroidswit hket o groups tothei ro - RESIDUE II pentafluorobenzyloxime (OPFB)derivatives . KETO-FUNCTIONS to oxims by MOX-Reagent These compounds are synthetized tointro (2% solution Methoxyamine HCl in Pyridine) duce electrophilic functions inth esteroi d molecules.Whe n steroids containboth ,hy HYDROXYL/PHENOLIC-FUNCTIONS to TMS-ethers by droxylan dket ogroup s also OPFB-TMS deri N-Methyl-N-Trimethylsilyl-Trifluoroacetamide vativeswer eprepared . Purification of the derivatives by liquid/liquid- extraction; Hexane-Aoetonitrile 10:1 (2.0ml ) Anoutlin eo f themetho d Extraction of steroids out of plasma Identification and quantification of the de rivatives PLASMA (0.3 - 1.5 ml) Adding: Internal Standards Cap. Gaschromatography-Negative Chemical - 2-Methoxyestradiol 2.0 ng Ionization Mass Spectrometry - llß Hydroxyetiocholanolone 10-60 ng Column: Sp 2100Fuse d silica, wide bore, 12.5 m BaCl„ 10%(0. 1 mlHPrecipitation of Programme temp, range: 160°-260 C. sulfates and phosphates) Programme rate: 2.0°C/min. Acetone (0.3 ml) and 4 N HCl (3 drops) Time 1 = 2.0 min., Carrier: Helium, flow 1.5 ml/min. (Precipitation of proteins) Transferline temp. : 275°C. H„0 up to a final volume of 3.0 ml. Source temp.: 200°C; Analyser temp.: 185°C. Reagent Gas: Methane _. Chloroform-Ethylacetate-Acetone 7.0 ml Source pressure : 0.5 Torr; 1.0 x 10 Torr. (70:28:2) Electron energy: 120 EV Extraction of the mixture for 2 min. on a Vibramix. Centrifugation (10 min.,3000 rpm) TOTAL ION MONITORING; sew. 400-1000 M/z EM 2600 Volt CLEAN-UP OF ORGANIC PHASE QUANTIFICATION by SELECTED ION MONOTORING Washing with 1) H.0,2) NH.OH (pH=8.3),3) H„0 using Hp 100 Ion-software, programme Hp 5985 GC/MS/DS; Drying by filtration ÈM 2800 Volt Evaporation by N„(0„-free) 50"C. Internal Standards: - 2-Methoxyestradiol l i RESIDUE I - lift Hydroxyetiocholanolone Isolation by ion-pair extraction Identification RESIDUE I dissolved in 2.0 ml Toluene (-20°C) Adding: Tetramethylammoniumhydroxide (TMAOH) Themor e concentrated steroids inth e (20% in methanol) 60 ul, - 20°C. Centrifugation (10 min., 3000 rpm) plasmawer e identified by comparing theob TMAOH-FRACTION TOLUENE-FRACTION tained spectrawit h thespectr a of standards atth eexpecte d GCretentio n times (fig.1) . TMAOH-FRACTION (Phenolic steroids; Bile acids) The identity of theothe r steroidswa sde Adding: N-Tricine (10% in H O)(0.5 ml, 0°C) Toluene-Diethylether 1:2 (2,1 ml) terminedb y confirmationo f selected ions Centrifugation (10 min. ,3000 rpm) outo fa total ionmonitorin g atdistinc t Organic phase is washed with 0.2 N HCl (0.5ml ) retention times. Adding: 2.0 Toluene Infirs t instanceattentio n ispai d toth e 107 17aHYDR0XY ,20/} DIHYDROPROGESTERONE presenceo fandrogen san doestrogen san d OPFB-DI-OTMS their intermediates.A secon d groupo fste MU=671. 3 roidsar eth eso-calle dmaturatio n inducing steroids.A thir d groupar esom emetabolite s ofandrostenedión ean dtestosterone . 621.5 £62.0 310.2 ,1 Quantification m 520 560 Toquantif yth esteroid sth eabundanc eo f selected ionsi.e .th emos t characteristic ionsan di nmos tcase sM-H F(M-20 )wer ecom paredwit hth eabundanc eo fselecte d ionso f 355.6 892.1 95S.5 987.7 theinterna l standards.Thes eselecte d ions 720 760 340 888 920 960 10' were takenou to fa tota lio nmonitoring . The resultsar esummarize di nTabl e1 . Acknowledgement 12 141 6 182 02 22 4 262 83 0 323 4 363 84 04 244 4 64 85 05 254 56S 8 Theseresult swer eobtaine dwit hth eai do f • 6 4 RET.TIME ! 41.53 TOT«BUNIi « 10091. BfiSE PK'ABUND: 651.4/ 1295. aH P5985B-GC/MS/D Sinstrumen tkindl ypu ta t M-HOTMS ourdisposa lby :Applie dMas s spectrometrical 100-1 M-CPFB+OTMS) '579.3 f andToxicologica lLaborator y (AMTOL B.V.), M pFB 50 401.3 - 1 5rd 300.1 356.2 458.3 . i Kleverparkweg 2023C A Haarlem, The 0- Netherlands. 280 320 360 400 449 480 520 560 600 100- «}-4l 7«HYDROX Y ,20/j DIHYDROPROGESTERONE F 50- "-." OPFB-DI-OTMS MW=671.3 1 679.6 7S0.2 783.0 333.2 874,9 ( flHT I ) 1 1 640 680 720 760 380 840 830 920 960 1000 Fig. 1. a. Mass spectrum (NCI) of the stan dard; b. Mass spectrum out of plasma. Table 1. Levels of steroids (ng/ml) in plasma of Salmo gairdneri three days before (1), one week (2) and four weeks (3) after ovulation. 2 3 Estrone 15.9 2.0 17a Hydroxy,20/5 -dihydroprogesterone 4.9 126.8 9.7 Estradiol 1.2 1.0 17a Hydroxy,20a -dihydroprogesterone Estriol _ Deoxycorticosterone 23.0 40.1 26.0 2-Methoxyestrone 1.8 2-Methoxyestradiol 11a Hydroxyprogesterone 42.5 15.6 31.0 16-Ketoestradiol Cortieosterone 23.9 33.6 37.7 20/5 Dihydrocortisone Pregnenolone 5.2 17. 10.0 17a Hydroxypregnenolone 15.2 12. 4.3 5/5 Pregnane 3a ,20a -diol Dehydroepiandrosterone 3.5 30. 15.5 5a Pregnane 3/5 ,17a ,20/5 -triol Androst-4-ene 3/5 ,17/5 diol 1 . 1,6 5a Androstanedione 118.5 37.3 50.3 Progesterone 42. 16.2 5a Androstane 3/5 ,17/5 -diol + + + 17a Hydroxyprogesterone 49.4 245. 29.0 5/5 Androstane 3a ,17/5 -diol Androstenedione 148.3 66. 28.4 Androsterone Testosterone 167.3 25. '17,5 Etiocholanolone 57.2 42.0 lip Hydroxyandrostenedione + + + 11/5 Hydroxyetiocholanolone 11- Ketoandrostenedione 42.3 71 . 30.3 11-Ketoetiocholanolone 13.7 60.6 24.4 11/5 Hydroxytestosterone 13.3 5. 3.6 5a Dihydrotestosterone 90.5 49.6 41.8 11-Ketotestosterone 36.6 10. 6.2 5/5 Dihydrotestosterone 27.6 1 1 .9 12.0 108 TOPOGRAPHICAL DISTRIBUTION OFSTEROIDOGENI C ENZYMES INRELATIO NT OSPERMATOGENESI S INTH E TESTISO FSQUALU S ACANTHIAS. * Jeffrey Pudney,Glori aV .Callard ,an dJaco b Canick. * Departmento fBiology ,Bosto n University,and , Laboratoryo fHuma n Reproductionan d Reproductive Biology,Harvar d Medical School,Boston ,M AUS A The processo fspermatogenesi si nverte The steroidogenic potential ofthes e zones brates is,structurall y andfunctionally ,a n was also investigated byincubatin g micro extremely complex phenomena. Althoughth e someswit h various radioactive substrates. morphogenesis ofspermatozo a isb yno wwel l The topographical distribution ofsteroido understood,th efunctiona l changes which genic enzymes associated with eacho fth e occur during thevariou s stageso fger m cell zones issummarize d inTabl e II. Asger m developmentar estil l largely unknown. Thus cell controls,fre e sperm stripped fromth e despiteth efac t thati ti swel l established vas deferenswer e analyzedfo rth esam e that spermatogenesis isandrogen-dependent , enzymes. iti sstil l uncertainwha t role steroids play duringth ematuratio n ofger m cells. Iti sessential ,therefore ,t ono tonl yde TableI I termine,qualitativel yan dquantitativel y the steroidal micro-environment associated Stero idogenic activities with specific germ cell stages,bu tt oals o identifyth ecell s responsible forsteroi d Uct C-17.C-20 production. Itis ,however , technically hydroxylase lyase/ aromat as e very difficultt oanalyz eth esteroidogeni c (pmol/min/mgj (fmol/min/m{ potential ofth evariou s germ cell stagesi n Zone the testeso fmos t laboratory animals. In I 5.84 2.90 2.93 these speciesth edifferentiatio no fger m +0.81 +1.33 +0.50 cells isno tsynchronize d inan yon etesti cular regionan dsinc eal lstage so fger m II 4.26 6.89 7.10 cell'developmen tar epresen t simultaneously +0.61 +1.10 +0.40 any changes insteroidogenesi s associated with these stages wouldb ehar dt odetect . III 10.76 24.18 2.83 To alleviate this problemw eselecte da n +2.05 +5.61 +0.40 animal model,th eshar k Squalus acanthias, where discrete germ cell stagesar edis Free tinctly separated within thetestis . This sperm 2.35 -0-L spatial segregation ofth edifferentiatin g germ cellsca nactuall yb evisualize di n aProduct yield perm gmicrosoma l protein thick transverse sectionso fth etestis , (means+ sem )fo rseparat e microsomal prep with theai do fa dissectin g microscope. arations from3 animals . Usingth egros s criteriao fopacity , color and relationship toth eepigona l tissueth e Productyiel d perm ghomogenat e proteinfo r testis sections were separated into3 zones . a single pool offre e sperm. Thecompositio n ofth eseminiferou s lobules presenti nthes e isolated regions,observe d Light microscopic observations indicated by"ligh tmicroscopy ,i sshow n inTabl eI . thatth einterlobula r tissue appeared poorly developed. Thus Leydig cells,i fpresent , wouldb eundifferentiate dbu tcoul d possibly synthesize small butimportan t quantitieso f TableI steroids. Iti smor e probable,however , thatth eseminiferou s lobulesar eresponsibl e forth emajorit yQ fth e androgenan destro Morphological componentso fcyst s gen synthesizing activityo fth eshar k tes tis. This study indicates,therefore, ,tha t Zone the steroidogenic activityo fth eshar k tes I Spermatogonia tisma yvary ,dependin g upon thestag eo f germ cell development. II Spermatocytes. Some cysts contain round spermatids (Supported byHD-15595 7 HD-16715). III Mixtureo fcyst s containing sperma tidsan dspermatozo a 109 PLASMA STEROID PROFILE DURING THE SPAWNING SEASONO FPIK E C. Simontacchi,D .Casciott i &C;* . Boiti Instituteo fVeterinar y Physiology and Instituteo fAnima l Production^ Universityo fPerugia , Italy Summary Iti swel l knowntha t female piked ono t Even ifprogesteron ei sa significan t pre spawn incaptivity ,eve n ifcapture d after cursor inth e ovarian steroid biosynthesis completiono fvitellogenesis . Ovarianatresi a of pike, itsrol ei ngonada l maturationi s ofpik e canb esuccessfull y prevented by not remarkable,a sovulatio n causesn ochang e several hormonal treatments which have been in itsperiphera l plasma profile. found effectivei nstimulatin g spawningi n captivity (De Montalemberte tal , 1978). However,th e reproductive physiologyo fpik e is largely unknown.Th e aimo fthi s work was to investigate plasma profileso fsex-steroi d hormonesi nconnectio n with gonadal matura tiono ffemal e pike during the reproductive | | OV (n.11) cycle. [HHÏÏH GVBD (n= 2) P | Spawning In- 9) Plasma progesterone,testosteron e and 17B - oestradiol were assayedb yhig hspecifi c RIA methodsi n2 2femal e pike captured from lake Trasimeno shortly beforean ddurin g the natu ral spawning season.Bloo d samples,obtaine d by branchial artery puncture onth e capture ofeac h pike,wer e centrifuged within 1- 2 hours andth e plasma storeda t-2 0 °Cunti l Progesterone Testosterone 17B-oestradio processing.Th e pike were subdivided into three groups depending onth e stageo f gona fig. 1.Sex-steroi d hormonal profiles during dal maturation accordingt ohistologica l ovarian maturation and spawning in pike. findings:a post-vitellogeni c groupwit hth e germinal vesicle (GV)locate d ina periphera l Testosteronei sa nimportan t precursort oC-1 8 position (GV stage:1 1fish) ;a preovulator y steroid biosynthesis inth e ovary andi s groupi nwhic h theG Vwa s not visiblean d synthetized and released only inth e preovu the ova were clear (GVBD stageo rG V break latory phases.However ,it sphysiologica lrol e down:2 fish )an da n ovulatory group (spawn in femalepik ereproductio ni sstil luncertain . ing stage:9 fish) . 17B-oestradio l hasa stimulatin g effecti n vitellogenesis buta probabl e inhibitory Plasma levelso f progesterone averaged effect onth e resumptiono fmeiosi s andovu 0.4±0. 2 ng/ml (Mean±SD)withou tan y lationa sdemonstrate db yit s constantly significant change during the three gonadal decreasing plasma level during the last sta maturation stages investigated. ges ofth e reproductive cycle. Plasmatestosteron e levels weresimila ri n The decreasing oestradiol profile,i ncontras t both groups during the preovulatory stage to the constant levelo ftestosteron e.suggest s resulting2. 6+0. 9ng/m l and 2.6+ 0. 7 ng/ml that inth e preovulatory stages the aroma respectively inth eG Van d GVBD stages,bu t tizing enzymes are partially inactivated. significantly (P<0.01 )fel lt o0. 2± 0. 2 ng/ml Ovulationi sassociate d witha significan t at spawning. dropi nbot h androgen and oestrogen plasma Plasma oestradiol concentration averaged levels suggesting that the biosynthetic ste 5.7±2.3 ng/ml inpik e having oocytes with roid pathway isblocke d between progesterone theG Vi nperiphera l position,wa s signifi andtestosterone . cantly (P<0.05)lowe r inth e GVBDstag egrou p being 2.0+ 0. 3 ng/ml,the n dropped (P<0.001) to 0.3±0. 2 ng/ml,i nth e spawning pike. References RIA methodologyi sparticularl y suitable De Montalembert G., B. Jalabert, C. Bry, 1978 forendocrinologica l investigations infis h Precocious induction of maturation andovu because its high specificity and sensitivity lation in northern pike (Esox lucius). allow independent assayso fsteroi d hormones Ann. Biol.anim. Bioch. Biophys. on plasma samples assmal la s0. 1t o0. 2ng/ml . 18(4):969-975. 110 SEASONALVARIATION SO FPLASM AANDROGEN SAN DGONA DHISTOLOG Y INMAL E SPOTTEDSEATROUT , CYNOSCIONNEBULOSU S (FAMILY: SCIAENIDAE). By: P.Thomas ,N.J .Brow nan d C.R.Arnol d Marine Science Institute,Por tAransa sMarin eLaboratory ,Universit y ofTexa sa tAustin ,Por t Aransas,Texa s78373 ,U.S.A . Severalmember so f thepercifor m family Sciaenidaear eimportan t recreationalan d Fig- 1 commercial fishes incoasta lwater so fth e southeasternU.S.A . Inaddition ,tw osciae - 11-KETOTESTOSTERONE nids, spotted seatrout (Gynoseionnebulosus ) and red drum (Sciaenopsocellatus) ,hav e D -»re beenpropose d asspecie s suitablefo rmari - H running , culturei nTexas . However,th ereproductiv e physiology of thesespecie s islargel yun known. Thepurpos eo f thisinitia lstudy , therefore,wa s todetermin ebot h thehisto logicalan d theendocrin e changeswhic h occurdurin g gonadalmaturatio n and spawning innatura lpopulation so fmal e spottedsea - trout. Spotted seatroutwer e caughtwit hartifi I ciallure sfro m earlyApri lunti l theen do f October 1981an d bledwithi ntw omi no fcap ture. This speciesha sa prolonge d spawning X season inth esubtropica lwater so f south Texas (Aprilt oOctober) . Over83 %o fth e males collected betweenApri lan d September (meantemp .27. 2 °C)wer erunnin g ripe. In June July Aug Sept. Oct. contrastal lth efis hobtaine d after theon seto f colderweathe r inmi d Octoberwer e ofplasm a fluctuations inmal e fishdurin g spent (meantemp .20. 4 °C). thespawnin g season (Fig.2) . Maximum Histological examinationreveale d thetyp levels (2.4ng/ml )wer edetecte d inApril . icalteleostea npatter no f spermatogenesis PlasmaT concentration s gradually felldur in C_. nebulosus. All stageso fspermatogen ing thesumme ran db y Septemberwer eonl y esis (fromspermatogoni a to spermatozoa) 0.2ng/m l inrunnin g ripe fish. Prelimary werefound . Theteste so frunnin g ripefis h dataindicate d thatplasm a levelso f17a , collected inApri l contained largenumber s 20ßOH P inmal e spotted seatroutwer elo w ofprimar y and secondary spermatocytes,a s (<0. 6 ng/ml)an d did not fluctuatedurin g wella sspermatid san d spermatozoa,wherea s thereproductiv eseason . thoseo frunnin g ripeindividual s collected inJun e containedmainl y spermatidsan d Fig. 2 TESTOSTERONE spermatozoa. Thelobule so frunnin g ripe I I mature fishobtaine d inSeptembe rwer efille d pri marilywit h spermatozoa and thoseo fspen t l-^i running rip individuals collected at theen do fOctobe r were emptyan d partially collapsed. lü spent Testosterone (T),11-ketotestosteron e(11 - KT)an d 17a-hydroxy-20ß-dihydroprogesterone (17a,20 ßOHP )wer emeasure d byradioimmun oassayusin gantiser awhic hdi dno tcross - 1hI April June July Aug. Sept. Oct. react significantlywit hothe rsteroids . 19e i Alterations inth ecirculatin g levelso fT Conclusions and11K Twer eobserve d during thereprod Theseresult s suggest thathig hplasm a uctiveseason . Highest levelso f11K Twer e concentrations of11K Tan d Tma yb eimport detected infis hbloo d collected during the ant inth esprin g for testesmaturatio nan d peakspawnin gperio d inApri l (Fig. 1). theearl y stageso f spermatogenesis inspo During thisearl ypar to f thespawnin g sea tted seatrout. However,i nmidsumme r the sonmatur e fishha dhighe rplasm a 11KTcon final stageso f spermatogenesis occur inth e centrations thenrunnin g ripeindividuals . absenceo fhig h circulating androgenlevels . 11KT subsequently declined inbot hmatur e Highlevel so f17a ,20 ßOH Pma yno tb enece and running ripespotte d seatrout toles s ssary forspermatogenesi s tooccu r inspot than2 ng/m li nJuly . tedseatrout . Testosterone exhibited a similar pattern Ill OVARIANSTEROI DMETABOLIS MI NTH EIMMATUR EEUROPEA NEE L (Anguilla anguilla). B.QUERAT ,J .LELOUP-HATEY ,A .HARDY . Laboratoired ePhysiologi e généralee tcomparé ed uMuséu mnationa ld'Histoir e naturelle; Laboratoired'Endocrinologi e comparée associéa uC.N.R.S . (L.A. 90),7 ru eCuvier , 75231Pari s cédex05 ,France . Summary Other compoundsar eye tt ob eidentified , someo fthe mbein gprobabl y5- ßreduce dC 1 9 The steroid production pattern of the steroids.I nou rexperimenta l conditions,n o ovary in the immature silver eel is directed estrogenswer efound .- E 2seeme dt ob epre towards the synthesis of 11 oxoderivative an senti nth eplasm abu tit sleve l (0.05,0.5 ) drogens : no aromatisation of androgens has waslowe rtha ntha to fT (0.8,3.5) .Th eMC R been found in the ovary ; the affinity of ofE 2 (0.57ml/k gx h )wa s2. 5time slowe r plasma binding proteins (PBP) for estradiol thantha to fT (1.48ml/k gx h) . -Th eaffi (E2) and testosterone (T) is very high ; the nityo fPB Pfo rE andT i sver yhig h (97 % metabolic clearance rate (MCR)of E is 2.5 2 2 E2bound ,9 8% T bound) .- Afte r intraveinous times lower than that of T, despite plasma injectiono fa dos eo flabelle d tracer,w e level (PL) of E (0.05, 0.5) lower than that 2 observed thatbot hhormone swer emainl ymeta of T (0.8, 3.5 ng/ml) . bolized inth eliver .T gav eris et oa meta The European eel differentiates sexually bolite thatappeare di nth eplasma ,it scon in continental waters but supposedly spawns centration increasinga sa functio no ftim e; in the vicinity of the Sargasso sea. In nor itschromatographi cpropertie swer edifferen t mal freshwater female eels, oocytes are at fromtha to fE 2. the prophase stage of the first meiotic di vision. The gonadal development occurs only Discussion- I nou rexperiments ,yield so f during their marine migration or when trea 11-oxoandrogensincrease d continuouslywit h ted with fish gonadotropin hormone. Colombo timewhil eyield so fA 4an dT decrease da t & Colombo-Belvedere (1976) showed that the theen do fth eincubatio n (Fig.).Sinc e the ovary of immature silver eel synthesizes an selas tcompound sar eke yintermediates ,i t drogens and 11-oxoderivatives. We performed canb esuppose dtha tthei rconversio n into a comparable kinetic study of ovarian synthe estrogensi sreduce db yth elarg eproductio n sis and investigated sexual steroid metabo of 11-oxoderivatiyes.Th eoccurenc eo fa n lism. extraovarian aromatisationo fT ha sno tbee n shown.However ,a loca laromatisatio ni n 14 Results - Incubation of (4- C) progesterone centralnervou s systemo rhypophysi swhic h with slices of ovarian tissue from immature hasbee n showni nsom eTeleost s (Callarde t silver eel yielded androgens (androstenedio- al., 1981)coul doccu ri nee lwithou tmodi ne (A 4) , T) and their 11-oxoderivatives ; fyingP Lo fE 2. Despiteth eprotectio no fE , againstperiphera l catabolism (highaffinit y 11- hydroxyandrostenedione (11 OH A4) , 11- hydroxytestosterone (11 OH T) and 11-keto- ofPBP )th eP Lo fthi shormon ei sver ylow . testosterone were the major transformation This confirmsou rhypothesi s thatth eovaria n products. The yield-time curves of the cha productionpathwa yi sno tdirecte d towards racterized products are given in the figure the synthesiso festrogens .Th elarg econver above. sionint o 11-oxoandrogensindicate s thatth e ovarianproductio npatter ni scomparabl ewit h medium thatgenerall y admitted (rev.Ozon ,1972 ) for ovary thetesti so ffishes . • References Jn. V... O Callard,G.V. ,T .Petro ,K .Ryan ,J.B .Clair - O bone, 1981.Estroge nsynthesi si nvitr o andi nviv oi nth ebrai no fa marin eTe - "*• leost (Myoxooephalus). Gen.an dComp . Endocrinol.,43_ ,243-255 . 13 5 10 13 5 10 Colombo,L. ,P .Colombo-Belvedere , 1976.Ste time |hour| -—»11OHT roidbiosynthesi sb yth eovar yo fth e . ,110HA4 European eel,a tth esilve rstage .Gen . * *A4 andComp .Endocrinol. ,28 ,371-385 . G -® T Ozon,R ., 1972 .Androgen si nFishes ,Amphi bians,Reptile san dBirds .I n"Steroid si n Yield-time curveso fth echaracterize dpro nonmammalia nVertebrates" .D.R .Idle red. , ductso fth eovar yo fth eimmatur esilve r Acad.Press ,N.Y. ,328-389 . eel, incubatedwit h (4- c)progesteron 14 e 112 CONSERVATION OF GAMETES 113 PRESERVATION OFFIS HGAMETE S Joachim Stoss andEdwar dM . Donaldson WestVancouve rLaboratory ,Fisherie s ResearchBranch ,Dept .o fFisheries-an dOceans ,416 0 MarineDrive ,Wes tVancouver ,B.C .V7 V 1N6 Canada. Summary asartificia l propagation of theindigenou s fish stocks,transplant s or selective In thisrevie wa discussio n of the fishery canalte r gene frequencies biology ofteleos t spermcell san d the considerably or lead toth ecomplet e losso f factors regulating theirmotilit y is somegene s (seeRyman,1981 ;Hyne s et al. followed bya description of techniques 1981; Ricker,1981) . Thesear e for the short-term preservation of teleost justifications forth eestablishmen t ofgen e spermatozoa and ova. Subsequently, bankswhic h preserve the genetic originality general aspects of thefreezin gan d of populations andkee p them available for thawing of cellsar e discussed and studies re-introduction when generalcondition s for onth e cryopreservation of teleost survivalhav e improved. spermatozoa are reviewed with particular emphasis onsalmonid s including Infis hcultur e systems,selectiv e pre-freezing effects,freezing ,thawing , breeding,a si nothe r domestic animals,i s post-thawing and insemination. essential for theimprovemen t of production Cryopreservation techniques are described traits. Awel lknow n examplei sth e for salmonid spermatozoawhic h provide Atlantic salmonindustr y inNorway . fertility rates close tothos e obtained Cryopreservation ofspermatozo a providesa with fresh spermatozoa. Factorswhic h useful toolt omaximiz e thenumbe ro f require further study include, 1) offspring froma fixe d number of selected evaluation of gametequalit y priort o broodstock bytim ean d locationindependen t preservation and also fertilization,2 ) gamete availability. reduction of celldensit y offrozen - thawed spermatozoa required forsuccessfu l The production ofmono-se x all-female fertilization,3 )extensio no f the stocksha sbecom e feasible byusin g a post-thawmotilit y of cryopreserved so-called indirect feminizationtechnique , spermatozoa. i.e.,sperm-cell s from phenotypic males Keywords:Oncorhynchus ,Salmo ,gametes , havinga female genotype result in cryopreservation,spermatozoa ,ova . genetically all-female offspring (Huntere t al., 1982a,b). Cryopreservation ofthes e Introduction sperm cellsha s theadvantage s of 1) identifying the genetic sexo fth edono r Considerable progress has beenmad e fishpos tmorte man d 2)permittin g themos t recently in the development of techniques efficientus e ofth emilt . forfis hgamet epreservation . This applies inparticula r tath e A finalexampl e refers tosom e tropical cryopreservation of sperm cells. There specieswher ecatc h ofwil d spawners often area numbe r of reasons for this increased isa nessentia l step forth ecollectio no f interest. The intensified cultivationan d ripe gametes (Kuo,1982) . Ifsper m cells propagationo f fishrequire s proper were constantly available,an y limitationi n techniques forgamet ehandlin g which thesucces so fartificia l reproductionwoul d include intermediate storage. Hereshort - bereduce d toth efemales . term preservation isindicate d to facilitateartificia l reproductiono r Inthi spaper ,n oreferenc e willb emad e increase itsefficiency . toth ecryopreservatio n of ovaan d embryos since thissubjec t isreviewe d elsewhere in Cryopreservation asa storage-technique thispublicatio n byB .Harvey . with practicallyn o time limit,ha sa muc h greaterapplicatio n potential thanshort - Thebiolog y ofteleos t spermcell s termpreservatio n techniques particularly with respect tofis hgenetics . Examples Teleost spermatozoa alllac ka hea d cap, ofmassiv e losses of fish stocks duet o theacrosome . Major morphological waterpollution ,habita t destructiono r differences have beenobserve d between overfishing arewel lknown . Recent spermatozoa from specieswit h externalo r research inpopulatio ngenetic sha s internal fertilization (Billard,1970) . The clearly demonstrated that theabov e former category is characterized by mentioned factors aswel la smeasure s such primitive sperm cells. Headsar eofte n 114 ovoid and themid-piec e issmall ,no t activating properties. Alkalinep Hha sbee n visiblewit h theresolutio n of light reported repeatedly tointensif y and prolong microscopy. Itcontain s afe wofte nfuse d motility invariou s fresh and saltwater mitochondria. Thetai li srelativel y long species (Petite t al., 1973;Hine s & (10-20time s thehea d length)an d hasmos t Yashouv,1971 ;Billard ,1980) . In often the typical 9+2 arrangemento f salmonids,ovaria n fluid and fishRinge r' s microtubules (seeBaccett i et al., 1979). are superior towate r (Ginsburg,1963) . Inviviparou s fish,hea d andmidpiec ear e elongated (Gardiner,1978a) . Themidpiec e Short-termpreservatio n of spermatozoa cancontai na substantial numbero f mitochondria asdescribe d inPoecili a Spermatozoawhic h areimmotil e insemina l reticulata. There issufficien t evidence plasmaar e suitable for short-term to believe that these structural preservation sincen oenerg y isrequire d for differences alsoreflec t differences in locomotion. Therefore,diluent s toreduc e themetaboli c activity of thesecells . motility ort osuppl y substratear eno t Spermatozoa from oviparous fishsho w necessary. limited glycolytic activity and depend on oxidativemetabolis m (Mounib, 1967). Themos t important factors determining the Spermatozoa fromviviparou s species can successo f storageare : convert exogenous sugars tolacti caci d - a reduction in temperature and use theenerg y toprolon g theduratio n - provision of gaseous exchange ofmotilit y (Gardiner,1978b) . However, - prevention of bacterial growth oneha s tocriticall y remark that - prevention of desiccation experimental data fromonl ya fewspecie s Lowtemperature s just above freezing point areavailabl e tofor m this generalized haveneve rbee nreporte d tocaus e anyhar m picture. tofis hspermatozoa . Thus storage around 0°C ismos tsuitable . Teleost spermatozoaar e immotllei nth e testis and ofteni nth esemina lplasma . Theeffec t ofvariou sgase s isknow nfo r The induction ofmotilit y canb erelate d salmonid spermatozoa. Storage underaerobi c toth eparticula r environmental conditions conditions,preferabl yunde rO 2maintain s during spawning. Infreshwate r spawners the ability tobecom emotil e for2 weeks . sperm cells become activated byth e CO2,whic h iscommonl y used inmammalia n hypotonicity of freshwater. Insaltwate r spermatozoa,kill s thecell s aftera spawners,hypertonicit y inducesmotilit y relatively brief exposure (Buyukhatipoglu & (Morisawaan d Suzuki,"1980) . Holtz, 1978). This reflects thecell' s dependence onaerobi c metabolism. By far themos twor kha s been donei n salmonids. There islittl e doubt thatK + Forpractica l reasons aunifor m from seminal plasma blocksmotility . availability ofO 2t oal lcell swithi na Dilutiono fK + or interactionwit hothe r sample has tob eensured . SinceO 2ha st o ions removes thisbloc k (seeBayne se t diffusea trathe r lowtemperature s intoth e al., 1981). sample,th ecritica l diffusiondistanc ei s ofimportance . Thiswa s demonstrated ina n The stimulationo fmotilit y in experimentwit hrainbo w trout sperm. When viviparous fish isles sunderstood . Both milt-height within thestorag e containerwa s a relative change between ions and the either2. 5 or6. 5 mm,motilit y couldstil l mechanical breakdown of spermatozeugma beinduce d after 17an d 28day s ofstorage , have beensuggeste d aspossibl emotilit y respectively. Storage at3 1mm ,i n inducing mechanisms (Morisawa andSuzuki , contrast,retaine d somemotilit y to4 day s 1980; Billard, 1978a). only. Whenrepeatin g thisexperimen twit h6 mm samples fertility wasunchange d relative Motility islimite d toa perio do f tocontrol s atDa y3 9 (Stosse tal. ,i n seconds tominute s in freshwater preparation). spawners because of lysis (Billard, 1978a). Motility isconsiderabl y longer As fara sothe r speciesar e insaltwate r spawners,1 5minute s in concerned,requirementsfo rgaseou sexchang e Atlantic cod (Davenport et al.,1981 )o r are lessclear . Therear e indications that several days inherrin g (Yanagimachi, somewhatanaerobi c storage conditions 1957). These examples showho wwel l maintained motility relatively well,suc ha s spermatozoa from saltwater species are inwhit ebass ,Atlanti c codan dmilkfis h adapted tohypertoni c conditions possibly (Clemens &Hill , 1969;Mouni b et al.,1968 ; explaining theirhardines swhe napplyin g Pullin& Kuo ,1980) . cryopreservation procedures (vide infra). Storage diluentshav e beenuse d Spermatozoanmotilit y canb eenhance d in occasionally.Dilute d sperm fromAtlanti c some physiological solutionswit h cod,gre ymulle t orchanne l catfishi n 115 isotonic media retained theabilit yt o shouldb ekep ti nno tmor e thanfou r layers becomeactivate d for20 ,2 3an d6 3days , (approx.0. 8mm )unde rai ra t1° Can d respectively (Mounibe tal. ,1968 ;Cha oe t protectedb yantibiotics . Apreviou s al., 1975;Gues te tal. , 1976). Aclea r comparison betweenvariou s gases showed advantageo fdilute d versusundilute d little difference betweenÛ £o rai rbu t storageha showeve r not been demonstrated. demonstrated theunsuitabilit yo fN 2o ra mixtureo fO 2(95% )and'C0 2 (5%)(Pueschel , Another modeo fstorag ei sb y 1979). Inthi sstud y70 %fertilit ywa s supercooling. Spermatozoa arekep ta t retained for2 0day s (Fig. 3). severaldegree sbelo w0° Can dfreezin go f the suspensionmediu m and cellsi s Cryopreservationo fspermatozo a preventedb yth epresenc eo f cryoprotectants. Afew ,quit epromisin g 1.Genera lAspect s attempts have beenreporte d from salmonid spermatozoa. Modified Cortland' smedium , Inthi ssection ,event sassociate d with whichi sbase do nth eminera l contento f the freezingan d thawingo fcell swil lb e blood plasmaan dsemina l plasmaIC " briefly reviewed. Formor edetaile d contentwa suse di ncombinatio n withDMS O information,paper sb yMeryma n (1971a,b), or ethylene glycol. Inbot hAtlanti c Mazur (1977)an dFarran t (1980)shoul db e salmonan d rainbow trout,cell sstaye d consulted. Whentalkin g about viable fora tleas t5 weeks . Fertilityo f cryopreservationw eusuall ymea nstorag eo f cellswa sno tteste d beyond this period cellso rtissu ea t-196°C ,th e temperature (Truscotte tal. ,1968 ;Sanchez-Rodrigue z of liquid nitrogen (Fig.2) . Thisi sfa r & Billard, 1978). Gloss transition Liquid Solid CO temperature for nitrogen (storage) Short-term preservationo fov a Justa sth epreventio no fmotilit yi sa n important prerequisite for successful Possibility for cell injury by: I. Formation of intracellular spermstorage ,th epreventio no f ice (fast freezing) activationi so fimportanc e forova . This 2.Osmotic shock by extra mayb erathe r difficulti na numbe ro f cellular solutes (slow freezing) species sucha scarp si nwhic hov a autoactivate after naturalo rartificia l Fig.2 . Cellinjur y during freezing. spawning (Yamamoto,1961) . Therei si ngenera l little available below theglas s transitiontemperatur efo r datao nstorag eo fov aan dsom eresult s water,indicatin g that moleculesar ei na obtainedi nrainbo w trout arepresente di n fixed statean dmotionless . Storage timei s Fig.1 . only limited byexterna linfluence s sucha s background irradiationwhic h theoretically STORAGE OF OVA: 1 layer a limitsth eperio do fstorag et osomewher e 2 layers between20 0- 32,00 0year s (seeAshwood - 4 layers 8 layers Smith,1980 ;Whittingham , 1980). Cellinjur y therefore isno t relatedt o the lengtho fstorag ebu tt oth etransitio n ofth ecell s fromambien t temperaturesa t collectiont oapproximatel y -80°C. Iti si n thisrang etha t3 type so fcel linjur yma y occur (Fig.2) . Thefirs tone ,referre dt o ascol d shock,i scause db ycoolin ga tabov e freezing temperatures. Thisappear st ob e of little importancei nfis h spermatozoa, butma yb emor e importanti nfis hova .Tw o typeso finjur yar e possible between approximately 0°Can d-80° Cbein g related eithert oth eformatio no fintracellula ric e DAVS OF STORAGE orosmoti c shock. Thiswil lb eexplaine d further. Fig.1 .Storag eo frainbo w trout ovai n oneo rsevera l layers. Air,1°C ,12 5 IU Ata fe w degrees below0°C ,th ecel l penicillinan d 125u gstreptomyci n perg suspensionmediu m freezes. Thecel l ova. X± SD ,n = 4 (Pueschel ,1979) . however,stay sunfroze nsinc eth eic e crystals cannot growthroug h thecel l membrane. Because theintracellula r liquid These dataindicat e thatrainbo w troutov a waterha sa lowe r chemical potential than 116 the frozenextracellula rwater , larger crystals,whic h thenca ncaus e intracellular water leaves thecel lan d irreversible damage. Bythawin g quickly, freezes externally. Thisproces so f recrystalization canb eprevented . dehydration canvirtuall y continue until thecel li sdehydrate d and thusha s Attempts tocryopreserv e fishspermatozo a reacheda suitable state for storagea t have primarily focussedo n salmonid species low temperature. Thisproces so f andw ear eno wabl e toachiev ehig h dehydration istime-dependent . fertility inthes e spermatozoa (Mounib, 1978; Stein& Bayrle ,1978 ;Buyukhatipogl u & When cooling proceeds toofast ,th e Holtz, 1978;Bayrle ,1980 ;Erdah l &Graham , temperature atwhic h the intracellular 1980; Stoss& Holtz ,1981a,b ; Stoss& water freezes,wil lb ereache d beforeth e Refstie, 1982). celli sentirel y dehydrated. Formationo f significant amounts ofintracellula r ice Successha s also beenachieve d inth e isassociate d with thedeat ho fth e saltwater spawners Sparusauratus , cell. Dicentrarchuslabra x (Billard, 1978b),Gadu s morhua (Mounib et al.,1968) , Pleuronectes During freezing,solute s sucha ssalt s platessa (Pullin,1972 )an d Clupeaharengu s concentrate severalfold ina smal lvolum e (Blaxter,1953) . High fertilitywa s ofremainin g liquid. If freezing is reported inLabe orohit a (Withler,1982 )bu t carried out ata slo wrate ,th ecell sma y resultsi nothe r freshwater spawners arei n become sufficiently dehydrated but generalquit e variable (seeSi n1974 ; theexposur e toconcentrate d solutesma y Moczarski,1976 ,1977 ;D eMontalember t et cause osmotic trauma. Further,ther ei sa al.,1978 ;Bayrle ,1980 ;Withler , 1982). possibility thatboun dwate rwoul d alsob e removed from thecells . Ina numbe r of speciesw etherefor estil l stand atth ebeginnin g ofadaptin g Thismode lunderline s theimportanc eo f cryopreservation procedures. Experience the freezing rate. Optimum freezing rates obtained insalmonid shas'show nho w are specific fora particular celltype . importantal lstep s ofth eentir e They depend onparameter s such asth e cryopreservation procedure aret oensur e cell' spermeabilit y forwater ,th e goodsurvival . specific temperature coefficient,th e amount of freewate rwithi na cello rth e Table 1list s thevariou sphase s ofth e diffusion surface. Knowing these cryopreservation procedure and variables parameters,a mathematica l modelca nb e withineac hphas e whichma y influence applied tooptimiz e freezing rates (Mazur, post-thaw survival. Thehighl y interactive 1963, 1977). nature ofal linput-variable s involvedwa s ponted out byFarran t (1980). Table1 wil l Undermos t circumstances,injur yb y servea sa guideline todiscus s factors osmotic shockan d byintracellula rice , influencing post-thawsurvival . i.e., fast freezing and slowfreezin g injury overlap,allowin g nosurvival . 2.Pre-freezin g effects Thereare ,however ,example s wherea smal l windowfo r survivalexists . Differences inpost-tha w fertility between Cryoprotectants openo rwide n thiswindo w differentmale shav e beenreporte d several by reducing primarily injury associated times (Ott& Horton ,1971a) . Poolingo f with slow freezing. Themechanism s rainbow troutmil t from several individuals involved areno t completely understoodbu t improved thefertilit y drastically according therei sevidenc e thatcryoprotectant s do toLegendr ean dBillar d (1980)bu tn osuc h not alter thecel l' ssusceptibilit y to effect wasfoun d inanothe r study (Stoss& slow freezing injury but that theyalte r Holtz,i n preparation). Itha sbee n thepropertie s of theextracellula r suggested by theforme rauthor s thatagin g medium. Further,cryoprotectantslowe rth e ofsper m cellsi nth etesti s during the temperature forfreezin g of intracellular period of spermiationi son ereaso nfo r water. Consequently, freezing ratesca n differences betweenmales . Similar effects not bedeal t inisolation ,bu t inrelatio n have beenfoun d tooccu r inshort-ter m toa specific cryoprotectant andit s preserved seabas sspermatozo a (Billarde t concentration. al., 1977). Thawing isth erevers e of theevent s By collecting milt from coho salmona t just described. Inth ecas eo f intervals of5 day s fromth e beginningo f spermatozoa,fas t thawing isalway s spermiation,w ecoul dno t findan y attempted. Usually,smal lamount so f differences related toeithe r thestag eo f intracellular icear epresen t inth ecel l spermiationo rth eparticula rmal e (Stosse t (Leibo et al. 1978). If thawing proceeds al.,i n preparation). Since the spermiation slowly,thi sic eca nrecrystallz e to periodwa slimite d to3 week s by thenatura l 117 Table 1. Variables affecting post-thaw during freezing and thawing (Mazur & Miller, fertility of cryopreserved spermatozoa. 1976). 00 r o con trol Equilibration Time Pre-freezing Males selected -o 0-1 min ^\^°'\ :•- • 1-2 min Nature and concentration of cryoprotectant 5-6 min Equilibration time and temperature 60-61 min Interval cell collection-freezing f ^\ 50 Freezing Freezing rate Cryoprotectant \ Extender Dilution rate 10 Storage Temperature 3.6 6.8 9.1 9.8 12.5 DMSO-concentration [%) in suspended sperm Background irradiation Fig.3 .DMSO-concentratio nan d equilibration Thawing Thawing rate timei nrainbo w trout spermatozoa (X,n = 5 , Thawing solution pooled SD= 5.1 ) (Stoss &Holtz ,i n preparation). Post-thawing, Motility induction and duration insemination Removal of cryoprotectant Theimportanc e ofusin g freshcell s for Incubation cryopreservationha s beenshow ni nrainbo w Cell density for insemination trout (Stoss& Holtz ,i npreparation) . Females selected for fertilization Short-term storage (0°C,air )o fmil t betweencollectio nan d freezing foronl yon e death of the fish,advance d stagesi n hourresulte d ina decrease in post-thaw spermiationsuc ha s thoseoccurrin g in fertility whencompare d to2 0mi no f rainbow trout,ma y never bereache d by storage. species of thegenu sOncorhynchus . 3.Freezin gan d thawing Cryoprotectantsma y affect spermatozoa atabov e freezing temperatures. Sucha n Freezing ratesi nconjunctio n withth e influence hasbee nobserve d whenusin g cryoprotectant have beeninvestigate d in glyceroli nvariou s salmonid species spermatozoa from sea bassan d seabream (Truscott etal. ,1968 ;Ot t& Horton , (Billard 1978b). Optimal conditions were 1971a;Erdah l& Graham ,1980 )o ri nth e achieved whenfreezin g cellsa t 10°C/minan d grouperEpinephelu s tauvina (Wlthler& using between5-15 %DMS Oi nth ediluent . Lim, 1982). Sperm cells from the Slower andhighe rrate swer e lesssuitable . saltwater spawnersGadu smorhu aan dMugi l InAtlanti c coda rat eo f5°C/mi nwa s cephaluswere ,however ,no t effected by applied with good successwherea sl°C/mi n glycerol (Mounib etal. ,1968 ;Cha oe t resulted inn osurviva l (Mounib et al., al., 1975). A toxic effect hasals o been 1968). observed whenusin g dimethylsulfoxide (DMSO)a tconcentration s between 6.8-12.5% Ina number of freshwaterspecies , in the final dilutionan d equilibrating freezing hasbee ncarrie d outb ypelletin g trout sperm for 1mi no r longer (Fig. 3). suspended spermont o dry ice (Nagasean d Dataobtaine d insockey e salmon Niwa, 1964). Thetransitio n from 0°Ct o (Oncorhynchus nerka), showed however,tha t -70°C lastsapproximatel y 2mi nresultin gi n DMSOa t6.8 %wa s tolerated wheni twa s a rateo f35°C/min . Since therat e added slowly toth ecells . decreases above-60°C ,othe r estimatesma y belower . Allowing no equilibration resulted inbes t fertility (Fig.3) . A similar finding has Thefreezin g rateachieve d by thepelle t beenmad ei nvariou ssalmoni d species (Ott techniqueha s beensuitabl e for salmonid &Horton ,1971b ;Legendr e& Billard ,1980 ; spermatozoa,(Stei n& Bayrle ,1978 ; Bayrle,1980 )an d inse abrea m (Billard, Buyukhatipoglu &Holtz , 1978;Legendr e& 1978b)commo n carp (Moczarski,1977 )an d Billard,1980 ;Stos s &Refstie ,1982) , channel catfish (Gueste t al., 1976). It Coregonus species (J.Piirone n &H . also supports thehypothesi s that Hyvarinen,persona l communication)an d the cryoprotectants dono thav e topermeat e pikeEso xluciu s (DeMontalember t et al., thecell s inorde r toprovid e protection 1978). 118 The composition of thesper m extender relatively unimportant due to the fact that hasalway s beeno fconcer nan dmuc hwor k with the technique described sperm cells are has beencarrie d out tooptimiz e the exposed to the extender for only a few extender composition. Mineral seconds. This is the case both prior to concentrations have oftenbee nbase d on freezing and after thawing. analysis of seminalplasma . The exceptional fertilization resultsobtaine d A relatively simple extender based on witha ver y unconventional extender in NaCl, NaHCÛ3 and glycine was used Atlantic salmonb yMouni b in197 8le d toa successfully in Atlantic cod and plaice comparison ofextender s fromth e (Mounib et al., 1968; Pullin, 1972), while a literature (Fig. 4). mixture of diluted seawater and glycerol has produced good results in herring spermatozoa (Blaxter, 1953). 1 Stein and Bayrle (1978) "V2" 2 - - • y2e- Various dilution rates of milt with 3 Borchard (1978) "extender 6" extender have been tested. In salmonids,no 4 " • " T differences were found when using rates of 5 Truscott and Idler (1969) "rtfx No.»" 1:1 to 1:19 (Truscott &Idler , 1969; Ott & 6 Mounib (1978) Horton, 1971b; Buyukhatipoglu & Holtz, 1978; 7 Stoss and Holtz (l98l) Bayrle, 1980). Legendre and Billard (1980), however, reported inferior post-thaw fertility results at dilution rates of 1:1 1 and 1:9 compared to 1:3. In pike, sea bass and sea bream spermatozoa a decrease in xxx ± fertility was obtained when dilution rates exceeded 1:2 (De Montalembert et al., 1978; Billard, 1978b). Interpretation of these results is difficult. Assuming that the concentration of cryoprotectant was kept constant in the final dilution and that the different cell density was accounted for at insemination, little difference between dilution rates would be expected. The effect of thawing rates has been SPERM EXTENDERS FROM VARIOUS AUTHORS investigated in pellet frozen spermatozoa from chum salmon (Stoss et al., in preparation). By adjusting the temperature Fig. 4. Effect of extenders on post-thaw of the thawing solution, rates between fertility in rainbow trout spermatozoa (x 140°C/min to 1500°C/min could be obtained ± SD, n = 5) (Stoss &Holtz , in without overheating the sperm cells. preparation). Post-thaw fertility almost identical to fresh sperm controls was obtained with the highest thawing rate; rates of 600 or In all cases the procedure of freezing 140°C/min were less suitable. and thawing described by Stoss & Holtz (1981a) was carried out. Clearly, little 4. Post-thawing and insemination variability was caused by the various extender media. Only the results obtained After thawing, spermatozoa may show with the extenders by Truscott & Idler different motility characteristics than (1969) and by Mounib (1978) were.different prior to freezing. Spontaneous motility from each other. induction has been observed (Stoss and Holtz, 1981a). The duration of motility can In pursuing the question about the be reduced, as in grouper spermatozoa, from optimal extender composition further, it 1/2 hr in fresh cells to 30 sec in was subsequently found that even an frozen-thawed cells (Withler and Lim, 1982). aqueous solution of DMSO provided cryoprotection and post-thaw fertilities Motility in fresh pink salmon of 44% and 67% were obtained in Atlantic (Oncorhynchus gorbuscha) spermatozoa was salmon and rainbow trout spermatozoa prolonged from 30 sec to 10 min when (Stoss & Refstie, 1982; Stoss & Holtz, in activated with an 120 mMNaHC03-solutio n to preparation). An extender consisting of which IBMX (3-isobutyl-l-methylxanthin) had 300 mMglucos e and DMSO (6.8% final been added. An attempt to prolong motility concentration) has been used with good with IBMX in freeze-thawed cells failed success in Salmo and Oncorhynchus species (Stoss et al., in preparation). (Stoss &Refstie , 1982; Stoss et al., in preparation). The extender composition is The rapid loss of motility is correlated 119 witha rapi d decrease inpost-thawin g Scott & Baynes, this volume). The different fertility. Adela y ofonl y 30se cbetwee n capabilities of ova from individual females thawing and insemination reduced fertility to become fertilized by cryopreserved significantlyi nrainbo w troutsper m spermatozoa still requires clarification. (Fig. 5). 2) High cell densities.of frozen-thawed ÎOOr 0 control spermatozoa are still required to achieve fertility close to fresh sperm results. To assess further improvements, not only maximum fertility per se but also cell density at insemination should be reported. 3) Post-thaw motility can be rather brief which in turn limits the period of fertility. Enhancing motility may improve the consistency of fertilization results. Acknowledgement JS received a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada, funded by the Department of Fisheries and Oceans. The authors are grateful to M. Young who typed Interval Thawing-lnsemination (min] the manuscript. Fig. 5. Interval thawing-insemination in References pellet-frozen rainbow trout spermatozoa (X ± SD, n = 7; Holtz &Stoss , 1981a). Ashwood-Smith, M.J., 1980. Low temperature preservation of cells, tissues and organs. In: M.J. Ashwood-Smith and J. In testing the effect of thawing frozen Farrant, (Ed.'J: Low Temperature rainbow trout sperm pellets in various Preservation in Medicine and Biology. solutions, only the ones which induced Pitman Medical Ltd., Tunbridge Wells, motility (ovarian fluid, 120 mMNaHC0 3, Kent. p. 19-44. 120 mMNaCl ) resulted in appreciable Baynes, S.M., A.P., Scott &A.P . success. Dawson,1981. Rainbow trout Salmo gairdnerii Richardson, spermatozoa: Not all cells may survive the effects of cations and pH on motility. cryopreservation procedure. Densities of J. Fish Biol. 19:259-267. frozen-thawed sperm cells have therefore Bayrle, H., 1980. Untersuchungen zur to be relatively high to reach a plateau Optimierung der Befruchtungsfahigkeit von in fertilizing capacity. This is reached gefrierkonserviertem Fischsperma. at a dilution factor of approximately Dissertation, Tech. üniv. München. 1:100 in rainbow trout spermatozoa Billard, R., 1970. Ultrastructure comparée (Legendre &Billard , 1980; Stoss & Holtz, de spermatozoïdes de quelques poissons 1981a). In fresh spermatozoa dilution teleosteens. In: B. Baccetti, (Ed.): rates between 10*"^ to 10~* still result in Comparative Spermatology. Academic Press, high fertility (Billard, 1981). New York, London, p. 71-79. Billard, R., 1978a. Changes in structure Conclusions and fertilizing ability of marine and freshwater fish spermatozoa diluted in Relatively simple procedures can be media of various salinities. Aquaculture applied to short-term preserve immotile 14:187-198. spermatozoa and non-activated ova. Billard, R., 1978b. Some data on gametes preservation and artificial insemination Cryopreservation procedures have been in teleost fish. Actes Colloq. Centre refined for salmonid spermatozoa. These National pour 1 "Exploitation des Oceans illustrate the importance of each step (CNEX0) 8, 59-73. involved. A number of problems remain, Billard, R., 1980. Reproduction and relating to all species. artificial insemination in teleost fish. Proc. 9th Intern. Congr. Anim. Reprod. and 1) Gamete quality is still an Artif. Insem., Madrid 1980, RT-H-3, unpredictable influence. Both aging of 327-337. cells and nutritional factors can affect Billard, R., 1981. Short-term preservation spermatozoa (Billard et al., 1977; see of sperm under oxygen atmosphere in 120 rainbow trout (Salmogairdneri) . all-female and sterile coho salmon,an d Aquaculture23:287-293 . experimental evidence formal e Billard,R ., J. , Dupont,& G .Barnabe , heterogamety. Trans.Am .Fish .Soc .Ill : 1977. Diminution de lamotilit ee tl a 367-372. duréed econservatio n dusperm ed e Hunter,G.A. ,E.M .Donaldson ,J . Stoss& Dicentrarchuslabra xL .(poisson , I.Baker ,1982b . Production ofmonose x teleosteen)pendan t laperiod ed e female groupso fchinoo k salmon spermiation. Aquaculture11:363-367 . (Oncorhynchustshawytscba )b yth e Blaxter,J.H.S. ,1953 . Sperm storage fertilization ofnorma l ovawit hsper m and cross-fertilization of springan d from sexreverse d females. Presented at autumn spawning herring. Nature (Lond.) Int.Symp .Genetic s inAquaculture , 172:1189-1190. Galway,Ireland ,Marc h 29-April3 ,1982 . Buyukhatipoglu,S. ,& W .Holtz ,1978 . Hynes,J.D. ,E.H .Brown ,J.H . Helle,N . Preservation of trout spermi n liquido r Ryman &D.A .Webster ,1981 . Guidelines frozen state. Aquaculture14:49-56 . for thecultur e of fishstock s Chao,N.H. ,H.P. ,Che n& I.C .Liao , forresourc emanagement . Can,J .Fish . 1975.Studyo ncryogeni c preservationo f Aquat. Sei. 38:1867-1876. greymulle t sperm. Aquaculture Kuo,CM. , 1982. Progressi nartificia l 5:389-406. propagation ofmilkfish . ICLARM Clemens,H.P .& L.G .Hill ,1969 . The Newsletter 5,8-1 0 (InternationalCentr e collectionan d short-term storageo f forLivin gAquati cResource sManagmenf , milt ofwhit e bass. Prog.Fish-Cult . MCCP.O .Bo x1501 ,Makati ,Metr oManila , 31:26. Philippines). Davenport,J. , S.Lonnln g &E -Kjjorsvik , Legendre,M .& R .Billard ,1980 . 1981. Osmotic and structural changes Cryopreservationo f rainbowtrou t spermb y during early development of eggsan d deep-freezing. Reprod.Nutr .Bev .20 : larvaeo f thecod ,Gadu smorhu aL . J. 1859-1868. FishBiol .19:317-331 . Leibo,S.P. ,J.J .McGrath' SE.G .Cravalho , DeMontalembert,G. ,C .Br y &R . Billard, 1978. Microscopic observationso f 1978. Control ofreproductio n in intracellular ice formationi n northernpike . Am.Fish .Soc .Spec . unfertilized mouseov aa sa functio no f Publ.11:217-225 . cooling rate. Cryobiology15:257-271 . Erdahl,D.A . &E.F .Graham ,1980 . Mazur,P. ,1963 . Kinetics ofwate rlos s Preservation ofgamete s of freshwater fromcell sa t subzerotemperature san dth e fish. Proc. 9thIntern .Congr .Anim . likelihood of intracellular freezing. J. Reprod.Artif .Insem. ,Madri d 1980 Gen.Physiol .47:347-369 . RT-H-2,317-326 . Mazur,P. ,1977 . Therol e of intracellular Farrant,J. , 1980. General observations freezing inth e death ofcell scoole da t oncel lpreservation .In :M.J .Ashwood - supraoptimal rates. Cryobiology Smith &J .Farran t (Ed.): Low 14:251-272. TemperaturePreservatio n inMedicin ean d Mazur,P .& R.H .Miller ,1976 . Survivalo f Biology.Pitma nMedica lLtd. ,Tunbridg e frozen-thawed humanerythrocyte sa sa Wells,Kent .p .1-18 . functiono f thepermeatio no fglycero lan d Gardiner,D.M. , 1978a. Fine structureo f sucrose. Cryobiology13:523-536 . the spermatozoono f theviviparou s Meryman,H.T. ,1971a . Cryoprotective teleostCymatogaste raggregata . J.Fis h agents. Cryobiology 8:173-183. Biol.13 :435-438 . Meryman,H.T. ,1971b . Osmotic stressa sa Gardiner,D.M. , 1978b. utilizationo f mechanism of freezing injury. Cryobiology extracellular glucose byspermatozo ao f 8:489-500. twoviviparou s fishes-. Comp.Biochem . Moczarski,M. , 1976. Cryobiologicalfactor s Physiol.59:165-168 . ingras scar p preservation. Proc.8t h Ginsburg,A.S. , 1963. Sperm-egg Intern.Congr .Anim .Reprod .Artif . association and itsrelationshi p toth e Insem.,Krako w 19764 :1030-1033 . activation of theeg g insalmoni d Moczarski,M. , 1977. Deep freezing ofcar p fishes. J.Embryol .Exp .Morphol . Cyprinuscarpi oL . sperm. Bull.Acad . 11:13-33. Pol. Sei.Ser .Sei .Biol .15 :187-190 . Guest,W.C. ,J.W .Avaul t &J.D .Roussel , Morisawa,M .& K . Suzuki,1980 . Osmolality 1976. Preservation of channelcatfis h and potassium ion:thei rrole si n sperm. Trans.Am .Fish .Soc .105 : initiationo f spermmotilit y inteleosts . 469-474. Science10:1145-1147 . Hines,R .& A .Yashouv ,1971 . Some Mounib,M.S. ,1967 . Metabolism ofpyruvate , environmental factors influencing the acetatean dglyoxylat e byfis h sperm. activity of spermatozoa ofMugi lcapit o Comp.Biochem .Physiol .20 :987-992 . Cuvier,a gre ymullet . J.Fis hBiol .3 : Mounib,M.S; ,1978 . Cryogenic preservation 123-127. of fishan dmammalia nspermatozoa . J. Hunter,G.A. , E.M. Donaldson,F.W . Goetz& Reprod.Fertil .53 :13-18 . P.R. Edgell,1982a . Productiono f Mounib,M.S. ,P.C .Hwan g &D.R . Idler,1968 . 121 Cryogenic preservation ofAtlanti c cod betweenthawin g and insemination. (Gadusmorhua )sperm . J.Fish .Res . Aquaculture22:97-104 . Board Can. 25:2623-2632. Stoss,J .& W .Holtz ,1981b . Nagase,H. ,1964 . Deep freezing bull Cryopreservation ofrainbo w trout (Salmo semeni nconcentrate d pellet form. gairdneri)sperm . II.Effec t ofp Han d I.Factorsaffectin g survivalo f presence ofa bufferi nth ediluent . spermatozoa. Proc.4t hIntern .Congr . Aquaculture25:217-222 . Anim.Reprod .Artif .Insemin. ,Trent o Stoss,J . &T .Refstle ,1982 . Short-term 19643:410-415 . storage and cryopreservation ofmil t from Ott,A.G .& H.F .Horton ,1971a . Atlantic salmonan d seatrout . Fertilization of steelhead trout(Salm o Aquaculture (inpress) . gairdneri)egg swit h cryo-preserved Truscott,B .& D.R . Idler,1969 . An sperm. J.Fish .Res .Boar d Can.28 : improved extender forfreezin g Atlantic 1915-1918. salmonspermatozoa . J.Fish .Res .Boar d Ott,A.G .& H.F .Horton ,1971b . Can.26:3254-3258 . Fertilization of chinook and coho salmon Truscott,B. ,D.R . Idler,R.J .Hoyl e& eggswit hcryo-preserve d sperm J. H.D.Freeman ,1968 . Sub-zeropreservatio n Fish.Res .Boar d Can.28:745-748 . ofAtlanti c salmonsperm . J. Fish.Res . Petit,J. , B.Jalabert ,B .Chevassu s& Board Can.25 :363-372 . R.Billard, 1973. L'insémination Whittingham,D.G . (1980). Principleso f artificielle de la truite (Salmo embryo preservation. In:M.J .Ashwood - gairdneri Richardson). I.- Effet sd u Smithan dJ .Farran t (Ed.): Low tauxd e dilution,d up He t del a TemperaturePreservatio n inMedicin ean d pression osmotiqued udilueu r surl a Biology.Pitma nMedica lLtd. ,Tunbridg e fécondation. Ann.Hydrobiol .4:201-210 . Wells,Kent .p .65-83 . Pueschel,H. ,1979 . Eibildung und Withler,F.C. , 1982. Cryopreservationo f Eikonservierung beide r spermatozoa of some freshwaterfishe s Regenbogenforelle. M.Sc.Thesis , cultured inSout han d SoutheastAsia . University ofGoettingen ,Institut efo r Aquaculture 26:395-398 . AnimalBreedin g andGenetics . Withler,F.C .& L.C .Lim ,1982 . Preliminary Pullin,R.S.V. ,1972 . The storageo f observations of chilled and deep-frozen plaice (Pleuronectes platessa)sper ma t storage of grouper (Epinephelus tauvina) lowtemperatures . Aquaculture 1: sperm. Aquaculture 27:389-392 . 279-283. Yamamoto,T. ,1961 . Physiology of Pullin,R.S.V .& CM . Kuo,1980 . fertilization infis h eggs. Int.Rev . Developments inth e breeding ofculture d Physiol.12:361-405 . fishes. Paperpresente d at Symp.o n Yanagimachi,R. , 1957. Studieso f Advances inFood-Producin g Systemsfo r fertilization inClupe apallasii . Parts Arid andSemi-Ari d Lands,Kuwai tCity , I-III. Zool.Mag .(Tokyo )66:218-233 . 1980. Ricker,W.E. ,1981 . Changes inth e averagesiz ean d averageag eo fPacifi c salmon. Can.J .Fish .Aquat .Sei .38 : 1636-1656. Ryman,N. ,1981 . FishGen ePools . Ecol. Bull.NF R (Naturvetensk. Forsknlngsraadet)34 . Sanchez-Rodriguez,M . &R .Billard , 1977. Conservation de lamotilit ee td u pouvoir fécondant dusperm e detruit e arc-en-cielmainten u ade stemperature s voisines de0°C . Bull.Fr .Piscic .265 : 143-152. Sin,A.W. ,1974 . Preliminary results on cryogenic preservation of spermo f silver carp (Hypophthalmichthys molitrlx)an d bighead (Aristichthys nobilis). HongKon gFish .Bull .4 : 33-36. Stein,H .& H .Bayrle ,1978 . Cryopreservation of thesper mo f some freshwater teleosts. Ann.Biol .anim . Bioch.Biophys .18:1073-1076 . Stoss,J .& W .Holtz ,1981a . Cryopreservation of rainbow trout (Salmo gairdneri)sperm . I.Effec t of thawing solution,sper m density and interval 122 CRYOBIOLOGYAN DTH ESTORAG EO FTELEOS TGAMETE S BrianHarve y Departmento fBiology ,Universit y ofVictoria ,Victoria ,B.C . Canada Summary Thesucces so fcryobiolog y infis hrepro ductionha sbee n limited intw oways . The Theproble m ofgamet estorag e inteleosts , firsto fthes estem sfro mattempt s atwhole whetherb y chilling or freezing,i sto oofte n saletransfe ro fmammalia ntechnique swithou t attacked empirically. Thisapproach ,thoug h a corresponding understanding of fundamental understandable given thepractica lnatur eo f cryobiology; the second fromphysiologica l theproblem ,ha sproduce d alarg ean dcon pecularities of fishgamete stha tplac e fused literature documenting awealt ho f rathersever econstraint so n cryopreservation methodswithou t unifyingbiologica ltheme . success. Ishal ltherefor e firstrevie wth e Teleost embryosrepresen t aparticula r fundamentals ofcryobiology ,the nconside r cryobiologicalchallenge ;th efe wpublishe d thoseaspect so f themorpholog y andphysiolog y attemptst ofreez ethe mmak ei tclea rtha t ofspermatozo a andov atha trelat edirectl y extrapolationo fmammalia n results,whil e toapplicatio n ofthes eprinciples . Someo f basically effective forsper mpreservation , thediscussio nwil lreflec tm yow nexperienc e willno twor k forembryos . inth ecryopreservatio n ofsper m (andth e Thispape rreview scryobiologica lprin attempted cryopreservation ofov aan dembryos ) ciplesa sthe y apply toth echilling ,super fromwarm-wate r andcold-wate rteleosts . cooling and freezingo fteleos tspermatozoa , eggsan d embryos. Idiscus sth eproblem s- Cellularan dPhysica lConsequence so fCoolin g biologicala swel la stechnica l- involve d / infreezin g any celltype ,includin g therol e Several excellent recentreview scove rth e ofcryoprotectant ,formatio no f intracellular fundamentals ofcryobiology ;th ereade ri s ice,an dfreezin g andwarmin g rates,an d referred inparticula r tothos eo fMazu r attemptt orelat ethe mt osuc hpeculiaritie s (1981)an dAshwood-Smit h (1980). ofteleos tgamete san dembryo sa ssize , Whena cel li scoole d ina naqueou smediu m permeability,motilit y andactivation . thetemperatur ewil lgenerall y fallbelo wth e freezingpoin to fbot h cellan dmediu mbefor e Introduction freezing occurs;tha t is,bot hcel lan d mediumwil l supercool. Inmos tcase sth ecel l The impacto fcryobiolog y onanima lhus will supercool farthertha nth esurroundin g bandry isundoubted , and iti sno t surprising medium sothat ,whe n icefirs tform si nth e thata goo d dealo feffor tha sbee npu tint o medium (usually slightlybelo w 0C) , dis thetransfe ro fth eresultin gbenefit st o solved solutesbecom eprogressivel yconcen fishculture . Sucheffort shav eno talway s trated outsideth ecel lwhil eth ecel lwate r been rewarded,an d iti sth epurpos eo fthi s remainsunfrozen . Thecel lrespond sosmotic - paper tosugges treason sfo rthi sfailure . allyt oth eresultin g soluteimbalanc eb y Cryobiology canpla ya rol ei nfis hcultur e losingwate r toth e surroundingmedium . andresearc h inthre eways . First,th e Ifcoolin g issufficientl y slow,th ecel l problem ofnon-coinciden tmaturatio n ofmale s willlos eenoug hwate rt oremai n inosmoti c andfemale sca npartl yb eovercom ei fsperma equilibriumwit hth econcentratin gbrin e tozoa orov aar ehel d instorage . Artificial surrounding it. Ifcoolin g israpid ,however , fertilization thusbecome smor econvenient , thecel lwil lequilibrat eb y freezingintern andphysiologist s and fishfarmer salik eca n allyupo n seedingb y icecrysta lpunctur e benefit fromthi sapproach . Second,program s throughth ecel lmembrane . Undermos tcon ofselectiv ebreedin g canb eundertaken . ditions intracellular freezing isfatal . This includes notonl yth egradua l stock Avoidance ofintracellula r freezingb yslo w improvement thati sa greate ro rlesse rcon coolingi snot ,however ,a panacea ;lowerin g cernt oever ycommercia l fishfarmer ,bu t thetemperatur e tooslowl yprolong sth ehyper alsoattempts ,particularl y inth eThir d osmotic stateo f themediu m toth epoin t World,t obree dindigenou s species. Third, wherehig h external saltconcentration scaus e cryobiology canpla ya nimportan tpar ti nth e irreparablemembran e damage. Damagefro m conservation ofgermplasm . Inthi sappli intracellular freezing orextracellula r salt cation,gamete sfro m threatened specieso r concentrations ismos t likelyt ooccu rbet strainsar ebanke d asa hedg eagains tth e weenabou t-15° Can d-50°C ;beyon d -50°C,th e dwindling genetic variability causedb y temperature canb erapidl y lowered toa con environmentaldisturbance s and extensive venient storagepoint ,usuall y-19 6 C, the monoculture. temperatureo f liquid nitrogen. No thermally drivenreaction soccu r atthi stemperatur e 123 and storage is essentially indefinite; in diction of the optimal cooling rate for any fact, storage lower than -130 C, the glass teleost egg, experience in our laboratory has transition point of water, will likely be as shown rates as low as 0.01 C min to be good. Warming, of course, is equally hazar ineffective for rainbow trout eggs (Harvey dous, because the same physical processes and Ashwood-Smith 1982). Part of the blame must take place in reverse. for this failure can probably be laid to Fortunately, some of the problems problems of cryoprotectant penetration (see attendant on the threat of intracellular below), but observation of ice formation freezing and solute injury can be minimized within the ova makes it difficult to avoid by the addition of cryoprotectants to the the conclusion that dehydration is simply too cells and medium before cooling. Cryoprotec slow. tants may or may not permeate the cell; Size is again a problem with-the teleost permeating compounds include dimethyl- embryo, although paradoxically less severe sulfoxide (DMSO) and glycerol, while those for relatively early stages of development. that presumably simply coat the cell surface The gastrulating embryo, though roughly the include sugars and such high molecular weight same size as the unfertilized egg, is composed polymers as polyvinylpyrollidone (PVP) and of many cells of a cryobiologically manage various dextrans. The dependence of protec able size; it follows that, suitably protected tion on permeation is still a matter of at least some of these embryonic cells should -cryobiological debate (Mazur 1970); one survive freezing. We have shown this to be advantage of non-pèrmeating cryoprotectants true using half-epiboly embryos from Brachy- is their ease of removal upon thawing. The danio rerio: in embryos cooled at 0.05 Cmin protective action of permeating cryoprotec to -196 C, individual cell survival is high tants.i spresume d to be colligative. (Harvey 1982b). Yet the practical importance of this result is minimal, for reasons to be Factors affecting the cryobiological detailed below. survival of teleost gametes and embryos In fact there has been almost no success in any attempts to freeze macroscopic bits of Cell size, cooling rate, and water tissue. Rajotte and Mazur (1981) report permeability resumption of biochemical function in fetal rat pancreases cooled to -196 C, but this One of the most important theoretical tissue is sheetlike, and the distance covered developments in cryobiology has been the by water during dehydration is not more than quantitative description of the dependence a few cell layers. Luyet and Gehenio (1954) of cell survival on size, cooling rate, and have published accounts of cryopreservation Water permeability. Thé presently accepted of large tissue aggregates, such as embryonic model, first proposed by Mazur in 1963, chick hearts,b y very rapid freezing, and permits calculation of the intracellular have suggested that vitrification of cell water content as a function of temperature, water can permit some cell survival. We have so long as the cell's surface area, its only been able to preserve epithelia from permeability to water, and the dependence of B. rerio embryos using this method, although water permeability on temperature are known. modifications such as made by James (1980) In practical terms the model has produced for cryopreservation of schistosomulae of some useful rules of thumb, namely: Schistosoma mansoni may yet make ultra-rapid (a) the larger the cell (and the lower its freezing feasible. surface area:volume ratio) the slower it must be cooled in order to have time to dehydrate Cryoprotectants: kind, amount and permeation sufficiently to prevent intracellular freezing. There is a bewildering arsenal of cryo (b) the lower the 'cell'spermeabilit y to protectants of known or imagined efficacy, water, the slower it must be cooled. and present theories to account for intra Neither of these rules place any severe cellular and extracellular cryoprotection are constraints on the freezing of spermatozoa. shaky enough that the list can only getlonger , Fish ova, on the other hand, seem cryo- There is an understandable tendency for those biologically disqualified on both counts. attempting to cryopreserve a new cell type to Even the smallest teleost ovum is more than follow precedent and choose one of the most one hundred times the diameter of the average popular cryoprotectants in mammalian systems, spermatozoon,an dth e water permeability of DMSO or glycerol. This is what most investi the unfertilized ovum, if not low in an gators have done for fish spermatozoa (see absolute sense, is effectively minimal review by Scott and Baynes 1980), yet other because of the enormous distances water mole compounds may be more effective. We compared cules must travel in order to enter or leave methanol (when combined with milk powder as the cell (for a discussion of the problem of extracellular protectant) with DMSO and effective water permeability in teleost eggs, glycerol in the cryopreservation of Saro- see Loeffler and Ljzivtrup (1970) and Harvey therodon (Tilapia) mossambicus sperm, and and Chamberlain 1982). Although it is still found that, on a molar basis, protection was impossible to arrive at a quantitative pre more than twice that of the more commonly 124 used compounds (Harvey 1982a). Weals ofoun d solidC O ,a twhic h dehydrationwil lb ecom strong,an dunexpected , synergistic effects plete)o rbetwee n 0 and 5C (seebelow) . whenmethanol ,DMSO ,an dglycero lwer ecom Unfertilized ova,however ,tolerat esuper binedwit hmil kpowde r oreg gyolk , coolingwell ,an d techniques tostor ethe m suggesting thatvariou s combinationsb etrie d between 0 and- 5C ma yprov ever yusefu li n beforea cryoprotectan tmixtur ei schosen . alleviatingproblem so fshort-ter mconser Spermatozoa are sufficiently smalltha t vation (1-2months) . Harvey andAshwood - cryoprotectantpenetratio n israpid ,an dn o Smith (1982)an dStos san dDonaldso n (1982) 'equilibrationperiod 'i srequire d (Harvey reported theabilit yo fth eprotecte d sal- 1982a). Penetrationo fth eunfertilize d monideg gt osupercoo l aslo wa s-1 6C ,an d ovum,however ,i sslow ,an dpose s acryo - recentexperiment s inou r laboratoryhav e biologicalproble m atleas ta sgrea ta sth e shownrainbo wtrou t eggst ob efertil eafte r lowcoolin g ratetheoreticall y required. amonth' s storagea t- 2C . Thisi sno ta Trouteggs ,fo rexample ,reac honl y 23%o f surprising result- th edro pfro mnorma l theexpecte d equilibrium valueafte rincu ambienttemperatur e isno tgreat ,an dth e bation inmethano l for 2hours ;DMS Oan d major challengeappear st ob eprovidin ga n glycerolpenetrat e evenmor eslowl y (Harvey appropriateosmoti c environment. Theegg so f andAshwood-Smit h 1982). This lowleve lo f tropical fishesar eles sabl et owithstan d protection ensuresthat ,eve nthoug hcoolin g cooling;w ehav eneve r succeeded incoolin g ratesar ever y lowindeed ,surviva li s theunfertilize d eggso fBrachydani o lower unlikely. Theonl ywa you to fthi sdilemm a than1 0C befor efertilizatio n isseverel y ist oforc eth ecel lt otak eu pa sufficien t affected. Storagea t 5C fo rmor etha n3 0 amounto fcryoprotectan t bybathin g iti na minutesresult si ncomplet einfertility . veryhig hinitia l concentration,a swa sdon e byRajott ean dMazu r (1981)wit h fetalra t Motility,activatio n andoxyge n requirements pancreas. Thismetho ddoe swork ,bu tagai n thesiz eo fth etissu ei slimiting :no tonl y Teleostgamete sar ewonderfull y adaptedfo r arehig hconcentration s (>2M )o fmos tcryo - externalfertilizatio n andembryoni cdevelop protectants likely tob etoxic ,bu tcells , ment ina variet yo faquati cenvironments , onceloade dwit hthes e compounds,becom e yetsevera lo f thestrategie sadopte d for extraordinarily sensitiveosmoticall y sothat , survivalpos e realproblem s forth ecryo - uponthawing ,remova lo f cryoprotectantb y biologist. dilutionmus tb es oslo wtha ttoxi c effects begint o showup . Wehav e shownthi swit h Motility inteleos t spermatozoa isnormall y Brachydanio embryosprotecte dwit h25 % confined toa perio d of frenetic activity glycerol and cooled slowlyt o-19 6C :upo n whoseshor tduratio ni soffse tb yth eproduc warming,th eembry oshow slittl esig no f tiono fvas tnumber so fcells . Thesecell s damagean d ahig hpercentag eo fcell sar e arethu sno tspecificall y adapted (asar e alive. Yetremova lo fth ecryoprotectant , mammalian sperm) forutilizatio n of exogenous evenb yver y slowdialysis ,i simpossibl e energy sourcesonc emotilit yha scommenced , (Harvey 1982b). Nevertheless,individua l althoughthe y appearquit e capableo fs o cellso rsmal lclump so fcell sca nb esepar doingwhe nprovide dwit hth eopportunity .Fo r ated fromth ethawe d embryoan dremai nviabl e cryopreservation,though ,i ti sadvantageou s afterdilution ,suggestin g apotentia lmetho d tokee pth ediluen t assimpl ea spossible , forth epreservatio n ofgeneti c information andi nth eabsenc eo fadde d energy sourcesi t should techniques fornuclea r transplantation isimportan t toensur etha tspermatozo aar e bedevelope d forteleosts . notactivate dbefor e freezing starts. In many casesth eosmoti cpressur eo fth eadde d cryoprotectantwil lb e sufficient toinhibi t Storagea trelativel yhig hsubzer o motility;w ehav efoun dpowdere dmilk ,itsel f temperatures anexcellen t extracellular cryoprotectant,t o bea ninfallibl e inhibitoro fmotility . Some Storageo fteleos t spermatozoa isa prac degreeo fmotilit ymay ,incidentally ,b e ticalproblem ,an dsevera linvestigator shav e triggeredb y thefreeze-tha wprocess ;Stos s attempted tous eth emos tpractica lmean sa t (1982)ha snote d thisphenomeno n insalmoni d hand,namel y thefreezin g cabineto fa sperm,a shav ew e forBrachydanio ;membran e laboratory refrigerator. Unfortunately, damagean d consequent depolarizationma y cryobiological theory states thata tth e account forit . temperature likelyt ob eencountere d (approx. Spermatozoanmotilit ybecome sa mor esever e -15C )cell sar eonl ypartiall y dehydrated problemwhe nmil ti st ob e storedabov e 0C andtherefor ebath e ina concentrate d 'brine' forsevera lday so rweek s (chilled storage); ofsolute stha twil ldamag eth ecel lmembran e solittl ewor kha sbee ndon e inthi sare a ('solutioneffects') . Wehav econfirme d this thaton ecanno tye t saywhethe rmotilit y forspermatozo a ofBrachydanio ;n oamoun to f shouldb eblocke d fromth eoutse to rwhethe r augmented extracellular cryoprotectant seems spermatozoa shouldb e suppliedwit h complex ablet opreven t celldeat hwithi n afe whours , exogenous energy sourcesthroughou tth e andpreservatio n of spermatozoa canonl yb e storageperiod . Thesetw oapproache sessen donebelo wabou t-7 9C (thetemperatur eo f tiallyrepresen ta choic ebetwee nundilute d 125 anddilute d ("extended")storage . Ourow n bemad ebetwee nactivatio na sa resul to f experience suggests thatfe wassumption sca n osmotic,ioni co rp Hchanges ,an dmechanica l safelyb elef tuntested :w ehad ,fo rexample , activation. Thelatte ri smuc h lessunde r assumed that,i na tropica l fish sucha s thecontro lo fth eexperimenter ,an dca n Sarotherodono rBrachydanio ,motilit ywoul d likelyonl yb eprevente db yscrupulou s surelyb elimite db yth ever yac to flowerin g attentiont oth efemale' s stateo freadiness . thetemperatur et o0 C - a dro po f25-3 0 degrees fromth enorma lambien ttemperatur e forthes ecells . Yetobservatio no fsper m References suspensionswit hth ecryomicroscop e showed thatroughl y 50%o fth espermatozo awer e l.Ashwood-Smith,M.J .1980 .Lo wtemperatur e weaklymotil ea t0 , an dtha tisolate d cells preservationo fcells ,tissue san dorgans . werecapabl eo fsluggis htai lmovemen ta slo w In:M.J .Ashwood-Smit han dJ .Farrant ,Eds : as- 9C . Energy reservesar eclearl ybein g Lowtemperatur epreservatio n inmedicin e utilized atthes elo wtemperature sand ,fo r andbiology .Pitma nMedical ,Tunbridg e storaget ob esuccessful ,motilit ymus t Wells. eitherb einhibite d completelyo rencourage d 2.Epel,D .1980 .Ioni c triggersi nth efert byprovidin g asuitabl eenerg ysource . ilizationo fse aurchi neggs . Ann.N.Y . Inhibitiono fmotilit y cano fcours eb e Acad.Sei .339 :74-85 . achievedb ystorin g undilutedmilt ,ye tw e 3.Harvey,B .1982a .Cryopreservatio no f havefoun dth eviabilit yo fundilute dSaro Sarotherodon (Tilapia)mossambicu ssperma therodonmossambicu smil tt odro p severely tozoa. Inpreparation . within severaldays ,whil etha to fsper m 4.Harvey,B .1982b .Coolin go fembryoni c providedwit h exogenousnutrient s froma n cells,isolate dblastoderm san dintac t egg-yolk diluentt olas t3 o r4 time sa slong . embryoso fth ezebr afis ht o-19 6C .I n Inhibitiono fspermatozoa nmotilit yb yelev preparation. atingK + concentration iswel lknow n (Sneed 5.Harvey,B. ,an dAshwood-Smith ,M.J .1982 . andClemen s 1956),althoug hth eeffec ti s Cryoprotectantpenetratio nan dsupercoolin g complicatedb yth epresenc eo fCa ^ orMg ^ inth eegg so fsalmoni d fishes.Cryo - ions (Scottan dBayne s 1980). Wefoun d 25-50 biology 19:29-40 . mMK + toinhibi tmotilit yi nSarotherodo n 6.Harvey,B. ,an dB .Chamberlain ,1982 . mossambicus spermhel di na negg-yolk-citrat e Waterpermeabilit y inth edevelopin gembry o diluent,bu tthi sinhibitio n lastedn omor e ofth ezebr afis hBrachydani o rerio.Can . thana fe whours . J.Zool .60 :268-270 . Eggactivatio ni sanothe rpeculia rpropert y 7.Harvey,B. ,R.N .Kelle yan dM.J .Ashwood - thatmake s short-termo rchille dpreservatio n Smith,1982 .Cryopreservatio n ofzebr a difficult. Therei sa larg eliteratur eo n fishspermatozo a usingmethanol .Can .J . thisphenomeno ni nfishe s (Yamamoto1961 , Zool.i npress . Kalman 1959,Pott san dRud y1969 )an dfurthe r 8.Jaffe,L .1980 .Calciu m explosionsa s insightsma ywel lb egaine d fromrecen t triggerso fdevelopment .Ann .N.Y .Acad . summarieso fwor ko nechinoder mov aan dth e Sei. 339:86-101 . roleo fioni ctrigger si nfertilizatio nan d 9.James,E.R .1980 .Cryopreservatio no f activation (Epel1980 ;Jaff e 1980). Years Schistosomamanson i schistosomulaeusin g ago, Suzuki (1959)elegantl y demonstrated 40%methano lan drapi d cooling.Cryo-Lett . thategg so fcertai nwarm-wate r fishesbecam e 1:535-544 . infertilea ssoo na selevatio no fth echorio n 10.Kalman,S.M .1959 .Sodiu man dwate rex severedth eprotoplasmic-micropyla rconnec changei nth etrou tegg . J.Cell .Comp . tion. Wehav e foundmechanica lactivatio n Physiol. 54:115-162 . andsubsequen tparthogeneti c developmentt o 11.Loe ff1er , C.an dS .LjzSvtrup .1970 . Water bea majo r stumblingbloc ki nstorag eo fth e balancei nth esalmo negg .J .Exp .Biol . unfertilized ovao fB .rerio ,an dWithle r 52:291-298 . (1980)ha sreporte d similarproblem swit hov a 12.Luyet,B.J .an dP.M .Gehenio .1954 .Effec t ofTha icarp san dcatfishes . Interestingly, ofth erewarmin gvelocit yo nth esurviva l activationpose sn osuc hproble m insalmonids . ofembryoni c tissues frozenafte rtreat Wear epresentl y exploringway si nwhic h mentwit hethylen eglycol .Biodynamic a activationca nb elimited . Ficoll,a hig h 147:213-223 . molecularweigh tpolyme r (m.w.400,000 ) 13.Mazur,P .1981 .Fundamenta l cryobiology occasionally useda sa nextracellula rcryo - andth epreservatio no forgan sb yfreezing . protectant,delay sactivatio n inzebr afis h In:A.M .Karo wan dD.E .Pegg ,Eds :Orga n ovaa ta concentratio no f12% ,an dactivatio n preservation fortransplantation .Marce l canb ecompletel y inhibitedb yimmersin gth e Dekker,Ne wYor kan dBasel . eggsi nsilicon eoil . Ca^+-free solutions 14.Mazur,P .1970 .Cryobiology :th efreezin g areineffectiv ei ndelayin g activation. It ofbiologica l systems.Scienc e168:939-949 . isprobabl y safet osay ,however ,tha tsuc h 15.Mazur,P . 1963.Kinetic so fwate r lossfro m methodsonl yserv et olimi ta trai no fevent s cellsa tsubzer otemperature san dth e begunb yth eac to fforcibl y expressingegg s likelihood ofintracellula r freezing. J. fromth efemale ,an dtha ta distinctio nmus t Gen. Physiol.47:347-369 . 126 16.Potts,W.T. ,an dP.O . Rudy.1967 .Wate r balance inth eegg so fth eAtlanti c salmon,Salm osalar . J.Exp .Biol .50 : 223-237. 17.Rajotte,R.V .an dP .Mazur . 1981.Surviva l offrozen-thawe d fetalra tpancrease sa s a functiono fth epermeatio no fdimethy l sulfoxidean dglycerol ,warmin g rate,an d fetalage .Cryobiolog y18:17-32 . 18.Scott,A.P . andS.M . Baynes,1980 . A reviewo fth ebiology ,handlin gan d storageo fsalmoni d spermatozoa. J.Fish . Biol. 17:707-739 . 19.Sneed,K.E .an dH.P .Clemens .1956 . Survivalo f fishsper mafte r freezingan d storagea tlo wtemperatures .Prog .Fis h Cult. 18:99-103 . 20.Stoss,J . andE.M .Donaldson .1982 . Studieso ncryopreservatio n ofegg sfro m rainbow trout (Salmogairdneri )an dcoh o salmon (Oncorhynchuskisutch) .Aquacultur e inpress . 21.Stoss,J . 1982.Persona lcommunication . 22.Suzuki,R . 1959.Sper mactivatio nan d aggregationdurin gfertilizatio n insom e fishes II. Effecto f distilledwate ro n thesperm-stimulatin gcapacit y andfertil - izability ofeggs . Embryologia4:359-367 . 23.Withler,F . 1980.Chille d and cryogenic storageo fgamete so fTha icarp san dca t fishes.Can .Tech .Rep .Fish .Aq . Sei.No . 948. 24.Yamamoto,T . 1961.Physiolog y offertil ization infishes .Intern .Rev ,Cytol . 12:361-405 . 127 CRYOPRESERVATION OF RAINBOWTROU TSPERMATOZOA :VARIATIO N IN MEMBRANECOMPOSITIO NMA Y INFLUENCE SPERMATOZOAN SURVIVAL S.M .Baynes ,A .P .Scot t MAFF,Directorat e ofFisherie s Research,Fisherie sLaboratory , PakefieldRoad ,Lowestoft ,Suffol kNR3 3OHT ,U.K . F.D,F.E ,D. E andF.Ü.E . are theeffect s Summary duet ointeractio no f thefactors . After cryopreservationwit h asucrose - Results and discussion based diluent,th espermatozo a ofa sprat - fed groupo frainbo w trout gavea highe r fertilization ratetha nspermatozo a ofa group fed ona commercially-produce d pellet diet. With eggyol k included inth e diluent,however ,fertilizatio nwa sno tsig nificantly different. The results suggest that substances ineg gyol kbecom eassoci atedwit h thespermatozoa nmembran ean dtha t this association is influenced bymembran e composition. Introduction %egg yolkindiluent Inconsistent results fromcryopreservatio n pellet experiments aregenerall y accepted asinevi sprat 0 table. They do indicate,however ,tha tmor e factors are influencing theexperimenta l Figure. Mean fertilization rate asa material thanar ebein g controlled orexa percentage ofth econtrols ,wit h thesper m mined. Using factorialexperimenta ldesign , from the sprat and pellet-fed groups andth e wehav e examined theeffect s and interac four levelso feg gyol k inth ediluent . tionso f threevariables : eggyol k inth e freezing diluent,sper mvariability ,an d The fertilization rate forsper m from variation inth eeg gbatche s used totes t sprat-fed fishwa s significantly greater fertilizations. (P< 0.05 )tha n forth epellet-fe d group whenn oyol kwa s included inth ediluent . Methods Therewa sn osignifican t differencebetwee n the twogroup s of sperm,however ,wit heg g Spermwa sobtaine d from twogroup so f yolkpresen t atan yo f thethre e rainbowtrou t (Salmogairdneri) ,on e fedo n concentrations. chopped sprat,th eothe r ona pelle tdiet . Although the fatty acid composition ofth e Tosimplif yexperimenta l designw euse da spermatozoadi ddiffe rbetwee n thetw o constant freezing rate (30°Cmi n )an da groups (mostnotabl y 20:5u3- 8 % and 1U% for uniform fertilization techniquethroughout . sprat-an d pellet-fed respectively) iti s Asucrose-base d freezing diluent wasuse d unlikely that thedifference s observed in with0 ,5 ,1 0o r20 Zfres heg gyol k added. thecryopreservatio n work canb e attributed Frozen spermwa s tested by fertilizing tothi salone . batcheso fmixe d eggs ontw odifferen tocca The improvement insurviva l of thesper m sions. The survival of the spermwa s esti witheg gyol k inth ediluen t isclear ,bu t matedb y fertilization ratea sa percentag e analysiso fth edat awit hGLI Mshow ssigni ofcontro l fertilizations with excess fresh ficant interaction (P< 0.01 )betwee n feed semen. TheGLI M (Generalized LinearInter ingan ddiluen t (F.D), andbetwee ndiluen t activeModelling )syste mwa s used toanalys e and eggbatc h (D.E). Such interactioncon thedat a formai neffect s and interaction founds interpretationo fmai neffects . The with the linearmodel : causes of these interactions areprobabl y (1)tha teg gyol k inth ediluen t interferes P= U+ F + D + E + F. D +F. E +D. E +F.D.E . with fertilization (wehav e otherevidenc e + ^ijk forthis )an d (2)substance s ineg gyol k P ispercentag e fertilization, become associated with themembran e andcon u. isth epopulatio nmean , Sjîfc,th eerror , fera differen t degreeo fcryoprotectio no n F isth eeffec t due toth e feedingregime , the twotype so fsperm . Interaction ispro D isth eeffec t due tofreezin gdiluent , bably amajo r causeo funexplaine d variation E isth eeffec t duet oeg gbatch , incryopreservatio nwork . 128 CRYOPRESERVATION OFRAINBO W TROUT SPERM: EFFECTO FEQUILIBRATIO N H. Bayrle Bavarian State Institutefo rFishery , Starnberg, West Germany Introduction Immediately after diluting respectively after equilibration of up to 55 minutes This study presents theresult s ofthre e the samples were dropped with a syringe experiments carried out to evaluate the on carbon ice (-79°C) and frozen to influence of equilibration time on pellets of 0,2 cm3. The frozen pellets fertilizing capacity ofcryopreserve d rain were stored in liquid nitrogen (-195°C). bow trout (Salmo gairdneri Richardson) sperm. For thawing, 3 of the pellets were put in 10 cm3 of a thawing solution 3 (1 g NaHCO,i n10 0cm H ?0)an dimmediatel y Methods and strongly shaked. Just before the pellets were totaly thawed, the solution The sperm wascollecte d byhan d stripping was poured over the eggs. For control and stored in glass tubes for each male egg samples were also inseminated with separately. Before collecting the sperm fresh sperm.O naverag e 149,,+4 5( x+ s ;n= the glass tubes were inserted into the 135)egg s were inseminated pirreplicate . same water in which the males were kept and therefore had the same temperature For the analysis of variance the asth esperm . percentages of the fertilization rates were transformed with the arcus-sinus- The visually unobjectionable sperm samples transformation. F-test and multiple were mixed. range test (Duncan) were used to test the significance. For diluting the sperm the following extenders were used: Results V2e 750Im *J\J gNaCl lliy MW ,V 3i j 8m^^J g KClm y i\,u20 i ) i_0m wg NaHCOgm y •iw.iww^, 5 3 3 3 3 With fresh sperm the following 10— 0m gGlukose , 100cm H m0, H2 20,cm 2 0cm 2 fertilization rates were achieved: egg yolk (used in experiment 2) Experiment 1 95,7% + 0,6( x+ s ; n=2) V2f 750m g NaCl, 200m g NaHC0 100m g 3> Experiment 2 96,8 %+ 2,6( x+ s ; n=4) Glukose, 100 cm3 H 0, 20 cm3eg g 2 Experiment 3 82,9 % +15, 8( x+ s ; n=4) yolk (used in experiment 1 and3 ) The fertilization rates obtained with For cryoprotection dimethyl sulfoxide cryopreserved sperm are presented in (DMSO) was used. In experiment 1 and2 fig. 1- 3. 10 cm3 DMSO were added to 100cm 3o fV 2e respectively V2f . In experiment 3 9cm 3 DMSO were addedt o10 0cm 3o fV 2f . The temperature of the extenders was also adapted to that ofth e sperm inth e way mentioned above. = X+B The sperm wasdilute d with this solutions at a dilution rate (sperm : extender) of = 3 1 : 3 (experiment 1 and 2) respectively 1 :5 (experiment3) . For freezing the pelleting technique equilibration time (minutes) of Nagasee tal . (1964)wa sused . fig. 1. Experiment 1: Results of the fertilization tests with cryopreserved sperm 129 Nagase, H.,T . Niwa, 1964. Deep freezing bull semen inconcentrate d pellet form., I. Factors affecting survival of spermatozoa.- 5th. Int. Congr. Anim. Reprod.an dA.I. , Trejito. equilibration time (minutes) fig.2 . Experiment 2: Resultso fth efertilizatio n tests with cryopreserved sperm W A/ equilibration time (minutes) fig. 3. Experiment 3: Results of the fertilization tests with cryopreserved sperm highly significant difference (P< 0,001 ) respectively significant difference (P <0,01 ) incompariso n with the results without equilibra tion. Conclusion Rainbow trout sperm was cryopreserved using the egg yolk containing extenders V2 ean dV 2f (Bayrle 1980). Equilibration of the diluted sperm prior to freezing caused rising fertilization rates intw o experiments and a falling fertilization rate in a third one.Sper m qualityma y be the reason ofth edifferen t reactions on equilibration. References Bayrle,H. ,1980 . Untersuchungen zurOpti mierung der Befruchtungsfähigkeit von gefrierkonserviertem Fischsperma. Disser tationT UMünchen ,Weihenstephan . 130 PHYSIOLOGICAL STUDY ONTH EINITIATIO N OFSPER M MOTILITY INCHU M SALMON.ONCORHYNCHUSKET A 12 3 M;Morisaw a ,M .Okuno ,S .Morisaw a 1 2 Ocean Research Institute,Universit y ofTokyo ,Tokyo , Department ofBiology ,Universit y of Tokyo,Tokyo , Biological Laboratory,St .Mariann a University School ofMedicine ,Kawasaki,Japa n Introduction Much evidence hasbee n accumulated containing ATPan d cAMP,axonema lmovemen t was concerning thedetaile d mechanism ofsper m observed. Axonemal movement occurred witha motility inmarin e invertebratesan dmammals . velocity of 55/jm/sec. Theseresult s suggest However little isknow n about spermmotilit y thatth eaxonem eo fchu m salmon spermatozoa is inteleosts . Recentwor k byMorisaw a & functionally immotile and isconverte d toth e Suzuki(19B0)usin g fishes showed that sperm motile stateb yexposur et ocAMP . motility isinhibite d inth emal ereproductiv e organ bypotassiu m which isfoun d athig h concentration inth e seminal plasma inchu m Discussion salmon and areductio n ofpotassiu m concentration inth e surrounding environment Inth epreviou s paper(Morisawa &Okuno,1982) , at spawning isth efacto rwhic h initiates wedescribe d inth erainbo w trout thatth e spermmotility . Morisawa S0kuno(19B2 ] reduction ofpotassiu m concentration further demonstrated inth e spermatozoao f surrounding spawned spermatozoa affects another salmonidfish ,rainbo w trout,whos e membrane-mediated processes/i nintac t plasmamembran ewa sremove d byth edetergen t spermatozoa and then inducesth e synthesiso f TritonX-10 0tha tth ereleas eo f suppression intraflagellarcAMP . ThecAM P then converts bypotassiu m inducesth e increaseo fintra - the immotile axoneme toa motil e stateresultin g flagellar cyclicAM P and the synthesized inth einitiatio n of spermmotility . Inchu m cAMPconver t the immotileaxoneme(9+ 2 salmon spermmotilit y isals o initiated by structure;motil eapparatu s insper m thereductio n ofpotassiu m during natural flugellum)t oa motil e one,resultin g inth e spawningCMorisawa &Suzuki , 1980). Therefore initiation of spermmotility . an external signal,chang e inpotassiu m Herew edemonstrate d that cAMP isals oth e concentration also inducesa nintraflagella r factort otrigge r the initiation ofaxonema l factor cAMP through somechang e ofth eplasm a movement i.e. spermmotilit y inanothe r membrane. The synthesized cAMP then converts salmonid fish,chu msalmon . theaxonem efro m animmotil e statet oa motil e onean d themotil eaxonem e conveysmotilit y Methodsan dResult s toth espermatozoa . Inth ereproductiv e physiology offish ,th e Theplasm a membrane ofchu m salmon sperm, mechanism involved in spermmotilit y have which were collected byinsertin g apipett e been notwel l known. We have shown heretha t intoth e sperm duct,wer eremove d bymixin g fish spermatozoa area usefu lmateria lfo r onic e 1volum eo f semenwit h 2Dvolume so f investigating themechanis m of initiation the extracting medium[0.15 MKCl ,0. 5m M of spermmotility . Further studiesusin g CaCl ,0. 5 mM EDTA, 1m M dithiothreitol(DTT), fish spermatozoa may providemuc h valuable 2m MTri s buffera tp H 8.2 and 0.04 %Trito n evidencefo rmolecula r processes ofcel l X-100]fo r3 0seconds . Thesedemembranate d motility aswel l asfo rfis h culture studies spermatozoa are immotile becausethe y lack i.e.artificia l insemination offish . anenerg y supply(ATP)an d the components essential foraxonema lmovement . Wetrie d thereactivatio n ofimmotil eaxonem eb y References mixing atroo m temperature thedemembranate d spermatozoa withth ereactivatin g medium Morisawa,M .8 K .Suzuki ,1980 . Osmolality containing 0.15 MKCl ,2 m MPlgS O, 0. 5m M and potassium ion:Thei r roles ininitiatio n CaCl2, 2m M EGTA, 1m M DTT,2 0m MTri s of spermmotilit y inteleosts . buffer,p H 8.2 and 0.2 mPlAT P inth eabsenc e Science,210 :1145-1147 . orpresenc e of2 0;u McAMP . Observationswer e Morisawa,M . &M .Okuno ,1982 . CyclicAM P madewit h dark-field microscopy. inducesmaturatio n oftrou t sperm axoneme When cAMPwa sabsen tfro m the reactivating to initiatemotility . Nature,295:703-704 . medium,n oaxonema l movement occurred suggesting thata nenerg y supplyalon ei sno t enough to induceaxonema lmovement . Incontrast ,whe n thedemembranate d sperm were suspended inreactivatin g medium 131 NEW APPROACHES FORCRYOPRESERVATIO NO FSPER MO FGRE Y MULLET,Mugi l cephalus Nai-Haien Chao Tungkang Marine Laboratory, TFRI, Tungkang, Pingtung, Taiwan 916,Rep .o fChin a Summary Potency ofmil t extenders, sodium mixed with equal volume ofth emixtur e citrate,glucose , lactose,fructose , and frozen at29°C/mi nt o-196°C . The mullet serum,an dOtt-Horton' s exten best cryopreserved sperm recovered90 % ders12 9an d16 4supplemente d witho r motility,an dprovide d 28.64% fertili without potassium chloride orsodiu m ty (45.17%i ncontrol )an d62.60 %ha chloridewa scompared . Glucosean d tching rate (100%i ncontrol) . fructose were favorable andsupplemen t ofpotassiu m chloridewa sbeneficia l Methods andResult s forth emotilit y ofth emulle t sperm. Combination offreezin g mediaan d Extenders were solutions ofsalt , packing conditions ofth edilute d milt saccharide ormulle t serumwit h HEPES inP Estraw s provided aserie s offre buffer adjusted toa stabl ep Ho f 7.7. ezing rates ranged 1.5-876°C/min. Organic compounds were included in the case ofextender s 129an d16 4(Ot t § Extender andcryoprotectan t employed Horton, 1971)an don eo fth eingredi inth emos t satisfactory groupwa sa entKC lwa sconcentrate d forcompari mixtureo f80 %dilute d glucose solu sona sfunctio n ofion st oinfluenc e tion (5%)an d20 %DMSO . Miltwa s sperm motilitywa sa possibl e factor other thanp Han dosmoti c pressure (Hines §Yashouv ,1971 ) Table• Osmotic pressure of various extenders used for the Extenders with cryoprotectanto f study and their effect on motility of grey mullet better resultwer e choseni ncryopre - sperm servation test atth erati o of1: 1 with milt. After equilibration,th e Extender Concentration Averagedosmoti c Bestmotilit y mixturewa sdispense d intoP Estraw s (mM) pressure (mOsm/kg) and duration and frozena tdifferen t ratesb yusin g the freezing media ofdryice ,isopro - 100 220 •••* 30" panolwit h dryice,LN 2 vaporo rLN 2i n 200 435 ++ 1'17" combination with various packingo f Sodium 300 630 ++++ 1' 2" citrate 400 840 +++ 48" straws incaniste r ofbamboo ,plastic , 500 1150 ++ 10" PE,PB ,cellulos e thimbles,stainless , stainless wire,o rbein g nake. Guest 200 165 +++ 2' 3" 400 330 ++++ I' 6" et al. (1976) indicated that extended Glucose 500 360 +++++ 2'14" equilibration appear best forchanne l 600 660 +++ 1'16 " catfish while minimal equilibration 1000 900 +++ 41" and rapid freezing arebes t forsalmo - 200 210 +++ 1'57" nids. Itwa sfoun d thatfo rgre y mul 400 370 +++ 56" let equilibration within 1h an dmedi Lactose 500 455 +++ 1' 9" 600 560 +++++ 1'35 " ate freezing rate between 21an d119° C 1000 570 +++++ 1' 50 " /minar efavorable . 200 175 ++ 10" Motility reading andfertilit y eva 400 365 ++ 10" luationo fcryopreserved-thawe d sperm Fructose 500 435 +++++ 2'46" ofth ebes t group showed abette rre 600 530 ++ 28" 1000 910 ++ 10" sult (Fig.1 )tha n previous study (Chaoe tal . 1975). Mullet serum 465 ++++ 1'58 " •12 9 378 ++ 2'18 " •164 428 +++ 10" NaCl+KOl 100;100 383 +++ I'll" NaCl+Glucose 100;200 378 ++++ 1'15" KCl+Glucose 10;390 345 ++++ 1*18" KCl+Glucose 50;350 340 +++++ 1'17" KCl+Glucose 100;200 370 +++++ |i jn •12 9wit hKC l 15 290 ++ IMS" •129wit hKC l 20 395 ++ 1'35 " •129wit hKC l 25 487 ++++ 30" •129wit hKC l 40 690 +++++ 30" *+++++ver ystrong ;+++ +strong ; moderate; 132 S Motility Oll Fertility 0 Hatching rate -g 80. £ 40 60 Control Equilibrationtim e (min) References Chao, N.H.,H.P . Chen §I.e .Liao , 1975. Aquaculture. 5:389-406 . Guest,W.C. ,J.W .Avault ,Jr . §J.D . Roussel, 1976.Trans .Am . Fish. Soc. 3:469-474 . 133 THECHEMICA L COMPOSITIONO FTH E COMPANIONFLUID S OFTH EGAMETE S INTH E COMMONCAR P (Cyprinus carpio) M.G.Plouidy ,R .Billar d Laboratoire dePhysiologi e desPoissons ,I.N.R.A ,Campu s deBeaulieu ,3504 2RENNES ,Franc e Introduction Aknowledg e of thechemica l compositiono f The inhibitiono fsper mmotilit y inth emal e theovaria n fluid and the seminalplasm ai s genital tractwa s due toth erelativel yhig h necessary toa bette runderstandin g ofth e osmoticpressur e ofth esemina lflui d physiology of fishgamete s aswel la s toa (286mOsmoles/kg) .Motilit y initiated after morepractica l goal,tha to f composinga dilutionoccure d ina rang eo f 15o-200 diluent forartificia l insemination orfo r mOsmoles. thestorag eo rdeep-freezin g of thegametes . Thepresen tpape r describes the chemical Table. Composition ofovaria n fluidan d composition ofthes e companion fluids-i n seminalplasm a incommo n carp.Values±SD ; thecommo n carp. n = 10;(1 )% o f drymatter ; (2)poole d samples Material andmethod s Ovarian Seminal The fishwer e reared inou rexperimenta l fluid plasma facilities atJouy-en-Josa si nrecycle d water (temperature: 13t o 20°C)unde r Water% 97 98.5 naturalphotoperiod .The ywer e collected in Junean dgive n atreatmen t of carpcrud e Organicmatte r (1) 73 58 pituitary extracts (3mg/k gbod yweigh tfo r Ash (1) 27 42 femalesan d 1.5mg/k g formales) .Afte r gamete collection,th esemina l plasmawa s Na g/1 2.8±0,34 1.18 (2) obtainedb y centrifugation (20mi n at360 0 K g/1 0.48+0.2 1.7 (2) x g)an d theovaria nflui dwa s takenb ysim plypourin g off theexces s fluid. Osmotic Camg/ 1 *-. 105±3 28.5 (2) pressure (OP)an dp Hwer emeasure d immedia Mgmg/ 1 15±6 6.5 (2) tely after collectionan d the remaining fluidswer e stored at -20"C unti lanalysis . Pmg/ 1 113±60 33±20 The fertility of thegamete s ofeac hmal e PLmg/ 1 44±30 5.6±1 and femalewa s estimated after insemination and 3-dayincubatio no f theegg s at20"C . TP g/1 4.1+2.3 1.2+0.3 Analysis includedmeasuremen t of theas h Amino acids pM/m l 10.6 (2) 36.7 (2) after ashing at350° Cfo r 24h ando fth e cationswit h flamephotospectromete r OPmOsmole s /kg 3Q5±12.9 286±10 Eppendorf.Tota lphosphoru s andprotein s pH 8.51±0.11 7.96±0.1 weremeasure d by colorimetry.Aminoacid s weremeasure d according theMenez o eta l Fert,% 78±14 96±1.4 (1978)afte r 24-hourhydrolysi s (HCl6N) . Except forK ,whic hwa s 3time shighe r in carp thansalmonid s or cod,th eminera l Results compositiono fcar povaria nflui dwa scompa rable totha to fothe r fish species.Eg g The composition of thesemina l plasmao f fertilitywa snegativel y correlatedwit hK thecommo ncar pwa s characterized by ahig h (-0.69).Th eP concentrationwa s 4time s contento fpotassiu mwhic hwa s themajo r lower thani nth ebloo dplasm aan dth e cation found.Contrar y to trout.,,th eK di d protein contentwa s 10time s lower.Highl y not inhibit spermmotilit y andeve nseeme d significant correlationswer e foundbetwee n topotentiat e it.Th eprotei n content ofth e TP andP L (0.97),C aan dP (0.93),K an dM g seminalplasm awa s relatively low (1.2g/1 ) (0.93)an dorgani cmatte r andP L (0.97).Th e but the concentration ofamin o acidsan d osmoticpressur ewa s surprisingly high (305 smallpeptide swa sver yhig h (36uM/ml) . mOsmoles).,.bein geve nmor eelevate d thani n Theoveral l composition of the seminal thesemina lplasm a sothat ,contrar y toth e plasmawa sver ydifferen t fromtha to fth e casei ntrout., ,th espermatozo adi dno tmov e bloodplasma .Highl y significantcorre whendirectl y added toth e eggs.Whe narti lationswer e foundbetwee n totalprotei n ficial inseminationwa s carried out introu t (TP)an dphospholipid s (PL) (0.8),P an dP L withovaria n fluid,fertilizatio noccurre d (0.71),T P andP (0.94),bu t therewa sn o assoo na sth egamete swer emixed ,whil ei n correlationbetwee n thefertilizin g ability carp itoccurre d only aftera diluen tha d of thespermatozo a( % of fertilization)an d been,added . anyo f themeasure d compounds. 134 GONADAL GROWTH 135 VITELLOGENESIS INFISHE S MurrayD .Wiegan d Marine SciencesResearc hLaboratory ,Memoria lUniversit y ofNewfoundland? ,St .John's ,New foundland,Canad a Summary Traditionally,yol kmateria lha sbee n classifiedb y thehistochemica l reactionso f Eggyol k lipid andprotei n componentsar e thevariou s oocyte cytoplasmic inclusionsa s discussed alongwit hconsideration so f carbohydrate,lipi d andprotei nyol k (Raven theirorigins . Inrecen tyears ,plasm a 1961). Thisrevie wconcentrate s onth ena vitellogeninha sbee nconfirme d as thepre turean d origino fyol k lipidsan dproteins . cursor of themajo ryol kproteins . Several Vitellogenin and itsovaria n derivatives are vitellogenins andvitellogeni n derivatives discussed insom edetail . Severalothe r havebee ncharacterize d insom edetail . ovarian components areconsidere d asar e Endogenous synthesis ofsom eyol kcompon selected aspectso f sex steroidhormon e ents ispossibl e although thequalitativ e actionsrelate d tovitellogenesi s andimpli and quantitative importanceo fendogenou s cationso fth emeasuremen to fvitellogenin . vitellogenesis remains tob edetermined . Thefunction s ofgonadotropin s inprocesse s Recent studieso festroge n inductiono f related tovitellogenesi s arediscusse d vitellogenesis haveconsidere d therol eo f elsewhere inthi s symposium (Idler 1982). estronea swel la sestradiol ,primar yan d Oocyte synthetic activityoccurrin gprio rt o secondary responses toestroge nan dinduc vitellogenesisha sbee nrecentl y reviewed tiono fvitellogeni nmRNA . Annual cycles (Guraya 1979;Bruslé "1980 ;Wallac e &Selma n of sex steroids arediscusse d asar eimpli 1981)a sha s the synthesis of "yolkvesi cationso f themeasuremen t ofvitellogenin . cles" (Wallace &Selma n 1981). Readersar e Keywords: vitellogenesis,lipovitellin , encouraged toconsul t theseworks . Itis , Phosvitin,estrogen ,yol klipids . however,wort hreiteratin g Wallace &Sel - man's (1981)poin t that "yolkvesicles "ar e Introduction likelyprecursor s of cortical alveoliwhic h areextrude d atfertilizatio nan dhenc e shouldno tb econsidere d yolk. Raven (1961) Thedramati c growth inth eovarie so f previously pointed outtha t "carbohydrate many fishes,fro mabou t1 %o r lesso fbod y yolk"i nfishe s isconsidere d synonymous weightu p to20 %o rmore ,tha toccur s in with "yolkvesicles. " themonth sprio r tospawnin g islargel ydu e toth eaccumulatio nb y theoocyte s ofnutri entreserve s forth eembryos ,th eyolk . Yolkmateria l and itsorigin s Thesynthesi s ofyol kmaterials ,terme d vitellogenesis,i sbelieve d tooccu rbot h (i) Lipids withinth eoocyt e (endogenousvitellogene "Lipidyolk "accumulatio n commencesprio r sis)an d external toth eoocyt e (exogenous totha to fprotei nyol k (Raven1961 )an dit s vitellogenesis). Ingeneral ,endogenou s appearance canb econsidere d tomar kth e vitellogenesis isconsidere d toprecee d starto f endogenousvitellogenesi s (Shackley exogenousvitellogenesi s but there isevi &Kin g 1977). Since themajo ryol kprotei n dencefo roverla p ofthes ephases . Itma y precursor,vitellogenin ,an d itsmajo rde indeedb emor eaccurat et oconside r thetw o rivative,lipovitellin ,contai n lipid,i t typeso fvitellogenesi s solely asprocesse s shouldb eremembere d thatth eclassica l rathertha ndiscree t temporalphases . term "lipidyolk "i snormall y reserved for Themajo r development inrecen tyear s in lipid inclusions observed inanatomica l thestud y ofvitellogenesi s hasbee nth e studies tob edistinc t fromvitellogenin - demonstration ina variet y of specieso f derived proteinyol kgranules . Insom e thepresenc eo fth efemale-specifi cplasm a cases,however ,lik etha to fth egouram i lipophosphoprotein complex,vitellogenin , discussed below,substantia llipi d isac whichha sbee nconfirme d asprecurso ro f cumulated inth eovar y although critical themajo ryol kproteins . Itis ,however , details of itsorigi nar eunknown . Forthi s importantt orecal l that,unlik eth ecas e reason,I hav econsidere d yolk lipidsfro ma inXenopus ,precis equantificatio no fth e variety of sources,includin gvitellogenin , proportiono fyol kmaterial sderive ddirect inthi ssection . lyfro mvitellogeni nha sno tbee nperforme d Lipidbodie s appear initially inth eper infis h (Wallace &Selma n 1981). Asdis inuclear cytoplasm ofth eoocyte . They cussedbelow ,ther e isevidenc e fora num profiferate during thedevelopmen t andmi bero fpotentia l sources offis hyol k in gration ofBalbiani' s body and occur fre addition tovitellogenin . quently inassociatio nwit h thatbod y 136 (Droller& Rot h 1966;Beam s &Kesse l1973 ; mated tocompris e approximately 38% -o fth e Chopra 1958;Guray a 1963,1965 ;Nayya r1 9 dryweigh t of lesser spotted dogfish, 64). Inearl yvitellogenesis ,th elipi d Scyliorhinus caniculavitellogeni n (Craik bodiesconsis t largely ofphospholipid s 1978a). Ingeneral ,fis hvitellogenin shav e (Chopra 1958;Guray a 1963',1965 ;Nayya r beenfoun d tohav emuc hhighe r lipidcon 1964; Shackley& Kin g 1977). Agenera l tentstha ntha treporte d inXenopu s (Redshaw classificationo f theearl y lipidinclus & Follett 1971). Of theovaria nderivative s ionsa stru eyol k iso fquestionabl eaccur ofvitellogenin ,onl y lipovitellinha sbee n acya sthe yhav ebee n observed todisappea r found tocontai n lipid,wit h contentsrang insom especie sprio r to,o rduring ,sub ingfro m 15-29% (Ando 1965;Jare d &Wallac e sequent exogenousvitellogenesi s (Chopra 1968; Placke ta l1971 ;Campbel l& Idle r 1958; Guraya 1963,1965 )bu tma ypersis t in 1980). Using aconversio n factor öf25 X others (Shackley &Kin g 1977). Iti sals o lipidphosphoru s determinations,th ephos noteworthy that theendogenou s origino f pholipid content isestimate d at58 %o f lipidbodie sha sbee nascribe d solelyo n lipovitellin lipid incoh o salmon,Oncorhyn - thebasi s of anatomical criteria. Detailed chuskisutc h (Markert &Vanston e 1971)an d analysis ofoocyt ecytoplasmi c lipidsan da 66% incod ,Gadu smorhu a (Placket ^al _1971) . directdemonstratio n of thecapacit yo f Vitellogenin and itsderivative s areconsid oocytes tosynthesiz e themremai n tob e eredmor e extensivelybelow . performed. Ina recen t studyo frainbo w An interesting series ofpaper sha scon .trout ,Salm ogairdneri ,ovaria n lipogenesis sidered theaccumulatio n ofwa xeste r inth e invitro ,i twa s found that radioactive ovaryo f theopalin e gourami,Trichogaste r acetate incorporated into lipidswa sre cosby. Waxeste r isth emajo r ovarianlipi d covered"primaril y inth epola r lipidfrac inthi sfish ,comprisin g about 37%o fovar tion ofovarie s inearl y stages ofrecrud ianweigh t (Sand eta l 1969). Wax esteri s escence (Wiegand &Idle r 1982). Whether themajo r ovarian lipid insevera l other thelabelle d polar lipids inthi sstud y teleostsbu t comprises alowe rproportio no f correspond tooocyt e lipidbod yphospho total ovarianmas s (Iyengar& Schlen k1967 ; lipids identifiedb y othershistochemically , Kaitaranta &Ackma n 1981). Thesourc eo f ort ostructura lpola r lipids,o rboth , theextremel yhig hwa x ester content inth e remains tob edetermined . ovaries ofT .cosb y isinterestin g inth e Lipid inclusions described inmor ead overallvie wo fteleos tvitellogenesis . Of vanced oocytes during after exogenousvit particular interest isth eaccumulatio no f ellogenesis hascommence d consistprimaril y thealcoho lportio no fwa xesters . of triglycerides (Chopra 1958;Guray a1965 ; InT .cosby ,San d eta l (1969N,1971 )foun d Nakagawa &Tsuchiye r 1969 citedb yLege r thatovaria nwa x esteralcohol s canb esyn eta l 1981). Thispatter no f shifti n thesized inviv o fromdietar y acetateo r composition of oocyte lipid inclusionsi s derived fromdietar y carbonchains . Theob common toa wid evariet y ofvertebrate san d servation thatd enov o synthesis ofth e invertebrates (Nath 1960;Rave n 1961). The ovarianwa xeste r components canoccu ri n capacity of trout ovaries to synthesize vivo,althoug h thesit ei sunknow n (Sande t triglycerides ata tim ewhe nvitellogeni n al 1969),couple dwit h theobservatio ntha t uptake isoccurrin g suggests anendogenou s theovar y ofa t least oneteleos t species sourcefo r someo f thisyol k (Wiegand & can synthesize fattyalcohol s (Wiegand& Idler 1982). Anexogenou s contributiont o Idler 1982),suggest s that someovaria nwa x the triglyceridepoo l fromplasm alipopro estercomponent s mayb e synthesized endogen teins includingvitellogeni n isals o likely ously . Themajo rpotentia l exogenous source (Legere ta l 1981). ofovaria nalcohol s isplasm a fattyaci d Vitellogenin isa n important sourceo f which isreduce d toalcoho lafte ruptak eb y ovarian lipidsan d islikel y themajo r theovary . Sincefeedin g experimentsre source ofpola r lipids. Total lipidha s vealed that labelled fatty acidwa strans been found tocompris e 21.5%o f S^.gaird ported primarily as triglyceride (Sand eta l - nerivitellogeni n (Campbell &Idle r 1980) 1973; Rahne ta l 1973), iti spossibl e that and 21-22%o f goldfish,Carassju sauratu s either T_. cosbyvitellogeni n isver yunusua l vitellogenin (Horie ta l 1979). S_.gaird interm s ofbot h itstota lan dneutra l lipid nerivitellogenin ,separate d fromothe r content,o rothe r lipoproteinsbeside svit serum lipoproteins by centrifugation,ha s ellogeninar e involved the transport oflip been found tohav ea distinc t fattyaci d idt oth edevelopin g ovary. This species complement from thato f theothe rlipopro would appear tooffe runiqu e opportunities teins, (Legere £a l 1981). Anotherprepar for thecomparativ e studyo fvitellogenesis . ationo f S.gairdner ivitellogeni nha sbee n (iij Vitellogenin,ovaria nderivative so f found tocontai n 280u gpola r lipidsan d6 0 vitellogenin and otherovaria nproteins . ug triglyceridepe rm gprotei n {Wiegand & Vitellogenins havebee ndemonstrate d ina Idler 1982). Approximately 87%o fC . widevariet y ofteleos t (forreviews ,se e auratusvitellogeni n lipid isphospholipi d Wallace 1978;Wallac e &Selma n 1981;als o (Horie ta l 1979). Based onyol kgranul e Campbell &Idle r 1976,1980 ;L eMen n1979 ; analysis,tota l lipidha sals obee nesti Hara eta l1980 ;Nat h &Sundarara j 1981)an d 137 in'severalnon-teleos t fishes (Pickering methods (Hara& Hira i 1978;Hor i eta l 1976; Craik 1978a;Y ue ta l1980 ,1981) . 1979; Harae ta l 1980)an d the ironbindin g Thehepati c origin ofvitellogeni nha sbee n capacity of S.gairdneri ,chu msalmon , confirmed (Plack& Fraze r 1971;Campbel l& Oncorhynchusket aan dA .japonic avitello Idler 1976;d eVlamin ge_ ta_ l1977a ;L eMen n geninsha sbee ndemonstrate d using immuno & Lamy 1977;Larse n 1978;Y ue ta l1980 ; logicalan d autoradiographic techniques Idler& Campbel l 1980;Sundarara j &Nat h (Hara1976 ;Har aeta l 1980). 1981). Ina numbe r of theabov estudies , Whenvitellogeni n istake nu pb y oocytes immunological relationshipsbetwee nvitello inXenopus ,i ti sproteolyticall y cleavedt o geninan d eggyol kprotein s and inductiono f form theyol kprotein s lipovitellinan d vitellogenin synthesisb y estrogen inbot h Phosvitin (Wallace 1978). While thesepro sexesha sbee ndemonstrated . cesseshav eno tbee ndirectl y demonstrated Vitellogenin and their eggyol kdériva- ' inteleosts ,ther e issubstantia l evidence tiveshav ebee ncharacterize d insom edetai l thatthe yd ooccur . ina numbe r of teleost species. Molecular Gonadotropin-sensitive and selectiveup weightdetermination s byge l filtration takeo f S.gairdner i oocytesha sbee ndem (Emmersen& Peterse n 1976;Har a &Hira i1978 ; onstrated invitr o (Campbell 1978;Campbel l Campbell &Idle r 1980;Har a eta l1980 )an d & Jalabert 1979). Autoradiography of electrophoresis (Horie ta l 1979;d eVlamin g oocytes from sheepshead minnow,Cyprinodo n eta l1980 ;Campbel l sTTdïër1980 )hav e variegates,whic hha dbee n injected 24hrs . yielded valuesbetwee n326,00 0an d 600,000 earlierwit h tritiated Xenopusvitellogeni n for thevariou s intactmolecules . Molecular revealed graindepositio nove ryol k spheres weights ofdelipidate d polypeptidemonomer s and fusingyol kmasses, suggestin g incorpor determinedb y electrophoresis inth eprecenc e ationo fth eheterologou svitellogeni n into of sodiumdodecy l sulfate (SDS)rang efro m yolk (Selman& Wallac e 1982). Ng &Idle r 140,000t o220,00 0 (Hara& Hira i 1978;Hor i (1980)injecte d femalewinte r flounder, eta l 1979;d eVlamin g et_a l 1980;Roac h& Pseudopleuronectesamericanus ,wit h labelled Davies 1980). Cautionha sbee n advised in serumphosphoprotei n isolated fromestrogen - theus eo fge l filtration for determination treatedmale san d demonstrated gonadotropin- ofvitellogeni nmolecula rweight s (Horie_ t sensitiveuptak eo f label intoyolk . The al1979 ;d eVlamin g eta l 1980),althoug h clearance fromth eplasm a of phosphoprotein closeagreemen tbetwee nresult susin gge l (aninde xo fvitellogenin )an d itsappear filtrationan d gradientge l electrophoresis ance inyol kgranule sha sbee n studied in hasbee nfoun dwit h S^.gairdner ivitellogeni n theelasmobranch ,S ^canicul a (Craik 1978b). (Campbell &Idle r 1980). Usingnativ e,ge l Anumbe r ofultrastructura l studieshav e electrophoresis,tw ogroup s of investigators shown incorporation ofmateria l takenu pb y havefoun dmonomeri can ddimeri c forms ofC . pinocytosis intoyol k spheres (forreview , auratusvitellogeni nalon gwit ha third , seeWallac e &Selma n 1981). Immunological comparatively minor,componen t of intermed relationshipshav ebee ndemonstrate d between iatemolecula rweigh t (Horie ta l1979 ;d e vitellogenins andvariou s eggyol kprotein s Vlaming et al_1980) . DeVlamin ge_ t_a l' ina numbe r of species (above)an d close (1980)furthe r foundmultipl emolecula r similarities inamin o acid compositionbe weight formsö fdelipidate d C.auratu svit tweenvitellogeni n and themajo r yolkpro ellogeninmonomer . tein,lipovitellin ,hav eals obee nnote d (Hara& Hira i 1978;Campbel l &Idle r 1980). Alanine,glutami c acidan d leucinehav e been found tob e themos tabundan t amino Lipovitellinmolecula rweight shav ebee n acids in S_.gairdner i (Hara& Hira i 1978 found tob e lower than thoseo fthei rvitel Campbell &Idle r 1980),C .auratu s (de logeninprecursor s insevera l studiesi n Vlaming etal _1980 )an d Japanese eel,Anguil - whichbot hwer edetermine d (Hara& Hira i lajaponic a (Harae ta l 1980)vitellogenins . 1978; Campbell &Idle r 1980;Roac h &Davie s Incontras t toth eXenopu s vitellogenin 1980; deVlamin g eta l1980 )bu tno t inA . (Redshaw& Follet t 1971;d eVlamin g eta l japonica (Harae ta l 1980). Theselatte r 1980),th eteleos tvitellogenin swer epoor " authors suggested thatee lvitellogeni nma y inserine . Otherchemica lanalyse s oftele be incorporated intoyol kwithou tmodifica ostvitellogenin s havereveale d lower cal tion. Hori etal _(1979 )mad ea similarsug ciuman dprotei nphosphoru s concentrations gestion regarding C.auratu svitellogeni n thanthos efoun d inXenopu s vitellogenin but thisha sbee ndispute db y deVlamin ge t (Horie ta l 1979;Campbel l &Idle r1980 ;d e al (1980)wh o isolated lipovitellins and Vlaming eta l1980 ;Har a eta l 1980). It Phosvitins from£ . auratus indicating that waspreviousl ypointe d out (deVlamin g eta l typicalproteolyti cmetabolis m ofvitello 1980)tha tconcurren t lowlevel so fprotei n geninoccur supo nuptak eb y theoocyt ei n phosphorusan d serine inteleos tvitellogen thisspecies . DeVlamin g eta l (1980)fur insar econsistent ,sinc eth e serinehydrox - therfoun d twomolecula rweigh t classeso f ylgrou p isth eprincipa l siteo fphosphopro - lipovitellin subunits,eac ho fwhic hcon teinphosphorylation . Both carbohydrates sisted ofmor e thanon epolypeptide . The and ironhav eve< n identified ascomponent s presenceo fhig h and lowmolecula rweigh t of teleostvitellogenin sb yhistochemica l subunitsha sbee n indicatedusin g a2 - 138 mercaptoethanol-reduced preparation of^S . specieswit hmolecula rweigh t37,000-40,000 , gairdnerilipovitelli n (Hara& Hira i 1978). suggesting thepresenc eo f adime r (Mano& Lowo rundetectabl e levelso fprotei n Lipmann 1966). Twomolecula r formso fC . phosphorushav ebee nreporte d fora numbe r auratusphosvitin ,7,60 0an d 14,500wer e of teleost lipovitellins (Jared &Wallac e separated by gelfiltratio n (deVlamin g et_a l 1968; Markert& Vanston e 1971;Plac ke ta l 1980)an d a lowmolecula rweigh tphosvitin , 1971; Campbell &Idle r 1980). Lipovitellin 4,200b y sedimentation analysis,ha sbee n lipids arediscusse d inth epreceedin g demonstrated in C.pallasi i (Inouee_ t al_ section. 1971). Wiley &Wallac e (1981)provide da Thephosphoprotei nderivative s ofvitell detailed examination ofmolecula rweigh t ogenin,th ePhosvitins ,hav e receivedmor e determinations ofXenopu syol kprotein swit h extensive study. Theseprotein sma yb eiso particular emphasis onth edifficultie s in lated by redissolving ayol kdilutio npre determiningmolecula rweight s ofhighl y cipitate insalin ean d precipitating lipo phosphorylated proteins. Theseauthor srec vitellin (and ß-component)wit hammoniu m ommended theus eo f SDS-gradient gelelectro sulfate (Wallaceei tal _1966 ;Marker t &Van - phoresis insuc hcases . stone 1971). Thephosviti nca n thenb e Ingeneral ,amin o acid analyses ofteleos t furtherpurifie d bychromatography . Other Phosvitinshav ereveale d apredominanc eo f procedureshav ebee n employed involvinga serine. Proportions of serine inth epre series of extractions,précipition san d parationsnote d above range from42 %i nS^ . .dialyse s followed bychromatograph y (Mano& gairdneri (Campbell &Idle r 1980)t o75 %i n Lipman 1966)o r the initialpartitio no feg g _C.pallasi i (Inouee ta l 1971). Anexcep- 1- proteinswit hwate ran d phenol followedb y tion isth egrou p ofphosphoprotein s isolated ethanolprecipitatio n ofphosphoprotein s from shad inwhic h serineconten trange d from from theaqueou sphas ean d subsequent chrom 9%t o22 %(Man o& Lipman n 1966). Thegenera l atography (Mano &Yoshid a 1969;Inou ee ta l observation ofMan o &Lipman n (1966)tha t 1971). Phosvitins arepoo r inhistidine ,sulfur - Thephosviti n content of fishovarie s containing and aromatic arfîonoacid sha sbee n appears tob equit evariable . Jared &Wal verified insubsequen t investigations tlnoue lace (1968)foun dver y littlephosviti n in eta l 1971;Tabl e 3,Campbel l &Idle r 1980). theovarie so f severalmarin e teleostsan d Theeste r linkage ofphosphat e toserin eha s suggested thatdevelopin g eggscoul d obtain been studied inC .pallasi i (Inouee ta l their required phosphate from theenviron 1971)an dmultipl e forms of several teleost ment. Insuppor t of this,ther e islittl e Phosvitinswit hvaryin g degrees ofphosphor phosvitin inth eegg s ofG .morhu a (Plack ylationhav ebee n demonstrated (Mano& Lip eta l 1971)o rPacifi cherring ,Clupe a pal- mann 1966). ThePhosvitin s of 0_.kisutc h lasii (Inouee_ ta l 1971;Inou e &Iwasak i (Markert &Vanston e 1971)an d S.gairdner i 1980)althoug ha hig hphosphoprotei nconcen (Campbell &Idle r 1980)wer e found tohav en o trationha sbee n found inflounde r ovaries detectable levels of lipid. (Mano &Lipman n 1966). Phosvitinha sbee n Anotheryol kprotei n thatma yb ederive d found tocompris e 3%o fovaria nyol k inS . fromvitellogeni n isth e B1 component. Jared gairdneri (Campbell &Idle r 1980). ~ & Wallace (1968)chromatographe d yolkprotei n Molecularweight s of teleostPhosvitin s extractso fa variet y of teleostso nTEAE - havebee n found tob eextremel yvariable , cellulose. Inth ecas eo f thej^ .gairdner i and descrepancies existbetwee n preparations extract,2 distinc t components, aan d B,wer e from the same species. For example,mole detected byabsorbanc e at280m man d athird , cularweight s of 19,400 (Mano& Yoshid a Y,ha d lowabsorbanc ebu ta hig h contento f 1969)an d 24,000 (Wallacee ta l1966 )hav e proteinphosphorus . Thea an d y components, beendetermine d for S.gairdner i phosvitin lipovitellin and phosvitin,wer e precipitated by sedimentation analysis. Thesevalue sar e by dilution leaving the ßcomponen t insol comparable tophosviti nmolecula rweight s in ution. Itwa spropose d that thisprotein , other salmonids,a sdetermine d bysedimenta which containedneithe r lipidno rphosphoru s tionanalysi s (Schmidt eta l 1965;Man o& wasa serumprotei n (Jared &Wallac e 1968). Yoshida 1969)o rge l filtration (Markert& Incontrast ,Marker t &Vanston e (1968)re Vanstone 1971). However,Campbel l &Idle r covered asimila r protein from severalOncor - (1980), using aprocedur e for isolationo f hynchus species thatdi dprecipitat ewit h S^.gairdner iphosviti n similar totha to f lipovitellin and phosvitino ndilutio nan d Wallace eta l (1966), determined amolecula r coprecipitated with lipovitellin duringam weight of45,00 0b y gel filtration. Inad monium acetate fractionation. Thisprotein , dition todifference s intechnique ,par to f designated ß1-component,wa s further studied suchdiscrepancie s couldb edu e tointerac in 0_.kisutc han d found tohav e amolecula r tionso f subunits. Molecularweight srang weighto f 30,000an d tocontai nn o lipido r ingfro m 14,000t o16,00 0wer edetermine d by phosphorus (Markert &Vanston e 1971). Its sedimentationalalysi s forthre ePhosvitins , presence in serumfro mmaturin g femalesan d ofvaryin g degreeso fphosphorylation ,iso estrogenized prepuberal fishwa s demonstrated lated from ling (Mano& Lipman n 1966). A- by immunodiffusion. An immunologicalrela bout 30%o fon efractio n consisted ofa tionshipbetwee n B1-componentan dvitello - 139 geninha sbee ndemonstrate d inj5 .gairdiier i thepresenc e inth eeg g ofa protei ncommo n suggesting that ß1-componenti sa par to f tobot hmale san d females andhenc eno t thevitellogeni ncomple x (Campbell &Idle r stictly avitellogenin . A similarprotei n 1980). common tomale ,non-vitellogeni cfemal ean d Thepossibilit y ofa nendogenou scontri vitellogenin femalehagfish , (Eptatretus bution toyol kprotei n shouldno tb e ignored. stouti)plasm aha sbeen-foun dusin g ananti InBrachydani o rerio,larg eyol k crystalso f serum toeg gyol k (Yue ta l 1980). exogenous originhav ebee nobserve d tob e enclosed ina namorphou smatri x thatappear s Sex steroids andvitellogenesi s tob e synthesizedwithi n theoocyt e (Ulrich 1969). Ina serieso f studies onfis hgon Theinductio n ofvitellogeni n synthesis adotropins (seeIdle r& N g 1979),hormona l byestrogen sha sbee n demonstrated ina wid e effectso novaria nmetabolis mhav ebeen , variety of teleosts (forreview ,se eWallac e investigatedb ymeasurin g uptake ofH 3 POi, & Selman1981 ;als oNat h &Sundarara j1981 ; and H-leucine_i nvivo . Itwa s consistently Campbell &Idle r 1976,1980 ;L eMen n1979 ; found thatmor e isotopewa s incorporated in Karae ta l 1980;Idle r &Campbel l 1980). toovaria nprotein swhic hwer e insoluble in Most studieshav e entailed theus eo fa sin trichloroacetic acid,tha ncoul db eaccoun gleestrogen ,normall y estradiol. As isth e tedfo rb yuptak eo f labelledvitellogenin . casei nXenopu s {Redshawe_ ta l 1969), treat Itremain s tob e investigated whether the mentwit h estradiolha sbee nfoun d tob e additional labelled protein istru eyol kan d morepoten t inth einductio no fvitellogeni n whether iti so fendogenou s or exogenous thantreatmen twit h estrone inS .gairdner i origin. (vanBoheme n eta l 1982a). However,highe r Inadditio nt oth emajo r eggyol kconstit plasmalevel so f estronetha nestradio l uentsdiscusse d above,som eothe rmaterial s during earlyphase s ofexogenou svitellogen havebee n studiedwhos eorigi n isno tknow n esis in_S .gairdner i (vanBoheme n &Lamber t tob evitellogeni n andma y represent pro 1981)an d theobservatio n thatpretreatmen t ducts ofendogenou s vitellogenesis orexo with estrone canpotentiat e the subsequent genousmateria l ofa non-vitellogeni nsource . vitellogenic response toestradiol ,sugges t Sialoglycoproteins havebee n isolated from thatestron ema ypla y arol ei nprimin g the theegg so fJ3 .irideu s (Inoue& Iwasak i 1978) liverfo r estradiol stimulation (vanBoheme n and C^.pallasi i (Inoue& Iwasak i 1980). The eta l1982b) . Estradiolwa s found tob e JS.irideu s sialoglycoprotein hasa siali c morepoten t ininducin gvitellogeni n synth acid content of59 %an da tota lamin oaci d esis inhypophysectomize d catfishHetero - content of 21%. Iti sabou t onequarte ra s pneustes fóssilistha neithe r estroneo r abundanta sPhosvitin . Afamil y of sialog estriol (Sundararaj &Nat h 1981). Therela lycoproteinswa s found in C.pallasi iegg s tivepotencie s of estronean d estriolvarie d with sialic acid contents of 5-6.5%an d tot with dose inthi slatte r study. Conversely, alamin oaci d contents 40-44%. Theyar ea - inovariectomized ,Tilapi a aurea,estrio l bout 10time s asabundan t asPhosvitin . was found tob eth emos tpoten t of thethre e Neutral andN-acetyl-amin o sugars comprised estrogens inincreasin g plasma calciuman d theremainde r of thepreparations . Although totalprotei n levels (Terkatin-Shimony & thefunctio n of these sialoglycoproteins is Yaron 1978). unknown,i ti swort hnotin g thatbot hth e Therespons eo fplasm aphosphoprotein ,a n corticalalveol i (Wallace &Selma n 1981)an d indexo fvitellogenin ,t othre e successive thechorio n (Tesoriero 1977,1978 )contai n injections ofestradio lha sbee n studied in glycoprotein and at least onerepor t exists H.fossili s (Sundararaj &Nat h 1981). Max of thedetectio no f sialoglycoproteins in imalphosphoprotei n levelswer erecorde d 2 teleost corticalalveol ib yhistochemica l daysafte r eachinjection . Sucessiveinjec means (Shackley &Kin g 1977). Whether orno t tionswer eadministere d afterplasm aphos the sialoglycoproteins isolated by Inouean d phoproteinha d returned tobasa l levelsan d collaborators arecomponent s ofeithe ro f eachproduce d ahighe rmaximu m than itspre these structures orar etru eyol kremain st o decessor. Femalesgav ea stronge r response bedetermined . thanmales . This study supports thehypoth Therear ea fe wreport s indicating the esistha t initial exposure toestroge nca n possibility ofuptak eo fprotein s other than prime thelive r forsubsequen t stimulation. vitellogenin from theblood . £[.gairdner i Theprimar y and secondary responses ofXeno oocyteswil l sequesternon-vitellogeni n ser pus toestroge ntreatmen thav ebee nexamine d umprotein s invitr o (Campbell &Jalaber t insom edetai l (forreview ,se eTat a &Smit h 1979). Uptake of theseprotein s isno tsen 1979),bu t similar extensivewor kremain st o sitive togonadotropi n and occurs ata muc h bedon e infishes . lowerrat e thanuptak e ofvitellogenin . In Theinductio no fvitellogeni n infis hb y theayu ,Plecoglossu s altevelis,on ecros s estrogenha srecentl y received attentiona t reactionbetwee neg gextrac tan d antiserumt o thenuclei c acid level. VitellogeninmRN A maturing femaleplasm a iseliminate d byab hasbee n isolated from livero festrogen - sorbing theantiseru mwit hmal eplasm a (band treated bullhead,Americanu snubulosa ,an d C,Fig . 7o fAid a eta l 1973). This suggests itscell-fre e translationproduc t confirmed 140 asvitellogeni nmonome rb y electrophoretic year (Craik1978a ;Sumpte r &Dod d 1979), and immunological criteria (Roach &Davie s theamplitud eo f thefluctuation s ismuc h 1980). VitellogeninmRN Aha sals obee niso less thantha tobserve d inteleost s like lated from estrogen-treated SL gairdnerian d thesalmonids . Areflectio n of thespread itscell-fre e translationproduc t confirmed ingou to freproductiv e effort isth eobser (Chene ta l 1982). vation thatplasm avitellogeni n levelsar e Highdose s ofandroge n inducevitellogeni n stable throughoutmos t of theyea r andar e synthesis inGobiu snige r (LeMen n 1979)an d much lower thanthos efoun d inothe rovipar C.auratu s (Horie ta l 1979). Binding stud ousvertebrate s (Craik 1978a). Inaddition , ies indicate that theandroge nactio ni s themagnitud e of thevitellogeni c response mediatedb y theestroge n receptor (LeMen n toestroge n inlowe r thani nothe rverte e_ta l 1980),an d theandroge neffec t iscon brates (Craik 1978c). Anannua lcycl eo f sidered pharmaciological rather thanphysio plasma estradiolha sbee nreporte d inthi s logical. specieswit hmaxima l levels inwinte r (Sump Therelatio n ofovaria ncycle s tothos eo f ter& Dod d 1979). plasma sex steroid levelshav ebee nstudie d Inadditio n toinducin g the synthesiso f ina numbe r of teleosts (forreview ,se e vitellogenin,ther e isevidenc e thatse x Crim 1982;als o Crim &Idle r 1978;Billar d steroidshav eothe r roles related toth e e£a l1978 ;Dind o &MacGrego r 1981;va n process ofvitellogenesis . Estrogens,wit h Bohemen &Lamber t 1981;MacGrego r eta l19 - estradiolbein g themos tpotent ,ar ebe 81). Aspointe d outpreviousl y (Crim 1982), lieved tohav edirec tprotectiv e effectso n sex steroid levels tend toincreas ewit h follicles asevidence db y theirabilit y to ovariandevelopmen t and declineafte rspawn delay atresia inducedb yhypophysectom y ing. An interesting finding inth eplaice , (Sundararaj &Goswam i 1968;Anan d &Sundar - Pleuronectesplatess a (Wingfield &Grim m araj 1974). Inadditio n tovitellogenin , 1977), S^.trutt afari o (Billard eta l 1978), estrogen canals o increaseplasm a levelso f 0.kisutc h (Jalaberte ta l 1978),an d£ . very lowdensit y lipoproteinwhic h isa n gairdneri (Whitehead e£a l 1978;va nBoheme n important carrier of triglyceride (Skinner & & Lambert 1981)i sth edeclin e inplasm a Rogie 1978). Estrogenmobilizatio n oflipi d estrogen levelswhic hcommence s amont ho r intoth eplasm a inexces s of thatcontaine d moreprio r tospawnin g andwhil eth eovar y invitellogeni nha sbee n suggested inth e isstil l growing. Thisoccur s inconcer t elasmobranchj3 .canicul a (Craik1978c )an d witha declinin g rateo f estrogen synthesis estrogen-induced increases inplasm aphos inth eovar y (vanBoheme n& Lamber t 1981) pholipid levels inC_ .auratu swer efoun d to andma ypermi tgonadotropi n secretiont o be independent from thoseo fprotei nphos rise toovulator y levelsb yreducin g feed phorus,a ninde xo fvitellogeni n (Bailey back inhibition. Thedeclin e alsoreduce sa 1957). Iti sals onoteworth y thatresponse s potential inhibition of gonadotropin-induced ofplasm a or serum levels ofcalciu m (Wood- maturation (Jalabert 1975)an dminima l levels head 1969;Ellio t et_a l 1980)an dneutra l may indeed signalmaturatio n (Fostier eta l lipids (Wiegand &Pete r 1980a)t oestroge n 1978). Pertinent toth e study ofvitello - havebee n found tovar ywit h season,repro genesis isth eobservatio n thatmaxima l serum ductive statean d temperature. Estradiol estradiol levelsprecee d maximal serumphos - treatmentha sals obee nfoun d toinfluenc e phoproteinphosphoru s levelsb y 3month s in enzymeactivitie s tofavou r glycolysisove r S. gairdneri (Whitehead eta l 1978). The gluconeogenesis inPlatichthy s flesus (Sand persistencean dmagnificatio no f thephysio eta l 1980). This issimila r tochange s logical effect of estrogen longafte rreduc observed innatur ea tth e starto fvitello tiono f theleve l of stimulus isconsisten t genesis. Similarly,estrogen-stimulate d with experimental findingsi nXenopu s (Dol- depletiono f liverglycoge nha s alsobee n pine ta l1971 )an d_S .gairdner i (Elliote t reported (deVlamin g et^aj ^1977a , b). al 1979). Annualcycle s ofplasm a testosterone that Estrogen inductiono fvitellogeni nsynth coincidewit h theovaria ncycl e"hav ebee n esisha sals obee nreporte d incylostome s noted insevera l teleosts (Campbelle_ ta l (Pickering 1976;Y u et_a l 1981). Estradiol 1976; Wingfield &Grim m1977 ;Scot te ta l hasbee n reported inth eplasm a of thehag - 1980)an da nelasmobranc h (Sumpter& Dod d fish E. stouti (Matty et al 1976),but ,t o 1980)althoug h theirphysiologica l roleha s myknowledge ,seasona lvariation s ofplasm a notbee ndetermined . Experimental evidence estrogens incyclostome shav eno tbee nre suggests that testosteronema yhav e arol e ported. inseasona l stimulation ofgonadotropi n pro Vitellogenesis inth eelasmobranc hS^ . duction (Crim& Pete r 1978;Cri m &Evan s caniculaha sreceive d detailed examination. 1979),a sa precurso r ofbrai nan dpituitar y Unlike thecommonl y studied temperatezon e estrogens (Callard eta l 1981a,b )and ,per teleost fishes,ovipositio nma yoccu r through tinent tovitellogenesis ,i n.th emobiliza mosto ral lo f theyea rwit hpea kactivit y in tiono ffre efatt y acids (Wiegand &Pete r winter (forreview ,se e Sumpter &Dod d 1979). 1980b). Therefore,althoug h significantvariatio ni n gonadosomatic indices.ma yoccu rdurin gth e Assayo fVitellogeni n 141 Thedevelopmen t ofassa y systemsfo r findings inXenopu s (Wiley &Wallac e 1981), vitellogeninma yprov evaluabl e formanage isth efindin g ofmultipl e forms ofvitello mento ffis hfo rwhic hvisua l sexdetermin geninpolypeptid emonome r and its derivatives ation isdifficul t or impossibledurin g inC auratus (deVlamin g eta l 1980). earlyreproductiv e development. Theabilit y Whether orno t thisphenomeno n iswidesprea d toasses s theproportio no f femalesunder remains tob edetermined . Similarly,th e goingvitellogenesi s ina populatio nca n demonstration ofprimar y and secondaryre allowestimatio no f thereproductiv epoten sponses toestroge n inH .fossili s (Sundar- tialfo ra give n season. On thebasi s ofa araj &Nat h 1981)merit s comparative study. tagan drecaptur e study,i twa spredicte d Furtherwor kwil ldoubtles s addressth e thatAtlanti c salmon,Salm o salarwit h plas possibility ofmultipl evitellogeni n genes mavitellogeni n levelsgreate r than25 0yg / infis han d comparemolecula r aspectso f ml, asdetermine d byradioimmunoassay ,woul d primary and secondary responses toestroge n spawnwithi nth eyea r (Idler et_a l 1981). with theXenopu smodel . Idlerji ta_ l(1979 )reporte d plasmavitello With severalbasi c aspects ofvitellogen geninlevel so f90 7ug/m l in'_S.sala r that esisno wwel lunderstood ,furthe r study were4- 5month sàwa yfro m spawning. Taken should attempt tointegrat evitellogenesi s together,thes edat a suggest thatfemal e with otheraspect s ofphysiolog y sucha sth e spawners ofth eyea rma yb e identifiable depositionan dmobilizatio n ofnutrien tre evenearlier . Animmunoagglutinatio nsyste m serves. This iso f special interestwit h based ona nantibod y raised toS .gairdner i regards tothos efis h thatfas tdurin govar vitellogeninha spermitte d theidentifica ianrecrudescence . Therol eo fthyroi dhor - tiono ffemale sdurin gApri lan dMa y ina 'monesi nvitellogenesi s isno twel lunder populationo fj> .trutt atha tspaw ni nDecem stood althoughexperimenta l evidence (Lewis ber (LeBai l &Breto n 1981). Their system & Dodd 1974)indicate s arequirement . Final alsoallow s identificationo ffemal ej5 . ly, considering thegrea tdiversit y offis h salara t similar early stages ofvitello species,i tshoul dno tb e surprising iffu genesis. Anothermetho d ofvitellogeni n turecomparativ e researchuncover suniqu e detection,determinatio n of serumphospho - strategies inth ephysiolog y ofvitellogene proteinphosphorus ,woul d appear toallo w sisi nspecie s facedwit huniqu edemands . sexing ofS^ .gairdner iabou t 4-5month s prior tospawnin g (Whitehead ejta_ l1978) . Thepresenc eo f lowlevel so f phosphoprotein Acknowledgements phosphorus inmal e serumpreclude s earlier sexdeterminatio nb y thismethod . Anadvan Iwoul d like tothan kDr .CM . Campbellan d tageo fa n immunoassay system issensitiv Dr. D.R. Idler forvaluabl e discussionsan d ity. For example,a S_.gairdner ivitello critical reviews of themanuscript . Thisre geninradioimmunoassa y issensitiv e toa - viewwa s supported byNatura l Sciencesan d bout 2-5n gvitellogeni n (Fig.4 ,Campbel l& Engineering Research Councilo f Can. grant Idler1980 )an d theJ5 .sala rradioimmunoas no. A-6732t oDr .D.R . Idleran d iscontri sayi ssensitiv e toabou t 0.1 ngo fyol k butionnumbe r 488fro m theMarin e Sciences protein (likely g1-component),representin g ResearchLaboratory . about4. 7 ngo fvitellogeni n (Idlere ta l 1979). Incontrast ,a n electrophoretic References assay for S.gairdner ivitellogeni n issen sitive to3 0n go fmateria l (vanBoheme ne_ t Aida,K ., P.-V . Ngan &T .Hibiya ;1973 . al 1981). An Immunoelectrophoresis system Physiological studieso ngonada lmatura hasbee nuse d todemonstrat e arelatio nbe tiono ffishes-I . Sexualdifferenc e incom tweenplasm avitellogeni n levelsan dgonado - positiono fplasm aprotei no fay u inre somaticinde xbetwee nOctobe ran dJanuar y lationt ogonada lmaturation .Bull .Jap . for spring spawningpik eEso x lucuis (Goed- Soc. Sei.Fish .39 :1091-1106 . emakers& Verboo m 1974). Anand,T.C .& B.I .Sundararaj ,1974 .Ovaria n maintenance inth ehypophysectomize d cat fish,Heteropneuste s fossilis (Bloch),wit h Conclusions mammalianhypophysea l andplacenta lhor mones,an dgonada l and adrenocortical ster Thefunctio no fvitellogeni na s thepre oids.Gen .Comp .Endocrinol .22 :154-168 . cursor of themajo r yolkproduct s iswel l Ando,T. ,1965 .ültracentrifuga lanalysi so f established. Thequalitiativ e and quant yolkprotein s inrainbo w trout eggan d itative importanceo fothe r sources ofeg g their changes during development.Can .J . yolk Sucha sendogenou s synthesis orplasm a Biochem. 43:373-379 . sourcesothe r thanvitellogeni n isno twel l known. Thederivatio no fyöl k fromothe r Bailey,R.E. ,1957 .Th eeffec t of estradiol sources inadditio n totypica l vitellogenins onseru m calcium,phosphorus ,an d protein mayb eo fspecia l importance inthos efis h ofgoldfish .J .exp .Zool . 136:455-469 . whichhav ea hig hneutra l lipid content in Beams,H.W .& R.G .Kessel ,1973 .Oocyt e theovar y sucha sT .cosby . An interesting structure and earlyvitellogenesi s inth e development,especiall y inligh to f similar trout,Salm ogairdneri .Am .J .Anat .136 : 142 105-122. pression inrainbo w trout.Proc . Can.Fed . Billard,R .,B .Breton ,A .Fostier ,B .Jala - Biol. Sei. 25:45 .Abst .169 . bert &C .Weil ,1978 .Endocrin e controlo f Chopra,H.C , 1958.A morphologica l andhis - theteleos t reproductive cyclean d itsre tochemical study of theoocyte so f thefis h lationt oexterna l factors:salmoni dan d Ophiocephaluspunctatus ,wit hparticula r cyprinid models.In :P.J . Gaillard &H.H . reference tolipids .Quart .J .Microscop . Boer (Ed.): Comp.Endocrinol .Elsevier / Sei.99 :149-158 . North-Holland,Amsterdam . 37-48. Craik,J.CA. ,1978a .Plasm a levelso fvitel vanBohemen ,CG . &J.G.D . Lambert,1981 . logenin inth eelasmobranc h Scyliorhinus Estrogen synthesis inrelatio nt oestrone , caniculaL . (lesser spotted dogfish).Comp . estradiol,an dvitellogeni nplasm a levels Biochem.Physiol .60B :9-18 . during thereproductiv e cycleo f thefe Craik,J.CA. ,1978b .Kineti c studies ofvit malerainbo w trout,Salm ogairdneri .Gen . ellogeninmetabolis m inth e elasmobranch Comp.Endocrinol .45 :105-114 . Scyliorhinus caniculaL . Comp.Biochem . vanBohemen ,CG. ,J.G.D .Lamber t &J .Peute , Physiol. 61A:355-361 . 1981.Annua l changes inplasm aan d liver Craik,J.CA. ,1978c .Th eeffect so festro inrelatio nt ovitellogenesi s inth efe gentreatmen t oncertai nplasm aconstitu malerainbo w trout,Salm ogairdneri .Gen . entsassociate dwit hvitellogenesi s inth e Comp.Endocrinol .44 :94-107 . elasmobranch Scyliorhinus caniculaL .Gen . vanBohemen ,CC , J.G.D. Lambert,H.J.Th . Comp.Endocrinol .35 :455-464 . Goos &P.G.W.J .va nOordt ,1982a .Estron e Crim,L.W. , 1982.Environmenta l modulationo f and estradiolparticipatio n duringexogen annual anddail yrhythm s associatedwit h ousvitellogenesi s inth efemal erainbo w reproduction inteleos t fishes.Can .J . trout,Salm ogairdneri .Gen .Comp .Endoc Fish.Aq . Sei. 39:17-21 . rinol. 46:81-92 . Crim,L.W . &D.M . Evans,1979 .Stimulatio no f vanBohemen ,CC , J.G.D.Lambert ,P.G.W.J , pituitary gonadotropinb y testosterone in vanOordt ,1982b .Vitellogeni n induction juvenilerainbo w trout (Salmogairdneri) . by estradiol inestrone-prime d rainbow Gen.Comp .Endocrinol .37 :192-196 . trout,Salm ogairdneri .Gen .Comp .Endoc Crim,L.W . &D.R . Idler,1^78 .Plasm agonado rinol.46 :136-139 . tropin,estradiol ,an dvitellogeni n andgo Bruslé,S ., 1980 .Ein e structureo fearl y nadphosviti nlevel s inrelatio n toth e previtellogenic oocytes inMugi l (Liza) seasonal reproductive cycleso f female aurattus.Risso ,181 0 (Teleostei,Mugili - brown trout.Ann .Biol .anim .Bioch .Bio dae). Cell.Tiss .Res .207 :123-134 . phys. 18:1001-1005 . Callard,G.V. , Z.Petro ,K.J .Rya n& J.B . Crim,L.W . &R.E .Peter ,1978 .Th e influence Claiborne,1981a .Estroge n synthesis in oftestosteron e implantation inth ebrai n vitroan d inviv o inth ebrai no f amarin e andpituitar y onpituitar y gonadotropin teleost (Myxocephalus)• Gen.Comp . Endocri- levels inAtlanti c salmonparr .Ann .Biol , nol. 43:243-255 . anim.Bioch .Biophys .18 :689-694 . Callard,CV. , Z.Petr o &K.J .Ryan ,1981b . deVlaming ,V.L. ,M.J .Vodicnik ,G .Bauer ,T . Biochemical evidence foraromatizatio no f Murphy &D .Evans ,1977a .Estradiol-17 @ef androgen toestroge n inth epituitary . fects onlipi d and carbohydrate metabolism Gen.Comp .Endocrinol .44 :359-364 . and onth e induction ofa yol kprecurso r in Campbell,CM. , 1978.I nvitr o stimulation thegoldfish ,Carassiu s auratus.Lif e Sei. ofvitellogeni n incorporation intotrou t 20:1945-1952 . oocytesb y salmonpituitar y extracts.Ann . deVlaming ,V.L. ,J . Singh,G .Paquett e &R . Biol.anim .Bioch .Biophys .18 :1013-1018 . Vuchs,1977b .I nviv oan d invitr o effects Campbell,CM . &D.R . Idler,1976 .Hormona l ofoestradiol-17 ßo n lipidmetabolis m in control ofvitellogenesi s inhypophysecto - Notemigonuscrysoleucas .J .Fish .Biol .10 : mizedwinte r flounder (Pseudopleuronectes 273-285. americanusWalbaum) . Gen.Comp .Endocrinol . deVlaming ,V.L. ,H.S .Wiley ,G .Delahunt y& 28:143-150 . R.A.Wallace ,1980 .Goldfis h (Carassius Campbell,CM . &D.R . Idler,1980 .Character auratus)vitellogenin :induction ,isolation , izationo f anestradiol-induce d protein rooertiesan d relationship toyol kproteins . fromrainbo w trout asvitellogeni nb yth e Comp.3iochem . Physiol.67B :613-623 . crossreactivit y toovaria nyol kfractions . Dindo,J.J . &R .MacGrego r III,1981 .Annua l Biol.Reprod .22 :605-617 . cycleo f serumgonada l steroids and serum Campbell,CM . &B .Jalabert ,1979 .Select lipids instripe dmullet .Trans .Am .Fish . iveprotei nincorporatio nb yvitellogeni c Soc.110 :403-409 . Salmogairdner i oocytes invitro .Ann . Dolphin,P.J. ,A.Q .Ansari ,C.B .Lazier ,K.A . Biol. anim.Bioch .Biophys .19 :429-437 . Munday &M .Akhtar ,1971 .Studie so nth ein Campbell,CM. , J.M. Walsh &D.R . Idler, duction andbiocynthesi s ofvitellogenin , 1976. Steroids inth eplasm a of thewinte r anoestrogen-induce d glycolipophosphopro- flounder (Pseudopleuronectes americanus tein.Biochem .J . 124:751-758 . Walbaum). Gen.Comp .Endocrinol .29 :14-20 . Droller,M.J .& T.F .Roth ,1966 .A nelectro n Chen,T.T. ,L.B .Agellon ,P.C .Rei d &D.A . microscope study ofyol k formationdurin g Howard,1982 .Estroge ncontro l ofgen eex oogenesis inLebiste s reticulatusGuppyi . 143 J. CellBiol .28. :209-232. . din,1981 .Determinatio n of sexualmatura Elliott,J.A.K. ,N.R .Bromag e &C .White tionstage so fAtlanti c salmon (Salmo sal- head,1979 .Effect s of oestradiol-17ßo n ar)capture d atsea .Can .J .Fish .Aq .Sei . serumcalciu man dvitellogeni n levelsi n 38: 405-413. . rainbow trout.J . Endocr. 83:54P-55P . Idler,D.R . &T.B .Ng ,1979 .Studie so ntw o Elliott,J.A.K. ,N.R .Bromag e &C .White types ofgonadotropin' s frombot h salmon head,1980 .0estradiol-17 gan dvitello - and carppituitaries .Gen .Comp .Endocrino l genesis inth erainbo w trout.J .Endocr . 38:421-440 . 87:65P-66P . Inoue,S .& M . Iwasaki,1978 .Isolatio n ofa Emmersen,B.K . &I.M .Petersen ,1976 .Natur novelglycoprotei n from theegg s ofrain aloccurrence ,an d experimental induction bowtrout :occurrenc e ofdisialosy l groups by estradiol-17-ß,o fa lipophosphopro - onal l carbohydrate chains.Biochem .Bio tein (vitellogenin)i nflounde r (Platich- phys.Res .Comm .83 :1018-1023 . tysflesu s L.). Comp.Biochem .Physiol . Inoue,S .& M . Iwasaki,1980 .Sialoglycopro - 54B:443-446 . teins from theegg so fPacifi cherring . Fostier,A. ,C .Weil ,M .Terqui ,B .Breto n Eur.J . Biochem. Ill:131-135 . & B.Jalabert ,1978 .Plasm aestradiol-17 ß Inoue,S. ,T .Kaneda-Hayashi ,H . Sugiyama& and gonadotropin during ovulation inrain T.Ando ,1971 .Studie s onphosphoprotein s bow trout (Salmogairdner i R.). Ann.Biol , fromfis h eggs.I .Isolatio n andcharacter anim.Bioch .Biophys .18 :9 29-936 . izationo fa phosphoprotei n from theegg s Goedmakers,A .& B.L .Verboom ,1974 .Studie s ofPacifi cherring .J .Biochem . 69:1003 - onth ematuratio n and fecundity ofth e 1011. pike,Eso x luciusLinnaeus ,1758 .Aquacul Iyengar,R . &H . Schlenk,1967 .Wa x esterso f ture.4 :3-12 . mullet (Mugilcephalus )ro eoil .Biochemis Guraya,S.S. ,1963 .Histochemica l studieso n try. 6:396-402 . theyolk-nucleu s infis hoogenesis .Z . Jalabert,B. ,1975 .Modulatio npa r différents Zellforsch.Mikrosk .Anat .60 :659-666 . steroidsno nmaturant sd el'efficacit é de Guraya,S.S. ,1965 .A comparativehistochem la17-a-hydroxy-206-dihydroprogesteron eo n ical study offis h (Channamaruleus )an d d'un extrait gonadotrope sur lematuratio n amphibian (Bufo stomaticus)oogenesis .Z . intrafolliculaire invitr ode sovocyte sd e Zellforsch.Mikrosk .Anat .65 :662-700 . latruit earc-en-cie l Salmogairdnerii .C . Guraya,S.S. ,1979 .Recen t advances inth e R.Acad . Sei.„Paris .Ser .D . 281:811-814 . morphology,cytochemistr y and functiono f Jalabert,B. ,F.W . Goetz,B .Breton ,A .Fos Balbiani'svitellin ebod y inanima loo tier &E.M .Donaldson ,1978 .Precociou sin cytes. Internat.Rev .Cytol . 59:249-321 . duction ofoocyt ematuratio n and ovulation Hara,A. ,1976 .Iron-bindin g activity offe incoh o salmon,Oncorhynchu s kisutch.J . male-specificseru mprotein s ofrainbo w Fish.Res .Bd .Can .35 :1423-1429 . trout (Salmogairdneri )an d chum salmon Jared,D.W . &R.A .Wallace ,1968 .Compara (Oncorhynchusketa) . Biochim.Biophys . tivechromatograph y ofth eyol kprotein s Acta.427 :549-557 . of teleosts.Comp .Biochem .Physiol .24 : Hara,A .& H .Hirai ,1978 .Comparativ e stud 437-443. ieso nimmunochemica lpropertie s offe Kaitaranta,J.K . &R.G .Ackman ,1981 .Tota l male-specific serumprotei nan d eggyol k lipids and lipid classeso f fishroe .Comp . proteins inrainbo wtrou t (Salmogaird Biochem.Physiol .69B :725-729 . neri). Comp .Biochem .Physiol .59B :339 - Larsen,L.O. ,1978 .Subtota lhepatectom yi n 343. intact orhypophysectomize d river lampreys Hara,A. ,K .Yamauch i &H .Hirai ,1980 . (Lampetrafluviatili s L.): Effects onre Studies onfemale-specifi c serumprotei n generation,blood glucoseregulatio nan d (vitellogenin)an d eggyol kprotei ni n vitellogenesis.Gen .Comp .Endocrinol .35 : Japanese eel (Anguilla japonica).Comp . 197-204. Biochem.Physiol .65B :315-320 . LeBail ,P.Y . &B .Breton ,1981 .Rapi ddeter Hori, S.H., T.Kodam a &K .Tanahashi ,1979 . minationo fth e sexo fprepubera l salmonid Inductiono fvitellogeni n synthesis in fishb y atechniqu eo fimmunoagglutination . goldfishb ymassiv edose s ofandrogens . Aquaculture.22 :367-375 . Gen. Comp.Endocrinol .37 :306-320 . Leger,C. ,L .Fremont ,D .Marion ,I .Nassou r Idler,D.R. ,1982 .Som eperspective s onfis h &M.-F .Desfanges ,1981 .Essentia l fatty gonadotropins.Thi s symposium. acids introu t serumlipoproteins,vitello Idler,D.R . &CM . Campbell,1980 .Gonado geninan d egg lipids.Lipids .16 :593-600 . tropinstimulatio n of estrogen andyol k LeMenn ,F. ,1979 .Inductio nd evitellogén - precursor synthesis injuvenil e rainbow inepa r1'oestradio le tpa rde sandrogene s trout.Gen .Comp .Endocrinol .41 :384-391 . chezu ntéléostée nmarin :Gobiu snige rL . Idler,D.R. , S.J.Hwan g &L.W . Crim,1979 . CR. Acad. Sc.Paris .Ser .D . 289:413-416 . Quantificationo fvitellogeni n inAtlanti c LeMenn ,F .& M.M .Lamy ,1977 .Protein svit salmon (Salmo salar)plasm ab yradioimmun ellines sériques ethépatique s chezu n oassay.J . Fish.Res .Bd .Can .36 :574 - poisson téléostéen,Gobiu snige rL. ,sou 578. mis ade s traitements hormonaux.Bordeau x Idler,D.R. ,S.J . Hwang,L.W . Crim &D .Red - Médical. 10:213-216 . 144 LeMerm ,F. ,H .Rochefor t &M .Garcia ,1980 . gaster cosby). J. Nutr.103 :1441-1447 . Effecto fandroge nmediate d by theestro Raven,C.P. ,1961 .Oogenesis :Th e storageo f genrecepto r offis h liver:vitellogeni n developmental information.Pergamo nPress , accumulation. Steroids.35 :315^-328 . NewYork . Lewis,M .& J.M . Dodd,1974 .Thyroi dfunc Redshaw,M.R . &B.K .Follett ,1971 .Th ecry tionan d theovar y inth e spotted dogfish stallineyolk-platele t proteins and their Scyliorhinus caricula.J . Endocr.63 :63P . solubleplasm aprecurso r ina namphibian , MacGregor,R . Ill,J.J . Dindo &J.H .Finu - Xenopus laevis•Biochem .J . 124:759-766 . cane,1981 .Change s inseru mandrogen san d Redshaw,M.R. ,B.K .Follet t &T.J .Nicholls , estrogens during spawning inbluefish , 1969.Comparativ e effects of theoestro - Pomatomus saltator,an d kingmackerel , gensan d other steroid hormones onseru m Scomberomoruscavalla .Can .J . Zool.59 : lipidsan dprotein s inXenopu s laevis 1749-1754. ~~~~ Daudin.J . Endocr.43 :47-53 . Mano,Y .& F .Lipmann ,1966 .Characteristic s Roach,A.H .& P.L .Davies ,1980 .Catfis h ofphosphoprotein s (phosvitins)fro ma vitellogenin and itsmessenge r RNAar e variety offis hroes .J . Biol.Chem .241 : smaller than their chickenan dXenopu s 3822-3833. counterparts. Biochem.Biophys .Acta . Mano,Y . &M .Yoshida ,1969 .A nove lcompos 610:400-412 . itiono fphosvitin s from salmonan d trout Sand,D.M. , J.L.Heh l &H . Schlenk,1969 . roe. J. Biochem. 66:105-108 . Biocynthesis ofwa x esters infish .Reduc Markert,J.R . &W.E .Vanstone ,1968 .Eg g tiono f fatty acids and oxidation ofalco proteins ofpacifi c salmon,genu sOncor - hols.Biochemistry . 8:4851-4854 . hynchus:protein s inth e solublefraction . Sand,D.M. , J.L.Heh l &H . Schlenk,1971 . Can.J . Biochem. 46:1334-1337 . Biocynthesis ofwa x esters infish .Metab Markert,J.R . &W.E .Vanstone ,1971 .Eg g olismo fdietar y alcohols.Biochemistry . proteins of coho salmon (Oncorhynchus 10:2536-2541 . kisutch): chromatographic separationan d Sand,D.M. , C.H.Rah n &H . Schlenk,1973 . molecularweight s of themajo rprotein s in Wax esters infish .Absorptio n andmetabo thehig hdensit y fractionan d theirpres lismo foley lalcoho l inth egouram i ence insalmo nplasma .J .Fish .Res .Bd . (Trichogaster cosby). J.Nutr .103 :600 - Can. 28:1853-1856 . 607. Matty,A.J. ,E .Tsureki ,W.W .Dickhof f &A . Sand,0. ,I.M .Peterse n &B .Korsgaard-Em - Gorbman,1976 .Thyroi d and gonadalfunc mersen,1980 .Change s insom ecarbohydrat e tion inhypophysectomize dhagfish ,Epta - metabolizing enzymesan d glycogen inlive r tretus stouti.Gen .Comp .Endocrinol .30 : glucosean d lipid inseru m duringvitello - 500-516. genesis and after inductionb yestradiol - Nath,P . &B.I .Sundararaj ,1981 .Isolatio n 17-ßi nth eflounde r (Platichtys flesus L.) and identificationo ffemale-specifi cser Comp.Biochem . Physiol.65B :327-332 . um lipophosphoprotein (vitellogenin)i n Schmidt,G. ,G .Bartsch ,T .Kitagawa ,K . thecatfish ,Heteropneuste s fossilis Fujisawa,J .Knolle ,J . Joseph,P .D e (Bloch).Gen .Comp .Endocrinol .43 :184 - Marco,M .Lis s &R .Haschemeyer ,1965 . 190. Isolationo fa phosphoprotei n ofhig h Nath,V. ,1960 .Histochemistr y of lipidsi n phosphorus content from theegg so fbrow n oogenesis. Int.Rev .Cytol . 9:305-320 . brook trout.Biochem .Biophys .Res .Comm . Nayyar,R.P. ,1964 .Th eyol knucleu s offis h 18:60-65 . oocytes. Quart.J .Micr . Sei. 105:353-358 . Scott,A.P. ,V.J .By e& S.M . Baynes,1980 . Ng,T.B .& D.R . Idler,1980 .Gonadotropi n Seasonalvariatio n inse x steroids offe regulationo fandroge nproductio n infloun malerainbo w trout (Salmogairdner iRich der and salmonids.Gen .Comp .Endocrinol . ardson). J .Fis hBiol .17 :587-592 . 42: 25-38. Selman,K . &R.A .Wallace ,1982 .Oocyt e Pickering,A.D. ,1976 .Effect s ofgonadec - growth inth e sheepshead minnow:uptak eo f tomy,oestradio l and testosterone onth e exogenous proteinsb yvitellogeni c oocytes migrating river lamprey,Lampetr afluvia - Tiss.Cell , (inpress) . tilis (L).Gen .Comp .Endocrinol .28 :473 - Shackley,S.E .& P.E .King ,1977 .Oogenesi s 480. ina marin e teleost,Blenniu spholi sL . Plack,P.A . &N.W .Fraser ,1971 .Incorpora CellTiss .Res .181 :105-128 . tiono fL-f 1 C]leucin e into eggprotein s Skinner,E.R .& A .Rogie ,1978 .Th eisola by liverslice s from cod.Biochem .J .121 : tionan dpartia l characterization ofth e 857-862. serum lipoproteins and apolipoproteins of Plack,P.A. , D.J. Pritchard &N.W .Fraser , therainbo w trout.Biochem .J . 173:507 - 1971. Eggprotein s inco d serum:natura l 520. occurrence and inductionb y injectionso f Sumpter,J.P .& J.M .Dodd ,1979 .Th eannua l oestradiol 3-benzoate. Biochem.J .121 : reproductive cycleo f thefemal elesse r 847-856. spotted dogfish,Scyliorhinu s canicula L., Rahn,C.H. ,D.M . Sand &H . Schlenk,1973 . and itsendocrin e control.J . Fish.Biol . Wax esters infish .Metabolis m ofdietar y 15:687-695 . palmidylpalmitat e inth egouram i (Tricho- Sundararaj,B.I .& S.V . Goswami,1968 .Ef - 145 fectso f estrogen,progesterone » andtest ectesplatess aL .Gen .Comp .Endocrinol . osteroneo nth epituitar y andovar yo fcat 31: l-TT. fish,Heteropneuste s fossilis (Bloch).J . Woodhead,P.M.J. ,1969 .Influenc eo foestra - Exp. Zool.169 :211-228 . diol 3-benzoate upon theplasm a contento f Sundararaj,B.I .& P .Nath ,1981 .Steroid - calciuman dvitami nA aldehyde s inth e induced synthesis ofvitellogeni ni nth e cod,Gadu smorhua .J;-mar .biol .Ass .U.K . catfish,Heteropneuste s fossilis (Bloch). 49:939-944 . Gen. Comp.Endocrinol .43 :201-210 . Yu,J.Y.-L. ,W.W .Dickhoff ,Y . Inui& A . Tata,J.R .& D.F . Smith,1979 .Vitellogene - Gorbman,1980 .Sexua lpattern s ofprotei n sis:A versatil emode l forhormona lregu metabolism inlive ran dplasm a ofhagfish , lationo fgen e expression.Rec .Prog .Hor . Eptatretus stoutiwit h specialreferenc e Res. 35:47-95 . tovitellogenesis .Comp .Biochem .Physiol . Terkatin-Shimony,A .& Z .Yaron ,1978 .Estro 65B:111-117 . gensan d estrogenic effects inTilapi a Yu,J.Y.-L. ,W.W .Dickhoff ,P . Swanson& A . aurea (Cichlidae,Teleostei) .Ann .Biol , Gorbman,1981 .Vitellogenesi s and itshor anim.Bioch .Biophys .18 :1007-1012 . monal regulation inth ePacifi chagfish , Tesoriero,J.V. , 1977.Formatio no fth e Eptatretus stoutiL .Gen .Comp .Endocrin chorion (zonapellucida )i nth eteleos t ol.43 :492-502 . Oryzias latipes. II.Polysaccharid ecyto chemistry of early oogenesis.J .Histochem . Cytochem. 25:1376-1380 . Tesoriero,J.V. , 1978.Formatio n ofth e chorian (zonapellucida )i nth eteleos t Oryzias latipes.III .Autoradiograph y of [3H]prolin e incorporation.J .Ultrastruct . Res.64 :315-326 % Ulrich,E. ,1969 .Etud ede s ultrastructures aucour sd el'ovogenès ed'u npoisso n télé- osteen,l edanio ,Brachydani o rerio (Ham ilton-Buchanan).J .Microscopie .8 :447 - 478. Wallace,R.A. , 1978.Oocyt e growth innon - mammalianvertebrates .In :R.E .Jone s (Ed.1:Th evertebrat e ovary.Plenu mPress , NewYork .p .469-502 . Wallace,R.A. ,D.W . Jared &A.Z .Eisen ,1966 . Agenera lmetho d for the isolationan d purification ofphosviti n fromvertebrat e eggs. Can.J . Biochem.44 :1647-1655 . Wallace,R.A . &K .Selman ,1981 .Cellula r and dynamicaspect s ofoocyt egrowt hi n teleosts.Am .Zool .21 :325-343 . Whitehead,C. ,N.R .Bromag e &J.R.M .Forster , 1978. Seasonal changes inreproductiv e functiono f therainbo w trout (Salmogair - dneri). J. Fish.Biol .12 :601-608 . Wiegand,M.D .& D.R . Idler,1982 .Synthesi s of lipidsb y therainbo w trout (Salmo gairdneri)ovar y invitro .Can .J .Zool . (inpress) . Wiegand,M.D .& R.E .Peter ,1980a .Effect s of sexsteroid s inplasm a lipids inth e goldfish,Carassiu s auratus. Can.J .Zool . 58:967-972 . Wiegand,M.D .& R.E .Peter ,1980b .Effect s of testosterone,oestradiol-17 ßan dfast ingo nplasm a freefatt yacid s inth egold fish,Carassiu s auratus.Comp .Biochem . Physiol.66A :323-326 . Wiley,H.S .& R.A .Wallace ,1981 .Th estruc tureo fvitellogenin .Multipl evitellogen insi nXenopu s laevisgiv e rise tomulti pleform so f theyol kproteins .J .Biol . Chem.256 :8626-8634 . Wingfield,J.C .& A.S .Grimm ,1977 .Seasona l changes inplasm a Cortisol,testosteron e andoestradiol-17 ßi nth eplaice ,Pleuron - 146 EFFECTS OF FEEDING LEVELS AND TEMPERATURE ONTH EDEVELOPMEN TO FTH E GONAD INTH EAFRICA N CATFISHCLARIA SLAZER A (C& V ) C.J.J.Richter ,E.H .Eding ,S.E.W .Leuve nan dJ.G.M .va nde rWijs t Department of Fish Culture and Inland Fisheries,Agricultura l University, Wageningen, Netherlands. Summary Theeffec twa s studied oftw o temperatures efficient feed conversionan d the othertw o 25 and 30°C and three feeding levels onth e levels were derived from it:Leve l 1wit ha development ofth egona d ofth eAfrica ncat third as much and Level 3wit hthre etime s fish Clarias lazera.Fo r males,th e gonado asmuc ha sLeve l2 .Th e following groupswil l somatic indexwa smor e affectedb ytemperat be distinguished in this paper: Groups 1-3 ure thanb y feeding level.Th e combination and 4-6 were raised at awate r temperature of high temperature and high feeding level of 30 and 25°C, respectively. Thetw oset s had a negative effect on the male gonado of groups received, in order of ascending somatic index.A t 25°C, the proportion of groupnumber ,foo d corresponding toLeve l 1, suitable male spawnerswa sth ehighest ,ir 2 and 3respectively .Ever y twoweeks ,feed respective of feeding level.Fo rfemale sth e ing levels were adjusted according to the gonado somatic index was more affected by observed growth rates.Developmen t ofgonad s feeding level than by temperature. A low in 10 males and 10 females per group was feeding level had a positive effecto nth e studied for5 samplingperiod s atabou t120 , female gonado somatic index.Th ebette rfe d 160,200 ,24 0an d 300day s afterhatchin go f femalesha d ahighe rultimat eabsolut efecun theeggs . ditytha npoorl y fed fish.Proportio nhatch ing of fertilized eggs was much higher in the females kept at 25°C, indicating that Somatic and gonadic growth 30°C is above optimum forcontrolle d repro duction. Egg quality didno talte rwit hde Formale san d females,somati c growth (wet creasing feeding levels.A temperature of body weight)wa s more affected by feeding 25°Can d afeedin g level giving themos tef level than bytemperature .A ta nag eo f30 0 ficient feed conversion is recommended for days the largest fishwer e found inGrou p 6, conditioning brood fishfo rbreeding . in which mean weights of males and females were80 0an d 600g , respectively (Fig.1 an d 2). Introduction The effects offeedin g levelsan dtemper ature on thegona d ofC .lazer awa s studied under N. Europeanligh tperiodicit y (march- january)t odevelo p anoptimu mprocedur e for conditioning brood fish for breeding. The relationship between the amounto f foodad ministered and reproductive output wases 0 100 200 300days 0 100 200 300 days timated. GROUP 2 GROUP 5 Characteristics studied included gonadal 1000 growth,maturatio n stage oftesti san d ovary, absolute and relative fecundity of females andproportio n offertilize d eggstha tdevel oped into normal larvae. In thisbrie fac O 100 200 300days 0 100 200 300days count statistical analysis isno tpresented . GROUP3 Raising offis h r 1000 The fishwer e raised fromeg g tofingerlin g intrough swit h recirculatingwate r at29°C . Under optimum feeding conditions, a mean 0 100 200 300day s 0 100 . 200 300d ; weight of4 0g wa s reached inabou t6 0days . Attha tage ,th e fingerlingswer edistribute d Fig. 1. Growth of males (wet body weight) of over fibre-glass tanks and fedo ntrou tpel c.lazera kept at 30 °C (left) and lets (45%protein ) added to thewate rfro m 25 C (right) and various feeding automatic feeders.Th e effectwa s studiedo f levels.(For explanation see text.) two temperatures 25an d 30°Can d threefeed ing levels onth edevelopmen t ofth egonad . Level 2wa sdefine d astha tgivin gth emos t 147 O 100 200 300days 0 100 200 300 days 0 100 200 300 days GROUP2 GROUP 5 1000 100 200 300days 0 100 200 300days GROUP3 1000 0 100 200 300days 0 100 200 300days 0 100 200 300days 0 100 200 300days Fig.2 .Growt ho ffemales .Fo rexplanatio nse e Fig.4 .Ovar ygrowth .Fo rexplanatio nse eFig .3 . Fig. 1. gonado somatic index. For females the gonado Testis growth was more affected by temper somatic index, during most of the sampling ature than by feeding level. At the higher periods, was affected by feeding level and feeding levels, this effect was pronounced not by temperature. A low feeding level had (Fig.3) . a positive effect on the female gonado som Ovary growth was with the exception of the atic index. last sampling period more affected by temper ature than by feeding level (Fig.4) . Development of testis and ovary The testis and ovary were examined histol ogically following autopsy of 10 males and 10 females per group in each sampling period. Central parts of the testis and ovary were fixed in calcium formaldehyde and embedded in paraffin wax. Sections 10 (jmthic k were 100 200 300days 0 100 200 300days stained with haemalumeosine. C. lazera has a tubular testis which is characterized by the presence of spermatog- enic cysts attached to seminiferous tubules. Testis development could be classified accor ding to the following index: an undeveloped, 100 200 300days 0 100 200 300days spent or regressed testis; a developing tes tis with cysts mainly containing spermatog onia, spermatocytes, spermatids and occasion ally spermatozoa; a ripe testis with cysts and tubules completely filled with spermat ozoa. The developed (lateral) part of each testis was studied and scored according to 0 100 200 300day s 0 100 200 300day s the index. Fig. 3. Testis growth (wet weight). For The proportion of males with undeveloped explanation see Fig. 1. and ripe testes was estimated (Fig.5) . The highest proportion of suitable male Gonado somatic index spawners was encountered during the last four sampling periods at 25°C, irrespective The gonado somatic index of a fish was cal of feeding level. culated as follows: The ovary of C. lazera is a hollow sacklike _ total gonad weight structure consisting of an outer wall with GSI x 100 body weight - total gonad weight lamellae penetrating the central lumen. The lamellae contain oogonia and oocytes in fol For males the gonado somatic index was more licles at various stages of development: pre- affected by temperature than by feeding level. vitellogenic, vitellogenic, post-vitellogenic The combination of high temperature and high and atretic follicles (Richter and Van den feeding level had a negative effect on the Hurk, 1982). The proportion of females with 148 The ovaries of fish kept at 25°C contained few atretic oocytes. This agrees with our ex perience in recent years that adult C. lazera females can reproduce successfully throughout the year. The present histological examin 100 200 300days 0 100 200 300day s ations suggest a continuous slow "turnover" from vitellogenic through post-vitellogenic to atretic oocytes in the ovary of females kept at 25°C. Absolute and relative fecundity of females 100 200 300days 100 200 300day s 0 The total number of vitellogenic eggs per GROUP 6 fish was estimated by counting the number of vitellogenic eggs (diameter > 0.35 mm) per 0.5 gram of ovary tissue. Absolute fecundity was defined as the total count of vitellog enic eggs at a given fish age. The number of 0 100 200 300days 0 100 200 300days fish containing vitellogenic eggs was not identical in all groups (Fig. 7). Fig. 5. Development of the testis. For expla nation see Fig. 1. meannumbe ro f „„„ ^. „„ . , . Proportion of males containing ripe spermatozoa (%) . . Proportion of males with undeveloped, spent or regressed testes (%) predominantly post-vitellogenic oocytes (di 9 to ameter > 1 mm') and with predominantly atretic 100 200 300 days 0 100 200 300day s oocytes in their ovaries was estimated (Fig. GROUP 2 H ^ GROUP 5 9 6). A high temperature (30°C) resulted in an earlier onset of vitellogenesis at the lower feeding levels. The high proportion of fe males with atretic post-vitellogenic oocytes 100 200 300days 0 100 20M-0 3O0days (Fig. 6, Groups 1-3) and the fluctuating GROUP3 GROUP 6 ntimberofeggsl proportion of females with "healthy" post- • , 10000 I vitellogenic oocytes (Fig. 6, Group 3) indic m • 7000 ! ated that complete regression of ovaries oc curred at 30°C. 0 100 200 300 days 0 100 200 300days Fig. 7. Absolute fecundity of females (10 fish per group). The figures on top of the standard deviation bars refer to the number of fish containing vitellogenic eggs. 100 200 300days 0 100 200 300days The absolute fecundity atal l feedinglev els reached a maximum on the lastsamplin g day (300days) . Attha ttim eth ebette r fed females (Groups 100 200 3O0days 0 100 200 300days 3 and 6)ha d ahighe r absolute fecundity than thepoorl y fed ones (Groups 1an d4) . The relative fecunditywa sdefine d asth e totalnumbe r ofvitellogeni c oocytespe rgra m bodyweight .Thes e resultsan d thenumbe ro f fishcontainin gvitellogeni c eggsar epresen 0 100 200 300day s 0 100 200 300day s ted inFig .8 . In both temperature groups the relative Fig. 6. Development of the ovary. For explanation fecunditywa snegativel y affectedb yincreas see Fig. 5. ing feeding level (compare Group 1wit h 3 ». » Proportion of females containing ovaries and Group 4wit h6) . with predominantly postvitellogenic oocytes (%) • • Proportion of females containing ovaries with predominantly atretic oocytes (%) 149 In all groups, the highest proportion hat Fertility of females ched occurred in the last sampling period (300 days). The proportion of deformed lar The egg quality of all groups was studied vae was fairly constant in all groups. during the last three sampling periods. Ten The proportion of eggs hatched was much females per group and per sampling period higher in the groups kept at 25°C, indicat were reproduced by artificially induced ing that 30°C is above optimum for control breeding with HCG (Eding et al., poster) led reproduction of C. lazera. Egg quality After stripping, a sample of 200 eggs per did not alter with decreasing feeding levels. brood fish was fertilized with milt from ma les of the brood stock of the hatchery and Discussion subsequently incubated in stagnant water at 30°C. The proportion of eggs hatched and the The relationship between food supply and proportion of deformed larvae were calcul reproductive output has received relatively ated (Fig. 9),th e difference being taken as little experimental study (Wootton, 1979). an expression of female fertility, i.e. the Ration size was positively correlated with proportion of fertilized eggs that develop the percentage of yolk bearing oocytes per into normal larvae. ovary but not with the atretic oocytes in mean numbero f the winter flounder Pseudopleuronectes amer- eggspe rgra m body weight icanus (Tyler & Dunn, 1976). The present study on C. lazera has likewise shown that the better fed females were more fecund but has also established that egg qual ity was not impaired by low feeding levels. The poorly fed females of C. lazera became 100 200 300days 0 100 200 300days mature at an unusually low body weight and had the highest relative fecundity. This means that food reduction in C. lazera leads to investment in the ovaries at the expense of somatic tissue. The three-spined stickle back (Gasterosteus aculeatus) has the same 100 200 300days 0 100 200 300days strategy (Wootton, 1979). The present feeding experiments with C. laz number of eggs era were carried out in the temperature range «s,* x 100 7 of its natural habitat. It is surprising that an increase of 5°C in temperature causes a ! 10 h marked disturbance in reproduction manifested 6 1 T Jà- by pronounced cyclic atresia of vitellogenic 0 100 200 300day s 0 100 200 300day s oocytes and regression of the testis. A tem perature of 25°C and a feeding level giving Fig.8 .Relativ e fecundity offemales .Fo r the most efficient feed conversion is recom explanation see Fig.7 . mended for conditioning brood fish for breed ing. Under these conditions hatchery raised GROUP 1 C. lazera can be reproduced successfully 100 - throughout the year. ] 50 References Richter, C.J.J, and R. van den Hurk, 1982. 0 , •• i L, 100 200 300d 100 200 300day s Effects of 11-desoxycorticosterone-acetate GROUP 5 and carp pituitary suspension on follicle T maturation in the ovaries of the African catfish, Clarias lazera (C S V). Aquacul ture 29, 53-66. Tyler, A.V. and R.S. Dunn, 1976. Ration, il i growth and measures of somatic and organ 100 200 300day s 0 100 200 300day s condition in relation to meal frequency in winter flounder, Pseudo pleuronectes amer- icanus, with hypotheses regarding popula tion homeostasis. J. Fish.Res.Bd.Can. 33: J T 50 63-75. li_L Wootton, R.J., 1979.Energ y costs of egg pro 0 100 200 300day s 0 100 200 300day s duction and environmental determinants of fecundity in teleost fishes. Symp.zool.Soc. Fig.9 .Fertilit yo ffemales .Fo rexplanatio n Lond. (1979) No. 44, 133-159. seeFig . 7. Intervet International B.V., Boxmeer, Nether f Proportiono fegg shatche d lands is gratefully acknowledged for supplying | Proportiono fdeforme dlarvae . the human chorionic gonadotropin. 150 THE INTER- AND INTRACELLULAR PASSAGE OF PROTEINS THROUGH THE OVARIAN FOLLICLE IN TELEOSTS K. Selman, R.A. Wallace Department of Anatomy, University of Florida, College of Medicine, Gainesville, FL 32610 U.S.A. Summary the major process contributing to formation of the relatively large teleost egg. Oocyte growth in teleosts primarily in Several ultrastructural studies have volves the sequestration and processing by described micropinocytotic activity at the the oocyte of afemale-specifi c protein that surface of vitellogenic oocytes concomitant is hepatically derived. The structure of with the appearance of yolk bodies. How vitellogenic follicles of two closely ever, only one previous study has described related euryhaline cyprinodonts, Fundulus the incorporation of an extracellular tracer heteroclitus and Cyprinodon variegatus, is into yolk spheres in teleost oocytes described. The electron-dense tracer, (Wegmann & Götting, 1971). In the present horseradish peroxidase, and a larger study of vitellogenesis in two closely- heterologous protein, Xenopus laevis related Cyprinodonts, the sheepshead minnow, [3H]vitellogenin, were used in this study Cyprinodon variegatus, and the ki11if is h to demonstrate the pathway by which exogen Fundulus"iïeteroclitus , we a)presen t experi- ous proteins leave the maternal circulation, mental evidence that growing oocytes incor and pass through the follicle to yolk porate exogenous proteins into developing spheres within the growing oocyte. Proteins yolk spheres and b) describe the pathway leave the perifollicular capillaries, and traversed by these proteins from the mater traverse the theca, the follicular epi nal circulation through tne ovarian follicle thelium, and the vitelline envelope. At the into yolk spheres within the oocyte. oocyte surface they are incorporated via micropinocytosis and translocated to enlarg Materials and methods ing yolk spheres. In contrast to studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study Mature sheepshead minnows (C^ variegatus) substantiates the importance of heterosyn- were collected in the vicinity of Crescent thetic processes during oocyte growth. Beach, Florida,durin g their breeding season Keywords: follicle, micropinocytosis, (April through September). Breeding oocyte, teleost,vitellogenesis . killifish, F^_ heteroclitus, were collected in May and early June Tn~th e vicinity of Woods Hole, Massachusetts. Introduction Pieces of ovary were placed in fish saline (F0 solution; Wallace & Selman, 1978) and Oocyte growth among nonmammalian verte follicles were manually dissected, measured brates is associated with the accumulation and rapidly placed in fixative for micro of a hepatically derived yolk precursor pro scopy. The procedures used for specimen tein, vitellogenin (VTG). This female- preparation and electron microscopy have specific protein is the major phosphoprotein been previously described (Wallace & Selman, in female serum, is easily identified by la 1979). Most tissues were embedded in Epon- beling with [32p]Pi, and can be induced Araldite although some were embedded in JB4 in males by estrogen injection (Wallace and plastic (Polysciences)fo r light microscopy. Selman, 1981). Subsequent to incorporation Horseradish peroxidase (HRP;Type II, by the oocyte, VTG is cleaved into yolk pro Sigma) was dissolved in F0 solution at a teins which accumulate in membrane-limited concentration of 30-40 mg/ml. Female C^_ bodies ("extravesicular yolk"). variegatus, weighing approx. 2 g, were Previous studies have indicated that injected intraperitoneal^ with 6m g HRP; F. oocyte growth in teleosts occurs primarily heteroclitus females, weighing approx. 3 g, by the accumulation of both endogenously and were injected with 9 mg HRP. At periodic exogenously synthesized yolk (e.g., intervals up to 12 hr, ovaries were removed Korfsmeier, 1966;t e Heesen, 1977). Endogen and follicles were measured, routinely ous or "intravesicular" yolk resides within fixed, and washed for 2 hr. Follicles were yolk vesicles which appear to be the precur sliced into halves or quarters, and were sors of cortical alveoli (Wallace & Selman, rinsed in 0.05 M phosphate buffer (pH 7.6) 1981). These structures release their con prior to incubation for 20 to 40mi n at room tents to the perivitelline space at the time temperature in DAB-reaction mixture (Graham of fertilization and hence do not represent & Karnovsky, 1966). Follicles from saline- yolk in a strict sense. This consideration injected fish were run as controls; in addi serves to focus on the mechanism of exogen tion, follicles from HRP-injected fish were ous or "extravesicular" yolk formation as incubated in DAB-reaction mixture which 151 lacked HgOg. Following incubation, tis females. HRP (MW 40,000) was used since it sues were post-fixed and embedded. Thin can be readily visualized by both light and sections were stained with lead solution electron microscopy. X^_ laevis [3H]VTG prior to examination in the electron micro (MW 465,00) has been chosen since its fate scope; 1u m sections were examined unstained would mimic the'fate of native VTG (Wallace in the light microscope. et al., 1980). Females were sacrificed up [3H]VTG was prepared from Xenopus laevis Tö~ ÜÄ hr after injection and vitellogenic by injecting afemal e with 4m g 17ß-estradiol follicles (C^_ variegatus: 0.4-0.7 mm; F. on day 0 and 5 mCi [3H]leucine on day 7 heteroclitus: 0.6-1.2mm )wer e examined. (Wallace et al., 1972). .[3H]VTG was iso Figs. 3-7 show the location of HRP within lated from pTäsma (Wiley et_ al_., 1979) on vitellogenic follicles. HRP reaction prod day 8 and had a specific activity of0.5-1. 3 uct is found within the lumen of the peri x 103 cpm/yg protein. Fish were injected follicular capillaries, lining the lumenal intraperitoneal^ with 1.25-2.7 mg [3H]VTG surface of the endothelial cells and within and maintained for 24 hr, after which the intercellular spaces between adjacent endo ovaries were removed, washed in FO solution thelial cells (figs. 4,5). HRP reaction and follicles were prepared for microscopy. product is found amongst the stroma of the Slides dipped for light microscopic autora theca and within the basal lamina of the diography were stored for 1-10 months at 4°C. follicle cells. Materials must traverse the follicular epithelium and vitelline envelope Results and discussion to reach the oocyte surface. HRP reaction product is found within the intercellular The structure of vitellogenic follicles spaces of the follicular epithelium (fig. 4) and the process of oocyte growth in both and within the pore canals of the vitelline C. variegatus and F^ heteroclitus are simi- envelope. These canals appear to provide a Tär and will be treated together in this direct means of communication between the study. Fig. 1 shows oocytes in various size intercellular spaces of the follicular follicles in the ovary of C. variegatus. epithelium and the oocyte surface. Oocytes within vitellogenic"Follicle s are The surface of vitellogenic oocytes is surrounded by several cellular and acellular projected into long microvilli which extend layers (figs. 2,4). The oocyte is immedi through the pore canals of the vitelline ately invested by the acellular vitelline envelope and terminate deep within the envelope (chorion) which is penetrated by intercellular channels of the follicular cytoplasmic processes from both the oocyte epithelium. Frequently, they are heavily and surrounding follicle cells. The fol stained and obscured by reaction product licular epithelium is comprised of a single within the intercellular spaces of the fol layer of columnar cells which are periodi licular epithelium (fig. 4). The surface of cally interrupted by large intercellular vitellogenic oocytes displays extensive spaces. Within these spaces reside long micropinocytotic activity (fig. 7). By 20 microvillar processes from the oocyte and min after HRP injection (our shortest time fibrillar appendages which extend from the interval), reaction product is found within outer surface of the vitelline envelope. A endocytotic vesicles and smooth-surfaced distinct basal lamina separates the follicle tubules near the oocytes surface which cells from the overlying theca. The thecal appear to fuse to form peripheral yolk layer is composed of a scant connective spheres (figs. 6,7). Yolk spheres contain tissue stroma and an extensive capillary ing HRP are readily visualized with the network which supplies the entire follicle light microscope (fig. 3). Several hr after with maternal nutrients. A surface epi HRP injection, the disposition of HRP reac thelium covers the entire follicle and con tion product remains the same, except that sists of a single layer of squamous cells. larger yolk bodies residing deeper in the oocyte also contain HRP. Control follicles By the time vitellogenesis begins, the from uninjected fish show endogenous peroxi oocyte has already elaborated numerous lipid dase activity only in erythrocytes within inclusions, yolk vesicles, and secondary the capillaries; control follicles from lysosomes. Most characteristically, the HRP-injected fish were negative for HRP oocyte begins to sequester material at its reaction product. surface by micropinocytosis and yolk spheres form due to fusion of endocytotic vesicles The distribution of [3H]VTG within and smooth-surfaced tubules. Yolk spheres vitellogenic follicles was examined auto- migrate centripetally and fuse to form radiographically 24 hr after [3H]VTG in larger yolk bodies which subsequently coal jection. Grains were found localized over esce to form a continuous yolk mass (fig. peripheral yolk spheres and over fusing yolk 2). As this occurs, yolk vesicles are dis masses (figs.8,9) . placed towards the periphery and become the We conclude from these studies that macro- definitive cortical alveoli. molecules leave the maternal circulation via In order to follow the route traversed by an intercellular route. Upon reaching the maternal protein during vitellogenesis, HRP follicular epithelium, they pass between, and ]L_ laevis [3H]VTG were injected into rather than through, adjacent follicle cells 152 en route to the oocyte. Circulating macro- Fig. 2. A cross section through a vitel molecules, such as VTG and other serum logenin follicle of C. variegatus showing proteins, are incorporated into the oocyte the oocyte (o) and its surrounding vitel via micropinocytosis and translocated to line envelope (v) and follicular epitheli yolk spheres in less than 20 min. These um (f). y, yolk; ca, cortical alveolus. studies substantiate the importance of Epon-Araldite, toluidine blue. heterosynthetic processes during oocyte Fig. 3. Light micrograph of an unstained growth in teleosts. Èpon-Araldite section of a follicle of C. variegatus from an animal sacrificed 20 References min after HRP injection. HRP reaction product is seen within small spheres in Graham, K.C. & M.J. Karnovsky, 1966. The the cortical ooplasm (o) and within the early stages of absorption of injected vitelline envelope (v). ca, cortical horseradish peroxidase in the proximal alveolus;y , unstained yolk sphere. tubules of mouse kidney: Ultrastructural Fig. 4. Outer region of a follicle from cytochemistry by a new technique. J. F. heterocljtus which had been sacrificed Histochem. Cytochem. 14:291-302. 'S-Vir after HRP injection. Dense reaction Korfsmeier, K.H., 1966. Zur Genese der product is seen within the extracellular Dottersystems in der Oocyte von Brachy- components of the theca (Th), the basal danio rerio. Z. Zellforsch . Mikrosk. lamina (B) overlying the follicular epi lïïâTT7TT28J-296 . thelium (F) and within the intercellular te Heesen, D., 1977. Immunologische Unter spaces (black arrows) between the follicle suchungen an exo- und endogenen Dotterpro cells. The lumen (L) of a perifollicular teinen von Brachydanio rerio (Teleostei, capillary contains dense reaction product. Cyprinidae) und verwandten Arten. Zool. SE, surface epithelium; V,vitellin e enve Jb. Anat. Bd.97:566-582 . lope;whit e arrow, pore canal containing a Wallace, R.A. & K. Selman, 1978. Oogenesis microvillar process extending from the oo in Fundulus heteroclitus. I. Preliminary cyte surface (0);whit e asterisk, fibril observations on oocyte maturation in vivo lar appendage of the vitelline envelope. and in vitro. Dev. Biol. 62:354-3617 Fig. 5. A perifollicular capillary from Wallace, R.A. & K. Selman, 1979. Physio C. variegatus, which was sacrificed 30mi n logical aspects of oogenesis in two "äTteT HRP injection. A thin layer of species of sticklebacks, Gasterosteus reaction product is found on the lumenal aculeatus L. and Ape!tes quadricus surface of the endothelial cells (En) and (Mitchell). J. Fish Biol. 14:551-564. within the intercellular spaces of the Wallace, R.A. & K. Selman, 1981. Cellular endothelium (arrows). SE, surface and dynamic aspects of oocyte growth in epithelium; L,capillar y lumen. teleosts. Am. Zool. 21:325-343. Fig. 6. Outer oocyte region from F. hetero Wallace, R.A., J.M. Nickol, T. Ho & D.W. clitus which was sacrificed 30"mi n after Jared, 1972. Studies on amphibian yolk. HRP injection. Reaction product is vis X. The relative roles of autosynthetic and ible within small Vesicles and tubules heterosynthetic processes during yolk near the oocyte (0) surface and within protein assembly by isolated oocytes. peripheral yolk spheres. An unlabeled Dev. Biol. 29:255-272. yolk sphere (Y) is closely associated with Wallace, R.A., R.A. Deufel & Z. Misulovin, a labeled sphere. CA, cortical alveolus. 1980. Protein incorporation by isolated Fig. 7. Outer oocyte region from C. vari amphibian oocytes. VI. Comparison of egatus which was sacrificed 20 mm after autologous and xenogeneic vitellogenins. HRP injection. Reaction product is vis Comp. Biochem. Physiol. 65B:151-155. ible within tubules, small vesicles and a Wegmann, I. & K.J. Götting, 1971. Unter peripheral yolk sphere, which appears to suchungen zur Dotterbildung in den Oocyten have recently fused with reaction-contain von Xiphophorus helleri (Heckel, 1948) ing vesicles. Unlabeled vesicles and (Teleostei, Poeciliidae). Z. Zellforsch. tubules are also present. V, vitelline Mikrosk. Anat. 119:405-433. envelope; 0, oocyte. Wiley, H.S., L. Opresko & R.A. Wallace, Fig. 8. Autoradiogram of a follicle from 1979. New methods for the purification of F. heteroclitus sacrificed 24 hr after vertebrate vitellogenin. Anal. Biochem. injection of pHlVTG. Grains are con 97:145-152. centrated as hot spots over small yolk spheres. y, unlabeled yolk spheres. ******* Toluidine blue. (40w k exposure). Fig. 9. Autoradiogram of a follicle from Fig. 1. The ovary of C. variegatus showing C. variegatus, sacrificed 24 hr after in- oocytes in various size follicles. a, jection of L^H]VTG. Grains are concen primary growth phase; b, early yolk ves trated over yolk spheres of varying sizes icle stage; c, late yolk vesicle stage; d, and over fusing yolk masses (white early vitellogenesis; e, late vitellogene- asterisk). Toluidine blue. (4w k exposure). sis. JB4-plastic, toluidine blue. 153 154 BIOCHEMICAL COMPOSITIONO FOVULE SAN DFECON DEGG SO FSE ABAS S (DicentrarchusLabrax) ,SOL E (Soleavulgaris )AN DTURBO T (Scophthalmusmaximus ) Devauchelle,N . Brichon**,F .Lamour*** ,G .Stephan * * CentreOcéanologiqu e deBretagne ,B P337 ,2927 3Bres tCedex , ** InstitutMiche lPacha ,8350 0Tamaris-Sur-Mer , ***L.N.P.A . COB,B P337 ,2927 3Bres tCede x Studieso nth eoveral lbiochemica lcompos itiono ffis hegg san d ovules areo finteres t forreachin ga bette r knowledge ofdifferen t aspectso fth ereproductio nbiolog y anddev 54,18 52,64 elopmento ffis hunde rartificia lconditions . 69,94 73.77 62,33 For instance, theviabilit yo fth eegg san d 17,34 15,57 15,67 26.23 larvae depends ofdifferen twel lknow nfac »=2,17 °'= 2,I 3 ff =0,9 4 »i0,7 4,16 11.30 6,19 tors such asth echaracteristic s offemale s ^=0,16 «•'6.6 (Blaxter,1969 )maturit y ofth eegg s forar tificial spawning and environmental condi tions especially nutrition (Bagnenal,1979 ; 90 Fontaine etOllivereau , 1962).Bu teve nwhe n I- optimal conditions foreg gdevelopmen t seem §70 tob e realized,n o fertilization orembryol - UJ S50j ogicaldevelopmen t occurs.A possibl eexplan >- ation forthi sphenomeno n couldb evariation s • 30 in the biochemical composition of ovules 10 (Konovalov, 1980). Consequently, iti simpor d tant to know the average content of their La. sM- reserves involved inth efertilizatio npro Table 1an dFigur e 1: Global composition cesses of eggs and development ofembryos . infis hovule s oreggs . But at thetime ,mor e information isavail ableabou t freshwater fishspecie s (Ginzburg, TURBOT SOLE E +C 1?- SEA-BASS 1972)tha nabou tmarin efis hspecies . m Q Hereafter our purpose ist opresen tbasi c T SPH0 L data concerning biochemical composition of 10- 5+WA X three marine fishspecie s actually involved inAquacultur e attempts:Turbo t (Scophthal LU r ; Ê8- -J (D A PH PH mus maximus), Sole (Soleavulgaris )an dSe a r' Bass (Dicentrarchus labrax). o Ë E a 1x1 c 0 4. f . The analysis concernovule s atth een do f <>6- _i E+C oogenesis fromwil d fishestrawle d orangle d . along Breton coasts,an d naturally spawns Ë4- v. (atmorul a stage)lai db y captivespawners . Q -- 0 Spawns are eithernaturall y fertilized eggs 3$ ?- ' CH CH orunfertilize d ovules.Captiv e spawnersar e all fed on the same trashfish .Ovule sar e 1 •1 laid undernatura l lightan d temperaturecon _ M <• Ji—Li \XMn ditions ("natural spawning season")an dar Figure 2: Lipids and different classes of tificial conditions (7month s shiftedspawn lipids ovules and eggs. ing season)- (Devauchelle, 1980). Several 40g batche s ofovule so regg swer e rinsed with distilled water andw emeasure d A «,,™ TURBOT n SOLE SEA BASS their content inwate r (drying at95°C ), pro i|2- teins (method ofKjedahl) ,ashe s (byoxidiz UJ ationa t55°C) ,tota l lipids (method ofFolc h 5 W3 and Sloane, 1957),differen t classes oflip 3 W3 1 ids (Beninger, 1982)fatt yacid s (Flanzye t •ä W7 *9 W3 ri. " la !(l al., 1976), cholesterol (method ofLieberman - IMJ Burchard, 1975),phospholipid s andminerals . 0 IIni l£1 1 All the results arepresentage s ofovule s or eggs dryweigh t (Tables 1,2 ,3 ,Figure s Figure 3:Fatt y acids contents infis hovule s 1,2 , 3). oreggs . 155 % DRY WEIGHT PPM DRY HEIGHT CALCIUM PHOSPHORES MAGNESIUM ZINC COPPER I RON MANGANESE (CA) (P) (*) (ZN) ( Cu) (FE) (MN) 0,396 1,17 0.39 201 70.2 16,3 31 I DRY HEIGHT 10"3 DRY HEIGHT PS PI PE 572 501 17 940 1463 2 390 324 437 (O- 60) (0 = 132) (6 = 2390) <«-74) (O = 595) («•-572) (6- £ (0 = 272) 7000 17 700 176 1580 466 (0 = 81) C-2350) (6 = 2720) (0-1(9) (0-672) (0-822) (0 = 132) (6 = 1371 19 E 246 3 670 134 (6 = 229) (0-32 (0 = (0-61) (6 = *84) (O-1020 ) (0-115) (6 = 29) 3 766 i 900 3177 611 0.276 455 20 690 774 474 99 (0=0.002) (0-11) «J-112) (0= 275) (0-51) (6 = 107) (0-752) (6= 51) (6= 27) 0.245 2158 18 390 326 115 132 (O-0.01) (0 = 387) (0 = 113) (6 =1390 ) (0 = 51) (0= 62) (0-795) (6= 21) («= 25) 0.29 847 961 22173 192 736 560 165 (O-0.02) (6 = 277) (6=261) (0-2157) (6 = 80) (6-165) (6- 74) 0.22 396 579 15 000 176 1119 316 168 (0 = 0,05) (0-159 (0- 20) (0 = 319) (6=38) (0 = 437) (0-362) (0 = 129) (6= 27) Table3 :Phosphatide s infis hovule s oreggs . LPC:Lysophosphatidylcholine ;SPH :Sphingomyelin ;PC :Phosphatidylcholine ; PS: Phosphatidyl Serine;PI :Phosphatidy l Inositol;PE :Phosphatidy lEthanolamin e DPG:Diphosphatidy l Glyerol;AP :Phosphatidi caci d Themai nconclusion s arepresente dbelow : Concerning theanalyse s offatt yacid s - Specific differences ofbiochemica lcompos (Fig. 3), wenotic e that saturated fattyaci d ition of eggsan d ovulesappear ,i.e .flat rate is generally loweri novule s fromwil d fishmateria l contains lesstota llipid san d fishes. On theothe rhand ,pol yunsaturate d fatty acids,bu tmor eprotein s andmineral s fattyaci d seemst ob emor e frequent inthes e ascompare d tose abass . ovules. -Th e ovules fromwil d fishcompare d toth e Mineral contents,an d especially phosphorus spawnso fcaptiv e fishcontai nmor eprotein s content (Table2 )diffe r among differentov and lipids (especially cholesterol andphos ules oreg gbatches . pholipids. On theothe rhand ,thei rminera l contenti slower . Phosphatides are given inTabl e 3suc ha s basicdata . - Shifted spawning seasoninduce stota llip idsan dminera l contentdecrease . Allthes e results canb euse d inth efutur e as indicators of the quality of the eggs, -Las to fall ,generall ya highe r totallip and consequently, indicators ofth equalit y idconten t corresponds tover y lowviabilit y of larvae,a s suggested by Nassour (1980) rateo feggs . 156 working onSalm ogairdneri .O nth eothe rhand , Nassour, I. (1980). Influenced el acarenc e iti swel lknow ntha tth e compositiono fdiet s en acides grasessentiel s surle slipide s caninfluenc eth esurviva lan d the fecundity de l'oeuf et du tissu adipeux chez la of spawners (Hilge,1979 ;Wooton , 1973).So , truiteArc-en-cie l (Salmo gairdneri)•Mém when there is very little information for oired eDEZ. :2 1pp . dietary requirements of spawners,th e data Wooton,R.J . (1973). The effect ofsiz eo f about general composition of eggs couldb e food rationo neg gproductio n inth efemal e used asa guidelin e forformulatio no fspaw three spined sticklebackGasterosteu s acul- ners feedstuffs aspropose d byKetol a (1980) eatusL . foramin oaci dpattern s ofdiets . References Bagenal, T.B. (1969). The relationship be tween food supply and fecundity inbrow n trout Salmo trutta L. J. Fish.Biol. ,1, : 167-182. Beninger,P.G . (1982).Etud ebiochimiqu e com paréd edeu zpopulation s debivalve sRudi - tapes decussatus etRuditape sphilippinarum . Thèse d'Etat:Universit é deBrest ,19 3pp . Blaxter,J.H.S . (1970).Developmen tegg san d larvae. In:Fis h physiology, Blaxter Ed. AcademicPress .Inc. ,Ne wYork ,3 :178-271 . Devauchelle, N. (1980)Etud e expérimentale surl a reproduction,le soeuf s etle slar ves de bar Dicentrarchus labrax, daurade Sparusaurata ,Mullet ,Liz a ramada,Rouget , Mullus surmuletus, Sole, Soleavulgaris , Turbot, Scophthalmusmaximus .Thès e de3 è cycleUBO : 194pp . Flanzy,J. ,M .Bourbon ,C .Lége r andJ .Pihe t (1976).Applicatio n ofcarbawa x2 0M a sa n open tubular liquid phase in analysiso f nutritionally important fatsan doils . J. chromatogr.Sei . 14:17-24 . Folch,J.L . andS.G.H .Sloan e (1957).A sam plemetho d forth e isolationan dpurifica tion of total lipids fromanima ltissues . J. Biol. Chem.,226 :497-509 . Fontaine,M . etM .Oliverea u (1962).Nutri tione tsexualit é chezle spoissons .Annal es de la nutrition et de l'alimentation T 16:A 125-A152 . Ginzburg, A.S. (1972) In:Fertilizatio n in fishes and theproble m ofpolyspermy .Ed . by B. Golek,Kete r Press Jerusalem: 366 pp. Hilge, V. (1979): Möglicher Einfluss ver schiedener,Kombinierte rprotei nun dFett gehaltede s futtersau fda sGonaden-Wachs - tumjunge r Spiefeikarpfen (Cyprinus carpio L^ Arch.Fischwiss ,29:(3 ): 113-118 . Ketola,H.G . (1980).Amin oaci d requirements of fishes:A review. Proc. CornellNutr . Conf.Feed .Manuf .Nov . 1980:43-48 . Konalov,Y.D . (1980).Viabilit y ofcarp sem bryos and larvaea sa fractio n ofth econ tento ftota lprotei nan d sulfhydrylgroup s inth egastrulatio n stage.Biolog y ofFish es: ISSN0018-8166/79:65-68 . 157 STRUCTURAL STUDIES OFBON Y FISH VITELLOGENIN A. Hara,H .Hira i Department of Biochemistry, Hokkaido University School ofMedicine , Sapporo,Hokkaido ,Japa n Summary Vitellogenin(VTG), originally named forth e nents,which yet consisted oftw osubunit so f female-specific insect protein hasbee n well 90,000 and 15,000 daltons associated bydi characterized inavia n andamphibia n species sulfide bonds ina mola r ratioo f 1t o1 . as a serum protein specific forfemale ,in - E2wa sdisulfide-1inke d dimer of 15,000 duible inanima l by treatment with estrogen dalton subunit. Using antisera toVTG ,E l and an immediate precursor ofeg gyol k pro and E2,i twa sdisclose d that thetrou tVT G tein. It isals o known that theVT G isa contained both El andE2 . This, together 1ipoglycophosphoprotein innatur e and isa with theabov e m.w.analyses , indicated that complex with lipovitellin and phosvitin the trout VTGmonome r iscompose d ofon eE l (Bergin ksWa llace,1974,Christman ne tal .,1977) . and oneE2 . Thiswa sfurthe r confirmed by Here,w epresen t identification, isolation comparison ofth eamin o acid compositions and structural characterization ofVT Go ftw o among thethre e proteins (Table1) . bony fish, rainbow trout andJapanes eeel . . EelVT Gan deg gyol k protein were also Schematic structural models ofVTG s ofth e isolated from Japanese eel (Angui1l a above twofis h arepropose d incompariso n japonica)• Structural analysis indicated with thoseo fAmphibi a and Ayes. that both theVT Gan dth eeg gyol k proteino f eel have thesam e molecular form withth e We purified a female-specific serum protein m.w. of 350,000,consistin g of four identical (FS)an dtw oeg gyol k proteins (El andE2 ) polypeptideso f 85,000 daltons. Schematic from serum andeg gyol k ofmatur e rainbow structural models ofVT Gfro m trout andeel , trout (Salmo gairdneri) byprecipitatio n with together with thoseo famphibia n and avian low ionic strength buffers andge l filtration. VTG arranged from thestructura l datao f The FSha d iron-binding activity andwa sa Bergink andChrisj:man n' sgroup s areshow n in 1ipoglycophosphoprotein. Itwa s inducedb y Fig. 1. Structural similarity isapparen t administration ofestradio]-I7 ß inseru mo f among VTGs presented, except forth eee lVTG . both sexes. From these characteristics,th e This indicate that thephylogeneti c diver FSwa sdefine d tob ea homologue ofVT Go f gence ofVT G that occurred between Amphibi a rainbow trout. Thetrou t VTGha da m.w .o f and Aves. around 600,000, consisting oftw o identical polypeptides of 220-240,000 daltons associat ed by non-covalent forces ando f some phos _E1_ pholipids andcarbohydrates . 9: Theeg gyol k proteins, El andE2 ,o f trout as were precipitable together ina low ionic strength buffer asa E1-E 2complex . Them.w . of El andE 2wer e approximately 300,000an d Rainbow trout 35,000, respectively. Elwa s non-covalently Lv _LY__ bound dimer of two identical 130,000 compo- Table 1.Amin o acid composition ofEl ,E 2an d Pv VTG of rainbow trout. Amino acid El E2 VTG/2 E1+E2 Amphibian (mol.wt.) (210,000) (30,000) (240,000) Avian Fig. 1.Schemati c models ofvariou s VTGmole Asp 145 42 187 185 Thr 101 11 112 109 cules. Numbers inth eschem e representth e Ser 91 29 120 165 m.w.fxlO1*). Lv:lipovite l1 in , Prphosvitin Glu 220 27 247 253 Pro 102 9 111 115 Gly 77 15 92 93 References Ala 270 9 279 257 Cys/2 15 7 22 26 Val 146 21 167 156 Bergink E.W.an dR.W .Wallace , 1974. Precur Met 49 8 57 56 sor-product relationship between amphibian lie 112 14 126 120 vitellogenin andth eyol k proteins, lipo Leu 197 19 216 208 Tyr 55 11 66 66 vitellin and phosvitin. J. Biol. Chem. Phe 88 5 93 89 249, 2897-2903. His 42 6 48 47 Christmann J.L.,M.J .Grayso n and R.C.C. Lys 119 29 148 156 Huang, 1977- Comparative study ofhe nyol k Arg 84 9 93 100 Total 1913 271 2184 2201 phosvitin andplasm a vitellogenin. Biochemistry 16,3250-3256 . 158 OESTRADIOL-INDUCED FEMALE SPECIFIC PROTEINS IN THE PLASMA AND IN THE OOCYTES OF GASTEROSTEUS ACULEATUS F. TRACHURUS. AN IMMUNOLOGICAL APPROACH. F. OLLEVIER & M. COVENS Zoological Institute of the University, B-3000 Leuven, Belgium Vitellogenesis was induced in non-sexually An injection with 1 .32 ;ug or 13.2 pg medroxy- mature dd and oo three spined sticklebacks by progesterone-acetate/g fresh body weight did injecting 6 times 0.66 ;ug oestradiol-3-ben- not induce vitellogenesis 12 days after the in zoate/g fresh body weight. After Polyacryl jection. This confirmed the results of Horî et amide gradient electrophoresis four new bands al. (1979) with goldfish. Antibodies prepared appeared in the plasma of the treated fishes : against Vgl did not reactwith plasma of non- two lipoprotein bands (fig. 1) and two bands oestradiol treated oo, but after electrophore with very high lipid concentration. These tic separation positive results were obtained at bands also appeared if only 1/10 of the above the level of Vg 1 with plasma from fishes of mentioned hormone concentration was injec both sexes showing oestradiol-induced vitello ted, but the response was very weak if only genesis. Homogenates of ripe eggs showed, 1/100 was injected. After bidimensional after agarose electrophoresis, two immunolo electrophoresis the strongest lipoprotein band gical cross reactions with tjie Ab : one with a (Vg 1)wa s split up into three different proteins lipovitellin component and one with an anodic (640,000 D, 550,000 D and 520,000 D). migrating component (pH 8.8) which we be lieve represents phosvitin. Tromp-Blom (1959) distinguished 6 stages in the development of the oocyte of the stick leback. In the non-oestradiol treated oo of this experiment, stage 1 to 4 were present. Stage 5 was only present in the oestradiol treated oo but stage 6 was lacking in both groups. The stage 4 oocytes of oestradiol treated oo U-Vg 1 reacted positively with Ab against Vgl at the level of the thecacells and the oolemma. This was not the case for the control fishes. Positi vely reacting 'vesicles' were never found in the cytoplasm of stage 4. Peripherally located 'vesicles' in the cytoplasm of stage 5 oocytes reacted positively with Ab against Vgl (fig. 2). This indicated that during exogenous vitello .a1, -v genesis only from stage 5 on proteins related with Vg 1 were accumulated in the oocytes. The proteins accumulated in the oocytes during the endogenous vitellogenesis were therefore immunologically different from Vg 1 ; this being the most concentrated vitellogenic lipoprotein présent in the plasma during exogenous vitel logenesis. Although chemically changed, as shown by electrophoresis, the components de rived from this lipoprotein and present in the oocytes were still immunologically reactive fig. 1 Electrophoretic pattern of plasma with Ab against Vg 1. proteins from oo Gasterosteus aculeatus. A. non vitellogenic control B. oestradiol induced vitellogenic o 159 fig. 2 Oocyte (stage 5) of oestradiol injected Gasterosteus acule atu s treated with Ab against vitellogenin 1 and stained with PAP. References Hori,S.H., T. Kodoma & K. Tanahaski, 1979. Induction of vitellogenin synthesis in gold fish by massive doses of androgens. Gen. Comp. Endocrin. 37 : 306-320. Tromp-Blom, N., 1959. The ovaries of Gas terosteus aculeatus before, during and after the reproductive period. Proc. K. Ned. Acad. Wet. 62 : 225-237. 160 A NEW PROCEDURE FOR THE ISOLATION OF INTACT VITELLOGENIN FROM TELEOSTS R. A. Wallace, K. Selman Whitney Marine Laboratory, Rt. 1, Box 121, St. Augustine, FL 32084, U.S.A., and Department of Anatomy, College of Medicine, University of Florida, Gainesville, FL 32610, U.S.A. Vitellogenin (VTG), the hepatically synthesized Comp. Biochem. Physiol. 24: 437-443. yolk protein precursor, is particularly difficult to Wallace, R. A. 1978. Oocyte growth in obtain in a pure and unaltered form from many nonmammalian vertebrates. In: The Vertebrate teleosts. Teleost VTGs are generally less Ovary, Ed. R. E. Jones. Chr. 13: 469-502. phosphorylated than those of other vertebrates (de Plenum, New York. Vlaming et aL, 1980), so that existing methods for Wiley, H. S., L. Opresko, & R. A. Wallace 1979. VTG isolation which rely on its high surface New methods for the purification of vertebrate charge, such as EDTA-Mg2+ precipitation or vitellogenin. Anal. Biochem. 97: 145-152. chromatography on DEAE-cellulose in Tris-Cl buffer with a NaCl gradient (Wiley et aL, 1979), either fail to precipitate VTG or do not resolve it adequately from other blood proteins. Furthermore, teleost VTGs are highly susceptible to proteolysis during isolation, generally yielding Phosvitin (PV)-like and lipovitellin (LV)-like cleavage products. The former peptide, which contains almost ' all the protein-phosphorous orginally associated with VTG, absorbs light at 280 nm either poorly or not at all (Jared & Wallace, 1968) and cannot be stained with protein-binding dyes such as Coomassie blue (unpublished); hence it is not detectable by the usual monitoring procedures. Analysis of the LV-like peptide without an appreciation of its proteolytic origin would lead one to conclude that plasma VTG is similar to yolk LV and hence is not proteolytically processed within the growing oocyte as has been found for other vertebrates (Wallace, 1978). The problem is illustrated in figs, la and lb, 0.2 0.4 which represent chromatographic profiles of Elution Position 1—Gradient started plasma from male and female Fundulus ~Sampl e ploced on column heteroclitus, an animal for which previously published methods have proven to be particularly fig. 1. Chromatography on DEAE-cellulose of inadequate. VTG, identified as the UV-absorbing approx. 0.5 ml plasma from (a) males, (b) females, [32P]protein present in females but absent in and (c) aprotinin-injected females. All animals males (fig. lb, elution position 0.72), elutes in were injected with 75 yCi[32p]pi 20 hr previously. association with the penultimate protein fraction After the plasma sample was placed on the column and is mostly degraded to yield a [ 32p]py-iike (30 ml bed volume) of microgranular DEAE- peptide (fig. lb, position 0.82), even though cellulose (Sigma), it was rinsed with starting buffer phenylmethylsulfonyl fluoride was added to the until most of the nonadherent protein percolated blood during collection. The problem was solved through. A 400-ml, 3-bottle gradient, which by injecting fish with the nontoxic proteolytic consisted of 134 ml starting buffer in each of the inhibitor, aprotinin (0.2 ml/10 gm fish; 17 TIU/ml; first 2 bottles and 132 ml limit buffer in the third Sigma), 15 min prior to bleeding and by using a bottle, was then pumped onto the column (flow phosphate rather than NaCl gradient to develop rate = 2.4 ml/min). The effluent was monitored the Chromatograph (fig. lc). VTG is well resolved for absorbance at 280 nm and 100 yl samples of from other plasma proteins and generally less than effluent fractions were placed on filter paper 10% is cleaved to yield the PV-like peptide. disks, processed to remove small molecular weight substances and lipid, and counted for protein- References associated radioactivity. Starting buffer: a and b = 50 mM Tris-Cl, pH 7.5; c = 2.5 mM KH2P04 de Vlaming, V. L., H. S. Wiley, G. Delahunty, & - 10 mM K2HP04. Limit buffer: a and b = 50 R. A. Wallace 1980. Goldfish (Carassius auratus) mM Tris-Cl, pH 7.5-0.35 M NaCl; c = 100 mM vitellogenin: Induction, isolation, properties, and KH2P04 - 400 mM K2HP04. The column could relationship to yolk proteins. Comp. Biochem. be regenerated for another run by washing Physiol. 67B: 613-623. sequentially with 0.2 M Na3 citrate-0.3% Brij-35, Jared, D. W., & R. A. Wallace 1968. Comparative water, limit buffer, and starting buffer (100 ml chromatography of the yolk proteins of teleosts. each). 161 MATURATION OF GONADS 163 PHYSIOLOGICAL BASISO FPRACTICA LMEAN S TO INDUCEOVULATIO N INFIS H A.Fostier ,B .Jalaber t Laboratoired ePhysiologi ede sPoissons ,I.N.R.A. ,Campu sd eBeaulieu ,3504 2Rennes ,Franc e Summary tionsma yb e somewhat differentbetwee nfis h inwhic ha massiv e synchronous ovulationoc Inorde r tobette rappreciat e thebes twa y cursafte r along-lastin g vitellogenesis inwhic htechnique s to induce spawning could (trout,carp ,catfish),an d fishwhic hpresen t beimproved ,a concis ean d criticalrevie wo f awide-sprea d breading seasoncharacterize d theirphysiologica l basisha sbee nmade .Th e by successivewave so foocyte sa tdifferen t endocrineregulatio no fmaturatio nan dovula stageso fdevelopment ,thu sovulatin gmor eo r tion,th eproces so facquisitio n ofovaria nre lessregularl y by "clutches"(medaka ,zebra - ceptivityan d competencean d themechanis mo f fish,killifish ,gilt-hea d sea-bream).Concer actionso fpresen t treatmentshav ebee nsucces ning oocytematuratio n and ovulation,thes e sively considered. mustb econsidere d astw odistinc t processes Keywords :rainbo w trout,hypophysation ,matu whichar enormall y linked but canb eartifi ration,ovulation ,review . cially dissociated (Jalabert,1 976 ;Iwamats u '1978a;Goet z& Bergman , 1978). Oocytematura I.Introductio n tioni smor eofte n morphologically characte rized bya clarificatio no fth eyol k (duet o Inorde rt oobtai n eggsfro m somefis h spe theseparat efusio no f proteic and lipidic cies, itwoul d beusefu l tocontro ldifferen t yolk),an d finallyb y thegermina lvesicl e phaseso foogenesis ,mainl yvitellogenesi sand / breakdown (GVBD).Ovulatio n sensusstrict o oroocyt ematuration-ovulation . Oneo rth e isth emechanica l expulsiono f theoocyt eou t othero rbot ho f thesephase sma yb edisturbe d of thefollicula r envelopswhic hca noccu ron byth eartificia l environment of fishfarmin g lyafte ra phas eo fdetachmen t between theo - whichma y imposestressfu l conditionso nth e ocytean d thefollicula r cells (Jalabert& fish (Billard etal. , 1981a),o r inwhic hin SzSllosi,.1975)."Finally,th e interpretation dispensable specific stimuliar e sometimeslac of invitr o experimentsmus t carefully take king.Eve nwhe n thewhol egametogenesi s canoc intoaccoun tho wan dwha t kind of ovariancom cur spontaneously itmigh tb emos tusefu l to ponentswer e incubated :piece so fovary ,iso beabl et ocontro l theovulatio n time inorde r lated follicles,oocyte ssurrounde d onlyb y tosynchroniz egroup so f spawnersan d produce granulosa cells,and/o rnake d oocytes.Th e equal-aged fry,whic h should help fish-farm distinction isneve r so easy :isolate dfolli management. clesma yretai n some interstitial tissuewhic h canb eo f importance (Iwamatsu& Katoh , 1978); Thiscontro l ispossibl e intw oway s :ei andnake d oocytesar eusuall y obtainedb yme ther throughth emanipulatio n of environmental chanicaldissociation ,whic hma y leavesom e factors,o r throughth eadministratio n of exo granulosa cells,o rb y enzymatic dissociation, genous substancesdirectl y actingupo nphysio whichma ydommag e theoocytes . logicalprocesse s tofavour ,o r even inducego nadaldevelopment .Today ,inducin g completevi tellogenesisappear s tob erealisti c infar m practiceonl y through environmentalfactors , II. Endocrineregulatio n ofmaturatio nan d when theyca nb emanipulated . Onth econtrary , ovulation treatmentswit h exogenous substances (especial lyhormone so rhormona l substitutes),eventual Thoughhypophysectomy ,performe d incompa ly inassociatio nwit h environmentalcontrol , risonwit hovulatin g intact fish,ha sclearl y havebee nwidel yuse d to induce thefina l demonstrated thatmaturatio n and ovulationar e stepso f oogenesis(i.e.oocyt ematuratio nan d under thecontro lo fth epituitar y gland (Vi ovulation).Thes etechniques ,generall yname d vien, 1941;Yamazaki ,1962 ;Iwamatsu , 1978b), "hypophysation",hav ebee n extensivelyrevie therole so fal lth edifferen t hypophysial wed (Pickford &Atz ,1959 ;Harve y &Hoar ,198 0 hormonesar efa rfro mbein gwel lunderstood . among others),thu sthi spape rwil lb elimi However,i t isknow nfro mdifferen t kindso f ted toa concis eanalysi so f thepresen tknow experiments thatth eglycoprotei ngonadotro ledgeabou t theirphysiologica l basis inorde r pin (adsorbed onconcanavali nA-sepharose )i s tobette r appreciate thebes twa y inwhic h apredominan t and essentialregulatin gfactor . theycoul db eimproved . Thus,a nantiseru m tothi shormon eadministe red inviv oblock s thematuratio n processi n Itma yb eusefu l torecal lparticularietie s salmono r inflounder ,whil ea nantiseru mt o whichcoul d limit somecomparison s betweenda a "carbohydrate-poor"protei n claimed asvi - taobtaine dwit hdifferen t kindso fmaterial s tellogenic gonadotropin,i swithou t effect andmethods ,an d tospecif y somedefinitions . (Nge tal. , 1980). Furthermore thehomologou s Sexual endocrinology and intraovarianregula 164 glycoprotein induces,i nvitro ,intrafollicu - Suchrefractorines smigh t bedu et oth econ laroocyte-maturatio n inrainbo w trout,com trolo fGT Hreleas eb ya gonadotropin-release - moncarp ,goldfis h (Jalabert,1976 )o rstur inhibiting factor (GRIF), probably originating geon (Burzawa-gerard etal. , 1976)anieve ni n inth eanterio r preoptic region,,a spropose d vivo ovulation inth ehypophysectomize dfloun ingoldfis h (Peter &Paulencu , 1980). Itma y der (Idler &Ng , 1979).Morphologica lobserva bea dopamin etyp efacto r (Peter etal. , 1982) tionso fth epituitar y parsdistali s (Ecken- though,i nothe r respects,prostaglandin sPG E grene tal. ,1978 )an dmeasurement s inplasm a andPGF„ a inhibit GTH secretion ingoldfis h bymean s ofradioimmunoassay s confirm thephy when injected into thehypothalamu s (Peter& siological implication of thisgonadotropi n Billard, 1976). (GÏH)i nth eregulatio no f theen do fth ese Therol eo fhypophysea lhormone s othertha n xualcycle .Th eplasm aGT H levelsreac hthei r GTH ismor equestionabl e (seeth erevie wb y highestvalue s during theperiovulator y period Fontaine, 1976).Probabl yACT H canac t though insalmonid s (Billard etal- , 1978;Breto ne t itscontro l of interrenalgland ,whic hprodu al., 1982;Cri m etal. ,1975 )an d cyprinids cescorticosteroid s hypothesized eithera sma (Billard etal. ,1978 ;Breto n etal. ,1980) . turation inducing steroids (MIS)i nth eIndia n However,whe n serial samplings havebee nper catfish (Sundararaj &Goswami , 1977), ora t formed around thetim eo fovulation ,th esal leasta spoten tmodulator so fGT H actionupo n monids and cyprinidspattern s appear tob edif thefollicl e (seepar tIII) .Margina leffects , ferent. Introut ,th ebasa lleve lrise sslowl y invitro ,o fmammalia n thyrotropin (TSH)o r over severalday st oreac h 10t o2 0ng/m la t prolactinhav ebee n sometimes reported (Iwa- ovulation (Bretone t al., 1982;Fostie r etal . matsu, 1978b;Hirose ,1980) ,bu tth eus eo f 1978, 1981a), (fig.)the na hig hleve li smain nonpiscin ehormones ,th epossibl econtami tained (between5 0an d 100ng/ml ,sometime s nationo fTS Hb yL H or thehomolog y between higher)durin g aperio d of severaldays ,de GTH andTS H (Fontaine,1976 )coul d explain pending onth eovule sretentio n inth eperito suchactions .Howeve r TSHmigh tb e efficient nealcavit y (Jalabert & Breton, 1980).Mor e via thethyroi dhormone s (seepar t III), thoug h accurately,a recen t study (Zobare tal. ,1982a ) dataavailabl eo nth eleve lo fplasm athyroi d ofth eshort-ter m profiles ofplasm aGT Hi n hormonesdurin g thespawnin g seasonar econ rainbow troutha sshow na circardia nrhyth m tradictory (Osborn etal. , 1978;Sowe r& inthos elevel scharacterize d by largean d Schreck, 1982;Pickerin g &Christie , 1981). slowfluctuations .Th ehighes t GŒHconcentra tion,whic hma yreac habou t twiceth ebasa lle Pinealha sbee n suggested asplayin g asti vel, occursdurin g thenigh t and earlymornin g mulatory roleupo ngonada lactivit y during Ina cyprinid ,th egoldfish , thepreovulator y spring ingoldfis h (DeVlamin g &Vodicknick , basalleve lo fplasm a GTHals o fluctuates (Hon- 1978)b ypromotin g adail y cycle inplasm a tela& Peter ,1978 )bu ta tru eovulatin g surge GTH (Hontela& Peter , 1980).However ,a stud y wasdetected ,reachin g atleas t tentime sthi s involved strictlywit h theoocyte smaturatio n basalleve lan d lasting lesstha n 12hour s process inth emedak aha s shownneithe r the (Breton etal. , 1972;Stace y etal. , 1979). inviv o effectso fpinealectomy ,no r thei n Thus, if insalmonid s thehypophysea l signal vitro direct actiono fmelatoni n (Iwamatsu, seemst ob ea relativel y slowprocess ,whic h 1978c). could beaccompanie d bya progressiv e change Analyticalstudie so f theovaria nsteroido inhormona lbalance ,i ncyprinid sthi ssigna l genesisan ddescription s ofhormona lprofile s seemst ob emor elik ea switch . inplasm a during thespawnin g period havecon firmed thepreviou s invitr oobservation s a- Theregulatio no fGT Hsecretio nb y theneuro bout theeminen t roleo f somesteroid s (MIS) endocrinological systemha srecentl ybee nre inth emediatio n ofGT Hactio nupo nfina lma viewed (Peter &Crim , 1979). Thegonadotropi n turationan d ovulation.I nsalmonids ,th epre releasing hormone (Gn-RH)activit y infis hhy ovulatory follicle isabl et oproduc eproges pothalamus (Breton etal. , 1975)ha sbee ncon tins (Fostier etal. ,1981b ;Suzuk i etal. , firmed (seeth erevie wb yBall , 1981). Though 1981a,b;Theofan , 1981),bu tals oandrogens , LH-RHan d itsanalogue shav ebee nuse dwit h estrogens and corticosteroids (Suzukie t al., somesucces st oinduc eovulatio n insevera l 1981a;Theofan ,1981 ;Zoha r etal. , 1982b). species (seereference s inDonaldso n etal. , In theamag o salmon,i tha sbee npropose d that 1981a), highdose sar eusuall ynecessary .Thi s bothgranulos aan d thecal layerswer einvol mayb edu et ostructura ldifference sbetwee n ved inth esynthesi so f theMI S (i.e.17a-hy - LH-RH and thefis hGn-R H (King &Millar ,1980 ; droxy-208-dihydroprogesterone= 17a,20ß-OH-P ) Idler &Crim , 1982;Barnet t etal. , 1982). (Nagahamae tal, , 1982).Als o identified in Furthermore,th ehypophysea l sensitivityt o plasma (Idler etal. , 1960;Campbel l etal. , LH-RH appears tochang econsiderabl y throu 1980), thissteroi d showsa shar pris ea tth e ghout thesexua lcycle .Thu s incar p (Weile t precisetim eo foocyt ematuratio n inrainbo w al., 1975)an d trout (Weile tal. , 1978)fe trout (Fostier etal. ,1981a ;Scot t etal. , malesth ehighes t sensitivity inviv o isfoun d 1982; Wright &Hunt ,1982 ) (Fig.). Thisin during thespawnin g period,an d even,i nth e crease ischaracterize d by large fluctuations last species,th erespons et oLH-R H increases (Zohar etal. ,1982b) . Steroidswhic hac tsy - during thever y lastpreovulator y stages,whi nergicallywit hGT Hupo noocyt ematuratio ni n leth epituitar y GTH content remainsunchanged . vitrohav eals obeen ,measure d (seepar tIII) - 165 responsest odifferen t exogenousfactors .Tha t ist osa ytha t informationo nth eaccurat e hormonal statusbefor ean d atth etim eo f sampling canhel p tointerpre t invitr odata . Inothe rwords ,i tma yb ea mean so fappre ciating follicular receptivity andcompetence . Ill.Acquisitio n ofovaria nreceptivit yan d competence Beforereceivin g thefina l signal(s)whic h trigger(s)maturatio nan d ovulation,th eso matic cellso f theovar yan d theoocyte sthem selvesar e involved ina preparatio nproces s whichma y bemor eo r lesslength y depending onth especies .Lookin g atth ematurationa l responseo foocyte s invitro ,tw omai nlevel s of sensitivity canb edefined . 1.Th e"oocy -t8 -16 -14 -12 -10 -8 -6 -4 -2 Ô .2 »4 days [11 ID (3) |5) f5) 19) [121 116) (20) (20) (19) (19) tesensitivity "o fnake d oocytest o exogenous MIS. 2.Th e"follicula r sensitivity"evalua tedfro m therespons eo foocyte s incubated within intact follicles (possiblywit hsom e Figure.Periovulator y changeso fplasm ahor ovarian interstitial tissue)t oexogenou s moneslevel s inrainbo wtrou t (mean s± S E) substances.Whe n thesear e steroids,th e"fol Plasma gonadotropin (o o o),estradiol-17 ß licular sensitivity"ma yreflec t theabilit y (AAA), 17a-hydroxy-20ß-dihydroprogesterone of thefollicl e (oocyte included)t ometabo (• • •)an d testosterone (A • A)hav ebee n lize them.Wher egonadotropin s areconcerned , measured everytw oday s inindividua lfish . the "follicular sensitivity" integratesth e Ovulationwa sdetecte d atda y0 .Th enumber s sensitivity of somatic celllayer st oth ego of sampled fish (inbrackets )wer e thesam e nadotropin (through thestimulatio no fMI S foral lth ehormone s except testosterone(4) . secretion)an d theprimar y sensitivity ofth e oocytet oMIS . Inrainbo w trout,estradiol-17 3 (E2-176)levels ,whic har ehig hdurin g exogenousvitellogene - sis,decreas edrasticall y beforematuratio n Table.Media n efficientdos eo fhormone sne (Fostier etal.,1978 ;Scot t etal,1980 )whil e cessary toinduc eoocyt ematuratio n invitr o testosterone levels,thoug hdecreasin g still inth efollicle s ofdifferen t femalessacri - appear high (Scott etal.,1980 )o reve nsho w fied at twodifferen t stages (seetext) .Re a littlepea kbefor eovulatio n (Fig.).Usin g lationwit h theestradio l level inplasma .In a cautiousprotoco lo f sampling,Pickerin g& cubations carried out invitr o at 12°5,p H Christie (1981)hav econfirme d an increasei n 7.9-8 for7 2h .a spreviousl y described (Ja- Cortisolconcentratio n during thisperiod .I n labert, 1978). goldfish,Cook,Stace y &Pete r (1980,i nGen . Comp.Endocrinol .40 :507-510 )hav edescribe d Oocyte Fish Media n Efficient Estradiol a preovulatory surgeo fCortiso lanalogou s stage dose (ng/ml)fo r in plasma toth eGt Hsurge . of donnor s-GTH 17a20ßP Toconclud e ifw econside r allth edifferen t fish hormonal eventsrecorde d inviv o aroundth e Endo f 1 393 101 30.0 timeo fmaturatio nan d ovulationw emust,poin t vltel- 2 183 419 29.9 out thedifficult y indiscriminatin g essen loge- 3 91 87 17.2 tialdominatin g factorsan dmodulatin g ones. nesis 4 128 62 12.8 What isth eprimar y signalwhic h switcheso n (E.V) 5 60 190 19.3 irreversibly thewhol ehormona lproces s ?I n trout thedecreas eo fplasm a E--176,relate d G.V. 6 18 21 4.3 toa fal l inaromatas eactivity ,ma yb econ peri 7 39 5.4 22.9 trolled byGT H (Sire &Depêche , 1981;Zoha r pheral 8 40 20 2.8 etal. ,1982b) ,an d could releasea negativ e (GVP) 9 29 4.7 - feedbackupo n theGT H secretion (Bommelaer 10 15 - 6.6 etal. ,1981 )necessar y tostimulat e theMI S synthesis (Fostier etal. ,1981b) .Furthermo re, thebalanc eestablishe d between theste The "follicular sensitivity"t osteroid s roidsothe r thanMI S (i.e.testosterone ,E2 - hasbee n shownt odepen d onth efollicula r 173,Cortisol )coul d exert apositiv esynergi c size (Iwamatsu,1978a ;Wallac e &Selman ,1978 ; action (seepar tIII) .Thus , if suchhormone s Hirose, 1980), o nth e season (Iwamatsu &Ka - act synergically by successive steps,th ehor toh, 1978),o ro nth ehou r ofsamplin g inth e monalsigna lwhic hha sbee nreceive d inviv o daily spawning cycleo f theMedak a (Hirose, by theovar ybefor eth ebeginnin g ofa n invi 1972). I tma y alsob erelate d toth estag eo f tro experiment,ma yb edecisiv eo nth efurthe r germinalvesicl e (GV)migratio nfro m thecen - 166 tret oth eperipher y of theoocyt ei nsom e nalstage so f GVmigratio n (Fostier etal. , species.Fo r example,withi n thesam estrai n 1981b)an d appeared allth emor e importanta s ofrainbo w trout,w e compared themedia nef GVwa smor eperipheral .Thi sabilit y iscer ficientdos e (MED)o fth eMI S 17a-20g-OH-P tainlymor eprecociou ssinc eintrafollicula r for intrafollicularoocyt ematuratio n invi oocytesa tth eE V stagear ealread yabl et o tro,usin g folliclesfro m differentindivi reachGVB D invitr o althoughth efollicle s duals inth esam econdition s ofincubation , thenrequir ea highe r doseo fGT H (Table). I n eitherwhe nth eoocyte s showed thegermina l Atlantic salmon,Saku n (1966)wa sabl et oin vesicle (GV)wel la tth eperipher y (GVP,a ducea maturationa l response,althoug hcharac stage estimated roughly at lesstha ton ewee k terizedb y theformatio no fabnorma lmetapha - from thetim eo fexpecte d naturalovulatio n ses,b y injectiono fpituitar ypreparations , at 10°C), orwhe n theG Vcoul dno tye tb esee n asearl ya s3 1/2month sprio rt o expected without fixation (stageE V= "endo fvitello - naturalovulation ,bu tmor eprecociou spitui genesis")estimate d herea taroun d 4t o6 tary treatmentsonl ypromote d anacceleratio n weeksprio r toexpecte d ovulationtime ,an d ofvitellogenesis .I nmos t speciesa transi corresponding toa hig hleve lo f estradioli n toryperio d probably occursdurin gwhic hth e theplasm ao f the sameanimal s (Table),whic h ovarycoul d possessa bipotentialit yregar might beinterprete d asa nindicatio ntha tvi - ding steroidogenesis,oriente d either toth e tellogenesis isstil lgoin g on.Th eoocyte sa t controlo fvitellogeni n synthesis (estrogens) theE V stagealread y appeared torespon dt o ort oth econtro lo foocyt ematuratio nan do - 17a-20ß-OH-Pbu t theME Dwa sthe nmuc hhighe r vulation (MIS).Recen tdat a inou r laboratory thana tth eGV P stage,thu s indicating lower tend todemonstrat e that themodificatio n in sensitivity (Table).Moreover ,injectin grain ovarian steroidogenesis could beamplifie d by bow troutwit h 17a-20f3-OH-Pa tabou t thesam e theshap ean d intensity ofth ematurationa l precocious stage induced oocytematuratio n gonadotropin (GTH)signa l (Zohar etal. ,198 2 inviv owithou t ovulation,an d althoughovula a,b) .However ,th eovaria ncompetenc ean d tioncoul d thenb einduce d invitro ,eithe r sensitivity iscertainl y theresul to fcom manually orb yactio no fprostaglandin gF^a , plex interactionbetwee nnumerou s factors.I n developmental capacity ofth eegg swa slo w rainbow troutE--17 6lower' swhil etestostero (Jalabert etal. , 1978a). Inth emedaka ,Ory - nean d Cortisolenhanc eth eefficienc yo fGT H ziaslatipes ,whic hexhibit sa precis edail y upon intrafollicular oocytematuratio n invi rythm ofspawning ,th epercentag eo foocyte s tro (Jalabert,1975 )whic hma yb epartl yre incubatedwithi n intactgranulos a cellstha t lated respectively toa ninhibitio no ra sti reachGVB Dafte r invitr o stimulated bypro mulationo fth eproductio no fMI Sb y thefol gesterone,increase su p tonearl y 100whe nth e licle (Jalabert &Fostier ,unpublishe d data). timea twhic h theyar etake ndraw snea r to1 5 Inth ecatfish ,th eactio no fgonadotropin s hoursbefor e expected naturalGVBD ;however , orCortiso lupo n intrafollicularoocyt ematu inthes econdition smaturatio n israrel yfol ration isreduce db yE„-17 6an d testosterone lowedb yovulation ,an d oocytescleav eabor (Sundararaj etal. , 1979).However ,othe ro - tivelywhe nactivate db y insemination (Iwa- varian functions thansteroidogenesis ,parti matsu, 1974).Altogethe r thismean stha teve n cularly related toth econtro lo fovulation , though theoocyte sar ealread y ablet oexhi areprobabl y regulatedb y GTH.Thi scoul d ex bitver y earlya maturationa lrespons e (GVBD) plainwh y hypophysectomizedfemale so fmeda toa nexogenou sMIS ,th efollicl e itselfwil l ka, injected witha MI S sucha sprogesterone , notnecessaril y ovulate,becaus euncompletel y undergooocyt ematuratio n butd ono tsubse differentiated,an d eveni fovule sar eobtai quentlyovulat e (Iwamatsu,1978a) .Besides , ned theywil lno tnecessaril y beabl et ob e taking intoaccoun t thata massiv e injection fertilized or todevelo pnormally . ofgonadotropi n insalmonid sa tprecociou s ovarian stagesca n induceoocyt ematuratio n_ Concerning the"follicula r sensitivity"t o without subsequent ovulation (Jalabert etal. , gonadotropins,itdepends ,fo ra part ,o nth e 1978b),a swel la sa n injectiono f 17a,20ß-OH- ability ofa steroidogeni c tissuet osynthe P, itma y behypothesize d that thegonadotro sizeth eMI S inrespons e toGTH .Thi ssteroi pinactio nnecessar y tobrin g about thecon dogenic tissueappear sgenerall y tob eloca venientdifferentiatio no fovaria nfunction s tedwithi n theovar y inmos t of thespecies , related toovulatio nmus t beprogressive . asdemonstrate d by thepositiv eactio no fgo Thiscoul d explainth erelativ e successo f nadotropinsupo n intrafollicular oocytematu priming treatments toobtai novulatio n invi ration invitr o inadequat econdition s (Jala vo (Jalabert etal. ,1978b ;Hunte r etal., bert, 1976;Wallac e& Selman , 1980).Howeve r 1981;Donaldso n etal. ,1981a) . Finallyendo inth eIndia ncatfish,'Heteropneusta sfossili s crinefactor sothe r thansteroid so rGT Hb y whereth efollicula r sensitivity togonadotro itselfals oparticipat e inth eregulatio no f pinsalon e invitr o appears extremely low,an d the"follicula r sensitivity"t ogonadotropins . wherecorticosteroid s arepoten t inducerso f Thus,i nth estellat esturgeo n exposed to GVBD itwa shypothesize d that theMI Smigh t cold temperature,o rkep t inprolonge dcapti beproduce d by theinterrena l (reviewedb y vityprio r toth eonse to fmaturation ,th e Sundararaj &Goswami , 1977).I ntrou t theabi follicular sensitivity topituitar y extracts lityo f theovar y tosynthesiz eth eMI Si n inviv oo r invitr owa s impaired,bu ttha ti t responset oGT Hwa s showni nvitr o atth efi 167 wasrestore d by injectiono f triiodothyronine byhig hlevel so f 17a-20ßP invitr o (Jalabert, (Dettlaff &Davydova , 1979). InOryzia slati - 1978). Thus, ifa clos erelationshi p between pes,a lo wmolecula rweigh t factorfro mrab oocytesan dgranulos a isnecessar y duringa bit serum inhibits steroid-induced oocytema parto fmaturation ,th efollicula r detachment turation invitro ,howeve r sucha facto rwa s should occuronl y after thisperiod .Thi s not sowel lcharacterize d inmedak aseru m could explainth edifference ,observe d invi (Iwamatsu &Takama , 1980). tro, betweenth ever y lowamoun t of 17a-20ßP Environmental factorsca nmodif y the"folli necessary toinduc eonl ymaturatio n on one cular sensitivity"bu tthei rmechanis m ofac hand,an d thever yhig hdose snecessar y to tionar efa r tob ealway swel lknown .W ehav e bring about oocytedetachmen t on theothe r alreadymentione d thelos so f ovariansensiti hand (Jalabert, 1978),an dwoul d becompati vity insturgeon s exposed tocol d temperature blewit h theprogressiv eris eo f 17a-20ßPob (Dettlaff &Davydova , 1979). InFundulu shete - served inviv o overa t last 2day s (Fostier roclituscessatio no f feeding eliminatesth e etal. , 1981a)an d the kinetics of secretion competenceo ffollicle s-t orespon d todeoxy observed invitr o (Fostier etal. , 1981b).Al corticosterone invitro ,bu thuma nchorioni c soinducin g tooabruptl y aris eo f theMI S gonadotropin (HCG)injectio no rrefeedin g res by exogenous means could lead todramati c toresth erespons e (Wallace& Selman , 1980). repercussionsupo n thequalit y of theeggs . Ingre ymullets ,whic hnormall y reproducei n Inextrem ecase s theovulatio no f immature seawater,modification so f ovariansteroido oocytesha s evenbee nobserved , either invi genesispathway swer eobserve d infis hconfi troafte ractio no funappropriat e MISo nth e ned infreshwater ,i ncompariso nwit hseawa follicles(as seenb y Goetz &Bergman ,1978 ) ter fish.Thes emodificatio nwer ehypothesi inth eyello wperch ,o r inmargina ltempera zed to impair the synthesis and releaseo fpi turecondition s (ascite db yDettlaf f &Sko - tuitary GTH,thu s explaining thematuration/o blina, 1969)i nth esturgeon ,confirmin g that vulationbloc kactuall y observed infreshwa oocytematuratio nan d ovulationar eindepen ter (Eckstein, 1975). Inversely, incoh o sal dantpocesse swhic hca nb eartificiall y dis monretaine d insea-wate rbefor ematuration , sociated,a sals o showni ntrou t (Jalaberte t thepatter no f plasmahormone swa s impaired al., 1972). Concerning themechanica l process and ovarianrespons et o exogenous treatments ofovulation ,thi srequires ,i nsom especie s appeared reduced compared totha to ffres h thecontractio n offollicula r cells.I nJ3ry_ - water fish (Sower etal. , 1982). Probably, ziaslatipe s suchcell scontainin g anorgani osmoregulatory disturbancema y also interfer zed contractilesyste m differentiatei nth e with theproces so fovaria nhydratatio nwhic h thecawithi nonl ya fe whour sprio r toovula developsdurin gmaturatio n (Hirose etal. , tion (Pendergrass& Shroeder , 1976). Introu t 1974; Oshiro &Hibiya , 1981). themorpholog y of thecal cells looksidenti calt oauthenti c smoothmuscl e cells (Szöllö- Inothe rrespects ,ovaria n sensitivity,re sie tal. , 1978).However ,th epresenc eo f garding oocytematuratio nan d ovulation,mus t contractilecell s inth efollicl ewal lmigh t not beconsidere d exclusively froma nendocri notb erequire d inspecie swher ematuratio n nologicalpoin t ofview ,an d cell-to-cellin isaccompanie d bya nimportan t swellingo f teractionswithi n theovar ymus tb ever y im theoocyt ean d (or)th eproductio no f ajell y portant toharmoniz e themorphologica lan d layerb y thegranulosa ,an dwher eth ehydra functionnaldifferentiatio no f thevariou s tionproces sca nprovid eth eforc e toexpe l celltype swhic hcooperat e invie wo f thefi theoocyt ea spropose d inplaic e (Oshiro& nalovulatio no fmatur eoocyte sabl et ob e Hibiya, 1981).Whateve r themechanic s ofth e fertilized and developnormally .Th egranulo ovulationprocess ,thi sappear st orequir e sacell sprobabl y assumenumerou s functions theloca lsynthesi s and.actio no fprostaglan t&Dughthei rdirec t contact withth eoocyt e: dins,a sha sno wbee ndirectl yprove d inth e notonl yd o theycooperat e toprovid eth eo - pond loach (Ogata etal. , 1979), andpossi ocytewit hth eMIS ,bu t theymigh t also the blya mediatio nb y catecholamines (Jalabert, sourceo fdirec t inhibiting factorso fmatura 1976)whic hwoul d suggest arol efo ra nin - tion ifany ,a ssee nb yth eincrease d effi traovarian innervation. ciencyo fMI S invitr oafte r eliminationo f granulosa inth emedak a (Iwamatsu, 1980).Mo Incompariso nt o thecomplexit y ofmecha reover,oocyte so f this species,take ni nvi nismswhic hregulat e theovaria n competence vo after theendogenou s stimulation necessary formaturatio n and ovulation,th ecriteri a for further spontaneousmaturatio n invitro , used inth ecurren tpractic et ochoos ebroo acquirenorma ldevelopmenta l activityonl y derstha twil lb esubmitte d tohypophysatio n when surounded byth egranulos a celllaye r arever y simplist,fro m thesimpl eexterna l (Iwamatsu &Ohta ,1981 )whic h seemst oprovid e observation of theabdome naspect ,t oovaria n thematurin g oocytewit h"specia lfactors "de biopsy inorde r tochec kth efollicula rmor pendantupo ngonadotropi n stimulation.Ovula phology (seeHarve y &Hoar , 1980). Intha t tion impliesth eruptur eo f closerelation caseth ecriteri ause d aleeithe r theoocyt e shipsbetwee n theoocyt ean d thegranulos ala diameter,suitabl ei nspecie s sucha sth e yerwhic hwer eprevailin g duringvitellogene - greymulle t (Shehadeh etal. , 1973), orth e sis. Introut ,thi sappear s tooccu rdurin g position ofth egermina lvesicle ,a s incar p oocytematuration ,an d torequir e stimulation (Brzuska &Bieniarz ,1977 )o r thepositio no f 168 thenucleol i inth eG Va si ncyprinid s (Hor- tomized fish,usin g steroidsalone ,migh tb e vath, 1978).However ,thes ecriteri agiv eon related toth efac t that thefis hwer eactual lya partia l indicationan d arever y far,u - lya ta fina lstag ebefor enatura lovulation , sually,fro mgivin g a 100pe r 100success . butals o that thesteroid sused ,sometime sa t Even introut ,wher ew ehav ea lo to fexpe highdose san dwhic hmigh tno tb especifi c rience,w ekno wtha t theG Vpositio n isonl y MIS,coul d act synergically withth eendoge an indicatorwhic halthoug h itwork swel li n nousgonadotropi nand/o rb emetabolize d into more than 50pe r 100o f casescanno tgiv ea n specificMIS . absolute certitude concerning theactua lsen Hypophysationsensu s stricto,i.e . thead sitivity, invitr o or invivo . ministration ofvariou sgonadotropi cprepa rations,fro m crudepituitar y extracts to IV.Mechanis m ofactio no fpresen t treatments gonadotropins fromvariou s zoologicalorigins , havebee nth emos twidel y utilized sofar . Threemai n relaysappea r tofollo won eano Their action isprimarel y expected tooccu r therbetwee n thestimulu so f specific environ directlyupo n therela y of ovariansomati c mental factorsan d thestar t ofoocyt ematura tissues,t o inducesimultaneousl y thesynthe tion/ovulation process.An y treatmentapplie d siso fMIS ,an d thefunctionna ldifferentia toth efis h inorde r toinduc e successfulma tiono f thefollicl efo r ovulation.I nfact , turationan d ovulationmus t reach theoocyt e severalproblem saris ebot hfro m currentprac and thefollicl e (ovulation),eithe r directly ticean d laboratory observations :1 .th e orthroug h theactivatio n ofon eo r several problem ofth ezoologica l specificity of exo successiverelays .The yar e :th eCentra lNer genousgonadotropi c preparationha softe n vousSyste m (CNS)an d theHypothalamus ,th e beenunderline d (Burzawa-Gerard &Fontaine , pituitary and thesomati c tissueso f theova 1972;Breto ne tal. , 1973)bu t itsconsequen ry.Eac ho f theserelay ssend sou ta trigge cesar edifficul t toevaluat efo r inviv o ring informationwhic hma y becompose d ofth e applications.2 .Th eproble m of evaluating releaseo fa lon g lasting inhibiting signal theactua lactivit y ofth edifferen tgonado and of theproductio no f astimulator ysignal . tropinspreparation suse d inpractic e (Yaron Concerningmethodologie s for spawningcontrol , etal. , 1982)i so fgenera loccurence .More mainly stimulating factorshav ebee ntake nin overwhe n crudepituitar y extractsar eused , toaccount ,i.e . :gonadotropins-releasin g theyrepresen t acomple xmixtur eo fvariou s hormone (Gn-RH),gonadotropi c hormone (GTH) hormoneswhic h caninterfer e withth eexpec andMaturatio n Inducing Steroids (MIS).Howe ted stimulation (seeparagrap h II). 3.Exo ver thecompetenc e of eachrela y toreceiv e genous injectionsma y promatea bruta lsurg e and send the specific stimulating information ofGT H (amoung otherhormone swhe ncrud epi isregulate d byothe r endocrineorgan sthroug h tuitary extractsar eused) . Thisartificia l secondary signals.Th emai n onethes e ispro surgewa sobserve d inrainbo w troutan d coho bably emitted by theovar ya sa functio no f salmon (Jalabert etal. , 1978a,b) ,appearin g Itsstat eo fdevelopmen t (feed-back). completelydifferen t inshap et otha twhic h Theoritically themos t simpletreatmen t wasfoun d innaturall y occuring ovulation would consist ina short-circuit ofal lrelay s (Fostier etal. , 1982;Zoha r etal. , 1982a). bydirec tactio no fth eMI Supo nth eoocyte . Incarp ,althoug ha patter no fGT Hfollowin g Taking intoaccoun t the elementsdiscusse d in hypophysationwa sals o described (Jalabert et paragraph III,suc ha napproac hma y encounter al., 1977;Wei l etal. , 1980), completena somerisk s :1 -inducin g thematuratio no f turalreference sar elackin g toappreciat e noncompeten t oocytesto oprecociousl y could whether thesevariation sar esimila r toth e be inefficient orproduc eegg sunabl e tode naturalprocess .4 .I twa sreporte d thatre velopnormally .2 -eventhoug h theoocyt ewa s peated injectionso fHC G could inducea "dru g competent,th etreatmen t couldb e inefficient resistanceeffect "relate d to theproductio n ifth ehypothesi swa svali d thata meiosis - ofantibodie sagains t foreignprotein s (The inhibiting factor couldb eproduce d byth e Freshwater CommercialFis hArtificia lPropa folliclean dunde rgonadotropi n control.3 - gationWor kGroup , 1977).T oa lesse r extant maturatlonmigh t occurwithou t subsequentovu wemigh t expect sucha phenomeno n tooccu r lation,becaus eo fa n insufficient function- against crudepituitar y extract. naldifferentiatio no f thefollicle ,unde rgo nadotropin control,a sobserved ,fo rexample , Concerning thepossibl eus eo fhypothalami c insalmonids .I ntha t caseth enegativ efeed hormonesactin g upon thepituitar y relay,th e backexerte d by 17a-20ßPupo n thegonadotropi n present limitationsar erelate d tosevera l secretion (Jalabert &Breton ,1980 )probabl y points :1 .Th esensitivit y of thepituitar y accentuated thegonadotropi ndeficiency .Howe evenwel lloade dwit h GTH,ma yb eGRIF-depen - ver,a convenien t "priming"wit ha gonadotro dant. 2.Th esyntheti cmolecule s ofmammalia n pinpreparatio nwa s shown tobrin g asolutio n Gn-RHo ranalogue sma y be inappropriate in toth eproble m of ovulation,whil e theproble m fish.3 .Th ecatabolis m of thesemolecule s ofoocyt ecompetenc e forfurthe r development inviv oma yb equic kdependin g onth eplac e couldb esolve d by theresearc ho fappropria ofinjection . tecriteria .Practically ,th esuccessfu lovu Themechanis m ofactio no fclomiphem ecitra lationsobtaine d sometimes onnon-hypophysec - teupo nfina lmaturatio n (recentlyrevieve d byDonaldso n etal. , 1982)i sno t completely 169 understood. Considering that estradiolappear s nol.25:405-415 . tomaintai n anegativ efeedbac ko nGT Hrelea Breton,B. ,L .Horoszewicz ,K .Bieniar z &P . sea tthi stim e (Bommelaere t al., 1981)clo - Epier, 1980.Temperatur ean d reproduction miphenema y probably acta sa nantiestroge n intenc h :effec to fa ris e inth eannua l involving Gn-RH synthesis (Dixit,1967 ;Vis - temperatureregim eo ngonadotropi nlevel , wanatan& Sundararaj ,1974 ). gametogenesisan d spawning.II .Th efemale . Reprod.Nutr .Develop' .20:1011-1024.: . V. Conclusion Breton,B. ,A .Fostier ,Y .Zohar ,P.Y .L e Bail& R .Billard ,1982 .Gonadotropin egly - Most of thepresen t knowledge,althoug hve coproteiquematurant e etoestradiol-17f >pen ry incomplete,concern sth enatur eo ftrophi c dant lecycl ereproducteu r chezl atruit e factors involved inth eregulatio n ofmatura fario (Salmo trutta)femelle .Gen .Comp . tion/ovulation. Thepresen tus eo f thesefac Endocrinol,i npress . tors ispurel y empiric concerning themetho d Brzuska,E .& K .Bieniarz ,1977 .Determina ofadministratio n and generallybase dupo n tiono f thestag eo f ovarianmaturit y in inaccuratecriteri ao f ovariandevelopment . femalecarps ,i nrelatio n toinjection so f Further progress involves: crudepituitar y extracts (inPolish) . Ins- - amor eprecis eanalysi so f the shapeo fphy tytutRybactw aSroladowego ,105 . siological trophicsignals . Burzawa-Gerard,E .& Y.A . Fontaine,1972 .Th e - abette r knowledgeo f themechanism s ofne gonadotropins of lowervertebrates .Gen . gativeregulation . Comp.Endocrinol. ,Supp .3:715-728 . - aresearc ho faccurat ecriteri a forovaria n Burzawa-Gerard,E. ,B .Goncharov ,A .Duma s & competence. Y.A. Fontaine,1976 .Furthe rbiochemica l studieso ncar pgonadotropi n (c-GTH);bio References chemicalan d biologicalcompariso no fc-GT H anda gonadotropi n fromAcipense r stellatus Ball,J.N. , 1981.Hypothalami c controlo fth e Pall- (ehondrostei)•Gen .Comp .Endocrinol . Parsdistali s inFishes ,Amphibia n andRep 29:498-505. tiles.Gen .Comp .Endocrinol .44:135-170 . Campbell,C.M .,A .Fostier ,B .Jalaber t& Barnett,F.H .,J . Somn,S .Reichlin ,I.M.Ill - B. Truscott,1980 .Identificatio nan dquan Jackson 1982.Thre eluteinizin ghormo tificationo f steroids inth eseru mo fsper - ne-releasing hormonelik e substances ina miating orovulatin g rainbow trout.J .En teleost fishbrai n :non e identicalwit hth e docrinol.85:3,71-378 . mammalianLH-R Hdecapeptide .Biochem .Bio - Crim,L.W. , E.G.Watt s &D.M . Evans,1975 . phys.Res .Commun .105:209-216 . Theplasm a gonadotropin profiledurin gse Billard,R. ,B .Breton ,A .Fostier ,B .Jala - xualmaturatio n ina variet y ofsalmoni d bert &C .Weil , 1978.Endocrin e controlo f fishes.Gen .Comp .Endocrinol .27:62-70 . theteleos t reproductivecycl ean d itsre Dettlaff,T.A . &M.N .Skoblina ,1969 .Th e lationt oexterna lfactor s :salmoni dan d roleo fgermina lvesicl e inth eproces so f cyprinidmodels , inCompar .Endocrinol .Gail - oocytematuratio n inAnur aan dAcipenserida e lardP.T .an dBoe rLL H eds.Elsevier/Nort h Ann.Embryol .Morphol. ,Supp .1:133-151 . HollandBiomedical,Press-Amsterdam ,37-48 . Dettlaff,T.A . &S.I .Davydova ,1979 .Diffe Billard,R. , C.Br y& C .Gillet ,1981 .Stres s rentialsensitivit y ofcell so ffollicula r environment and reproduction inteleos tfis h epithelium and oocytes inth estellat estur in"Stres san dFish "Edite d byA.D .Picke geont ounfavorabl econditions ,an dcorre ring.Academi cPres s- London/N.Y . lating influenceo f triiodothyronine.Gen . Bommelaer,M.C. ,R .Billar d &B .Breton ,1981 . Comp.Endocrinol .39:236-243 . Changes inplasm agonadotropi n afterova DeVlaming ,V .& M.J .Vodicnik ,1978 .Seaso riectomyan d estradiol supplementationa t nal effectso f pinealectomy ongonada lac different stagesa tth een d of therepro tivity inth egoldfish ,Carassiu sauratus . ductivecycl e inth erainbo w trout (Salmo Biol.Reprod .19:57-63 . gairdneri R.). Reprod.Nutr .Develop .21 : Dixit,V.P. , 1967.Neurosecretio n andfee d 989-997. backmechanis m inclaria sbatrachu s Linn. Breton,B. ,R .Billard ,B .Jalaber t &G .Kann , Ovariectomy andadministratio n of exogenous 1972.Dosag eradioimmunologiqu ede sgonado - sexhormones .Cellul e68:213-221 . tropinesplasmatique s chezCarassiu sauratu s Donaldson,E.M. , G.A.Hunte r &H.M . Dye,1981 a au coursd unycthémèr e etpendan tl'ovula - Induced ovulation incoh osalmo n (Oncorhyn- tion.Gen .Comp .Endocr .18:462-468 . chuskisutch) . II.Preliminar y studyo fth e Breton,B. ,R .Billar d &B .Jalabert ,1973 . useo fLH-R Han d twohig hpotenc y LH-RHa - Spécificitéd'actio ne trelation simmunolo- - nalogues.Aquaculture ,26:129-141 . giquesde shormone sgonadotrope sd equelque s Donaldson,E.M. , G.A.Hunte r &H.M . Dye,1981 b téleostéens.Ann .Biol .Anim .Biochim .Bio- Induced ovulation incoh o salmon (Oncorhyn- phys.13:347-362 . chus kisutch)• III.Preliminar y study onth e Breton,B. ,B .Jalaber t &C .Weil , 1975.Ca - useo fth eantiestroge n tamoxifen.Aquacul ractérisation partielled'u nfacteu rhypo - ture26:143-154 . thalamiqued eliberatio nde shormone sgona Eckstein,B. ,1975 .Possibl ereason sfo rth e dotropesche zl acarpe .Gen .Comp .Endocri infertility ofgre ymullet sconfine dt o 170 freshwater. Aquaculture5:9-17 . corhynchusnerka ). Biö.ehem .B iophy s. Res . Ekengren,B. ,J .Peut e& G .Fridberg ,1978 . Commun ,2:133-137 . Gonadotropic cells Inth eAtlanti cSalmon , Idler,D.R . &B.T .Ng , 1979.Studie so ntw o Salmosalar .Cel lTigs.Res .191:187-203 . typeso fgonadotropin s frombot h salmonan d Fontaine,M. , 1976.Hormone s and thecontro l carp pituitaries.Gen .Comp .Endocrinol . ofreproductio n inaquaculture .J .Fish . 38:421-440. Res.Bd . Canada33:922-939 . Idler,D.R . &L.W . Crim,1982 .Gonadotropi n Fostier,A. ,C .Weil ,M .Terqui ,B .Breton , releasing factor(s) (GTH-RF)fro mhypotha & B.Jalabert ,1978 .Plasm a estradiol-17ß lamuso fwinte r flounder.Proceeding so f and gonadotropin during ovulation inrain theIXt hInternationa l Symposium onCompa bowtrou t (Salmogairdnerii) .Ann .Biol , rativeEndocrinolog y -Hong-Kong .Abst .20 . anim.Biochim .Biophys .18:929-936 . Iwamatsu,T. ,1974 .Studie s onoocyt ematura Fostier,A. ,B .Breton ,B .Jalaber t &0 .Mar - tiono f themedaka ,Oryzia s latipes.II . cuzzi,1981a .Evolutio nde sniveau xplasma - Effectso f several steroidsan d calcium tiquesd el agonadotropin e glycoproteique ionsan d therol eo f folliclecell so ni n etd el a17a-hydroxy-20f5-dihydroprogestero - vitromaturation .Annot .Zool .Japon ,47 : nea ucour sd el amaturatio n etd e l'ovula 30-42. tionche zl atruit earc-en-ciel ,Salm ogair - Iwamatsu,T. , 1978a.Studie so noocyt ematu dneri. CR. hebd.Seance sAcad . Sei.29 3D : ration inth emedak a Oryzias latipes.V . 817-820. Onth estructur eo f steroids that induce Fostier,A. ,B .Jalabert ,C .Campbell ,M . maturation invitro .J .Exp .Zool. ,204 : Terqui& B .Breton ,1981b .Cinétiqu ed eli 401-408. bérationi nvitr od e17a-hydroxy-206-dihy - Iwamatsu,T. , 1978b.Studie so noocyt ematu droprogesterone par desfollicule s detruit e rationo fth emedaka ,Oryzia slatipes .VI . arc-en-ciel,Salm o gairdnerii.C.R .Acad . Relationship between thecircardia n cycle Se.292:777-780 . of oocytematuratio nan d activity ofth e Goetz,F.W . &H.L .Bergman ,1978 .Th eeffect s pituitary gland.J .Exp. 'Zool .206:355-363 . of steroidso nfina lmaturatio n andovula Iwamatsu,T. ,1978c .Studie so noocyt ematu tiono foocyte sfro mbroo ktrou t (Salveli rationo fth emedak a Oryziaslatipes .VII . nusfontinalis )an d yellowperc h (Percafla - Effectso fpinealectom y andmelatoni no n• vescens)• Biol.Reprod .18:293-298 . oocytematuration .Annot . Zool.Japon. ,51 : Harvey,B.J . &W.S .Hoar ,1980 .L areproduc 198-203. tionprovoqué e chezle spoisson s :théori e Iwamatsu,T. ,1980 .Studie so noocyt ematura etpratique .IDRC-Ottawa-4 8p . tiono fth emedak a Oryzias latipes.VIII . Hirose,K . 1972.Biologica l studyo novulatio n Roleo ffollicula r constituents ingonado invitr oo ffish .IV .Inductio no fi nvitr o tropinan d steroid inducedmaturatio no f ovulation inOryzia slatipe soocyt eusin g oocytes invitro .J . Exp.Zool .211:231-239 . steroids.Bull .Jap .Soc .Sei .Fish .38:457 - Iwamatsu,T .& T .Katoh ,1978 .Maturatio ni n 460. vitroo foocyte so fth eloach ,Misgurnu s Hirose,K. , 1980.A bioassa y forteleos tgona anguillicaudatus by steroid hormonesan dgo dotropinusin g thegermina lvesicl ebreak nadotropins.Annot . Zool.Japon. ,51:79-89 . down (GVBD)o foryzia s latipesoocyte si n Iwamatsu,T .& K . Takama, 1980.Inhibitor y vitro.Gen .Comp .Endocrinol .41:108-114 . effecto frabbi t serum factor on invitr o Hirose,K. ,T .Hiran o &R .Ishida ,1974 .Ef oocytesmaturatio no fth eteleos tfish , fecto f salmongonadotropi n onovulatio ni n Oryziaslatipes .Develop .Growth .Differ . theayu ,Plecoglossu s altivelis,wit h spe 22:229-235. cialreferenc e towate rbalance .Comp .Bio - Iwamatsu,T .& T .Ohta ,1981 .O na relation chem.Physiol .47A:283-289 . shipbetwee noocyt ean d folliclecell sa - Hontela,A .& R.E .Peter ,1978 .Dail ycycle s round thetim eo fovulatio n inth emedaka , inseru mgonadotropi n levels inth egolfis h Oryziaslatipes .Annot .Zool .Japon. ,54:17 - effectso f photoperiod,temperatur ean dse 29. xualcondition .Can .J . Zool.56:2430-2442 . King,J.A .& R.P .Millar ,1980 .Comparativ e Hontela,A .& R.E .Peter ,1980 .Effect so f aspectso f luteinizing hormone-releasing pinealectomy,blindin g and sexualconditio n hormone structurean d function inverte onseru mgonadotropi n levels inth egold bratephylogen y Endocrinol.106:707-717 . fish.Gen .Comp .Endocrinol .40:168-179 . Jalabert,B. , 1975.Modulatio npa rdifférent s Horvath,L. , 1978.Experience s inpropagatio n Steroidesno nmaturantsd el'efficacit é del a of thecommo n carp (Cyprinuscarpi oL. )ou t 17a-hydroxy-20ß-dihydroprogesteroneo u d'un ofth espawnin g season.Aqu .Hüng . 1:66-72. extrait gonadotrope surl amaturatio nintra - Hunter,G.A. ,E.M .Donaldso n& H.M .Dye ,1981 / folliculaire invitr o desovocyte sd el a 1982. Induced ovulation incoh o salmon (On- truitearc-en-ciel ,Salm o gairdnerii. C.R. corhynchus kisutch). I.Furthe r studieso n hebd.Seance sAcad . Sei.281D:811-814 . theus eo f salmonpituitar ypreparations . Jalabert,B. , 1976 .I nvitr ooocyt ematura Aquaculture26:117-127 . tionan dovulatio ni nrainbo wtrout ,(Salm o Idler,D.R. , U.H.M.Fagerlun d &A.P .Ronald , gairdneri)norther npik e (Esoxlucius )an d 1960. Isolationo fpregn-4-ene-17a,20ß-diol - goldfish (Carassius auratus). J. Fish.Res . 3-onefro m theplasm a ofpacifi c salmon (On- Bd.Canad a33:974-988 . 171 Jalabert,B. , 1978.Productio no ffertiliza - 30:451-456. bleoocyte sfro m follicleso frainbo w trout Peter, R.E. &L.W . Crim, 1979. Reproducive en (Salmogairdnerii )followin g invitr omatu docrinology of fishes : gonadal cycles and rationan d ovulation.Ann .Biol .anlm .Bio - gonadotropin in teleosts. Ann. Rev. Physiol. ch.Biophys .18:461-470 . 41:323-335. Jalabert,B. ,B .Breto n& C .Bry ,1972 .Matu Peter, R.E. &C.R . Paulencu, 1980. Involvement ration etovulatio n invitr o desovocyte sd e of the preoptic region in gonadotropin re latruit earc-en-cie lSalm o gairdneri. C.R. lease-inhibition in goldfish, Carassius au Acad. Sc. Paris275 ,séri eD:1139-1142 . ratus. Neuroendocrinology 31:133-141. Jalabert,B .& D .Szöllösl , 1975.I nvitr o Peter, R.E., J.P. Chang & A.F. Cook, 1982. maturation oftrou t oocytes :effec to fpros Neuroendocrine regulation of gonadotropin taglandins on smoothmuscle-lik ecell so f and growth hormone secretion in goldfish. thetheca .Prostaglandin s 5:765-778. Proceedings of the IXth International Sym Jalabert,B. ,B .Breton ,E .Brzuska ,A .Fos - posium on Comparative Endocrinology- Hong- tier &J .Wieniawskl ,1977 .A ne wtoo lfo r Kong. Abst. 64. induced spawning :th eus eo f 17a-hydroxy- Pickering, A.D. &P . Christie, 1981. Changes 20ß-dihydroprogesteronet o spawncar pa t in the concentrations of plasma Cortisol lowtemperature .Aquacultur e10:353-364 . and thyroxine during sexual maturation of Jalabert,B. ,B .Breto n &A .Fostier ,1978a . the hatchery-reared brown trout, Salmo trut- Precocious inductiono foocyt ematuratio n ta L. Gen. Comp. Endocrinol. 44:487-496. and ovulation inrainbo w trout (Salmogaird Pickford, CE. &J.W . Atz, 1959. The physio neri): problem swhe nusin g 17ct-hydroxy-20f>- logy of the pituitary gland of fishes. New dihydroprogesterone.Anh .Biol .anim .Bioch . York Zoological Society. NY, 613 p. Biophys.18:977-984. Sakun, O.F., 1966. Transition to meiotic di Jalabert,B. ,F.W . Goetz,B .Breton ,A .Fos vision of oocytes with uncomplete vitello- tier& E .Donaldson , 1978b.Precociou sin geny under the influence of hormonal stimu ductiono foocyt ematuratio n and ovulation lation in Salmo salar (Russian). Dokl. Akad. incoh o salmon,Oncorhynchu skisutch .J . Nauk. 169:241-244. Fish.Res .Bd .Canad a35:1423-1429 . Scott, A.P.-, V.J. Bye &S.M . Baynes, 1980. Jalabert,B .& B .Breton ,1980 .Evaluatio nd e Seasonal variations in sex steroids of fe lagonadotropin eplasmatiqu et-GT Haprè sovu male rainbow trout (Salmo gairdneri Richar lationche zl atruit earc-en-cie l (Salmo dson) . J. Fish. Biol. 17:587-592. gairdneriR. ) et influenced el arétentio n Scott, A.P., E.I/S Sheldrick & A.P.F. Flint, desovules .C.R .Acad . Sei., SérieD 290 : 1982. Measurement of 17a,20ß-dihydroxy-4- 799-801. pregnen-3-one in plasma of trout (Salmo Nagahama,Y. ,G .Youn g &A .Kagawa ,1982 .Ste gairdneri R.). Seasonal changes and respon roidogenesis inth eamag o salmon (Oncorhyn se to salmon pituitary extract. Gen. Comp. chusrhodurus )ovaria nfollicl e :a tw ocel l Endocrinol. 46:444-451. typemodel .Proceeding s of theIXt hInterna Shehadeh, Z.H., CM. Kuo & K.K. Milisen, tionalSymposiu m onComparativ eEndocrinolo 1973. Induced spawning of grey mullet Mugil gy,Hong-Kong . cephalus L. with fractionated salmon pitui Ng,T.B. ,CM . Campbell &D.R . Idler,1980 . tary extract. J. Fish. Biol. 5:471-478. Antibody inhibition ofvitellogenesi san d Sire, 0. &J . Depêche, 1981. In vitro, effect oocytematuratio n insalmo nan dflounder . of a fish gonadotropin on aromatase and Gen.Comp .Endocrinol .41:233-239 . 178-hydroxysteroid dehydrogenase activities Ogata,H. ,T .Nomur a &M .Hata ,1979 .Prosta in the ovary of the rainbow trout (Salmo glandinF„ a changes inducedb y ovulatory gairdneri Richardson). Reprod. Nutr. Dev. stimuli inth epon d loach,Misgurnu sanguil - 21:715-726. licaudatus.Bull . Jap.Soc . Scient.Fish . Sower, S. &C.B . Schreck, 1982. Steroid and 45:929-931. thyroid hormones during sexual maturation Osborn,R.K. ,T.H . Simpson &A.F .Youngson , of Coho salmon (Oncorhynchus kisutch) in 1978. Seasonalan d diurnalrhythm so fthy seawater or freshwater. Gen. Comp. Endocri roidal status inth erainbo w trout,Salm o nol. 47:42-53. gairdneriR .J . Fish.Biol .12:531-540 . Sower, S.A., C.B. Schreck &E.M . Donaldson, Oshiro,T .& T .Hibiya ,1981 .Wate r absorption 1982. Hormone-induced ovulation of Coho ofoocyte si nth eplaice ,Limand ayokohama e salmon (Oncorhynchus kisutch) held in sea- duringmeioti c maturation and itsrol ei n water and fresh water. Can. J. Fish. Aquat. rupturing follicles.Bull .Jap .Soc .Scient . Sei. 39:627-632. Fish.47:835-841 . Stacey, N.E., A.F. Cook &R.E . Peter, 1979. Pendergrass,P .& P . Schroeder,1976 .Th eul Ovulatory surge of gonadotropin in the gold trastructureo fth etheca lcel lo f thetele - fish Carassius auratus. Gen. Comp. Endocri ostOryzia slatipes ,durin g ovulation invi nol. 37:246-249. tro.J .Repröd .Fertil. ,47:229-233 . Sundararaj, B.I. &S.V . Goswami, 1977. Hormo Peter,R.E .& R .Billard ,1976 .Effect so f nal regulation of in vivo and in vitro oocy thirdventricl e injectiono f prostaglandins te maturation in the catfish, Heteropneus- ongonadotropi n secretion ingoldfish ,Caras - tes fossilis (Bloch). Gen. Comp. Endocr. siusauratus .Gen .Comp .Endocrinol . 32:17-28. 172 Sundararaj, B.I., S.V. Gosvami & V. Lamba, Wright, R.S. &S.M.V . Hunt, 1982. A radioim 1979. Some aspects of oocyte maturation in munoassay for 4-pregnene-17a,20ß-diol-3-one catfish. J. Steroid Biochem. 11:701-707. its use in measuring changes in serum level Suzuki, K., Y. Nagahama &B . Tamaoki, 1981a. at ovulation in atlantic salmon, Coho sal In vitro synthesis of an inducer for germi mon and rainbow trout. Gen. Comp. Endocri nal vesicle breakdown of fish oocytes, 17a, nol. 46:p.396, Abst. 145. 20(3-dihydroxy-4-pregnen-3-one by ovarian Yamazaki, F., 1962. Effects of hypophysecto- tissue preparation of Amago salmon (Onco- my on the ovulation, oviposition and sexual rhynchus rhodurus)• Gen. Comp. Endocrinol. behavior in the goldfish, Carassius auratus 45:473-481. Bull. Fac. Fish. Hokkaido Univ. 13:39-46. Suzuki, K., B. Tamaoki & K. Hirose, 1981b. Yaron. Z., A. Bogomolnaya &V . Hilge, 1982. In vitro metabolism of 4-pregnenes in ova Application of an in vitro bioassay for ries of a freshwater teleost, the Ayu (Pre- fish GTH in improving harvest of carps pi- coglossus altivelis): Production of 17a-20ß tuitaries. Proceedings of the Internatio dihydroxy-4-pregnen-3-one and its 5ß-redu- nal Symposium on Reproductive Physiology of ced metabolites, and activation of 3(3-and Fish. Wageningen - Netherlands. 203-hydroxysteroid dehydrogenases by treat Zohar, Y., B. Breton &R . Billard, 1982a. ment with a fish gonadotropin. Gen. Comp. Short-term profiles of plasma gonadotropin Endocrinol. 45:533-535. levels in the female rainbow trout throu Szöllösi, D., B. Jalabert &E . Breton, 1978. ghout the reproductive cycle. Gen. Comp. Postovulatory changes in the theca folliculi Endocrinol. 46:p. 396, Abst. 146. of the trout. Ann. Biol. anim. Bioch. Bio- Zohar, Y., B. Breton &A . Fostier, 1982b. phys. 18:883-892. Gonadotropic function during the reproduc The Freshwater commercial fish artificial pro tive cycle of the female rainbow trout, pagation work groups in Fujian, 1977. Salmo gairdneri, in relation to ovarian Acta Biochim. Biophys. Sinica, 9:15-23. steroid secretion : in vivo and in vitro Theofan, G., 1981. The in vitro synthesis of studies. Proceedings of the International final maturational steroids by ovaries of Symposium on Reproductive Physiology of brook trout Salvelinus fontinalis and yel Fish, Wageningen, Netherlands. low perch Perca flavescens. Ph. D. Disserta tion - University of Notre Dame - USA Viswanathan, N. &B.I . Sundararaj, 1974. Res ponse of hypothalamohypophyseal ovarian system of the catfish Heteropneustes fossi- lis to administration of oestrogen and an drogen. Anat. Pec. 178:525 abstr. Vivien, 1941. Contribution à l'étude de la physiologie hypophysaire dans ses relations avec l'appareil génital, la thyroïde et les corps suprarenaux chez les poissons séla ciens et teleostéens Bull. Biol. Fr. Belg. 75:257-309. Wallace, R.A. &K . Selman, 1978. Oogenesis in Fundulus heteroclitus. I. Preliminary obser vations on oocyte maturation in vivo and in vitro. Dev. Biol. 62:354-369. Wallace, R.A. &K . Selman, 1980. Oogenesis in Fundulus heteroclitus. II. The transition from vitellogenesis into maturation. Gen. Comp. Endocrinol. 42:345-354. Weil, C, B. Breton &P . Reinaud, 1975. Etude de la réponse hypophysaire ä 1'administra tion de Gn-RH exogene au cours du cycle re producteur annuel chez la carpe, Cyprinus carpio L. CR. Acad. Sei. Paris, Série D 280:2469-2472. Weil, C, R. Billard, B. Breton &B . Jalabert, 1978. Pituitary response to LH-RH at diffe rent stages of gametogenesis in the rain bow trout (Salmo gairdneri)• Ann. Biol. anim. Bioch. Biophys. 18:863-869. Weil, C. A. Fostier, L. Hörvath, S. Marlot & M. Berscenyi, 1980. Profiles of plasma gona dotropin and 17ß-estradiol in the common carp,Cyprinus carpio L., as related to spaw ning induced by hypophysation or LH-RH treat ment. Reprod. Nutr. Dev. 20:1041-1050. 173 EXPERIMENTAL MATURATION OF EUROPEAN SILVER EELS I.Boëtiu s and J. Boëtius Danmarks Fiskeri-o g Havundersagelser,Charlottenlund ,Denmar k Abstract normally developed Ovaria. Such speci mens are as rare as calves born with Work on experimental maturation of two heads and they only confirm the eels has been made by us in our rule that silver eels delay their institute since 1958.I n our work on sexual maturation until their long HCG-matured male eels we have studied journey iswel l over. the effects of dose and environmental In the era before hormone treatment parameters (temperature, salinity of eels was established eel-workers etc.)» In our experiments on female havemad e many laborious experiments eels treated with carp pituitary ex in order to induce sexual maturity. tracts we arrived at ametho d which Thematte r was not simple.Environ resulted inmatur e eggs and partly mental parameters such as temperature, spawning.Als o sexual behaviour was salinity, light,pressur e and others studied.Attempt s at fertilization have been changed more or less system resulted in early embrynic development atically and also ingenious exercisers only.Finall y a brief account is given have been invented inorde r to make of recent experiments on repeated the eel feel as if itwer e on migration maturation of males and of an attempt or in the breeding area.Non e of these to compare the effects of carp-pitui efforts have resulted in the slightest tary and salmon-pituitary extracts in change in'the eel's gonadal structure. maturation experiments with female As early as 193o the late Anton Bruun eels. and his team started hormone experi Keywords: European Eel,maturation , ments with eels. You may say that these gonadotrophins. experiments afe still running. Since about 1958 they were taken over byus . The Danish eel catches are bigger Our intensions were (and still are) than those of other European countries. the following: by inducing sexual Economically evaluated the eel isou r maturity we try in our laboratory to fishnumbe r three among fishes for throw light on the almost completely consumption, only surpassed by cod and unknown phases in the eel's life-story: plaice.Mor e than 2ooo papers on the themigratio n and the breeding biology. biology of the eel have been written Our work isno t an endocrinological - but still we are almost ignorant work as such.W e make use of thehor about fundamental parts of its complex mones rather as tools for provoking life-cycle.Thi s istru e for age and results which could contribute to our growth innature , sex differentiation general understanding of the eel's etc., but especially as far as its natural history. oceanic life concerns:th e migration What isrelate d by us in the follow to the Sargasso Sea and the breeding ing ismainl y based on our two latest inthi s distant area. papers: that on male eels from 1967 Eels leave European continental and that on females from 198o. Finally waters in a condition well adapted for we give some hitherto unpublished migration (silver eels), but with results on repeated maturation of male quite immature gonads.W e join their eels and acompariso n between the cycle in the Sargasso Sea where tiny effects of carp- and salmon-pituitary larvaewit h rudiments of yolk sacs extracts upon female silver eels. are found in the upper water layers of Successfull experimental maturation an apparently well-defined area.Wha t of male silver eels was achieved by lies between isunknown .A n adult French and Italian workers during the European eel was never caught in the thirties of this century.Th e eels Sargasso Sea or in the open ocean at were treated with urin from pregnant all. women. From European coastal waters we have In our experiments we used a standar a few quite unique reports (2o r 3)o n dized preparation of HCG.Muc h effort sexually mature eels with fully deve was made inorde r to establish a proper loped spermatozoa and also a few dosage.W e arrived at a dosage of female eels with large,bu t not 25o IUHCG/eel/wee k given until the eel 174 gave off sperm when stripped. they were injectedwit h looo IUHC G The full maturation cycle was des and transferred towar m seawater cribed by us for male silver eels at (22°C). 283day s later (1979,Sept . body weights of about 80g treated 6th,D2 ) 15 out of the surviving 43 with the above mentioned "standard eels were re-injected with looo procedure" and under experimental IU HCG. "standard conditions" whichwere : In the period between Dl and D2 a temperature of 14 ± 2°C, 27pe r 14 of the eels had died (and 3 mille salinity and diffuse daylight. escaped). Inspection of their gonads We charactherized the sexual cycle by at death demonstrated that their seven developmental stages,stag e 1 sexual cycle from stages 1t o 7wa s being the immature Syrski organ, passed in about loo days. 14 eels we stages 4an d 5th e fully ripe stages sacrified for control at the D2-day. and stage 7th e post-spawned, depleted They were all in stage 7. testesresemblin g the original Syrski The 15 re-injected eels were strip organ very much.Whe n the matured eel ped for sperm 25 days afterD2 . starts to give off sperm when stripped Result: lo of the eels were re-matured. the developmental stage of testes will Stripping 4o days after p_2gav e still be close to stage 3. 2mor e ripe eels. The remaining 3 eels This standard maturation formed a had not responded upon the second starting point for experiments in hormone treatment.The y we sacrified which we changed experimental environ 69 days after D2 showing gonads in mental parameters such as light, sali stage 7. nity,temperatur e etc.Onl y tempera Out of the 12re-mature d eels 3 ture was seen to affect the rate of died 29,6 6 and 68 days afterD2 . induced maturation. The relation Their development of testes ranged between maturation rate and tempera from stage 3t o stage 6-7. The 9 ture was described as: survivors were sacrified atp_ 2= 69 . Developmental range: stage 5t o T = 13.2 (l+exp(-o.22(t-19.9))) stage 7. The induced repeated maturation where T is the number of days elapsed described does not necessarily for reaching stage 3an d t the tempe reflect the eel's natural habits. rature. Complete maturation was Fontaine, 1982',describe s cases in achieved in the interval 13.2 - 25.5°C which both male and female eels only.A possible thermal optimum for started feeding after induced spawning. thematuratio n process was indicated Hepropose s a hypothesis of "land to about 2o°C and a biological zero locked" eel populations and concludes was calculated to ll.o°C. that further research is required. The results described make it likely In our laboratory such work has been that the eel matures at temperatures initiated this year.Th e intention close to 2o°C.I n the Sargasso Sea of the workers (Dollerup and Graver) such temperatures are met with in the ist oprovok e feeding activity breeding season at depths of about between twoperiod s of induced 15ometres . maturity inmal e silver eels. Their We have several reasons to believe experiments are followed up by that the European silver eels do not studies of intestinal histology. feed at all during their migration The paper by Fontaine et al.,1964 , and spawning.Als o we have strong and the later appearing Japanese reasons to suppose that they spawn works inspired us to resume our work only once during their life-time. on female eels. Our results from the Recently we have been able to induce period 1973-79 are given in our paper repetition of full sexual cycles 198o. (stt. 1-7) inmal e silver eels without As in our work on the males we aimed intermediate feeding. These experi to establish a "standard procedure" of ments (not earlier published) are maturation.W e arrived at: 15m g carp mentioned shortly next. pituitary + 5oo IU HCG per eel (at Successfully repeated maturation body weights about 1kg ) given twice was established in two different a week until fully mature. Experiments experimental runs started 6.2.1976 were carried out in seawater (3op.m.S ) and 27.11.1978.Onl y the1978 - at 23°C.W e arrived atGSI-value s up experiment isdescribe d here. to 61.Attempt s at fertilization, 60mal e silver eels (38± 2c m long) however,wer e successful only ina were caught in the Baltic (Oct.1978 ) few cases,an d the embryonic develop and kept in seawater (15°C, 3o p.m.S) ment ceased at very early stages until 1978,Nov . 27th.Thi s day (Dl) (gastrula). An important biological parameter induction of themal e sexual cycle, was stated from the experiments:th e itsrelatio n to temperature and fecundity was shown to range between other factors.Meddr . Danm. Fiskeri- 0.7 and 2.6 million eggs for eels at og Havunders.N.S . 4:339-4o5 . body weights between o.5 and 2kg . Boëtius, I. &J . Boëtius,198o . The experimentally produced eggs Experimental maturation of female (about 1.1 mm indiameter ) gave us silver eels,Anguill a anguilla. a background for seaking up possible Estimates of fecundity and energy eel eggs in the classic Danishmate reserves for migration and spawning. rial from the Sargasso Sea.W e found Dana 1: 1-28. none. Fontaine,M. ,E .Bertrand , E. Lopez The sexual behaviour was studied by & 0. Callamand, 1964.Su r la bringing matured eels together in maturation des organes génitaux de separate tanks.Th e courtship was l'Anguille femelle (Anguilla described between the sexes both of anguilla L.) et l'émission spon which had considerably enlarged eyes. tanéede s oeufs en aquarium. Ovaries and somatic bodies of Comp. Rend.Acad . Sei. 259: matured female eels were analysed 29o7-291o. for contents of water, lipid and Fontaine,M. ,N . Delerue-LeBelle, protein.Base d on the calculated F. Lallier &E .Lopez ,1982 . energy reserves aroug h estimate of Toute les Anguilles succombent an energy budget was established. elles après la reproduction et Itwa s provided that the eel does frayent elles nécessairement en not feed during migration. mer? Comp. Rend.Acad . Sei. 294: The energy available for migration 809-811. and spawning was calculated to cover Tucker,D.W. , 1959.A new solution the costs of a 4ooo km active to theAtlanti c eel problem. migration. In his "new solution to Nature 183:495-5ol . theAtlanti c eel problem" Tucker, 1959, suggests that the European eel will never reach the breeding area in thewester n Atlantic. If so,th e reason isno t the lack of sufficient energy reserves. Since 197 9w e have continued our maturation work on female eels. We have focused at acompariso n between the effects of acetone dried carp and salmon pituitaries.Ou r background was the fact that the successful maturation and hatching results of Japanese workers were obtained by means of salmon pituitaries. Unfortunately our experimental eels were affected strongly by diseases (Vibrio, etc.) during the last two seasons. The infection,however ,di d not seem to influence the develop mental rate of the ovaries,bu t far the greater part of our eels died well before maturity. Four experimental runs (9o eels) were carried out inwhic h standard treatments with carp and salmon pituitary were run simultaneously. Very guardedly we give the conclusion that the two treatments have had very much the same effect. References Boëtius, I. &J . Boëtius,1967 . Studies in the EuropeanEel , Anguilla anguilla (L.).Experimenta l 176 APPLICATION OFLH-R HAN D LH-RHANALOGUE ST OTH EINDUCE DFINA LMATURATIO NAN DOVULATIO NO F COHO SALMON (ONCORHYNCHUSKISUTCH) . EdwardM .Donaldson ,Georg eA .Hunter ,Gle nVa nDe rKraa kan dHele nM .Dy e WestVancouve rLaboratory ,Fisherie sResearc hBranch ,Dept . ofFisherie s andOceans ,416 0 MarineDrive ,Wes tVancouver ,B.C .V7 V 1N6 Canada. Summary (SG-G100). Weals o investigated theus eo f twohig h potency LH-RHanalogue s preceded by Studies onth eus eo fLH-R Hanalogue s to priming injections of SG-G100(Donaldso ne t induce ovulation inth e coho salmon al., 1981). Inth esutdie s presented here Oncorhynchuskisutc har e reported. Two we report further studies onth eus eo fhig h potentLH-R Hanalogue s are shown toinduc e potency LH-RHanalogue s with the SG-G100 ovulation incoh o salmoneithe ralon eo r primeran d also present studies onth eus e witha primin g injection of partially ofhig h potency LH-RHanalogue salone . purified salmongonadotropin . Keywords: salmon,Oncorhynchus ,ovulation , Materialsan dMethod s LH-RH,gonadotropin . The experiments reported inthi sstud y Introduction wereconducte d atth eWes tVancouve r Laboratory ofth eFisherie sResearc hBranc h Since thefirs t studies oncontrolle d inth efal lo f19 79an d 1980. reproduction inteleost swer e conducteda half century ago inSout hAmeric a The experimental animals used wereadul t3 (Houssay,1930 ;Ihering ,1937 )significan t yrol d coho salmonOncorhynchu skisutc h from strides have beenmad e towardsmor e theCapilan oSalmo nHatchery ,Nort h efficient and reproducibleprocedures . Vancouver. Fishwer e transported bytruc k These "firstgeneration " techniques to thelaborator y and held in3 m fiberglas involved theus e ofhomologou so r tanks undernatura lphotoperiod . These heterologous pituitary extractsfro m tankswer esupplie d withaerate d wellwate r mature donor fish. Later refinements at10.0-10.5°C . involved theus e of preserved pituitary glands or partially purified gonadotropin Theexperiment s conducted in 1979 an d 1980 preparations (Donaldson,1973) . Also were initiated onOct .2 9an dOct .28 , during this period parallel procedures respectively. Fishwer e allowed to using mammaliangonadotropin s especially acclimate fora minimu m of3 day s priort o HCGeithe r aloneo ri nconjunctio nwit h treatment. Before treatment,fishwer e fish pituitary extractswer edeveloped . screened toselec t only those individuals Problemswit h theavailabilit y of whichha dno tundergon e germinal vesicle sufficient fishpituitar y glands andwit h breakdown (GVBD). Theprocedure s followed standardization have led toth e search for forth eanesthetization ,weighing ,taggin g synthetic alternatives togonadotropi nan d and interperitoneal (l.p.)injectio no f fish thedevelopmen t of "second generation" have been described byHunte r et al. techniques for theinductio no ffina l (1978). Similarily spawning proceduresan d maturation and ovulation. These methodsuse d for the determination ofeg g techniques involve theregulatio n ofth e survival toth eeye d stagean d tohatchin g production and/or release ofendogenou s have beenreporte d byHunte r et al. (1978). gonadotropin e.g.gonadotropi n releasing hormones and antiestrogens orth e Physiological salinewa suse d asth e provision of ovarianhormone s normally vehicle foral lpreparation s at0. 4ml/k g produced directly orindirectl y by bodywt . The partially purified salmon gonadotropin stimulation e.g.progestin s gonadotropinSG-G10 0use d wasprepare d andprostaglandins . according toprocedure s described by Donaldsone tal . (1972)an dDonaldso n (1973) The subject of thepresen t paper isth e from chinook salmonpituitaries . development of oneo f thesesecon d generation techniques,i.e .th eus eo f Inth e 1979 study ,th e syntheticLH-R H gonadotropin releasing hormones,fo r analogueD-Ala 6,de sGly 10-LH-RH(1-9 ) induced ovulation in thePacifi c salmon. ethylamide,(LH-RH A DAla6)wa s supplied by Inou r initial studies onthi ssubjec tw e AyerstResearc h Laboratorieswhil e investigated theus e of themammalia n the[D-Ser(Buü)6]-LH-RH(l-9)ethylamide , gonadotropin releasing hormone (LH-RH) analogue (LH-RHA DSer(But)6)wa s supplied by alone andwit ha primin g injectiono f Hoechst Canada Inc. TheLH-RH ADAla 6use d partially purified salmon gonadotropin in198 0wa s supplied byPenninsul a 177 Table 1. Induced ovulationI ncoh o salmonusin g thegonadotropi n releasing hormoneanalogue s LH-RHADAla 6an dLH-RH ADSerCBu') 6 preceded bya priming injection ofsalmo n gonadotropin (SG-G100)o rLH-RHA . Adult females Injection protocol Cummulative % Experiment Number Mean wt ± SD/kg fg/kg ovulation Day 0 Day 3 1979 LH-RH 7 2.25 ± .83 6. 6 DAla6 33 DAla6 100 primer 7 2.1 ± .35 20 DAla6 100 DAla6 100 6+ 2.3 ± .46 60 DAla6 300 DAla6 83.3 7 2.18 ± .61 6. 6 DSerCBu11)6 33 DSerCBu')6 85.7 7 1.82 ± .47 20 DSerCBu1)6 100 DSerCBu')6 100 7 2.49 ± .32 60 DSerCBu11)6 300 DSerCBut)6 100 Day 0 Day 3 1979 SG-G100 7 2.0 ± .53 100 SG-G100 30 DSerCBut)6 100 primer 7 2.25 ± .62 100 SG-G100 300 DSerfBu1)6 100 7 2.37 ± .73 100 SG-G100 30 DAla6 100 7 2.42 i .61 100 SG-G100 300 DAla6 100 7 2.22 ± .46 100 SG-G100 Saline 100 7 2.69 -t .82 Saline Saline 85.7 Day 0 Day 2,3,4 1980 SG-G100 8 2.5 ± .64 100 SG-G100 11 DAla6 100 primer 8 2.2 ± .57 100 SG-G100 33 DAla6 100 8 2.15 ± .37 100 SG-G100 100 DAla6 100 8 2.06 ± .23 100 SG-G100 .11 DAla6 100 8 2.14 ± .59 100 SG-G100 33 DAla6 100 8 1.95 ± .62 100 SG-G100 100 DAla6 100 8 1.95 ± .37 100 SG-G100 Saline 100 8 1.91 ± .42 Saline Saline 60 + One fish accidentally killed. Laboratories Inc.,an d SyndelLaboratorie s or10 0ug/k gbod ywt . Thesevent hgrou p Ltd. received saline onDay s2 ,3 an d 4. An eighthgrou preceive d salineo nDay s 0, 2,3 1979LH-R Hanalogu e primer and4 (Table1) . In1979 ,6 group s of 7fis hwer e Results administered priming injections of the LH-RHADAla 6o rLH-RH A DSer(Buc)6 at 6.6, SG-G100 primer 20o r6 0jig/k gbod ywt . Thesewer e followed onDa y3 b yinjection s ofth e The results of the experiments involving analogues at33 ,10 0o r30 0jig/k gbod ywt , the use of the SG-G100 primer are presented respectively (Table1) . in Table 1. Cumulative percent ovulations of test groups and SG-G100 and saline 1979SG-G10 0prime r control groups for 1979 are presented in Figs. 1 and 2, respectively, while those OnDa y0 ,5 group s of 7female swer e conducted in 1980 are presented in Figs. 3 administered ani.p . injectiono fSG-G10 0 and 4, respectively. In 1979, groups at 100pg/k g bodywt . OnDa y3 ,on eo f administered the SG-G100 primer followed by these groupsreceive d asalin e injection either the DAla6 or DSer(But)6 analogues; while theothe r4 receive d 30o r30 0ug/k g reached 100% cumulative ovulations by either injection ofeithe rth eDSer(Bu c)6 or Day 9 or 12. However, the group which DAla6analogues . A sixth groupwa s received the SG-G100 primer alone also administered salineo nbot hDa y 9an dDa y reached the 100% level by Day 9. The saline 3. This latter group served asa contro l control group was 57 and 71% ovulated by forbot hstudie s conducted in1979 . Days 9 and 12, respectively. 1980 In198 0th ethre egroup sadministere d 11, 33o r10 0ji go fth eDAla 6analogue/k g body In1980 ,7 group s of8 fis hwer e wt reached 100%cumulativ e ovulations on administered aprimin g injectiono f Days15 ,1 2an d 10,respectively . The SG-G100a t10 0ug/k g bodywt . OnDay s2 , groupsadministere d theSG-G10 0prime ralon e 3 and4 ,6 o f thesegroup s received didno t reachth e100 %leve lunti lDa y15 . injections of theDAla 6analogu e at11 ,3 3 OnDa y 1560 %o f thefis hi nth esalin e 178 controlgrou pwer eovulated . LH-RHprime r • 1 Cumulative %ovulation s of thegroup s administered aDSerCBu') 6 orDAla 6 analogue primer followed bya secon d injection of theanalogu e at 5x th e priming dosage arepresente d inFig .5 . Cumulative% ovulation s of thesalin e controlgrou par epresente d inFig .2 . With theexceptio n of the lowest dosageo f theDSer(Bu t)° analogue all testgroup s reached the80 %leve lo fovulation s byDa y 7o r 9(Fig .5) . Thegrou pwhic h received theSG-G10 0prime ralon e reached 71an d 100%cumulativ e ovulations onDay s 7an d Fig. 3. Cumulative percent ovulation in 9,respectivel y (Fig.2) . The percentages adult female coho salmon administered l.p. of ovulated salmoni nth esalin econtro l injections of SG-G100 at 100 fig/kg body wt groupo n these 2day swer e4 3an d57 % on Day 0 followed by injections of LH-RHA (Fig. 2). DAla6 at 11 £-X—}, 33 (-®—} or 100 ug/kg body wt on Days 2, 3 and 4. 1% t DAYS 3 4 5 6 7 e 9 10 n l'a 13 14 1 4 6 7 9 10 11 12 13 H 15 Fig.1 .Cumulativ e percent ovulation in adult female coho salmonadministere d Fig. 4. Cumulative percent ovulation in i.p.injection s of SG-G100a t10 0ug/k g adult female coho salmon administered i.p. bodyw t onDa y0 followe d bya secon d injections of either SG-G100 Ç-&—) at 100 injection ofLH-RH ADAla 6a t3 0( —©— ) or /ig/kg body wt or saline on Day 0 followed by 300^-B— )ug/k go rLH-RH A DSer(But)6a t saline injections on Days 2, 3 and 4. 30(—«— )o r30 0(—•— ) ug/kgo nDa y3 . Arrows indicateinjectio n days. % DAYS i—r »i4- DAYS Fig. 5. Cumulative percent ovulation in adult female coho salmon administered i.p. Fig. 2. Cumulative percent ovulation in injections on Days 0 and 3 of LH-RHA DAla6 adult female coho salmon administered at 6.6-33 ;ig/kg (-•—), 20-100 >ig/kg (-B—) i.p. injections of either SG-G100 f-A—) at or 60-300 jug/kg (-0—;), or LH-RHA DSer 6 100 jig/kg body wt or saline (—•—} on Day (But) at 6.6-33 Jjg/kg (—O—), 20-100 ^jg/kg 0 followed by saline injections on Day 3. 6-A—) or 60-300 ^g/kg (-«—). 179 Survival of theegg sfro m alltes t administration. Inrecen t studies conducted groups toth e eyed stagean d tohatchin g in 1981w ehav eobtaine d ovulation witha ranged from 89-100%an d 81-97%, single injectiono f LH-RHA. Aprime ro f respectively. LH-RHA followed bya single secondary injectiono fLH-RH Awa s evenmor e Discussion effective. On the otherhand ,a combination of SG-G100an dLH-RH A ina singl e injection Inou r initial studies on the induction wasmor e effective thana singl e injection of ovulation incoh o salmonusin gLH-R H ofLH-RH Aalon ebu t lesseffectiv e thantw o and itshig h potency analogues itwa s separate injections. These findingsar e evident thatLH-R Hwa sno t effective alone currently being correlated withconcommitan t for theinductio n ofovulation . Onth e changes inth e plasma gonadotropin profile otherhand ,i twa spossibl e toinduc e (VanDe rKraa k etal. ,1981 ,unpublished) . ovulationwit hLH-R Hanalogue s used in Collectively,th eresult s indicate thatth e conjunctionwit ha nSG-G10 0prime r synthetic LH-RHagonist s showconsiderabl e (Donaldson et al., 1981). Inth eabov e potentiala s second generation ovulation study the10 0Jig/k gSG-G10 0prime rwa sno t inducers inPacifi c salmonids. tested alone asi tha d been shownearlie r tob e below thecritica l dosagerequire d References toinduc e ovulation (Jalabert et al. 1978). Iti seviden t from thecurren t Donaldson,E.M . 1973. Reproductive study thata single injection of 100/ig/k g endocrinology offishes . Am.Zool . 13: SG-G100i scapabl e of inducing ovulation 909-927. incoh o salmonwhic h are close tomaturit y Donaldson,E.M. ,F .Yamazaki ,H.M .Dy e& with orwithou tLH-R Hanalogue . However, W.W.Philleo . 1972. Preparationo f in the 1980experimen t (Fig.3 )ther e gonadotropin from salmon (Oncorhynchus appeared tob ea dos erelate d synergism 6 tshawytscha)pituitar y glands. Gen. between the SG-G100an d theLH-RH ADAla Comp.Endocrinol . 18:469-481 . inwhic h thehig hdos e (100jig/k gLH-RH A 6 Donaldson,E.M. ,G.A. ,Hunte r &H.M .Dye , DAla )grou p ovulated faster thanth e 1981. Induced ovulationi ncoh o salmon other twogroups.Furthe r evidence forth e (Oncorhynchus*kisutch).II . effectiveness of the SG-G100prime r Preliminary studyo nth eus e ofLH-R Han d followed byLH-RH Awa s provided bya stud y twohig hpotenc y LH-RHanalogues . onth einduce d ovulation ofcoh o salmon Aquaculture. 26:129-141 . held inseawate r orfreshwate r conducted Houssay,B.A. ,1930 . Accion sexual del a inOrego n (Sower et al., 1982). hipofisise nlo s percesy reptiles. Rev. Soc.Argent .Biol . 6:686-688 . Furthermore,i n the experiment inwhic h Hunter,G.A. , E.M.Donaldson ,E.T .Ston e the twoanalogue s were tested alone(Fig . &H.M .Dye ,1978 . Induced ovulationo f 5)al l treatments except thelo wdosag eo f female chinook salmon (Oncorhynchus LH-RHA DSer(But)6 werea s effective as10 0 tshawytscha)a ta productio nhatchery . ug/kg SG-G100fo rinducin g ovulationi n Aquaculture. 15:99-112 . thecoh o salmon. Thestud yprovide s the Ihering,R .Von ,1937 . Ametho d for first experimentalevidenc e forinductio n inducing fish tospawn . Prog.Fish-Cult . of ovulation ina salmoni dwit hLH-R H 34: 15-16. analogue alonean d suggests thatLH-RH A Jalabert,B. ,F.W . Goetz &B .Breton , DAla"ma y be slightlymor e effective than A.Fostie r& E.M .Donaldson ,1978 . LH-RHA DSer(But)6. Wehav e recently Precocious induction ofoocyt e maturation obtained experimental evidence forth e and ovulation incoh o salmon(Oncorhynchus difference inpotenc y inth e salmonid kisutch). Fish.Res . Board Canada. 35: betweenLH-R Han d itspoten t analogues. A 1423-1429. single intraperitoneal injection of0. 2m g Sower, S.A., C.B.Schrec k &E.M .Donaldson , LH-RHresulte d ina plasm a gonadotropin 1982. Hormone induced ovulationo fcoh o (GtH)concentratio n of 14r«g/m la t1. 5h r salmon (Oncorhynchuskisutch )hel d inse a which declined toa bas e linevalu e of5 wateran d freshwater . Can.J .Fish . Mg/ml at24-4 8hr . On the other hand, Aquat. Sei. 39:627-632 . 6 injectiono f0. 2m gLH-RH A DAla resulte d VanDe rKraak ,G. ,H.R . Lin,E.M .Donaldson , ina Gt Hconcentratio n of2 07ig/m la t 1.5 H.M.Dy e &G.A .Hunter ,1982 . Effectso f hran d of2 7ng/m l at24-4 8hr . Fishi n LH-RHan d des-Gly10[D-Ala6]LH-RH - theforme r groupha d not undergone final ethylamideo nplasm a gonadotropin levels maturation after 96h rwhil emos t of those and oocytematuratio n inadul t femalecoh o inth e latter groupha d completed GVBD salmon (Oncorhynchus kisutch). Gen. (VanDe rKraa k etal. , 1982). Thisdat a Comp.Endocrinol , inpress . indicates that theeffectivenes s ofth e LH-RHanalogu e iscorrelate d withth e magnitude and duration of the increasei n plasmaGt Hconcentratio nwhic h follows its 180 INDUCED BREEDING OF GREY MULLET, MUGIL CEPHALUS L. Ching-ming Kuo International Center for LivingAquati c Resources Management MCC P.O. Box 1501, Makati, Metro Manila, Philippines ICLARM Contribution No.7 1 Abstract .70 - A: Treated, 24°-26°C This paperdescribe sth e presentstatu so f artificial propagation B: Untreated,24°-26° C of the grey mullet (Mugil cephalus) including manipulation of breeding time and induced breeding. Predictable spawningca nb e .65 induced by administration of piscine pituitary gonadotropin and . A human chorionic gonadotropin. The synthetic steroid,deoxycor 60 ticosterone (DOC) can also be used when oocytes have reached the subperipheral germinal vesicle stage (dosage 120 mg/kg fish) following a priming injection of gonadotropin, which is essential ,55 B for bringing oocytes at yolk globule stage to final maturation. _ •=.-"- " The resultso f usinggonadotropi n followed by DOCar ecompare d 50 with those usinggonadotropin s alone. Keywords: induced-breeding, hypophysation, gonadotropin, de , , , i 1 i ... 1 . . . i 1 . oxycorticosterone, Mugil cephalus. 0 10 15 20 Introduction Days From First Injection The initiation and course of gametogenesis is controlled by 1. Effect of s-GTH injections onoocyte development. environmental and endocrinological factors. In females vitello- Fig. genesis is the main developmental phase and in many captive teleosts oocytes remain at the tertiary yolk globule stage. Their final maturation often requires exogenous hormone treatments. Induced breeding Hypophysation and exogenous hormone treatments have been widely used since Houssay's work (19311-piscine pituitaries, 1. The stateo f recipients mammalian gonadotropins, steroids and synthetic luteinizing releasing hormone (LH-RH)—but have always suffered from a Recipient fish must be at the proper stageo f ovariandevelop lack of standardization between workers, particularly where ment. Identification of this stage iscritical .Variou s methodshav e spawning is induced by injections of fresh or preserved pitu been used: external anatomical characteristics, the microscopic itary material. Lack of standardization can be mainly attributed appearance of oocytes (Sundararaj and Goswami, 1969), and to inadequate descriptions of the potency of pituitary prepara physiological parameters associated with sexual maturation such tions, the sexual maturity of recipients,an dth e dosagean d injec as elevated plasma proteins and calcium concentration (Booke, tion proceduresused . 1964;Woodhead, 1968). Shehadeh et al. (1973a) and Kuo etal. (1974) described work For grey mullet, however,th e degreeo f sexual maturity isbes t with captive broodstock of grey mullet (Mugil cephalus). At expressed in terms of developmental stage of oocytes and their tempts at induced breeding of grey mullet by administration of mean diameter. exogenous hormones were first madeb y Tang (1964).Thi s paper Oocytes are removed by biopsy from unanaesthetisedfemale s summarizes recent progress using quantified gonadotropin prepa using a polyethylene cannula of 0.8 mm 1 D inserted 6 to 7 cm rations andth e synthetic steroid deoxycorticosterone. (depending on length) into the ovaries through the oviduct (Shehadeh et al., 1973b). Oocytes from the mid-portion of the Oogenesis andenvironmenta l factors ovary are the most representative. Samples from the extremities should beavoided . Vitellogenesis in captive grey mullet beginstoward sth e end of Hypophysation gives predictable spawningonl y if begunwhe n October in Hawaii andoocyte sdevelo pt oth etertiar y yolk globule the oocytes are at the tertiary yolk globule stage and the mean stage as in the breeding season,thoug h the rate of development oocyte diameter is at least 0.6 mm and preferably larger than varies among individuals.Vitellogenesi s can beinduce d by manip 0.65 mm. ulation of photoperiod and temperature regimes out-of-season: 6L/18D at 17-26°C initiates vitellogenesis after 7-9 weeks irre 2. Hormone treatments spective of preconditioning photoperiod; 6L/18D at a constant temperature of 21 C gives the best result for environmental The total dose of s-GTH required to induce spawningha sbee n advancement of vitellogenesis and prevents atresia. Vitellogenesis described elsewhere (Shehadeh et al., 1973a; Kuo et al., 1974; is accelerated at lower temperatures (17-19°C) butth e quality of Kuo and Nash, 1975). It is inversely proportional to oocyte oocytes is questionable asthei r yolk deposition is less.Suc hegg s diameter and can be estimated from a regression line (Fig. 2) to fail to develop beyond blastula stage. Oocyte development at be given in two injections: one-third of the total dose intramus 24 C can be accelerated by salmon gonadotropin injections cularly as a priming dose (which reinitiates final maturation) and (s-GTH) (Fig. 1). the remainder at attainment of subperipheral stage,usuall y 24 hr later. Ovulation occurs after about 8 hr and oviposition after about 12h r from the 2nd injection.Spawnin g behavior isinitiate d by the first release of asmal l number of ripe eggswhic h attracta male to respond and to releasespermatozoa .Spawnin g in a200- I 181 aquarium is very rapid, and is completedwithi n 30second s from mg DOC/kg. Spawnings were completed in 36 to 38 hr from the the first release of eggs. The first cleavage of the fertilized eggs priming injection. Mean oocyte diameters were 0.823-0.831 mm canb eobserve d in 55-65 minutes at 24°C (32 % salinity). and 0.923-0.944 mm at ovulation and oviposition, respectively. Thesam eprocedur e isfollowe dusin ghuma nchorioni c gonado The responses of functional mature mullet females to repeated tropin (HCG). A total of 50 lU/g body wt is required in two in daily DOC injections at 50 and -100 mg/kg are shown in Fig. 3. jections. A priming injection (16.7 lU/g body wt) is used.Th e No change in the oocyte diameter and appearance was observed effectivenesso f HCGdepend supo n its purity. Clinical grades (e.g., unlike obvious increaseswhe n usings-GTH . Antuitrin-Parke Davis and APL-Ayerst) are equally effective and spawning can be expected after about 10 hr. HCG-SIGMA,how Further work with DOC ever, gave spawning after 12h r andmultipl e oil-globule eggswer e frequently observed (thoughthes ewer e fertilizable). 1. DOCbindin g inviv o Administration of the priming dose,whethe r s-GTH or HCG, does not alter the mean diameter or diameter-frequency distribu A female containing oocytes at the tertiary yolk globulestag e tion of oocytes but at ovulation and oviposition, mean diameter was first injected with 4.7 mg/kg s-GTH,an d the oocytes atsub - increases dramatically to 0.825 and 0.923 mm, respectively, re peripheral stagewer e obtained in 24 hr,whe n 100ßC\ of3 H-DOC gardless of oocyte size at injection. Rapid increases in the water was administered intramuscularly and the 3H-DOC binding was content, electrolytes and total osmolarity of oocytes occur. followed for different subcellular fractions: amembran e fraction (precipitated at 800 x g), a mitochondrial fraction (precipitated at 10,500 x g) and the cytosol (supernatant at 10,500 x g). The highest binding level was noticed 15 min after the injection, Q 25 followed by astee p decline. A binding peakwa sals oobserve da t Y= 74.937-87.333 X 5 6 hr (Fig.4) . Ingeneral , the DOCwa smostl y bound by themem -o brane fraction (31.8-80.5% of total binding) followed by mito- o chrondria fraction (13.3-52.8%). m 20 i i- a. a> 5 0.90 Ovulation 8 lo Oviposition o 0.80 0.6O 0.65 0.70 0.70 - Initia) Mean Oocyte Diameter (mm) Fig.2 .Tota ldos eo f s-GTH requiredt o complete final maturation 0.60 A mixture of s-GTH and HCGfo r priming and spawning injec tions is also effective (Table 1): 1 mg s-GTH = 2250 IU HCG Days Spawning can also be induced by aprimin g injection of acetone dried carp pituitary homogenate (CPH: Stoller Fisheries, Iowa) Fig. 3. Response of mature mullet femalest o injections of s-GTH 50 mg/kgfollowe d by either s-GTH or HCG as above. and DOC. : s-GTH,thre e times aweek ; : DOC, 50Mg/ g daily; : DOC,10 0ßg/g daify . 3. Confirmation of the necessity for exogenous hormone treatments A female containing 0.668 mm mean diameter eggswa s left with two running males for three days. No change in the oocyte size or frequency distribution was observed and no spawning occurred. The female was then given s-GTH doses asabov e and spawning occurred 10 hr after the spawning injection (9.19 g s-GTH/g body wt: total dosage, 14.85 Mg/g body wt). This con firms that captive mature mullet are unable to complete final maturation and spawning without triggering by hormone admin istration. Induction of final oocyte maturation by manipulation of environmental conditions hasye tt o bedetermined . 4. Induced-breeding with Deoxycorticosterone (DOC) Spawning was induced by aprimin g injection of either CPHo r s-GTH, followed by aspawnin g injection of DOCa t 50-157.1mg / kg (Table 1). The response of recipients to DOC varied with the injection dose and spawning season, being more effective during the peak of the breedingseaso nan dwit h doseso f about 100-130 Fig.4 . DOCbindin g invivo . 182 2. DOC bindingwit h oocyte homogenatej n vitro Table 1. Injection dose, schedule and response of grey mullet females to gonadotropins and deoxycorticosterone (DOC). Homogenates containing 20 mg oocytes and 40 ng 3H-DOC were incubated at 24°C for various periods. Binding increased Initial Spawning rapidly during the first hour and reached equilibrium after 4-5 hr, Body oocyte hr followed by a decline. The binding of 3H-DOC (5 ng) with the wt diameter 1st 2nd after 2nd Fertilization membrane fraction of the oocytes isfurthe r shown in Fig.5 .Th e (g) (mm) injection injection injection (%) binding increases proportionally with the amount of oocyte tissue upt o 100 mghomogenate .Th e binding capacity of oocyte homo- A genate (50 mg wet wt) increases linearly with the concentration s-GTH1 s-GTH of H-DOC up to 5 ng,an ddoe sno t differ between4-h r and 5-hr (Mg/g> (Mg/g) incubation period. 872 0.651 5.7 11.4 12.0 98 To determine the specificity of the binding of the oocyte 980 0.711 5.1 10.2 11.8 83 homogenate with DOC,homogenate s containing 50m go f oocytes 714 0.623 7.0 14.0 12.3 92 and 2.5 ng of H-DOCwer e incubated for 4 hr with various con 1010 0.665 5.0 9.9 10.0 93 centrations of cold hormones,hydrocortison e andcorticosterone . 717 0.664 7.0 14.0 11.5 98 The inhibition curves of the steroids examined were very similar 862 0.613 5.8 13.9 10.5 87 (Fig. 6), though corticosterone seemed to compete slightly more B in the binding with oocyte homogenate to H-DOC.Th e specific HCG2 HCG binding of oocyte homogenate to H-DOC reduced to 50% of (lU/g) (lU/g) total binding when the corticosterone and hydrocortisone con 759 0.645 19.6 30.4 15.0 98 centration was increased beyond 10 ßgan d 100 jug,respectively . 908 0.621 19.3 38.6 13.2 90 1032 0.625 17.2 34.4 12.0 94 C s-GTH HCG 800 X g fraction (Mg/g) (lU/g) 600 0.679 6.7 33.3 ' 12.2 96 557 0.652 9.0 44.9 13.0 94 D CPH3 HCG (Mg/g) (lU/g) 900 0.632 55.6 33.3 10.5 95 750 0.646 66.7 33.3 10.0 91 900 0.640 55.6 33.3 10.2 90 1000 0.674 50.0 30.0 11.7 83 800 0.667 62.5 31.3 9.5 94 750 0.687 66.7 33.3 9.5 77 E CPH DOC (Mg/g) (Mg/g) 1000 0.704 50.0 100.0 12.0 68 850 0.662 58.8 117.7 12.5 86 Tissue (mg) 1000 0.690 50.0 100.0 10.8 74 Fig. 5. Binding of DOC (5 ng) with membrane fraction of oocytes 900 0.629 50.0 105.6 12.2 .90 in vitro. 1100 0.635 50.0 95.5 12.5 92 900 0.634 55.6 111.1 11.4 91 1000 0.657 50.0 130.0 13.5 92 900 0.677 55.6 122.2 10.2 72 850 0.674 58.8 141.2 16.0* 73 700 0.707 71.4 157.1 16.2* 94 60 1100 0.611 50.0 136.4 20.3* 82 F c S s-GTH DOC corticosterone c (Mg/g) (Mg/g) 600 0.662 7.8 110.0 11.5 91 55 hydrocortisone 745 0.618 6.7 50.0 23.0 82 I / 693 0.633 7.2 50.0 21.5 84 835 0.634 4.8 50.0 21.0 76 1. s-GTH:Salmo n pituitary gonadotropin (SG-G100). 2. HCG: Human chorionic gonadotropin 50 3.CPH :Car p pituitary homogenate (acetone dried) "Experiment was conducted toward the end of natural breeding I 2 3 4 5 6 season. log (ng) cold hormone Fig. 6. Competitive binding of hydrocortisone and corticosterone with membrane fraction of oocytes. 183 Discussion Houssay, B.A. 1931. Action sexuelle de l'hypophyse sur lespois sonse t lesreptiles . CR. Soc. Biol. 106:377-378. Ovarian development in grey mullet can be accelerated by Jalabert, B. 1976. In vitro oocyte maturation and ovulation in manipulation oftemperatur e(17-21°C ) or by injection of gonado rainbow trout (Salmo gairdneri ), northern pike (Esox lucius), tropin preparations, but without further hormone stimulation and goldfish (Carassius auratus ). J. Fish. Res. Board Can. captive females will not complete the final oocyte maturation 33: 974-988. in captivity, and oocytes will undergo atresia and degenerate. Jalabert, B., B. Breton,an dC . Bry. 1972. Maturation et ovulation Oocytes become responsive to exogenous gonadotropins and in vitro des ovocytes de la truite arc-en-ciel Salmo gairdneri . DOC when they have reached the subperipheral germinal vesicle CR. Acad.Sei . 275:1139-1142. stage. Jalabert, B., B. Breton, E. Brzuska,A . Fostier, andJ .Wieniawski . The action of piruitary gonadotropins on final oocyte matura 1977. A new tool for induced spawning, the use of 17a- tion isknow nt o be mediatedthroug h steroid hormones (Jalabert, hydroxy - 20/3 - dihydroprogesterone to spawn carp at low 1976). In vitro ovulation has been induced effectively by corti temperature.Aquacultur e 10:353-364. sone in Misgurnus fossilis (Kirshenblatt, 1959) and by deoxycor Kirshenblatt, I.D. 1959. Effect of cortisone on ovaries of the ticosterone acetate (DOCA) in Heteropneustes fossilis (Goswami loach. Bull. Eksper. Biol. Med.USSR .47:108-112 . and Sundararaj, 1971). The ability of gonadotropins to promote Kuo, C.-M., CE. Nash, and Z.H. Shehadeh. 1974. A procedural corticosteroidogenesis has been demonstrated by Sundararaj and guide to induced spawning in grey mullet (Mugil cephalus L.). Goswami (1969, 1971). They proposed that gonadotropins may Aquaculture 3:1-14. not act directly on the ovary to stimulate ovulation, but via the Kuo, C.-M., and CE. Nash. 1975. Recent progress onth e control interrenal tissue by stimulating corticosteroid production. By of ovarian development and induced spawning of the grey contrast, final oocyte maturation canb einduce dj n vitro by carp mullet (Mugil cephalus L.) Aquaculture 5:19-29. pituitary gonadotropin or progesterone in Salmo gairdneri (Jala Shehadeh, Z.H., C.-M. Kuo, and K.K. Milisen. 1973a. Induced bert et al., 1972). Progesterone or hydrocortisone in Oryzias spawning of grey mullet Mugil cephalus L. with fractionated latipes (Hirose, 1972), and in hypophysectomized goldfish salmon pituitary extract.J . Fish. Biol. 5:471-478. in vivo (Yamazaki, 1965). More recently, Jalabert et al. (1977) Shehadeh, Z.H., C.-M. Kuo, and K.K. Milisen. 1973b. Validation concluded that the action of gonadotropins on final maturation is of an in vitro method for monitoring ovarian development in mediated through steroid hormones; 17a hydroxy-20/î dihydro- the grey mullet (Mugil cephalus L). J. Fish. Biol. 5:489-496. progesterone (17a-200 P) appears to beth e most likely natural Sundararaj, B.I., and S.V. Goswami. 1969. Role of interrenal mediator of oocyte maturation in rainbow trout, northern pike in luteinizing hormone-induced ovulation and spawning in and goldfish (Fostier et al., 1973;Jalabert , 1976). the catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Spawning of grey mullet has been repeatedly induced by a Endocrinol. Suppl. 2: 374-384. priming injection of carp pituitary homogenate, s-GTH, or HCG, Sundararaj, B.I., and' S.V. Goswami. 1977. Hormonal regula followed by a spawning injection of DOC. The priming injection tion of in. vivo and 'mvitr o maturation in the catfish, Hete merely stimulates the oocyte developmentfro m thetertiar y yolk ropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 32: globule to the subperipheral germinal vesicle stage in 24 hr and 17-28. this can be achieved only by treatments with agonadotropi n pre Tang, Y.A. 1964. Induced spawning of striped mullet by hor paration. mone injection.Jap .J . Ichthyol. 12:23-38. The use of steroid hormones targeted at the gonadst o induce Woodhead, P.M.J. 1968. Seasonal changes in the calcium con final maturation and spawningafte r aprimin g doseo f gonadotro tent of the blood of arctic cod. J. Mar. Biol. Assoc. U.K. pin is a technique that merits further investigation. Steroids, 48:81-91. such as DOC and 17a-20 (3P,ar e relatively cheap and availablea s pure preparations. They could give culturists ausefu l method for timing the final stage leading up to spawning of captive brood- stock. Oocytes are capable of not only synthesizing DOC but also of binding exogenous DOC which is detectable in aver y short time after receiving in vivo administration. Furthermore, the exo genous DOC is bound in various subcellular fractions, though mostly inth e membranefraction . References Booke, H.E. 1964. Blood serum protein and calcium levels in yearlingbroo k trout. Progr. Fish Cult 26:107-110. Fostier, A., B.Jalabert , and M.Terqui . 1973.Actio n predominate d'un dérivé hydroxyle de la progesterone sur la maturation jn vitro des ovocytes de la truite arc-en-ciel, Salmo gairdneri. CR. Acad.Sei . 277 Sei.Ser . D.421-424 . Goswami, S.V., and B.l. Sundararaj. 1971. In vitro maturation and Ovulation of oocytes of the catfish Heteropneustes fossilis Bloch. Effects of mammalian hypophysial hormones, catfish pituitary homogenate, steroid precursors and metabolites,an d gonadalan dadrenocortica l steroids.J . Exp.Zool . 178:467-478 . Hirose, K. 1972.Biologica l study on ovulation in vitroo f fish. IV. Induction in vitro ovulation in Oryzias latipes oocytes using steroids. Bull. Jap.Soc .Sei . Fish. 38:457-461. 184 EXPERIMENTS ONARTIFICIA LMATURATIO N ANDFERTILIZATIO N OFTH EJAPANES EEE L (ANGUILLA JAPONICA) K.Yamauch ian dK .Yamamot o Department ofBiology ,Facult y ofFisheries ,Hokkaid oUniversity ,Hakodat e 041,Japa n Summary Experimentalmaturatio n ofmale s Gonadalmaturatio n ofJapanes eeel swa s Becauseo fth edifficult y inobtainin gmal e induced infemale sb y injectiono fchu m silver eels,cultivate dmale shav ebee nuse d salmonpituitar yhomogenat ean d inmale sb y inexperimenta lmaturatio n inJapan . Synaho injectiono fSynahori no rhuma nchorioni c rin (mixtureo fhypophysia l and chorionic gonadotropin. Miltwa s obtained after6 gonadotropin;Teikok u Zoki)ha sbee nuse da s injections and full-grownoocyte swer e thegonadotropi chormone ,bu towin g tosus obtainedafte r 8-15 injections. However, pensiono fit sproduction ,huma nchorioni c onlya smal lproportio no fth eoocyte so f gonadotropin (HCG:Teikok u Zokio rSanky o femalesunderwen t finalmaturatio n inrespons e Zoki)ha sbee n substituted (Motonobu et al., tothi streatment . Finalmaturatio nan d 1976; Matsumaruan d Ishii,1979 ;Satoh , 1979). ovulationcoul db einduce d in vitro by Cultivatedmal eeel s (200-300g inbod y steroid treatmentbu tno tb y salmongonado weight)wer epurchase d froma commercia lee l tropin (SG-G100)treatment . supplier inHakodate . Theywer ekep t incir Matureoocyte swer espherical ,whit ean d culating seawater tankswit ha capacit yo f transparentwit h adiamete r of1. 0mm . A 5.5 gallons at19°C . Intramuscularinjec highpercentag eo f larvaehatche d outfro m tionso f Synahorin (50RI//ish )o rHC G (250 artificially-inseminated oocyteskep t insea - IU/fish)wer e givenonc ea week . Fishwer e water at 23°C. Hatching occurred 38-45h r not fed throughout theexperimenta lperiod . after fertilizationan d thenewly-hatche d Thetesti so fcultivate d eelsprio rt o larvaemeasure d about 2.9m mi nbod ylength ; treatmentha drestin go rdividin g germcells , pectoralfin swer eno tvisibl ebu t themembra incontras t toth etesti so f silvermale s nousfi nwa swel l developed. Thelarva ewer e whichha d spermatocytes. However,i nculti kepta t 23°Co nth eda y ofhatching . Subse vatedmale s treatedwit h 2-3 Synahorininjec quently,th elarva ewer emaintaine d at19°C . tions,spermatocyte s couldb e found. These At 14day safte rhatching ,the ywer eabou t males showedmarke d secondary sexcharacter s 7.0m mlong ;teeth ,ja wan dpectora lfin s and after6 injection s spermatozoa couldb e weredeveloped . Thelarva edi dno t survive extrudedb y stripping theabdome n (Yamamoto forlonge rperiods . Thisma yb edu et oth e et al., 1972). Male eelstreate dwit hHC G lacko f suitable food and/or failureo fth e showed similar development of thetesti sa s yolksa ct oprovid esufficien tnutrition . tothos e treatedwit hSynahorin . Keywords:Japanes e eel,artificia lmaturation , However,thes emethod s of experimental gonadotropin,larvae . maturationo fcultivate dmale sar eno t totally satisfactory,particularl y since Introduction theseeel sha d spermo fhig hviscocit yan d of smallquantity . Ineel sfro mwhic hsper m Thelif ehistor y ofth eee li scomple xan d couldb eextruded ,th esper mrati orepre theecolog y ofmigratio n and spawningar e sentedb y therelativ eamoun t of spermatozoa stillope nt oquestion . Although thebreedin g toth etota lgermina lelemen t showedlo w place of theEuropea nee lha sbee ndetected , values (50-75%)relativ et oothe r fish (e.g., themorpholog y and ecology ofee llarvae , more than95 %i nth echu m salmon, Oncorhynchus especially inpreleptocephalu s stages,remai n keta; Hiroian dYamamoto ,1968) . Thislo w tob e investigated. Apart from thesebiologi valueseem s tob erelate d toincomplet e calinterests ,i nJapa ni ti simportan t that spermatogenesis and spermiation. Sincethes e a reliablesuppl y ofth elarva e isestab processes arebelieve d tob eregulate db y lished,sinc eee lcultur ei sextensive . gonadalsteroi dhormones ,particularl yandro Wean d other investigatorshav esucceede d gensan dprogestogens ,th emeasuremen to f inobtainin g eggsan d larvaeo f theJapanes e plasmasteroi dprofile s duringgonadotropin - eel, following artificialinductio no fmatu induced testicular developmentma y identify rationi naquaria . However,ther eremain s andhel p tosolv esom eo f theseproblems . numerousproblem s tob esolved ,a swil lbecom e apparent inth epresen t descriptiono fth e Experimentalmaturatio n of females experimentalmaturatio no fJapanes eeel si n Japan. Experimentalmaturatio no f femalesilve r Japaneseeel sha sbee n carried outb yusin g Observationsan d discussion differentmethods . .Thesemethod s canprinci pallyb eclassifie d asfollow s: method s 185 PRIMARY SECONDARYI TERTIARY MIGRATORY NIH YOLK STAGE YOLK STAGEIYQLK STAGEICLEUS STAGEl J30- •1100 •1000 _ E • 900 3 §20- NUMBER OF INJECTIONS 500Ï 400 - i 1 1 1 1 r 1 1 1 1 0123466789 NUMBER OF INJECTIONS Fig.1 .Correlatio nbetwee nchange s inbod y weight (solid line)an d oocytediamete r (brokenline )i nfemal eJapanes e eelsin jectedwit h chum salmonpituitar yhomogenate . using Synahorino rHC G (Ochiaie t al., 1974; Satoh,1979) ,an d thoseusin g fishpituitar y homogenate (Yamamotoe t al., 1974;Yamauch i et al., 1976;Motonob ue t al., 1976;Matsu - maruan d Ishii,1979) , thelatte rbein gmor e effective thanth eforme r inobtainin g PXVJ^W\^ matureeggs . 300 500 700 900 1100 OOCYTE DIAMETER ( (jm) Silver eelswer ecaugh t during theirdown streammigratio n inth eHiranum a river (Aomo- riprefecture )durin g October andNovember . Fig. 2.Change s inbod yweigh t (I)an d fre Fishwer e transferred toHakodat ean draise d quency distribution (II)o foocyte s infemal e inseawate r tanksi na manne r similar totha t Japaneseeel sinjecte dwit h chum salmonpitu described formal e eels. Intramuscularin itaryhomogenate . Biopsywa s carried outa t jections of chumsalmo npituitar yhomoge thetim eo f (a),2 day safte r (b),an d 5day s nate (2pituitaries/500 g BW)wer egive n after (c)th epea ki nbod yweight .Th eshade d weekly. area inI Isignifie s degeneratedoocytes . Bothbod yweigh t and oocytediamete r increased progressively during thetreatmen t theoocytes ,sinc e thewate r content ofth e (Fig.1) . Vitellogenesiswa s completed after ovary increased inparalle lwit h theris ei n 6-8 injections and theoocyt ediamete r bodyweight . Incontrast ,th ewate r content attained themaximu m sizeo fabou t 1.0m m of thelive ran dmuscl eremaine d unchanged. after 7-9 injections,whe nth egermina l Tohel punderstan d thefailur eo fth e vesiclewa s situated atth eperipher y ofth e majority offis ht ounderg omaturatio nan d ooplasmnea r theanima lpol e (migratory ovulation,th ediamete r ofth eoocyte s atth e nucleus stage). Theaverag enumbe ro f timeo f thepea ki nbod yweigh tan d during oocytesa tthi sstag ewa sabou t1,300,00 0pe r thedeclin ei nbod yweigh twa sassesse db y ovary. Serumvitellogeni n levelsassesse db y biopsy. As showni nFig .2 ,th eovar ycon thesingl eradia limmuno-diffusio ntechniqu e tained oocytesa tvariou s developmental (incollaboratio nwit hDr .Hara )showe da stages,fro mth eprimar y yolkstag e toth e marked increasefro mabou t 100ug/m lvitello migratorynucleu s stage. Alarg epercentag e genint o5000-10,00 0yg/m lafte r thefirs t of thebigges t oocyteswer edegenerate da t injectionan d apart froma tendenc y tode thepea ko fbod yweigh t and thispercentag e creasei nth emiddl eo f thetreatmen t these increased asth ebod yweigh t declined. Iti s levelswer eessentiall ymaintaine d throughout notclea ra tpresen twhethe r thissiz edis theexperimenta lperiod . Oneexceptio nt o tributiono foocyte swoul dnaturall y occuro r thispatter nwa si nfis hi nwhic hinitia l if iti sabnormal ,induce db y thehormon e controlvalue swer ever yhigh ;th etreatmen t treatment. Ifi ti sa natura lphenomenon ,i t appeared toinduc ea sligh t increase. Body may suggest thateel sspaw nmor e thanonc e weight increased slowlyunti l 9-10 injections ina reproductiv eseason . hadbee nadministere d and thensharpl yin creased. Ina smal lproportio n ofanimal s Radioimmunoassay analysiso fseru mfo r ovulationoccurre d 1-3 injections afterth e various steroidsdurin g thetreatmen tperio d beginning of theshar p increasei nbod yweight , showed thatestradiol-17f 5 (method ofKagaw a but themajorit y didno tunderg omaturatio n et al., 1981)level sremaine d low (1-2ng/ml ) and ovulationan dbod yweigh t subsequently inth eperio d inwhic hbod yweigh t remained repidly declined. Theincreas e inbod yweigh t essentially unchanged,an d levels increased seems tob edirectl y related tohydratio no f with theshar p increasei nbod yweight ; valueswer ehoweve r stikingly inconsistent 186 betweenindividuals ,rangin gbetwee n 2-10 sary forfina loocyt ematuratio n and ovulation ng/ml. Theseobservation s onEstradiol-17 ß tooccur . Cytological evidencesuggest stha t levelsar edifficul t toreconcil ewit hth e thisi sals oth ecas e inth eeel . Aftera numerousrepor t ofincrease dEstradiol-17 0 serieso f injections ofpituitar yhomogenate , levelsassociate dwit hvitellogenesi san d pituitary GTHcell saccummulate dhormon e decreased levels inth epost-vitellogeni c granuleswhic h disappeared immediately after period. ovulation (Fig.3) . Incontras t eelswhic h Full-grownimmatur eoocyte scoul db e didno tovulat epossesse d GTH cellsdisplay induced tomatur e in vitro withvariou s ingheav y granulation. Theseresult s suggest steroids,th emos t effectivebein gproges thatth eprocesse s controlling vitellogenesis terone. However,a subsequen t experiment and oocytematuratio nma yno tb e sequential showed that17a,20ß-dihydroxy-4-pregnen-on e inth eee lan d that suitablehormon etreat was themos t effectivematuration-inducin g ment foracceleratin gvitellogenesi s isno t steroid. 17a-Hydroxyprogesteronelevel s necessarily effective formaturatio nan d wereundetectabl e (lesstha n3 0pg/ml )i nth e ovulation. Thepotentia lus eo fLH-R H asa n ofbot hovulate d andnon-ovulate dfemales . agent forinduce dmaturatio n andovulation , Sinceprogestogen s areknow nt ob edirectl y by causing anGT Hsurg ewa s exploredbu t involved inth einductio no f finaloocyt e effortswer eunsuccessful . However,ther e maturation ina numbe r of teleosts,th elac k arereport so fa positiv e effect ofLH-R Ho n of evidencefo rprogestoge n synthesisi n maturation and ovulation inJapanes e eels, eelswhic hunderwen t finaloocyt ematuratio n although thesucces s ratewa s low (Motonobu isdifficul t toexplain ,particularl ya s et al., 1976;Satoh ,1979) . Furtherinvesti 17a-hydroxyprogesteronei seffectiv e forth e gationma yhoweve r allowth epresen tmetho d inductiono f finalmaturatio n inee loocyte s ofexperimenta lmaturatio n tob erefine dt o in vitro. Incontrast ,partiall y purified includethi sagent . chinook salmongonadotropi n (SG-G100)a ta As theforegoin g indicates,a numbe ro f concentrationo f1 an d 10yg/m lha dn o problems remain tob eovefcom ei nth eexperi effect onmaturatio n in vitro. This in mentalmaturatio n ofJapanes e eels. Further vitro datasuggest s thatth eoocyte shav e worko nth ephysiologica l changes thatoccu r thecapacit y torespon d tosteroida lmatura - aftertreatmen t overlon gperiod swit h tionalagents ,an d thatth efollicl ehoweve r gonadotropicpreparation s should helpiden isi nsom ewa y deficient,an d cannotsythe - tify theshortcoming s of thepresen tmethod . sizematurationa l steroids inrespones et o gonadotropin. Nonetheless ultrastructural Development ofegg san dlarva e observationso nth efollicl ereveale d special thecalcell so factiv eappearanc ewhic h Thespherica l ripeegg sobtaine dwer e possessed characteristics of steroidsecret transparentwit h adiamete ro fabou t 1.0mm . ingcells . The failureo f SG-G100t oinduc e Theywer e inseminated inlarg eglas spetr i finaloocyt ematuratio nma yb edu et osevera l dishesb y thedr ymetho dwit h spermcollecte d possibilities including aspecificit y ofth e from 2-3male s induced tomatur eb yhormon e folliclefo r eelgonadotropi n and/or the treatment. A suitabletemperatur e fordeve incomplete development of thefollicl ewit h lopmento f eggsseem s tob ei nth erang eo f regard toth esynthesi s ofmaturatio n inducing 20°-25°C,judgin g fromrat eo fdevelopmen to f steroid(s). Asestradiol-17 ßlevel sar e eggsfro m 3h rafte r fertilizationt ohatch highdurin ghydratio no f theoocyt e therei s ing. Hence,th edevelopmen t of theferti also thepossibilit y of inhibitiono fmatura lized eggs innorma l seawaterwa sobserve da t tionb y thissteroi db y anactio no nth e 23°C (Fig.4-a,b) . Theseegg skep t at23° C follicle,a sreporte d inrainbo wtrou t hatched out38-4 5hour safte rfertilization . (Jalabert,1976) , ifestradiol-17 ßi spro duced inrespons e toSG-G10 0 in vitro. Theoptimu m rearingwate r temperature showed atendenc y todecreas ewit h timeafte r Insevera l species,i tappear s thata hatching andlarva ewer ereare d at23° Co n preovulatory surgeo f gonadotropin isneces theda yo fhatchin g and subsequentlymain - Fig. 3..Electro nmicrograph s ofgonadotroph s inth epituitar y gland offemal eJapanes eeel s injectedwit h chumsalmo npituitar y homogenate. a,initia lcontrol ;b ,ee lwit hovar ycontain ingoocyte sa t themigrator y nucleus stage;c ,ee lsoo nafte rovulation . a-c,x 4800 . 187 \ *' K l.J /•% *a V/y"* * -',' -' - mg i~~ d Fig.4 .Livin g eggsat :a ,eight-cel lstage , x37 ;b ,hear t formationstage ,x 37 . Liv inglarvae :c ,jus tafte rhatching ,x 23 ;d , 14thda yafte rhatching ,x 19 . tained at1 9°C . normally foru p to14-1 7day san d thendie . Thelarva esurvive d for1 4days . Newly- Twopossibl ean d related factorshav ebee n hatched larvaewer eabou t 2.9m m inbod y tentatively identified,lac ko f suitable length,an d theymeasure d 7.0m m longb y1 4 external food supply,an d insufficient daysafte rhatching ;teeth ,ja wan dpectora l supply ofnutrient s fromth eyol k sacs, finswer edevelope d (Fig.4-c,d) . Thelarva e possibly duet oincomplet evitellogenesis . didno t survivefo rlonge rperiods . Satoh (1979)reporte d thatlarva egive na certai n zooplanktoncoul d survivefo r longerperiods , References up to1 7days . Themortalit y oflarva ema y thusb erelate d toth elac ko fsuitabl efood . Hiroi,0 .& K .Yamamoto ,1968 . Studieso n Related tothi si sth epossibilit y thatth e thematuratio n of salmonidfishes . I. short survivalo flarva ema yb elinke d toa n Changes inth etesti so f thechu msalmon , insufficient development of theyol ksuppl y Oncorhynchus keta, during anadromousmig duringvitellogenesis . This suggestion ration. Bull.Fac .Fish .Hokkaid oUniv. , arises from thebiops y datadurin goocyt e 19:173-184. growthwhic hreveale d oocytes inal lstage s Jalabert,B. ,1976 . In vitro oocytematu ofdevelopmen t and from theobservatio ntha t rationan d ovulation inrainbo w trout thesurviva lperio d oflarva evarie dbetwee n (Salmo gairdneri), northernpik e (Esox individualfemales ,rangin g from5-1 4 days. lucius), andgoldfis h (carassius auratus). Thisha sals obee nreporte db y otherworkers . J.Fish .Res .Bd .Can .33:974-988 . Thecause so flarva lmortalit yma y thusb e Kagawa,H. ,K .Takan o &Y .Nagahama ,1981 . rooted inth evitellogeni cperiod . Correlationo fplasm aestradiol-17 6an d progesterone levelswit h ultrastructure Conclusion andhistochemistr y of ovarianfollicle s in thewhit espotted-char , Salvelinus Spermatogenesisan dvitellogenesi s can leucomaenis. CellTissu eRes. ,218:315 - bestimulate db ygonadotropi c preparations 329. butsubsequen t "maturationprocess "o fth e Marumatsu,Y . &T . Ishii,1979 . Studiesa n germcell s (spermiationan d finaloocyt e artificialmaturatio n of femaleJapanes e maturation)onl yoccu r ina ver y lowpro eels. Bull.Chib aPref .Fish .Exp .Stn . portiono fanimals . Preliminaryphysiologi 3:13-17. (inJapanese). . caldat asuggest s thata lac ko fdevelopmen t Motonobu,T. ,K .Yamashit a &H .Oka ,1976 . of gonadal somaticelement s involved in Amemoi r onth ehatche d larvaeo fth e steroidogenesisma y account forthes e Japaneseeel , Anguilla maponoca, fromth e observations; Larvaeappea r todevelo p adultsmature d artificially. Bull. 188 ShizuokaPref .Fish .Exp .Stn .10:87-90 . (inJapanese) . Ochiai,A. , S.Umed a& M .Ogawa ,1974 . On theacceleratio n ofmaturit y of thecata - dromous female eelb yhormon einjection , and changes inth elive r andbloo dcharac ter. Bull.Jap .Soc . Sei.Fish. ,40(1) :43 - 50. (inJapanes ewit hEnglis h summary). Satoh,H. ,1979 . Towards thecomplet e cultivation ofJapanes e eels. Iden, 33(11):23-30 . (inJapanese) . Yamamoto,K. ,0 .Hiroi ,T .Hiran o &T .Mori - oka,1972 . Artificialmaturatio no f cultivatedmal eJapanes eeel sb y synahorin injection. Bull.Jap .Soc .Sei .Fish. , 38(10):1083-1090. (inJapanes ewit hEnglis h summary). Yamamoto,K. ,T .Morioka ,0 .Hiro i &M .Omori , 1974. Artificialmaturatio n offemal e Japaneseeel sb y theinjectio no fsalmoni d pituitary. Bull.Jap .Soc .Sei .Fish. , 40(1):l-7. (inJapanes ewit hEnglis h summary). Yamauchi,K. ,M .Nakamura ,H .Takahash i& K.Takano ,1976 . Cultivation of larvaeo f Japanese eel. Nature,263(5576):414 . Acknowledgement I thankDr .G .Young ,Nationa l Institute forBasi cBiology ,Okazaki ,Japan ,fo rhi s criticalreadin g ofth emanuscript . 189 GONAD DEVELOPMENT IN FOOD FISHES OF KUWAIT R.Abu-Hakima, C.El-Zahr, M.Al-Shoushani, S.Akatso, K.Abul-Ellah Mariculture and Fisheries Department, Kuwait Institute for Scientific Research, Kuwait. Summary The seasonal changes iin the gonadosomatic index (GSI) correspond closely to the matu A knowledge of the biology of food fishes ration stages in the three species. The GSI including reproduction, is important for the is low in the non-breeding season and maximal successful management and mariculture of in February and March during the spawning these organisms. The silvery croaker, period (Fig. 2). Otolithes argenteus (Fam.Soiaenidae), the silvery grunt, Pomadasys argenteus (Fam. Five-stages of oogenesis can be Pomadasyidae) , and the yellow-finned black distinguished in the ovaries of the thret porgy, Aoanthopagrus latus (Fam.Sparidae), species. Small oogenia, closely associated are among the most important food fishes in to the lamellar membranes, and oocytes in Kuwait. In our studies, aspects of the repro the primary growth phase predominate in the ductive biology of the three species are resting gonads. Oocytes in early vitello- examined and some of the results presented genesis and' active vitellogenesis are found here. mostly in maturing gonads. Hydrated, mature eggs occur just prior to spawning. In the Maturation of the gonads, in the three post-ovulatory gonads the lamellae are species, which can be divided into eight collapsed and oocytes undergoing breakdown arbitrary stages, following a distinct se and resorption occur. quential pattern over one annual reproductive cycle (Fig. 1). Immature and resting gonads are found predominantly in the late summer and early winter months. During the breeding season, from January to April, gonads in the maturing and spawning stages of development occur. F _ CÜ 1 D _ CUM m N _ 0_ i - A _ J _ M _ A _ M _ F -\—i—i—rri—i—r ' i i rrlTi i 12 3 4 5 6 7 8 12 3 4 5 6 7 8 $• Stages Fig. 1:Seasona l changes inth ematuratio n stages (1-8)o fth egonads . 190 ui Otolithesargenteu s 0 Acanthopagrus latus 70 15. 10. 1r 5_ ^*-.rf rSntrrf AMJJASONDJFM ASONDJFMAMJJ FMAMJJASONDJ Months Fig. 2. Changes in the GSI in male andfemal e fish. The distribution of oocytes of different sizes within the ovary is a good indicator of the spawning habits of fish. Oocyte development in all three species examined here is of the "group-synchronous" type. A hetero-genous population of small oocytes give rise to a synchronous population of larger oocytes released in the spawning season. 191 ON SOME PATTERNS OF REPRODUCTIVE PHYSIOLOGY INMAL E TELEOST FISH. R. Billard Laboratoire de Physiologie des Poissons, I.N.R.A., Campus de Beaulieu 35042 RENNES France Two main types of testicular structure, displacement during spermatogenesis. The associated with different patterns of sperma spermatozoa are free,movin g from the lobule togenesis, spermatology and reproductive to a short, non-secretory efferens duct and physiology, can be identified in teleost then to a sperm duct. The seminal fluid ori fish. A tubular type characterizes the testis ginating from the Sertoli cells and the secre of the Poecilidae (e.g. gupp.y) in which sper tory epithelium of the sperm duct is abundant. matogenesis is continuous. Type A spermato Sperm morphology is very simple. The sperm gonia (G.) are restricted to the proximal head is spherical or slightly elongated and blind end of the tube in which they divide the mid-piece is quite reduced. The process and organize into cysts (Gß) that move to of spermatogenesis is a subcontinuous process the distal extremity. The spermatids adhere in some species as carp, in which spermiation tightly to the Sertoly cells and, after may occur nearly all the year round, provided spermiation, the spermatozoa remain packed that such environmental conditions as tempe together in spermatozeugma. Spermiogenesis rature are satisfactory. In many other species is complicated, and the most complex sperma such as trout, spermatogenesis is seasonal tozoa are found in teleost fish. They have and discontinuous, even if the environmental highly condensed chromatin, an elongated conditions are satisfactory, and a new sper- head, a large mid-piece with glycogen stores matogenetic cycle starts only when the and a highly modified centriolar complex. spermatozoa from the previous cycle have Sperm motility is very long (a few hours or been released from the testis. days) and sperm survival can reach a few months in the ovarian cavity of the female The efficiency of spermatogenesis is quite (internal fertilization). Seminal fluid variable in fish. The maximal value of the secretion is very limited, being restricted GSI varies from 0.1% in Leporinus (Godinho, to the efferent duct; this seems to be rela personal communication) to 101 or sometimes ted to the existence of spermatozeugma which more in salmonids (see Table). Annual sperma- probably constitute a very efficient way of togenetic production, expressed in number of transferring the sperm to the female. spermatozoa per g of body weight for inter species comparison, varies from 100 to 7000 In the lobular-type testis, the seminife millions in the species listed on the table. rous tube is often anastomosed into a network The GSI in females is more stable (20-3o%) of connective tissue originating from the but fecundity is very variable because of the testicular capsule. In histological sections, great differences in egg size. When spermatp- this network appears in the form of a lobe, genetic production is expressed in 10° sper hence the term "lobule". In addition, this matozoa per egg laid by a female of similar type of structure seems to be homologous to size, the ratio varies from 3500 for trout to the lobule of the mammalian testis and may only 1.65 for Leporinus• Th e minimal number correspond to a primitive form of testicular of spermatozoa that need to be inseminated structure. to fertilize one egg is much lower, being about 2000 when fertilization is internal and These lobules have a permanent layer of about 10000-30000 when it is external. In the Sertoli cells with GA at the inner periphery latter case, the value is optimistic because and a central lumen in which-th e spermatozoa insemination would be carried out in a are released from the cysts at the end of diluent. Approximately 10 times more sperma spermatogenesis. The cysts are rather statio tozoa would be needed in fresh water. nary, showing only a slight centripetal Table.Efficiency of reproduction in some teleost fish species GSI (maxi) . Spermatogenet ic Sperm No of spe rmatozoa Fecunc it y , Egg Species production ameter moti1 it y per egg per egg 0* egg/g b.w Î 10° spz/g b. w mm min laid (109) insemimated Trout 10 25 7000 2 4 1 3 500 30 000 Carp 8 30 4000 100 1.2 10 40 10 000 Bike 2 20 600 30 2 2 20 30 000 Leporinus 2 20 100 90 1.1 — 1.65 -- Guppy 5 20 2700 30 1,5 120 90 2 000 192 ULTRASTRUCTURE OFTESTI S ORGANIZATION ANDRESTIN G CELLS INLiz a aurata Risso 1810, (Teleostei, Mugilidae) S. Bru s 1 e Laboratoire deBiologi e Marine, Universite dePerpignan , France Summary In Teleosts, thetubula r structure ofth e At theonse t of spermatogenetic activity, testes isno wwe l1 establishe d (Grier etal , spermatogonia divide several times and enter 1980; Grier, 1981). meiotic prophase.Th enes t ofthes e cells is surrounded by Sertoli cells, which constitu The testis of Liza aurata iscompose d of tes a cyst. seminiferous tubules bordered by basal lamina. Inside thetubule s areger m cells Grier eta l (1980) andGrie r (1981) and Sertoli cells (outside thetubule sar e describe twodifferen t tubular testis-types. interstitial cells or Leydig cells). Germ In the"unrestricte d spermatogonial testis cells aregathere d inseminiferou s cysts in type", typical ofmos t Teleosts, spermato which they differentiate synchronously. gonia are located along theentir e length CYSTO f SPERMATOGONIUM SPERMATOCYTES SPERMATIDS ORGANIZATION OFA SEMINIFEROUS TUBULE b.1. = basal lamina; cy.= cyst; PGC.= Primordial Germ Cell; Se.C. = Sertoli Cell; SPG. = Spermatogonium; SPZ.= spermatozoa; t.1. = tubule lumen. These cysts, bordered by Sertoli cells, of thetubule . Inth e"restricte d sperma contain primary spermatocytes, secondary togonia! testis-type", typical ofAtherini - spermatocytes, spermatides at different formes, spermatogonia aretotall y restricted stages andspermatozoa . Spermatogonia indi to thedista l terminus ofth etubul e beneath vidually surrounded bySertol i cells areno t the tunica albuginea. InLiz a aurata,i t gathered incysts ; they occur alongth e seems difficult tothin k that theteste sar e entire length ofth etubul e and inth e an intermediate type between those described apical part ofth etubul e immediately bene by Grier (1981) . Iti spossibl e that, apart ath theepitheliu m which borders thegonad . 193 from Atheriniformes, Teleosts present seminiferous tubules with parietal and apical spermatogonia as in Liza aurata. Moreover, in golden grey mullet, there is no germinal epithelium as is generally admitted for Teleosts.A t any moment of the gonadal cycle, in addition to spermatogonia we observe primordial germ cells (PGC), which are undifferentiated cells,characte rized by a high nucleus cytoplasm ratio,a n abundance of ribosomes and a scarcity of cytoplasmic organelles. These resting cells differentiate into spermatogonia as shown through transitional stages. Spermatogonia divide very actively and colonize the semi niferous tubule after the spawning period. The germ stock of the gonad is constituted by spermatogonia as dynamic elements and by PGC as latent ones. References Grier H.J., 1981. Cellular organization of the testis and spermatogenesis.^ Fishes. Amer.Zool., 21, 345-357. Grier H.J., Linton J.R., Leatherland J.F., de Vlaming V.L., 1980. Structural evidence for two different testicular types in Teleost fishes. Amer. J. Anat., 159, 331-345. 194 EFFECTSO FHUMA N CHORIONIC GONADOTROPIN(HCG ) with alatenc y timeo fabou t 16h a t2 5 °C ON MATURATION ANDOVULATIO N OFOOCYTE SI N was found.(Fig.1.)Sample so fabou t20 0egg s THECATFIS H CLARIASLAZERA (C & V). per female were subsequently fertilized with fresh miltan dincubate d inPetr i dishes with E.H.Eding,J.A.L.Janssen ,G.H.J.Klein e stagnantwater . Between3 0a n4 0h afte r Staarmanan dC.J.J.Richter . fertilizationa t3 0°C ,th eegg s hatched. Departmento fFis h Culturean dInlan d Maturation responseo ffemal e brood-fishwa s Fisheries,Agricultura l University, defined asth enumbe ro fnormall y developed Wageningen,Netherlands . larvae divided bytota l numbero fegg s incu Administration ofHC Gi snecessar y toprovok e bated.A noptimu m effective dosageo fabou t maturation andovulatio n ofoocytes .Measure 2.5 IU/ga ta latenc y timeo fabou t 16ha t mento fdosag eo fth einducin g agent (HCG), 25° Cwa sfound.(Fig.2. ) correct timingo fth elatenc y andselectio n MATURATION of relevant criteria forovulatio nan d maturation ofoocyte si sa majo r problemi n standardizing procedures forbreedin g experi ments. Selection ofadul t femalesfo rartifi cially induced breedingwa sbase d onextru siono fpostvitellogeni c oocytes (0> 1 mm ) after pressureo nth eabdomen . Upon intra muscular hormone injection,th efemale s were stripped. OVULATION placebo [1.o] [1.6] [2.5] [4.0] [6.3] log [dosage (IU/g)] Fig. 2. Maturation responses (see Fig. 1.) In a second trial, a dosage-response curve for ovulation and maturation was estimated for hypophysectomized and sham-operated fish at a constant latency time of 16 h and temperature 25 C. These two parameters were investigated for a future development of a placebo [to] [2.5] [4.0] [6.3] biological assay of gonadotropin in pituitary log [dosage (IU /g )] tissues. The brood-fish were stripped four Fig.1 .Ovulatio n responses with S.D.an d weeks before the trial, during that period, a latency timet oHC Gi n C.lazera. uniform stock of postvitellogenic oocytes was The effectwa sstudie d ofHC Gan dlatenc y built up. They were injected 8 h after the timeo fhormon e injection onovulatio no f operation and stripped according to the first oocyteso fadul t brood-fish 10-12month sol d trial. At a dosage of 4 IU/g, the ovulation ando fmea n weight 150g .Th eovulatio n and maturation response curves reached a maxi responseo ffish ,5 fis h pertreatment ,kep t mumo f about 70 and 80 %, respectively. The at2 5° Cwa sdefine da sth eweigh to fth e curves for hypophysectomized and sham-operated stripped eggs divided bytota l weighto f fish were similar in shape. This means that ovary estimated after stripping andautops y the tedious procedure of hypophysectomy is not ofth ebrood-fish .A noptimu m effective necessary to develop a biological assay for dosageo fabou t2. 5I Upe rgra m bodyweight gonadotropin; 195 EFFECTO FTRIIODOTHYRONINE(T. )AN DSOM EGONADOTROPI NAN DO FSTEROI DHORMONE SO N MATURATION OFCARP(CYPRINU S IJARPIOL. )OOCYTE S INVITR O P.Epier ,K .Bieniar z Academyo fAgriculture ,Departmen to fIchthyobiolog yan dFisheries , ul.Ambrosow a 6,Kraków-Mydlniki ,Polan d Summary The effects of triiodothyronine (T) a s Group II-Oocyte s incubated inmediu msupple wella so fgonadotropi n and steroid hormones mented with gonadotropin or with on carp oocytematuratio n invitr ower ein oneo fth esteroi d hormone+ T „ vestigated withth eus eo fovaria n fragments - nine subgroups, one of themwit h (from5 females )containin g theoocyte safte r T alone. completed vitellogenesis. Twenty-fourhour s The percentage of mature oocytes insub before the starto fovaria n fragmentculture , groups with T +steroi d hormone orwit hT females were injected carphypophysia lhom - + gonadotropin hormone was nearly twice as ogenate (chh)a t a dose of 0.5 mg/kg body high as thati nth esubgrou p incubatedwit h weight. only steroid oronl ywit hgonadotropi nhor The ovarian fragments from all femaleswer e mones (withoutT )(Fig.1) . divided into2 groups : Differences between firstan d second groups Group I- Oocytes incubated inmediu msupple proved tob estatisticall y significant mented with gonadotropin orwit h (p0.01) . • oneo fth e steroid hormoneswithou t Itma y indicate that insideth eoocyte so r T3 inside granulosa cells there are T,.recep - nine subgroups,on eo fthe mwith tors. outan yhormon econtrol , «KK PFLO«TM f m DOCA Fig. 1. Percentage of mature carp oocytesobtaine d inth emedi awit hT 3 andi n the media withoutT (chh-carp hypophysial homogenate;pp.c-GTH-partl ypurifie d carp gonadotropin Hormone; T-testosterone; A-androsterone; P-progesterone; 17a20ßP-17a20ßdihydroprogesterone ;DOCA-desoxycorticosteron e acetate;CA-cortis - oneacetate ;^-triiodothyronin e alone;C-control) . 196 GONAD MATURATION OFFEMAL E CAPELIN (MALLOTUSVILLOSU SVILLOSUS ) K.G .Forber g Institute ofFisheries ,Universit y ofTromsstf ,Norwa y Summary Developing capelin oocytesha sbee nstudie d growth isinitiall y slow (classII-III )an d using both histological and totalpreparat the datawer ewel l fitted toa linea r growth ions.A ne wmaturit y scale hasbee ncon function during June- October : structed andmaturit y offemal ecapeli ni s D =44.7 4+ 1.03t , r2 =0.72 ,n = 1 7 assessed according tomorphologica lstruc (t= tim ei n days from 1.1.1978). tures in fresh orconserve d oocytes,usin g Oocyte growth rate thenincrease s (classIV - lowpowe rmicroscopy .Maturit y byag ean d V)an d the datawer ewel l fitted toa nexpo length hasbee n studied using datacollecte d nential growth function during November- using thene wmaturit y scale.A comparisio n ismad ebetwee n estimates ofspawnin gstoc k March: D= 201. 4e D-001t,r 2 =0.96, n= =36 . of femalecapeli n using traditional andne w criteria ofmaturity .Th e growth rateo f Maturity rateb y length andag eo fth e fish oocytesha sbee n estimated,an d predictions ofmaturit y by timeca nb emade . Maturity data,usin g thene wscale ,wer e collected during September-October 1977-80. Introduction Fishles s than 12.0c mwer e immatures.Th e proportion ofmaturin g fish increaseswit h Thestud y ofth ematurit y cycle offemal e increasing size,an dwit h increasing age capelinwa sinitiate d duet ouncertaintie s within intermediatesiz egroup s (12.0-13.0cm). inmethod s forth eestimatio n ofth esiz eo f Fishlarge r than 14.0c mwer e allmaturing . thespawnin g stock.Thes e estimationsar e Therat e ofmaturit y increaseswit hincreas carried outi nSeptember ,abou t6 month s ing fishsize ,an dwit h agewithi nsiz e prior toth etim eth e fishbegi n tospawn . groupssmalle r than 16.0cm . Atthi stim eth egonad sar esmal l andstag e ofsexua l development isdifficul t toassess . Estimations ofth espawnin g stock Thepurpos e ofth estudy ,carrie d outa tth e Institute ofFisherie s Research inBergen,an d Traditional method-ofmaturit yclassifi the Institute ofFisherie s inTromsizi ,wa st o cation appearst ogiv ea larg e underestimate presenta maturit y scalewhic h could beuse d ofth e totalnumbe r ofth epotentia l inth e field,an dwould ,mor eprecisel y pre spawners (Table1) . dict "spawners"durin g early stageso f development. Table 1.Tota lstock ,an d spawning stock of-femal ecapelin ,i nnumber sx 10" ,usin g Resultsan d discussion five different definitions ofth epotentia l spawning stock:a )"maturing "b y traditional Thematurit y scale classification;b )clas s IIao rmor emature ; c)clas s libo rmor emature ;d )clas sIli a Oocytemorpholog y andhistolog y hasbee n ormor emature ;e)clas s Illbo rmor emature . studied and amaturit y classificationwa s developed from theappearanc e ofwhol e oocyte preparations using lowpowe r microscopy year totalstoc k spawning stock (Forberg,1983 ,i n press). Immaturecapelin , ? 12c m a b c d e orclas s I,hav e only firstgrowt hphas e oocytes. Idefin e them asmaturin g whenyol k 1977 13 4 9 7 5 4 vesicles appear inth ecytoplasm ,i nclas s 1978 13 4 10 8 6 4 Ila-IIb.Durin g class Illa-IIIc,fa tvesicle s 1979 16 1 10 7 3 2 appear in thecytoplasm ,an d during classI V 1980 18 6 14 12 10 7_ rapidaccumulatio n ofsoli dyol k takesplace . ClassV describe s fishwit h ovulatedoocytes , classV Iar espawning,.clas sVI Iar espent , References and classVII Iar espent/recoverin g capelin. Forberg,K .G . 1983.Maturit y classification Growth rateo foocyte s andgrowt h ofcapeli n oocytes.J .Fish . Biol, (inpress) . Increase inmea n diameter (D)o fsecon d growth phase (SGP)oocyte swa s studiedi n material from 1978an d 1979.SG Poocyt e 197 ULTRASTRUCTURE OF CELLULAR AND NON-CELLULAR LAYERS SURROUNDING THE OOCYTE OFA TELEOSTEA N GOBIUSNIGE RL .: PRELIMINAR Y OBSERVATIONS F. LEMEN N Laboratoire de Biologie Marine et Département de Microscopie Electronique Université de Bordeaux I, 33 405 Talence, France. SUMMARY From the early previtellogenic stages At Stage III, follicular cells appear oocytes of Gobius niger are surrounded by a as a continuous epithelium alternating with single layer of flattened follicular cells and transversal sections of adhesive filaments. separated by a basal lamina from several These filaments constitute a longitudinal layers of thecal cells which progressively network which covers the oocyte and is decrease to form a single very thin thecal attached to the zona radiata only at the layer. animal pole. As the adhesive filaments are formed the ultrastructure of the follicular At Stage II, the follicular epithelium cells takes on a secretory aspect. separates from the oocyte surface forming a narrow space filled with a slightly floculated During Stage III and IVa, the adhesive material. Microvillosities protrude from the filaments develop. Their outer part is deeply oocyte surface into this space. An electron- indented, with pin-like formations. dense material is then deposited between the At maturation (Stage IV"b),the follicular oocyte microvillosities and constitutes the cells separate from the surface of the zona beginning of the zona radiata. From this radiata which quickly fills in and frees the stage on, numerous mitochondria and Golgi complexes are present in the peripheral adhesive filaments . During spawning the ooplasm. They produce smooth dense-cored adhesive filaments break at the vegetative vesicles which deposit the electron-dense pole and later attach the animal pole of the material by exocytosis on the external oocyte to a support. surface of the oocyte . Microvillosities The zona radiata can be considered as a protrude from the follicular cells towards primary envelope and the adhesive filaments the oocyte. The zona radiata is gradually as secondary formations. built up. At Stage II , the zona radiata consists of two electron-dense layers, the higher homogenous and the lower finely reticulated, perforated by pores through which the oocyte and follicular microvillosities pass. "With the formation of the zona radiata, bristle- coated vesicles appear at the base of the oocyte microvillosities.They are pinocytotic and collect the exogenous vitellogenin which reaches the oolemma by passing between follicular cells, then along microvillosities towards the base of the zona radiata. Exogenous vitellogenesis occurs very early at the same time as endogenous vitellogenesis. Between the follicular cells lacunae appear inwhic h a material subsequently transformed into adhesive filaments is deposited. 198 STUDIESO N THEBREEDIN GBIOLOG YO PTH EEE L (MOUILLAJAPONIC ATEMKINC K& SCHLEGEL) tinHao-re n andLi n Ding Department ofBiology ,Zhongsha n (SunYatsen )University ,Guangzhou ,Chin a Summary Methodso fidentifyin g the sexo feel sa t lervesicle smeasurin g 450-1000 1 indiameter . the seawardmigratin gperiod ,ultrastructur e Most of theneurosecretor y fibresd ono tmak e ofpituitar y of female eels,developmen t of direct contactwit h theendocrin e cellsbu tar e gonadsan d artificial inducement of sexual separated from themb ya basementmembrane . maturity inmal e and female eelswer estu Therear e synaptic junctionsbetwee nneurose died.Th e resultsindicate d that theeel s cretory fibresan d pituicytes.Si x different collected at the seawardmigratin gperio d cell types(prolactin cell,ACT Hcell ,TS H cell, canb e induced sexualmaturatio n ofbot h GtH cell, GHcel l and the secretory cello fme - sexesb ymean s ofhormona l injectionsunde r ta-adenohypophysis)ca nb e recognized inth e pondconditions . adenohypophysis by thecharacteristic s oful trastructure and the sizeo fth egranule sthe y External characteristics ofmal e andfe contain.A t theleve lo fultrastructure ,pro male eelsa t seawardmigratin gperiods ,b y lactincells ,ACT Hcell san d TSHcell sca nb e comparing thediamete r of eyes, interorbital demonstrated inhig hactivit y during thesea space,th eshap eo fsnout ,etc ,ar esuffi wardmigratin gperiod ,whil eGt Hcell sha dno t ciently reliable forth e identification of yet ina functional state. both sexes.Thes emorphologica l dimensions Fullymatur emal ean d female eelswer ein whencalculate d with theai d of distinguish duced by injectionwit hcommo ncar p pituitaries function,confir m the differences between plusHC Gan dLHBH-A ,des-Gly 10(D-Ala6)LH-RH- the two sexes.Consideratio n ofmorphologi ethylamide.Th e eelswer ekep t inpond swit h caldifference so fth e400-60 0m m sizegroup , artificial seawate r at a temperature of permits separation of thesexe swit h9 0% 15-20°C.Pro mNovembe r tonex tMarch ,afte r accuracy.Plottin g of sizedistributio nse 6-10 injectionswit h10-2 0day sinterva lbe paratesnaturall yon e sex from theother , tweeneac h injection(4-5m g carppituitar y+ thoseabov e 600m m arenearl y all female, 1500-2000 IUHC G+ 40-50u g LHRH-AA« b°dy and those below40 0m m are almostal lmale . weight),mos t offemale swer ematured .Matur e Because thebod yweigh t (w)o fee lvarie sa s maleswer eobtaine d by4 o r5 injectionswit h somepowe ro f body length(L), thati s W=aLb, lowerdosage(0.5-1. 0m g carppituitar y+ 500 - the relationship between bodylengt h andbo 800 IUHCG/k gbod y weight). dyweigh to fbot h sexesma y be expressed as The oogenesisan d spermatogenesis aswel l as W=0.0000001285L3-015.Th e regressioncoeffi thedevelopmen t ofovar yan d testisunde rar cient ofthes e twovariâte so fee lbetwee n tificial inducementwer e also investigated his 400-600m m inbod ylengt h isdifferen t for tologically and cytologically. The development male and female.Th e formeri s0.887 ,tha t of testisi scompose d of six stages:l )earl y is, W=0.887L-280.20,an d the later,1.150 , multiplication ofspermatogonia ,2 )lat emul thatis ,W=1.150L-376.73 . Inth e caseo fth e tiplicationo f spermatogonia,3 )growt han d condition factor,th emal e is found tob e maturation of spermatocytes,4 )initia lappea 1.15*0.062,th e female1.42*0.053 ,differen rance of spermatozoa, 5)complet ematuratio n ceso fbot h figuresar e significant.So ,th e ofspermatozoa ,6 )degeneratio n ofspermato external characteristics and sex distinction zoa.Th eoogenesi s iscompose d of sixphases : ofmal ean d female eelsca nb euse d forth e 1)oogonia ,2 )oocyt ewit h follicularepithe selection ofbreeder s in theinduce d breeding lium consist ofa singlelaye ro f follicle ofeel . cells,3 )oocyt ewit hadipos e vesicles,4 )oo cyte filledwit hyolk , 5)oocyt ewit hmigra Theneurohypophysi s ofee l iscompose d of torynucleus ,6 )degeneratio n ofoocyte . neurosecretory fibres,pituicyte s andcapil Basedo n the sixphase so foogenesis, th ede laries. Thegenera l characteristics of the velopment ofovar y isals o composed ofsi x neurosecretory fibresar e typical of those stages correspondingly. offishes ,a fewwer emyelinated . Somemulti - This investigation showed it ispossibl e to lamellatebodie swer e present inneurohypo induce sexualmaturatio n in themal e andfe physis.Tw o typeso ffundamentall y different male eelb ykeepin g them inartificia l seawa neurosecretory fibres canb e clearlydistin teran d givinghormona linjections . guished. TypeA neurosecretor y fibre contain •spherica lmembrane-boun d vesiclesmeasurin g 1250-1750 1i ndiameter ,typ eB contain smal 199 EFFECTO FENDOSULFA NO NBIOCHEMICA L CHANGES INOVAR YAN D GSIO FTW O SPECIESO F FRESHWATER TELEOST R.B.Raizada ,K.K .Datt a andT.S.S .Dikshit h IndustrialToxicolog yResearc h Centre,Mahatm a GandhiMarg ,Pos tBo xNo .80 , Lucknow-226001 (INDIA) Summary Endosulfan a chlorinated insecticide was values. Singh and Singh, (1980)hav e also daily exposed to two species of freshwater reported no change duringpreparator yphas e teleost, Clariasbatrachu s andHeteropneus - in total lipids and cholesterol values in tesfossili s during the preparatory phase ovaryo fH .fossili sdu et ocythio nan dHex - (February-April) for aperio d of 30 days. adrin. Kapur et al., (1978)hav e reporteda Pesticide at a concentration of 0.0001pp m decrease in 3B-HSD level after fenitrothion significantly increased theglycoge n content treatment inth eovar y andtesti s ofCyprin - of ovary in C.batrachu s and H.fossilis . us carpio. In thepresen t study,endosulfa n The activity ofaci d and alkalinephosphat treatment could significantly enhanceglyco ase,tota lan d freecholestero l and ascorbic gen contents of ovary both inC .batrachu s acid and the value of gonadosomatic index andH . fossilis.Th eATPas eactivit y inovar y was unaffected duringthi sphas e inth etw o was slightlydecrease d inH .fossilis .I ti s species of fresh water teleost. It seems apparent fromthes e findings that endosulfan thatendosulfa na tthi sdos e level failedt o during thisphas e ofreproductiv e cyclecoul d induce bio-chemical changes inovar ydurin g notbrin g anydrasti c changes inth eovaria n preparatoryphase . biochemistry evenafte ra prolonge d treatment Endosulfan (6,7 ,8 ,9 , 10,10 ahexachloro , of3 0days . 5,5a ,6 ,9 ,9 ahexahydro-6 ,9 methan o 2,4 , Higher concentration ofendosulfa n ifgive n 3-benzedioxathiepin-3-oxide) is a non sys forth e sameperio dma yproduc e some changes temic contact insecticide and acaricide is inovar yo ftw o»specie so ffish . used extensively forth e control ofvariet y ofplan tpests .Endosulfa nwa s found tocaus e the death of fishi nth eRhin e riverdurin g References 1969 (Walker, 1971). Severalworker s havere portedvarie d toxicological effects ofendo C.Walke r (1971).Environmenta lpollutio nb y sulfan in specieso fanimal s likemice ,ra t chemicals.Hutchinson . and rabbit (Nath et al., 1978;Dikshit he t G. Nath,K.K . Datta,T.S.S . Dikshith,S.K . al., 1978;Dikshit h andDatta ,1978 ;Dikshit h Tandon and K.P.Pandy a (1978). Toxicology etal. , 1982a,b (unpublished)).Ver ylittl e n, 385. work is available onendosulfa n toxicityo f H. Singh andT.P . Singh (1980).Environmen fish (Krishna Gopale tal. , 1981;Mohanarang a talpoll . 23,9 . Rao andMurty , 1980). K.Kapur ,K .Kamaldee p andH.S .Too r (1978). Adult female C.batrachu s andH . fossilis Bull.Environ .Cont .Toxicology . 20,438 . were kept under laboratory conditions for R.B.Raizada ,K.K .Datt a andT.S.S .Dikshit h twoweek sbefor e experimentation and fedwit h (1982).Drug s and chemicalToxicolog y (com commercial fish food and livero nalternat e municated). days. Single concentration of endosulfan T.S.S.Dikshith ,G .Nathand ,K.K .Datt a 0.0001ppm )wa s added daily after changing (1978). Ind.J .Exp .Biol . 16,1000 . the aquaria water.O ncompletio no fda y30 , T.S.S.Dikshit h andK.K .Datt a (1978).Bull . fishwer e killed,ovar ywa s takenou timmedi Environ.Cont .Toxicology ,20 ,826 . atelyan dweighe d individually.Gonadosomat T.S.S. Dikshith, R.B. Raizada,K.K . Datta icinde x (GSI)wa s calculated bymultiplyin g andB.S .Kapali a (1982).Drug s and chemical theovar yweigh twit h 100an d quotientbein g Toxicology(communicated) . divided by bodyweight .Ther ewa sn ochang e in the GSIo fth etw ospecie s of freshwater teleost after exposurewit hendosulfa nwhe n compared to controlanimals ,10 %homogenat e ofovar ywa smad e andestimatio n ofth ebio chemical parameters were conducted as per thestandar dprocedures . Itha sbee nobserve d that endosulfan atth edos eused ,coul dno t produce changes in ovarian biochemistry of C.batrachu s andH . fossilis duringpreparat oryphas ewhe n compared to respective control 200 SPERMIATION RESPONSEO FCAR PT OHOMOLOGOU SPITUITAR Y GLAND S.Rothbar d andHamuta lRothbar d FishBreedin g Centre,Gan-Shmue l38810 ,Israe l Israeli carpreac h sexualmaturit y atth e Theseasona lfluctuation s ofpon dwate rtemp ageo f 6month s and differ inthi srespec t eraturedi dno t showan y significantcorre fromothe r carppopulations . Maturation-in lationwit h thepituitaries 'gonadotropi c ducingpotenc y inIsrael icar ppituitarie s potency (Fig.2) . werepreviousl y tested by thefro gspermia - tionassa y (Yashouve tal. , 1970). Thepur poseo f thisstud ywa s todetermin egonado tropicpotenc y incar ppituitaries ,b yth e spermiation responsei nth ehomologou s species. Pond temperaturewa srecorde d dailydurin g theperio d ofpituitarie s collection inorde r toasses spossibl erelationshi p between ambient temperaturean d gonadotropicpotenc y inth eglands . Recipient fish (500+ 100g rv.w. )wer ekep t 48hr sa t16° Cprio r totreatmen t toreduc e variability inrespons eamon g individual fish. The testwa sperforme d on1 3group s ofequa lsiz emal ecarp . Fisho f1 2group s were injectedwit hpituitar yhomogenat e (1 gland/kg)collecte d inanothe rmonth ,whil e thefis hi nth econtro l groupwer einjecte d withsaline . Twenty-fourhour slate rth e fishwer eweighe d and stripped 3times .Th e semenfro meac h fishwa scollecte d andit s volumerecorded . TheSN Ka posterior ites t S' • (Sokal& Rohlf ,1969 )wa sapplie d formult iplecomparison samon gmean so fseme n volumeso fvariou smonths . Anothergrou po f 18fis hwa sinjecte dwit h homologouspituitar yhomogenat ean d stripped Spermiation response of carp to homogenate thenex tda y inorde r toestimat e theassoc of homologous pituitaries collected at month iationbetwee nindividua lvolum eo f semen and corresponding bodyweigh t (Fig.1) . ly intervals (mean + SEM ; n-14). Maximal and minimal temperature of pond water during the same year. No significantdifferenc ei nspermiatio nre sponsewa snote d among thegroup streate d withpituitarie s collected along 12consecu tivemonth s asteste db y theSN Ktest . Semen released bymale s treatedwit hpituitarie s wasmor eflui d thanb y thecontro lfish . Asa consequenc eo f thepresen t studyw e suggesttha tcar ppituitarie scollecte d in Israela tan y timedurin g theyea rma yb e suitable for inductiono fspawning . References: 700 Sokal,R.R . &Rohlf ,F.J. , 1969. Biometry. Body Weight W.H.Freema nan d Co.,Sa nFrancisco .77 6p . (gr) Yashouv,A. ,Nussbaum ,M. ,Berner-Samsonov , Correlation between volume of semen stripped E. &Abraham ,M. ,1970 . Relativepotencie s and body weight of oarp treated with homolo of carppituitarie s collected for spawning gous pituitary homogenate (1 gland/1 kg b.w.) induction. Bamidgeh,20(4) :125-127 . y=0.0572x-0.82307 ; r = 0.5003. 201 INDUCED SPAWNINGO FTH ESHARPTOOT H CATFISHCLARIA SGARIEPINU SWIT HNOTE SO NTECHNIQUE S EMPLOYED INTH EREMOVA LO FEG GADHESIVENES S HJ SCHOONBEE ResearchUni t forFis hBiology ,Ran dAfrikaan sUniversity ,Bo x524 ,Johannesbur g SouthAfric a THECHT ,L POLLIN G andJ ESAAYMA N Dept ofZoology ,Universit y ofth eNorth ,P/ B X5090,Pietersburg ,Sout hAfric a SUMMARY Theus eo fmammalia n gonadotrophic hormones dicate that the sharptooth catfish isno t andpituitar y glands tospaw nth ecatfis h difficult to spawnartificiall ywhe na mix ClaMÂMM hasbee ndemonstrate d by anumbe r tureo f carppituitar y andHC G isemployed . ofresearc hworkers . Becauseo fth eadhe Indications aretha t ifsufficien t pituitary sivepropertie s of the eggs,hormone-injec material from CtcUtlcU canb eobtained ,onl y ted females areusuall y allowed tospaw n thisma yb erequire d to spawnthi sfish . naturally inspeciall y prepared ponds.Wher e specieso f C&VUJI&ar eartificiall y spawned, thefertilize d eggs areusuall y deposited onto nylon screens,t owhic hth eegg s stickafte r they comeint o contactwit hwater . These screens arethe nsubmerge d inwell-oxygenate d waterunti lth eegg shatch . Alternatively, fertilized eggs aredeposite d ina monolaye r inplasti c trays inth epresenc e of fresh water. These screens and trays takeu pcon siderable spacean d are impractical if CZxWLCH ist ob e spawned ona larg e scale. Theconti nuous removal ofdea d eggst oavoi dmoul d for mation isals o anuisance . Adaptations oftechnique s followed forth e removalo feg gadhesivenes s inth ecommo ncar p byusin gure a (Woynarovich, 1962;Rothbard , 1978), aswel l asful l creampowdere dmil k (Soin, 1977), was adapted and usedsuccessful lyfo r Clafu.cn> gcuu.2.plnui (Schoonbee it at., 1980). Eggs sotreate dwer e thenhatche d in breeding jars (Fig. 1). Thewor k described onth einduce d spawning andhatcher y procedures Fig. 1. Smalleffectiv ebreedin g jarsca nb e bythes eauthor s canb e seena sth efirs tste p made from \Z cordialbottles .Not eho wdensel y towards the large scale cultureo fC . QCUvit- theegg s of ClcOvùU goJviz.pi.ywJ> pack. p-tnoóa sa pon d fish. Theseresult s alsoin Table 1. Results of induced spawning experiments with the sharptooth catfish Clarias gariepinus Hormone injection times and dosagest No. of Date and season females used Day 1 Day 2 Remarks 18 October 1979 8 15h30 22h30 18h30 Gate spring) 350 I.U. Puberogen 150 I.U. Puberogen 4 females, complete spawn Gonads of 2 males used per female + 1gland/k g + 1 gland/kg 2 females, partial spawn spawner. Male gonads removed and milt stripped over eggs. 1* 15h30 19h30 Clarias pituitaries used. Fish spawned 1.3 gland/kg 1.3gland/k g in tank within 10 h of first injection. 1 November 1979 2 10h30 13h30 . 08h30 Only (early summer) 250 I.U. Puberogen 250 I.U. Puberogen Complete spawn of both one male of approximately 4 kg used + 1 gland/kg +• 1 gland/kg females, 22h after first to fertilize eggs. Approx. 80% injection fertilization success. 4 December 1979 4 08h30 13h30 08h00 (mid-summer) 750 I.U. Pregnyl/kg 1 gland/kg 3 females, complete spawn 1 female, no spawn. Gonads underdeveloped. •Pituitarygland s from female C. gariepinus donors, preserved inalcohol . In allothe r casespituitar ygland swer eobtaine d from ccnmon carp.tDosage s are given per mass of spawning fish. 202 INDUCTIONO FOVULATIO N INH .FOSSILIS-ANALYSI S OFSE XRELATE DDIFFERENCE S INTH E POTENCYO FHOMOLOGOU SAN DHETEROLOGOU S PITUITARYEXTRACT S I.J. Singh,H.N . Singh andT.P . Singh FishEndocrinolog y Laboratory,Departmen t ofZoology ,Banara sHind uUniversity , Varanaisi-221005,Indi a Inorde rt omak e the induced breedingtech Ovulatingpotenc y ofpituitar y glands col niquemor e successful and economic,attempt s lected inJul y fromeithe r sexo fH .fossili s are continuously being made to standardize and C.mrigal a was significantly greater than thedose s ofpituitar y gland extract forin thoseo fMay .Homologou spituitar y glandex ducting spawning in important food fishes tracts from female fishwer emor eeffectiv e (Jhingran, 1975).Report s on comparativeov thanhomologou s extracts ofmale s ininducin g ulatingpotenc y ofhomologou s andheterolog ovulation and heterologous extracts ofbot h ous pituitary gland extracts from malean d sexes. Bovine LHa tth eteste d dosesfaile d female fishes aremeagr e and debatable.Ef to induce ovulation inhypophysectomize d H. fect of homologous and heterologouspituit fossilis. ary extract collected from male and female specimens on induction o spawning hasbee n Theabov e findings suggest that (1)homol describedhere . ogous pituitary extract is more effective thanheterologou sone ,i ninducin g ovulation Pituitary glands frommatur e specimenso f inH .fossilis , (ii)ovulatin g response of either sexo fCirrhinu smrigal a andHeterop - pituitary glands of both species collected neustes fossilis were collected separately duringJul y indicatespresenc e ofhighe rgon duringprespawnin g (May)an d spawning (July) adotropic potency inth egland stha nthos e phases of reproductive cycle. Glands were ofMay , (iii)ther e isa se x relateddiffer dried in chilled aectone immediately after ence inovulatin gpotenc y inbot hth especie s removal. Mature female specimens ofH .fos studied which ishighe r infemal e thanmale , silis, collecte d frompond s (Varanasi,India ) (iv) least effectivesness of bovine LHma y in June and were hypophysectomized as per bedu et oth edistan tphylogeneti c relation technique ofSing h (1969)an duse d asrecip ship. ients for evaluation the ovulating potency ofdifferen tpituitar y extracts.Variou s dos eso fbovin eL H (5,10 ,15 ,2 0an d25|Jg/fish ) References and extracts ofaceton edrie dpituitar y glands (10,20 ,30 ,4 0an d 50(Jg/gish )wer e injected Jhingran,V.G. , 1975."Fis han dFisherie so f to hypophysectomized H. fossilis andovula India, Hindustan Publishing Corporation tion was checked by stripping 24hr safte r (India). theinjection . Singh,T.P. , 1969.Observatio n onth eeffect s of gonadal and adrenal cortical steroids upon thyroid gland in hypophysectomized catfish, Mystusvittatu s (Bloch). Gen. Comp.Endocrinol. ,12 :556-560 . 1 Ç H FOSSILIS MAY, Y'-10 00 2 rf H FOSSILIS MAV. V-9'87- 3 O C-MRföALAMAY. Y=-M0. U 0 Ç.MRIGALA MAV; Y>-9-67- Acknowledgement S Q H.FOSSILIS JULY Y=»5-7! 6 o"îj FOSSILIS JULÏ Y=-19-87 Financial assistance fromDS T (No.HCS/DST / 7 Q CMaSALA JULY, V< fl'96 a O Ç. MRIGALA JULY Y=-12•3! 928/80 and CSIR (No. 38 (389)/81-EMR-II), Governmento f India,Ne wDelh i isgratefull y acknowledged. RV DOSE l>ig) 203 REPRODUCTION REGULATED BY EXTRAOCULAR PHOTORECEPTOR INA SEASONALLY BREEDING PISH, ORYZIAS LATIPES Hiroshi Urasaki,Takeyuk i Abe and Sin-Iti Ohtake Department of Biology, Nihon University School ofMedicine ,Oyaguchi ,Itabashi , Tokyo 173,Japa n Summary The present study was carried out for aim at possible role of pineal in re- Enucleation of bilateral eyes(OPX) gulating gonadal development and loco- in teleost,Oryzia s latipes, enhances motor activity with respect to sp awn- gonadal development, e.g. increase of ing behaviour. Suppressive effec t of gonosomatic index(GSI), break down of pinealectomy to increase of GSI i nduc- germinal vesicle and completion ofoo ed by long photoperiod, was witho ut e- cyte maturation, during a period from ffect on ovulation and pvipositio n. On prespawning(March) through spawning(A- the view point of GSI value corre lat- prll to August)(Pig.1). Since blinded •ingt o plasma estradiol level(Yar on et female, furthermore,respond s to daily al.,'77), ovulation and spawnin after long or short photoperiod in regard to pinealectomy may-no t depend oocyt ema - an increase of GSI and progress of oo turation in regarding hormonal le vel. cyte maturation, and shows diurnal lo comotor activity being synchronous to the daylight cycle,i t emphasizes the -- INTACT? X OPXS presence of extraocular photoreceptor. •—• OPX 9 X INTACTS -— INTACT9 X INTACTS Pinealectomized(PINX) female revea f' \ led suppressed gonad(Fig.1,2), even in a long daylight condition, and without •«^a-*--^ ... 'T - \ synchronization of locomotor activity to the daylight cycle. Therefore, it -JlJlZZ-^^-t-iïi^*'- IT i« t) Pig. 3.Actograp h of a couple of fish. Blinded fish revealed GSI at high, but ovulation and spawning, suppressed and without punctuality(Urasaki et al. '8l). Moreover,th e blind female sho wed a actogram completely synchronous Pig. 1.Ovarie s of PINX(left) and OPX. to LD cycle,and , it is also given in is highly probable contribution of the Fig.3- Therefore, it likely indicate pineal in the photoreceptive mechanism the pineal play a role in oocyte matu with respect to progonadal function. ration,bu t not in spawning mechanism. Photoreceptive outer-segment organ ization of the pineal,an d pineal in References nervation of Habenula commissure(Uras aki,'74 )an d the third ventricular ep- Sathyanesan,A.G . &V.K.S . Sastry, endyma(Sathyanesan and Sastry,'82)li 1982. Pineal innervation of the kely,indicat e apresenc e of pineal ro third ventricular ependyma in the le Inmediatin g photic stimulation for teleost, Puntius sophore(Hams). J. hypophyseal gonadotropic-gonadal axis. Neural Trans. 53:187-192 . Urasaki, H. et al., 198I. Photoperio- dicity of reproduction modulated by 14- extraocular photoreceptive mechan 12 ism in Oryzias latipes. Bull. Lib. Arts &Sei . Sch. Med. Nihon Univ. £*> 9: 1-21. 3 8 Yaron, Z. et al., 1977- Occurrence and biological activity of estra- 6 diol-17ß in the intact and ovari- ectomized Tilapia aurea(Cichlidae, MAR ÄR ÜY JÙN Teleostei). Gen.Comp.End. 33: 45- Pig. 2.Seasona l difference ofGSI . 52. 204 SATURABLEGONADOTROPI N BINDING SITESI NTH ETESTI SAN DOVAR Y OF IMMATURE CHUMSALMON . GlenVa nDe rKraa kan dEdwar dM .Donaldso n Dept.o f Zoology,universit y ofBritis h Columbia,Wes tVancouve rLaboratory ,Fisherie s Research Branch,Dept .o fFisherie s andOceans ,416 0Marin eDrive ,Wes tVancouver ,B.C . V7V 1N6 Canada Summary uptake of radiolabelled hormonei nth e presence of0. 1 and 10u gSG-G100 . There isabundan t evidence thatpeptid e Saturable binding sites couldno t be hormones exert their effects ontarge t demonstrated innon-gonada ltissue s cells by binding tospecifi c high-affinity including theliver ,kidne y andmuscle . The receptor sites in theplasm amembrane . demonstration of saturable binding siteswa s Peptide hormonereceptor s havecommonl y dependent onbot h timean d temperature. been identified bycompetitiv e binding Bindingwa shighe r at20 °tha n8° Can d studies involving theuptak eo f increased with thetim eo f incubationfo r2 0 radiolabelled and unlabelled hormoneb y hr. Competition for125 I-s-GtH binding the target tissuewhe n incubated in siteswa seviden t atlo wlevel s ofs-Gt H vitro. Byus e of thisapproac h with significant displacement ofboun d gonadotropin receptors have beendescribe d hormoneoccurrin g with less than2 n g ingonada l tissues from allvertebrat e s-GtH. Gonadotropins ofmammalia n origin groupswit h theexceptio n of fish. A had onlya wea k effectivenessi ncompetin g similar approachwa s adopted to forovaria n gonadotropin bindingsites ; characterize thepropertie s of s-GtHhavin g at least30 0x thepotenc yo f gonadotropin binding sites ingonada l ovineL Han dFSH . tissues obtained from immature chumsalmo n Theaffinit y and capacity of ovarian (Oncorhynchus keta). gonadotropin binding were determined by Anecessar y prerequisite to competitive Scatchardanalysi s following correctionfo r binding studies involves evaluation ofth e themaxima l binding activity ofth e labelled biological properties of salmon hormonepreparatio n (Dufauan d Catt,1978) . gonadotropin (s-GtH)subjecte d to Data suitable forScatchar d analysiswa s radiolabelling. Iodinated s-GtHwa s obtained following incubationo f ovarian prepared by the lactoperoxidasemetho d tissuewit heithe r aconstan t amount of usingK Isupplemente d with100,00 0cp m l"i-s-GtHan d increasing amountso f l"i instead ofcarrie r free125 j unlabelled hormoneo r with increasing routinely used for Iodinations. The amountso f radiolabelled hormone. Saturable iodinationmixtur ewa s then serially gonadotropin bindingwa sdu e toa singl e diluted and aliquotswer eassaye d for classo fhigh-affinit y binding siteswit ha n their ability tostimulat e testosterone equilibrium association constant of2. 5an d production inmince d testispiece s 3.1 x10 % forth etw omethods , incubated invitro . The potency ofth e respectively. Bybot hmethod s thebindin g iodinated preparationwa s compared with capacities wereabou t4 0pg/m gwe tweigh to f intact s-GtHan d separate preparations of tissue. iodinated s-GtHi nwhic h eitherK Io rH2O 2 Thepropertie s of saturable gonadotropin had been omitted. Based onth e binding sites in chum salmon showa clos e incorporation of44.5 %o f the125 j^ t was parallelism to thepropertie s described for calculated thats-Gt Hwa s iodinated toa n gonadotropin receptors in other vertebrate averagemola r ratio of0.8 8mo liodine/mo l groups. However,th econventiona l s-GtH. The doserespons e curves for definition ofa hormon e receptor embodies intact s-GtHan d thevariou s iodinated thedua lfunctiona l propertieso f preparationswer e indistinguishable recognition plus translation of thehormone - suggesting thelac ko f aneffec to f receptor interaction into aspecifi c iodination on thebiologica l activityo f biologicalresponse . Further studiesar e s-GtH. necessary toestablis h the physiological Initialcompetitio n studies'wer e significance of thesesite s inth esalmon . conducted byincubatio no f •'•"I-s-GtHan d increasing amounts ofunlabelle d SG-G100 References with the300 0x gparticulat e fraction obtained from chum salmon testis orovar y Dufau,M.L .an dK.J .Catt ,1978 . homogenates. Thepresenc e of saturable Gonadotropin receptors and theregulatio n 125j_s_Qtjjbindin g siteswa s demonstrated of steroidogenesis inth etesti san d bygreate r thana 50 %reductio n inth e ovary. Vitam.Horn .36 :461-592 . 205 GROWTH HISTORY AND REPRODUCTION IN ATLANTIC COD (Gadus mor hua) K. G, Waiwood Fisheries and Oceans, Biological Station, St. Andrews, N.B., Canada. Summary Growth and reproductive condition of if the growth pattern in the year prior to mature and potentially first spawning cod spawning was related to maturation rate in were followed under laboratory conditions potentially maturing cod or to fecundity in for a period of 10 months (June to March). mature cod. These fish exhibited natural reproduction with respect to the timing of spawning, Methods percent maturation-at-length and age, and relative fecundity. Preliminary analysis Cod, 39 to 70 cm in length were caught indicates that immature cod which had the by otter trawl in Passamaquoddy Bay, trans potential to spawn for the first time had ported to the laboratory, and held in 2-m different growth histories with respect to diameter tanks, where they were kept under their ultimate reproductive state. Cod in natural photoperiod at 4 or 8°C and sub this category which did mature exhibited jected to a water current of about 0.2 to better early summer growth than did those 0.5 body length. The fish were fed 3 times which did not mature. In mature females, a day on a prepared diet consisting of fecundity was related to initial body size ground flounder, herring meal, shrimp meal, (June weight) and growth during June to herring oil'and binder. Apart from the September. These results are discussed in spawning period when appetite declined relation to the egg maturation cycle. markedly, the cod were fed prescribed rations varying from 0.5% body weight d~! to satiation? The fish were tagged and their weights and lengths were recorded Gadoids, including Atlantic cod (Gadus every 2 months (June to March). At the end morhua), exhibit considerable variability of the experiment, the gonads were removed in relative fecundity, age and size at ma and fecundity was determined in all females turity, egg size and spawning time. This having ripe ovaries. species is widely distributed in the North Atlantic and much of this variability can be attributed to inter-stock differences Results (May, 1967i Oosthuizen and Daan, 1974). However, intra-stock shifts in length-at- Although there was considerable varia maturity over time have been noted for the bility within tanks, growth increased with North Sea (Oosthuizen and Daan, 1974) and increasing ration, plateauing at a ration Gulf of St. Lawrence cod (Lett, 1978); in level of about 1% d'1. the latter case over a relatively short The cod showed normal reproduction period (15 y). Similarly, differences in with respect to the timing of gonad growth, relative fecundity have been observed among size and age at maturity and relative cod stocks and individuals of the same fecundity. No direct effect of temperature stock. Year-to-year variation within on reproduction was observed. The length stocks probably occurs also. It has often at which 50% of the males and females been suggested that this variation may be matured was 42 and 52 cm, respectively. an adaptive response to fishery-induced The percent of males maturing at ages 3, 4 changes in population density. A compensa and 5 was 70, 95 and 100, while the corres tory increase in egg production during ponding values for females were 50, 70 and periods of low population density would be 100. Two groups of fish were distinguished a potentially important aspect of the those with initial weights suggesting they stock-recruitment process. had the potential to mature during the ex Woodhead and Woodhead (1965) investi perimental period, and mature (previously gated the reproductive cycle in Barents Sea spawning) fish. Preliminary results indi cod and concluded from consideration of the cate that in potential first spawners, dif egg maturation cycle that feeding condition ferent growth patterns were observed be (and hence growth) in the summer prior to tween fish that did mature and fish that spawning could influence fecundity in the did not mature (the initial weights of next spawning season. these two categories were not significantly different). Males which did mature weighed The purpose of the following study was significantly more in all growth periods to determine, under laboratory conditions. than nonmaturing males (Fig, 1). 206 Since eggs are committed to mature as 220 Moles early as 11 months prior to spawning, I tested the hypothesis that fecundity was a Mature (g)-760 (568-897) function of initial size (June weight) plus 200 growth during various periods prior to Immature(g ) 775 (559-1200) spawning. Regression analysis was used with fecundity as the dependent variable. The _ 180 equation with June weight as the only inde 2 JZ pendent variable gave an r of 0.647. When o> a second independent variable was added .5 '6° (June-July growth), the r* increased to 'E 0.756. With June to September, June to November, or June to January growth as the ° 140 second independent variable, the corres 2 C ponding r values were 0.821, 0.822, 0.823, respectively. Since there was no improve a. 120 ment in fit when growth after September was included in the equation it was concluded that the equation with June to September 100 growth adequately represented the model. This relationship is presented in Fig. 3. _ I i i i i i i i i i i 80 J J A S 0 N D J F M A Fecundity = -52567 * 654W + 11637 Growth Months June June-Sept (n=37 RZ= 0.82 ) Fig. 1. Growth pattern of potentially first spawning males during the year prior to spawning. Starting weights are. indicated. Bars denote 95% C.I. Only during the first growth period (June to July) was growth in females which ultimately matured, significantly better than in those which did not mature. There after, there was no significant difference in size indicating that non-spawning fe males partially recovered from the poorer early growth (Fig. 2). 220 Females Mature(g ) -783 (581-1115) 200 Immature (g) 795 (586-1151) Nj _ 180 Fig. 3. The relationship between fecundity, Q--~ 1 '60 June weight and June to September 'c growth. o 140 Discussion c a> o 207 growth did not directly .influence matura References tion rate but that some other factor (such as stress from capture) may have had a Hester, F. J. 1964. Effects of food coincidental hut separate effect on growth supply on fecundity in the female and maturation. However, all fish were guppy, Lebistes reticulatus (Peters). held for at least one month prior to exper J. Fish. Res. Board Can. 21(4): imental use and they showed no obvious 757-766. signs of ill health. Hislop, J. R. G., A. P. Robb, and J. A. Relationships between fecundity and Gauld. 1978. Observations on effects ration have been demonstrated for several of feeding level on growth and repro species although the mechanisms involved duction in haddock, Melanogrammus may differ. In Salmo gairdneri, restricted aeglefinus (L.) in captivity. J. food supply resulted in a high proportion Fish. Biol. 13(1): 85. of atretic eggs (Scott, 1962), but in Lett, P. F. 1978.. A comparative study of Poecilia reticulata (Hester, 1964), Pseudo- the recruitment mechanisms of cod and pleuronectes americanus (Tyler and Dunn, mackerel, their interaction, and its 1976), and Gadus morhua, differential implication for dual stock management. recruitment of oocytes may be involved. My Ph.D. Thesis, Dalhousie University, 125 results indicate that the relationship be P- tween growth and fecundity was not a con May, A. W. 1967. Fecundity of Atlantic founded function of gonad growth since cod. J. Fish. Res. Board Can. 24(7): ovaries did not appreciably increase in 1531-1551. weight until after October. The growth Oosthuizen, E., and N. Daan. 1974. Egg period having the greatest influence on fecundity and maturity of North Sea fecundity corresponded to that period in cod, Gadus morhua. Neth. J. Sea Res. the egg maturation cycle in which oocytes 8(4): 378-397. reached the circumnuclear ring phase (maturation commitment). In this respect, Scott, D. P. 1962. Effect of food quantity the data are consistent with the hypothesis on fecundity of rainbow trout, Salmo of Woodhead and Noodhead (1965). gairdneri. J. Fish. Res. Board Can. 19(4J: 715-731. If the relationship between fecundity, Tyler, A. V., and R. S. Dunn. 1976. June weight and June to September growth Ration, growth, and measures of somatic applies to the natural situation it can be and organic condition in relation to concluded that, over the size and growth meal frequency in winter flounder, range observed in this experiment, initial Pseudopleuronectes americanus, with size had the greater influence on fecun hypotheses regarding population dity. However, much of the individual homeostasis. J. Fish. Res. Board Can. variability in relative fecundity, encoun 33: 63-75. tered in nature, might be explained by Woodhead, A. D., and P. M. J. Woodhead. differences in summer growth. 1965. Seasonal changes in the physiology of the Barents Sea cod, Acknowledgements Gadus morhua L. in relation- to its environment. I. Endocrine changes I am indebted to Drs J. S. Scott and particularly affecting migration and D. F. Markle and to Messrs P. Hurley and maturation. Int. Comm. Northwest Atl. P. Koeller who made many helpful sugges Fish. Spec. Publ. No. 6: 691-715. tions to improve this manuscript. Mrs. T. Parker and Mr. W. McMullon assisted in the preparation of text and figures. 208 ENVIRONMENTAL FACTORS AND REPRODUCTION 20? ENVIRONMENTALFACTOR S INFIS HREPRODUCTIO N R.J.Wootto n Department ofZoology ,Universit y Collegeo fWales ,Aberystwyth ,Wales ,U.K . Summary goalo fthi srevie w ist oconside r someo f these influences,a sfa ra spossibl ewithi n Environmental factorsac tbot h asselect theframewor k of abio-energetic s modelo f iveforce si nth eevolutio no f reproductive fishreproduction . strategies and asproximat e factorsdurin g thelife-tim eo findividuals . They affect Bioenergeticsmode lo f reproduction bothquantitativ e andqualitativ e components ofreproductio nb ymodifyin g thepartition Inth ebioenergetic smodel ,th e individual ingo ftim e andresource sbetwee nreproduct fishi sviewe d asa devic etha tconvert sfoo d ivean dnon-reproductiv e activities. The asa ninpu t intoreproductiv eproduct s asa n confidentpredictio n ofth eeffect so fsuc h output. Theneuro-endocrin e system iscon factorsrequire s anunderstandin g ofth e ceptualised asa mechanis m that achievesa n physiologicalmechanism sb ywhic hth eeffect s -adaptiv epartitionin g ofth eresource smad e aremediated . Becauseo fth ecomplexit yo f availableb y feedingbetwee n reproduction theenvironmenta l andphysiologica lcompo andth ecompetin gdemand so fsomati cmain nents,suc h anunderstandin gwil lrequir e tenance andgrowth ,i nth efac eo ffluctuat careful experimentationwit h attentionpai d ingenvironmenta l conditions,th emos t toth eappropriat e controls and statistical crucialo fwhic h isth efoo d supply itself. analysis. Inparalle lwit h theinput/outpu tmode lo f Keywords:feeding ,fecundity ,gametes , energybein gpartitione dbetwee n thegonad s timing. andth esoma ,ther e isa tim ebudge tmode li n whichth eneuro-endocrin e system isconcept Introduction ualised asa mechanis m thatachieve s anadap tivepartitionin g oftim ebetwee nreproduct Anecologis t studying fishreproductio ni s ivean dnon-reproductiv ebehavioura lactivit interested inth equantitativ e relationships ies. Measurements ofth eenerg y costso fth e betweenenvironmenta l factors andth erepro activities allowstranslatio nbetwee nth etw o ductiveoutpu to fth efish . The aim ist o models. predict thereproductiv e successfro ma knowledgeo f theprevailin g environmental There arerelativel y fewstudie so nth e conditions. Ifth eprediction s aret ob e energy costso freproductio n infishe s madewit hconfidence ,th ephysiologica l (Wootton, 1979). Reproductive effort (R.E.) mechanisms thatmediat ebetwee nth efactor s canb edefine d asth erati oo fth eenerg y and thereproductiv e outputmus tb eunder content ofth eegg s (orsperm )produce d ina stood. Mostphysiologica l studieso nth e giventim eperio d toth eenerg y contento f effectso fenvironmenta l factorshav einvest thefoo d consumed intha tperiod . Female igated theirrol ei ndeterminin g thetimin g medaka (Oryziaslatipes )ha d anR.E .o fabou t ofth ebreedin g season. Because themajorit y 5-25%dependin g onratio n andtemperatur e of suchstudie shav ebee no ntemperat e (Hirshfield, 1980). Theviviparou s species,th erole so ftemperatur e andphoto - Poeciliopsis occidentalis innatura lpopul period insynchronizin g thetimin go fbreed ationsha dR.E. sestimate d torang ebetwee n ingi nrelatio n toth eseasona l cyclehav e 3.1 and 9.8% (Constantz,1979) . Forfemal e receivedmos t attention (deVlaming , 1974). sticklebacks,Gasterosteu s aculeatus,th e R.E. infemale stha twer eno tchangin gi n Althoughth etimin go f reproduction weightwa s 14-27% (Wootton, 1977). Glebe& relativet oseasona lvariation s isa nimport Leggett (1981)estimate d thati nsemelparou s antelemen to f thereproductiv e strategyo f Alosasapidissim a andsalmonid smor e than70 % aspecies ,i t isonl yon eo fth ecomponents . ofth etota lenerg y reserveswa suse d inth e Thelife-tim ereproductiv eoutpu t ofa fis h spawningmigratio nwherea s initeroparou s_A . willdepen do nsevera llife-histor y character sapidissima andsalmonid s lesstha n60 %wa s isticsincluding : agea tfirs tmaturity , used. These figuressugges t thesignifican t intervalbetwee n successivebreedin gseasons , energy costsassociate d withreproductio nan d numbero fbreedin g attemptsdurin g abreedin g emphasise theimportanc e ofth ephysiologica l season,fecundit y perbreedin g attempt,siz e mechanisms thatensur ethes ecost s areex andqualit y ofth egamete s and theeffective pended atecologicall y appropriate timesan d nesso f anyparenta l care. Environmental inappropriat e environmentalconditions . factorsma y influence eacho f these,an dth e 210 Environmental effectso nfeedin g Neuro-endocrine effectso nfeedin g Environmental factorsma y limit therepro Theneuro-endocrin e system thatcontrol s ductiveoutpu tno tbecaus eo fan ydirec t reproductionma y also affect thefeedin g effecto nth ereproductiv e systembu tbecaus e motivationo f thefish . Experimentswit h ofthei rdirec t effectso nth erat eo ffoo d exogenous androgens such as11-ketotesto - consumption andthu so nth ematerial savail terone insalmonid s and carphav eshow ntha t ablefo rreproduction , hormonal treatments canincreas egrowt h rates,appetit e andth efoo d conversion Temperatureha s astron geffec t on the efficiency (Matty &Lone ,1979 ;Fagerlun de t rateo ffoo dconsumptio n infishe s (Elliott, al, 1979). Someprotogynou s sex-reversing 1981). In(J .aculeatu s therei sa monotonie , fishsho w aspur t ingrowt hdurin gth e positiverelationshi pbetwee nth emaximu m change from female tomal e (Shapiro, 1979). rateo ffoo dconsumptio n andtemperatur eove r As femaleG .aculeatu s comet oth een do f therat e5.0-19° C (Woottone t al, 1980). At thebreedin g season,thei r adli b foodin lowtemperatures ,th edifferenc ebetwee nth e take declines (Fletcher,unpub) , whichals o maximum ration thefis hconsume s andit s suggests thatth eneuro-endocrin e system maintenance ration ismuc hnarrowe r thana t thatcontrol sreproductio n exertssom e higher temperatures,th escop efo rgrowt h controlove r feedingmotivation . increaseswit htemperatur e (Allen& Wootton , 1982),henc eth escop e forth etransfe ro f Thusenvironmenta l factorswhic hac t material toth egonads . Ina natura lpopul directlyo »th ereproductiv e systemma y also ationo f(J .aculeatu s during thewinter ,th e cause changesi nfeedin gbehaviou rwhic har e rateo ffoo d consumption wasclos et oth e mediated by thereproductiv esystem . maintenance requirements andonl y asmal l proportiono f theenerg y incomewa s invested Ageo ffirs t reproduction inth egonads ,wherea s insprin gth emainten ancecost s formed amuc h smallerproportio n Short generation timesCa ngenerat ehig h ofth eenerg y incomean d therewa s arapi d rateso fnumerica l increase,s otha t afis h growth ingona d size inth efemale s (Wootton canincreas e itsgeneti c representation in et al, 1980;Alle n& Wootton , 1982). thenex tgeneratio n byshortenin g thetim e ittake st obecom esexuall ymature . Ina Theeffec t ofphotoperio d onth erat eo f strongly seasonalenvironment ,ther ema yb e food consumptionha s received relatively arestricte d period of time inwhic hbreed littlesystemati c study. Many speciesten d ingca ntak eplac e successfully sotha tth e torestric t theirnorma l feeding activityt o minimum ageo ffirs t reproduction iscon certainperiod sdurin gth edie lcycle ,an d strained. withseasona l changesi nth erelativ elengt h ofth eligh t anddar k componentso fth e Minimum ageo ffirs t reproductioni s cycle,th etim e available forfeedin gca n genetically determined, although ina fe w vary. In(5 .aculeatus ,feedin g inFebruar y casesi ti spossibl et oinfe r theenviron andDecembe rwa srestricte d toth erelativel y mental factorstha thav ele dt oth eevol fewhour so fdaylight ,wherea s inMay ,feed utiono f anobserve d age. A fieldstud yo f ingtoo kplac eove rvirtuall y a24 hperio d Poeciliareticulat ai nTrinida d hasimplic (Allen, 1980).' Thu seve ni fphotoperio dha d atedpredator y fisha sa nimportan t factor, nodirec t effecto nth ereproductiv e system fori npopulation s inwhic hadul t guppies of(3 .aculeatus ,th eeffect so fvariation si n wereheavil y predated,th esiz e andb yim food consumption imposedb yvariation s inth e plicationth eag ea tfirs t reproductionwa s timeavailabl e foractiv e feeding couldin reduced (Reznick8 sEndler , 1982). fluencebot hth etimin g andquantitativ e aspectso freproduction . Inmos t fishspecie s agean d size arecon founded becauseo f theindeterminat enatur e Waterqualit yma y alsoinfluenc eth erat e of fishgrowt h and iti slikel y thatwithi n of food consumption ands oth eresource s agive npopulation ,i t issiz erathe rtha n availablet ob epartitione d intoreproduct agewhic hdetermine swhe n reproduction ion, e.g. feeding rate inG . aculeatusseem s occurs. Aim (1959)conclude d thatwithi na tob edepresse d atlo wp H (Faris,unpub) . givenpopulatio nth efaste rgrowin gfis h tended tomatur e ata nearlie r agetha nth e Inassessin g theimportanc e ofenvironmen slowergrowin g fish. Growthrat ei s talfactor s ininfluencin g thereproductiv e particularly susceptible toeffec to fenvir biology of fishes,i ti snecessar y todis onmental factorssuc h asfoo d andtempera tinguishbetwee ndirec t effectsan d indirect ture. In G. aculeatus asmalle rproportio n effectswhic h aremediate d through thein of females fedo na lo wratio nbecam e fluenceo f thefactor so nfeeding . sexuallymatur e thanfemale s fedo nmediu m orhig hrations . Mostpopulation so fG_ . aculeatus reachsexua lmaturit y atth eag e 211 of ayear ,bu t insom epopulation smaturit y rate atwhic h thecritica l size isreached . mayb edelaye db y ayea r (Wootton, 1976), Thesecon dhypothesi s suggests thati ti s althoughth e factorswhic hcaus ethi sdela y thesocia lenvironmen t thatdetermine swhe n areno tknown . Food shortagema y causea n sexreversa l takesplace . Inprotogynou s increase inth eag eo fmaturit y becauseth e species,th eremova l of themal e inth e critical sizei sreache d aftera longe r groupresult si nth esë xreversa l ofon eo f period of time,bu tth elong-ter m selective thefemales ,ofte nth e largest. Themechan effect ofchronicall y lowfoo d levelsma yb e ismb ywhic hth esocia lcontro lo f there toselec t fora smal l sizea tmaturit y and versalproces s ismaintaine d iscontrover hencea decreas ei nth eag ea tmaturity . sial. Unfortunately theresee m tohav ebee n Populationso fG .aculeatu s ininfertil e nosystemati c experimental studieso nth e watersca nreac hsexua lmaturit y aton eyea r effectso fresourc e levelssuc h asfoo do n butd os oa t asmal lsiz e (Wootton et al, theproces so f sex reversal. Too littlei s 1978). knowno fth ephysiologica l controlo fse x reversal fora predictiv emode lo f thepro Inth ecyprinodont s Xiphophorusmaculatu s cesst ob edeveloped . andX .variatu sth esocia lenvironmen tin fluences the ageo f firstreproduction . Fecundity Largermale s tendt odela y thematuratio no f small andbehaviourall y subordinatemale s Thelifetim e fecundity ofa femal ei sth e (Sohn,1977 ;Borowsky , 1978). Infemal e product ofth efecundit y perspawning ,th e Sarotherodonmossambicus ,a cichlid ,socia l numbero fspawning spe rbreedin g seasonan d isolationdelaye d the first spawningb y thenumbe ro fbreedin g seasons. Eacho f about1 0day s (Silverman, 1975). thesecomponent si slikel y tob e dependent onenvironmenta l factors. Inthos especie s Thephysiologica lmechanis m which controls which deposit allth eegg sproduce dpe r theonse to f sexualmaturatio n forth efirs t breeding seasonove r ashor t timeinterval , time inrelatio nt oag ean d sizei sno t andi nsemelparou sspecie stha t aretota l understood,bu t theonse t ofmaturatio ndoe s spawners,an yenvironmenta l effectso n represent acrucia l change inth epatter no f fecunditywil lb eexerte d prior totha t partitioningo fbot htim e andmaterial sbet spawning. Inpartia lo rseria lspawners , weenreproductiv e and non-reproductive environmental factorsma ymodif y fecundity activities. within abreedin gseason . Sex ratios Thedetectio no fenvironmenta l effectso n fecundity iscomplicate d infis hbecaus e Populations inwhic hth ese xrati oi s there isa positiv erelationshi p between biased towardsfemale swil lhav ehighe r fecundity andbod y size. Fishusuall y show totalreproductiv e outputstha npopulation s indeterminategrowt hs otha ta sa femal e withmor eequa lse xratio si fal lth efemale s growshe rfecundit ywil l tendt oincrease . haveacces st oth efertilizin g capacityo f Appropriate statistical andexperimenta l themales . Twomechanism s throughwhic hen techniquesmus tb euse d topartitio nou tth e vironmental factors caninfluenc e sexratio s effectso f achang e inbod ysiz eo nfecund thatd ono t seem todepen d ondifferentia l ity from anydirec t effectso fenvironmenta l mortality rateshav ebee nreported . Inth e factorso nfecundity . Any environmental Atlantic silverside,Menidi amenidia ,a effectso ngrowt h insiz ewil lprobabl yb e specieswit h alif espa no f ayear ,th een reflected inchange si nfecundity . vironmental temperature influences these x ratioo f theprogen y ofsom e females. Warm Onceth eeffect so fsiz eo nfecundit y have temperatures tend tobia sth ese xrati o been removed,th eeffect so f therat eo f towardsmales ,th ecritica lperio d forse x foodconsumptio no nfecundit y canb edefined . determination apparently occurring latei n Finally,th eeffect so fothe r environmental larvaldevelopmen t (Conover& Kynard , 1981). factorso nfecundit ywhic hcanno tb eaccoun Sequentialhermaphroditis m hasbee nreporte d ted forsolel y interm so fth eeffect so f forman y species (Shapiro,1979 )an d inman y those factorso nth erat eo f foodconsump casesthi si sassociate dwit h abiase dse x tionca nb e analysed. OnlyHirshfield' s ratio. Inprotogynou s species,th ese x (1980)stud yo n0^ .latipe si nwhic hth e ratioi sbiase d towardsfemales . Twomajo r effectso fratio n andtemperatur ewer eana hypotheseshav ebee npropose d toaccoun tfo r lysedusin gcovarianc etechnique shav eapp theinitiatio no fse xreversal :th edevelop roached thiscomplet e analysis, j). latipes mentalhypothesi s and thesocia l control femalesspaw ndail y for aslon g asfou r hypothesis. The formerpostulate s thatse x months. At2 5 and2 9C a positiv ecorrel reversal isinitiate dwhe nth e fishreac ha ationbetwee nth edail yrat eo feg gproduct critical sizeo r age. Ifsiz e iscrucia l ionan dratio nwa sfound ,wherea s at2 7C , thenenvironmenta l factors such asfoo d therewa sn osignifican t correlation. The supplyma yhav e animportan t influenceo nth e rationsconsume d at2 7 and2 9C wer ecom - 212 parable,bu t at2 5C th ehighes tratio ncon thebreedin g seasonwa shighl y correlated sumedwa ssimila r toth elowes tratio nuse d withratio n (Wootton,1977) . Thisrespons e atth ehighe r temperatures,a strikin gexam tofoo d levelmean stha tth efemal eCi . pleo fth eeffec to ftemperatur eo nfoo d aculeatus cantrac kfoo d level interm so f consumption andhenc e fecundity (Hirshfield. theireg goutput . Young G. aculeatusstar t 1980). Theapparen t interactionbetwee n eatingsimila r food itemst oth eadult srel temperature and ration atth ehighe rtempera atively soonafte rhatchin g sotha tth efoo d tures raisesth epossibilit y that temperature availability experienced by theadult si s has adirec t effecto nth ereproductiv e sys likely tob e agoo dpredicto ro ftha texper temo fthi sspecies ,bu t themajo reffec to f iencedb y theiroffspring . temperature isprobabl ymediate d throughit s effecto nfoo dconsumption . Thephysiologica lmechanis m bywhic hfoo d supply affects fecundity isno tknown . Nor InG . aculeatus thebreedin g season lasts isi tknow nt owha t extentdifferen t stages twoo rthre emonth s inlat esprin g andsummer , ofoogenesi s aresensitiv e tofoo dshortage . with thefemale sspawnin g atinterval so fa Atresia andth erat eo frecruitmen t ofvit - fewdays . Theeffec t ofratio n andbod ysiz e ellogenic oocyteshav ebee nsuggeste da s onfecundit y perspawnin gwa s analysedusin g mechanisms forcontrollin g thenumbe ro feggs . mutiple regressiontechnique s (Fletcher & Inth ecyprini dPhoxinu sphoxinus ,ther ei s Wootton,unpub) . The twopredicto rvariable s someevidenc etha tbot hmechanism sma yoper accounted for55 %o f thevarianc e infecund atebu t atdifferen t stages inth e annualre ity andther ewa sn o significant interaction productive cycle. Femalescollecte d in between them. Thestandardise d regression autumn andhel d at1 0C an d a12L12 Dphoto - coefficientsindicate d thatbod ysiz ewa s period forsi xweek sha d ahighe rproportio n about twice asimportan t asa predicto r ofthei roocyte s inth estag eo fvesicula r variabletha nration . Thisresul t confirms yolkformatio n ifthe ywer efe dtha ni fthe y andextend stha t ofWootto n (1977)wh onote d werestarved . Theproportio no fatreti c theimportanc eo fbod y size,bu tusin ga oocyteswa sver y lowi nbot hfe d andstarve d smallernumbe ro freplicate sha dno tdetect fish. When femaleswer ecollecte d insprin g ed asignifican t effect ofration . andhel d at7 C an d aphotoperio d of14L8D , thefe d fishha d ahighe rproportio no f oocyteswit hgranula ryol ktha n starved fem InG .aculeatu s and in0 .latipe sa t2 7 ales,bu t thelatte rha d ahighe rproportio n and2 9C ,ther ewa s aninvers e relationship ofatreti coocytes . Insumme rcondition so f betweenR.E .an dration . Thusth elowe r 15C an d 16L8D,starve d femaleslos tal lthei r fecundity atth elowe rration snevertheles s oocytes containing granularyol k and thebul k involvedpartitionin g ahighe rproportio no f ofthei roocyte swer e inth epre-vitellogeni c thefoo d consumed intoeg gproduction . Fish stages. Sinceegg swer eno t spawned during on lowration swer esacrificin g growtht o thesumme rexperiment ,i twa s assumedtha t maintain theirreproductiv eoutput . theovarie so fth estarve d femalesha dunder gonemassiv e atresiadurin g thesi xweek so f Cichlasomanigrofasciatu m femalesshowe da starvation (Mehsin,1982) . Histological declinei nth efecundit y perspawnin gwhe n studieso nth eovarie so fwil d andexperimen kept onlo wrations . Thisdeclin ewa srel talM .aeglefinu s suggested thatth elo w ativelymuc h greater thanwa snote d inCI . fecundityo ffis ho n low rationswa sno tdu e aculeatus inwhic hth eeffec t ofratio no n toa chang e inth erat eo f atresiabu t are fecundity perspawnin gwa swea k (Townshend & duction inth enumbe ro foocyte s developing Wootton,unpub) . that season. Inwil d fishth enumbe ro f oocytesbein gresorbe d varied atdifferen t Themarin egadoid ,M e1anogrammu saeglefinus , stageso foogenesi swhic hsuggeste d thatad isa partia l spawnerwit h anextende dbreed justmentswer econtinuall ymad e toth enumbe r ing season. Femaleso n aratio nclos et o ofoocyte sundergoin gmaturation . Theuppe r maintenance retained thetrai to fpartia l limito fpotentia l fecundityma yhav ebee n spawning,bu t ateac h spawningproduce d far determined by theNovembe rprio r toth esprin g feweregg stha n femaleso nhig hrations . The inwhic h spawningtoo kplac ean dvariation s fecundity ofth eexperimenta l fishwa scom inth efoo d supply then caused reductions paredwit h thefecundit y ofwil d fisho fth e from thisuppe r limit (Robb,1982) . same length. Femaleso nth elo wratio nwer e producing anaverag eo f 16.9%o fth eexpecte d numbero feggs ,whil efemale so nth ehig h Thesocia lenvironmen tma y influencefecun rationproduce d anaverag eo f 89.1%o fth e dity (Dahlgren,1979 ;Kapur , 1981), butth e expected number (Hislope t al, 1978). experimental design fordetectin g sucha n effectmus t showtha t iti sno t solelya Incontrast ,femal eG . aculeatus onlo w result ofchange s inth erat eo ffoo dcon rationsalmos tmaintaine d thefecundit ype r sumption asa resul to fth esocia lenviron spawningbu tha d fewerspawning s thanfemale s ment. Infemal e G. aculeatushouse di n onhighe r rations. Thusth efecundit yove r pairsi n13 1tanks ,th eaggressivenes s ofon e 213 ofth efemale s frequently prevented accessb y onreproductio n through itseffec to nth e theothe r femalet oth eavailabl e food,an d resources availablet ob epartitione d into thesubordinat e femaledi dno tproduc eeggs . gonadalproductio nan dt owha t extent itha d But thisresul t isprobabl y anexperimenta l adirec teffec t onth ereproductiv e system. artefact,i nnatura lpopulation s femalesar e Oogenesis seemed tobe-sensitiv et owate r frequently seenfeedin g inschool s andma y quality,bu t iti sals osensitiv e tofoo d not form strongdominanc e relationshipsi n supply. such asituatio n (Wootton,1976) . Itha s beensuggeste d thatpopulatio n density can Theeffect so fenvironmenta l factorso n affect thefecundit y and fertility offemal e therat eo fgamet eproductio nb ymal efis h ]?.reticulata . Dahlgren (1979)foun dsom e havereceive d little attentionbecaus eo f evidencetha t density did affect fecundity thedifficult y inquantifyin g sperm. In and fertilitywhe n aconstan tweigh t offoo d male (5.aculeatu s theteste s aresmal lan d was fedbu t theresult swer eambiguous . probably lesssensitiv e tofoo d levelstha n When foodwa ssupplie d inexcess ,n oeffect s theovarie s (Stanley,unpub) . ofdensit y oneithe r fecundity or fertility weredetecte d (Dahlgren,1980c) . Socialin Therei sa nee d fora systemati cexperi hibitiono f theonse t ofsexua lmaturatio n mental studyo fth efactor swhic h determine seemst ob ea mor esignifican t factor than thepotentia l fecundity offishe s andth e thesocia lmodulatio n offecundity . mechanisms bywhic hth eactua l fecundity is determined including therelativ erole so f Pathogensparticularl y parasites interfere atresiaan dth erat eo frecruitmen t ofpop witheg gproductio n by females. Cr.aculeatu s ulationso fmaturin goocytes . femalesinfeste dwit hth epleurocercoi d stage ofth ecestod eSchistoeephalu ssolidu softe n Gametequalit y failt ospawn ,an dheavil y infested females have significantlysmalle rovarie s thanun - Environmental effectso ngamet equalit y infestedo rlightl y infested fish (Wootton, have received less attention thanthei r 1976). SJ.aeglefinu s infestedwit hth e effectso nfecundity . Twoaspect so feg g copepodLernaeocer abranchiali sha dsignific quality areeg gsiz ean dchemica lcomposit antly lower fecundities thanuninfeste d fish ion. InCi .aculeatu s therewa sa signific ofth esam e length. Thissignifican tdiffer antbu twea kcorrelatio nbetwee neg gdia encewa smaintaine d whenth ecompariso nwa s meter andratio n and apositiv e relationship forfemale so f agive ngutte dweigh t (Hislop betweeneg gdiamete r and thelengt ho fth e & Shanks,1981) . BothG . aculeatus andM . frya thatching . Nosignifican t relation aeglefinus showa clea rrelationshi pbetwee n shipwa sfoun dbetwee n frylengt h andth e fecundity and food supply and iti sprobabl e numbero fday stha t frycoul dsurviv eafte r thatth eeffect so fparasitis m inthes e hatchingwhe nstarve d (Fletcher,unpub) . No speciesar ea resul to fth eparasite sinter significant effect ofratio no nth echemica l cepting resources that shouldb e investedi n composition ofth eegg swa sdetected . Apart eggproduction . But insom especie sth e froma sligh teffec to fratio no neg gdia parasitema y interferedirectl ywit h there meter,femal e C. aculeatusmaintaine d both productive systemo fth ehos t toinhibi t thesiz e andqualit yo f theiregg si nth e gonadal development sotha t theparasite s are faceo f lowfoo d supply. Dahlgren (1980a,b) not competingwit hth egonad sfo rresources . detectedn osignifican t effect ofdifferen t Disentangling thedirec t and indirect effects levelso fprotei no rnon-protei n inth edie t ofparasitis m onth ereproductiv e systemwil l onth ediameter so fov ai nP .reticulata . requirecarefu lphysiologica lanalysis . Somefiel d studieshav esuggeste d thaten Aspectso fwate rqualit y such astempera vironmental factors,includin g food supply, ture,p Han d levelo f contaminationb y canhav edetectabl e effectso neg gquality . pollutants areknow n tob erelate d tofecund InRutilu srutilus ,Kuznetso v& Khalito v ity (Wootton,1979 ;Billard ,1981 ;Gerking , (1978)claime d thatth eamoun to f fati nth e 1980). Gerking'scomprehensiv e study ofth e ovarieswa shighe runde r favourable feeding cyprinodontCyprinodo n n. nevadensissuggest conditions,althoug h they found littleeff edtha treproductio nwa smor e sensitivet o ecto nth eweigh t ordiamete ro fth eeggs . waterqualit y thanothe r componentso fth e InCoregonu s albula,th echemica lcomposit lifehistory . Therewa sa relativel y narrow iono fth eegg s andthei rdr yweigh twer e rangeo foptimu m temperatures foreg gpro related toth elak e fromwhic h thefemale s duction,an d thelatte rwa s alsosensitiv et o weretaken . Thelak ei nwhic h fishgrowt h acidwater ,wherea s therewa s arelativel y wasslowes tha d thepoores tqualit y eggs widerang eo fsalinitie sove rwhic heg gpro (Kamler& Zuromska , 1979). But inthes e ductionwa smaintained . Sinceth erat eo f studiesth eabsenc eo fexperimenta l studies food consumption isrelate d totemperatur e makes itdifficul t toidentif y thecausa l andpossibl y alsot opH ,i ti sno t cleart o basis forth eeffect sreported . what extentwate rqualit y exerted itseffec t 214 Athig h temperature, CJQ. nevadensisprod pH isno tknown . uced anumbe ro f small softeg gshell scon tainingn oyolk ,whil ea tlo wp Hth epercent Theenvironmenta lrequirement sfo rsuccess ageo f abnormal eggswa s increased (Gerking, fulspawnin gmay , insom especies ,b erigor 1980). InGambusi aaffini sexposur eo f ous andsubtle . Aquaticvegetatio n isa femalest ofreshwate r inthei r firstwee ko f stimulus forovulatio n inCarassiu sauratu s lifecause d themt oproduc eheavie rtha n buti sa nessentia lcu efo rspawnin g (Stacey averagene wbor ncompare dwit h femalestha t et al, 1979). P.phoxinu sha sprove d werekep t inbrackis hwate r (Stearns,1980) . difficult tobree d reliably inaquaria , Infreshwate rth e (}.affini sha dt omee tth e althoughmanipulatio n ofth ephotoperio dan d energy costso fosmo-regulatio nbu tthi sdi d temperature canstimulat e gametogenesis notexplai nwh y theyproduce d largeroff (Scott, 1979). Kapur (1979)note dth e spring. difficulty ofobtainin g spawning insom eo f theIndia ncarp si ncaptivity . Thephysiologica lmechanism sb ywhic hth e size andqualit y ofegg sar econtrolle d are Termination ofth ebreedin g season obscure,an d soi ti sdifficul t topredic t thepossibl eeffect so fenvironmenta lfact Infish ,fecundit y isa functio no fbod y ors. Theevidenc e suggests that inth efac e size,bu tbreedin g involvesth e commitment ofenvironmenta l fluctuations,fis hten dt o ofresource s tospawnin g that couldhav e conserveeg gqualit y rathertha nfecundity . beencommitte d toincreasin gbod ysize . Terminationo fbreedin gpermit sth ealloc Reproductive behaviour ationo fresource s tosomati c growth. (3. aculeatushel dunde rpermissiv e conditions Behaviour associated with thesuccessfu l ofphotoperio d andtemperatur e alternates reproduction islikel y tob esensitiv et o betweenreproductivel yactiv ean d inactive environmental factors. Thereproductiv e phases,althoug hth eperio dbetwee nth e behaviouro fmal eG . aculeatusi scomplex ,i t starto fsuccessiv ephase so freproductiv e includes territorial defence,nest-building , activity isles stha nth eyea r thatwoul d courtship andparenta lbehaviour . Anexperi separate successivebreedin g seasonsi n mental analysiso fth eeffec t ofdensit yo f natural conditions (Baggerman, 1980). Thus malesan d foodratio n onsom e aspectso fmal e there isevidenc e fora nendogenou s control behaviour indicated thatdensit ywa s acru ofreproductiv e cycling inCi .aculeatu san d cial factor interritoria lbehaviou ran d probably otherspecie sto o (Sundararaj, nesting. Ina give nfixe d area,th epropor 1978). InG^ .aculeatu senvironmenta lfact tiono fmale sholdin g aterritor y andbuild orsca nmodif y thelengt ho fth ereproduct - ing anes twa slowe ra thighe rmal edensi ivelyactiv ephase . Femaleskep to nlo w ties,an dther ewa s agreate r chanceo fa rationshav eshortene dreproductiv ephase s nestbein g destroyed. Lowfoo dration sals o withprematur e regressiono fth eovarie s caused areductio n inth eproportio no f (Wootton,1977) . Inth eclosel y related malesholdin g aterritor y andbuildin gnests , Culaea inconstans,th een do fth ebreedin g butha dn oeffec t onth eproportio no fnest s seasoncoincide dwit h thetim ewhe nth e destroyed. Whenmale swer e transferred from water temperaturewen t above2 0C fo rpro thegroup st o isolation,a hig hproportio no f longedperiod si nsumme r (Reisman& Cade , themale s thatha dreceive dhig hration sthe n 1967). Both foodshortag e andhig htempera builtnests ,eve ni fthe yha dno tbee n turescause dgonada lregressio ni n successful inth egroup . Incontrast ,onl ya Gillicthysmirabili s (deVlaming , 1974), smallproportio no fth efis htha tha drec while food shortagecause drapi d regression eived alo wfoo d rationbuil t nests inisol inth eovarie so fP .phoxinu shel d at1 5C ation (Stanley& Wootton ,unpub) . Isolated (Mehsin,1982) . maleso nlo wration s spent lesstim efannin g eggs,althoug hthei rsucces s inhatchin ga Thechange s inenerg y partitioning during singleclutc ho fegg swa sno tdifferen t from gonadal regressionwoul db eo fconsiderabl e maleso nhighe rrations . Food shortage interest especially ifth ephysiologica l altersth etim ean dprobabl y theenerg ypar mechanismscontrollin g thechange scoul d titioning ofmal e sticklebacks,bu tth e alsob eelucidated . Therelativ e importance physiological controlmechanism s areno t ofendogenou s and exogenous factors inth e known. processneed sclarification . InC.n.nevadensi sth erang eo ftemperature s Timingo fbreedin g overwhic hmale s showed activechasin go fth e femalewa swide rtha n theoptimu mrang efo r Althoughth econtro lo ftimin go fbreedin g eggproductio n (Gerking, 1980). But,i n seasonsha sreceive dmuc hattentio nfro m general,th erelativ e sensitivity ofth ebe physiologists,ther ehav ebee nfe wecolo havioural components ofreproductio n toen gicalstudie swhic hsho wwh y relatively vironmental factors sucha stemperatur ean d precisecontro l of timingi sessential . 215 Nevertheless,i ntemperat especies ,th e ductivecycl ewhic har emor edemandin go f period oftim ei nwhic hth emajorit yo f resourceswhethe r forgametogenesi s orth e young fishbecom e free-swimming isrelat behavioural aspectso freproductio n aremor e ively short comparedwit h thetim epotent sensitive toth etimin gcue so fphotoperio d iallyavailable . andtemperature . Many specieshav ewid enorth-sout h geo Therelativ eimportanc eo ftemperature , graphical ranges,s otha t aspecie sma yex photoperiod andothe r factorsa stimin gcue s perience awid erang eo fseasona l patterns variesbetwee n taxa (deVlaming , 1974). It ofphotoperio d andtemperature . Some isno t clearwha t therelationshi p isbetwee n speciesma y alsoestablis h breedingpopul suchvariatio n and theecologica lrequire ations inenvironmenta l regimeswhic hthe y mentso fth eadults ,egg san d fry,bu tth e haveno texperience d inthei r evolutionary ecology ofth eearl yontogeneti c stagesma y history,e.g .th ebreedin gpopulation so f £[. beimportan t indeterminin gwhic h cuesar e trutta inuplan d equatorial streamso f most appropriate. Therat eo fdevelopmen t of Kenya. Thus speciesma yb e flexiblei n eggsi sparticularl y sensitive totempera theirus eo f cuest oensur e thecorrec t time ture,hig htemperature spermi t rapidescap e ofbreeding . fromth eeg gt oth efree-livin g stages.. In speciesi nwhic h therei sn oparenta lcar e Several cuesma yb e available fortimin g and theegg s areno t laid ina refug efro m andsom ema yb emor ereliabl etha nothers , predators,a perio do fincreasin gtempera but inman y casesth ecue s tend tob econ turesma yb eth emos t suitablecu e forth e founded. Inhig h latitudes,lo wtempera timingo fth efina lstage so foogenesis . In tures,shor tphotoperiod s and lowfoo d specieswit hparenta l caresuc h asG . availability mayoccu r together inth esam e aculeatus,photoperio dma yb ea mor ereliabl e timingcue . season. Init sevolutionar y history,a speciesma y neverhav eencountere d these factorsvaryin g independently, and soma y Disentangling theeffect so ftemperature , respond toa comple xo fcue srathe rtha na photoperiod andothe rfactor so nth etimin g single factor. P.phoxinu shide sunde r ofreproductio n requires considerationo f stones during theda y inwinter . Only after theenvironmenta l conditionsan d theecolog y thetemperatur eha srise n above 8°Cd oth e ofth especie sdve rth enorma l reproductive fishbecom eactiv e indaytim ean dstar tt o cycle. G.aculeatu sfr yhatche d inJun ean d experienceth elonge rphotoperiod scharact thenkep to na 16L8 Dphotoperio d at2 0C d o eristico fsprin gwhic hstimulat evitello - notbecom esexuall ymatur e (Baggerman, 1980). genesis inth eovarie s (Scott,1979) . If The firstmont h afterhatchin g isa perio d fishar epresente dwit h cueso r combinations ofrapi d somaticgrowt h (Allen,1980) . In of cuestha td ono toccu rnaturall y andhav e autumn,spermatogenesi s andoogenesi s toth e not beenencountere d during theirevolution stageo fvesicula ryol k formation takeplac e aryhistory ,the nth ephysiologica l response inthi sperio d ofdeclinin g temperatures and may,i nfunctiona l terms,b enonsensical . photoperiod. At lowrations ,somati cgrowt h Suchresponse sma yno t throwligh t onth e stillha spriorit y overgonada l growthwhil e normal control oftiming . Inexperiment si n athig h rationsth egrowt ho fth elive ri s which fishar eexpose d tovariou scombinat particularly high. Atthi sstage ,exposur e toa photoperio d of16L8 Da t2 0C wil lin ionso ffactor ssuc ha stemperatur ean d ducesexua lmaturatio nwithi n6 5 days,bu t photoperiod,carefu l thoughtha s tob egive n naturalpopulation swil lno texperienc esuc h towha tstimulu scombination s represent conditions. Shorterphotoperiod s dono tin controlconditions . ducesexua lmaturation . Over thewinter , thegonads ,especiall y theovarie s continue Thebes t indicationtha tth erol e ofen tomatur eslowl yeve na tlo wrate so ffoo d vironmental factors inth etimin go fa consumption. Asth ewinte rprogresses , breeding season isreasonabl ywel lunder sexualmaturatio n after thefis h areexpose d stood istha tth etimin g canb eprecisel y to2 0C canb e stimulatedb y shorteran d controlledb yth eexperimenta l manipulation shorterphotoperiods ,s otha tb y theen do f offactors . Someprogres s inachievin gthi s February,a photoperio d of8L16 Di ssuffic hasbee nmad e for(5 .aculeatu s (Baggerman, ient,bu tunde r suchcondition s thelengt h 1980)an dSalm ogairdner i (Whitehead et al, ofth eperio d of activesexua l activityi s 1978; Billard, 1980). Inbot h species,on e short. Innatura l conditions,th elengthen alat esprin g theothe r alat ewinte rspawn - ingphotoperio d and theincreasin gtempera er, there isevidenc etha t different stages tureo f springpermi t sexualmaturation , inrecrudescenc eo fth egonad s aresensitiv e then thelon gphotoperio d andwar mtempera todifferen t environmental conditions. The tureo f latesprin g andsumme rmaintai na n phaseso f gametogenesis thatrequir e the extended breeding season. Throughout the least investment ofmaterial s appear tob e annualcycle ,unti lth eonse to fspawning , themos t independent ofenvironmenta lcon thegonad ssee mt ob erelativel y insensitive ditions,wherea sth estage s inth erepro - 216 tofluctuation si nth efoo d supply,th e Physiological experiments should alsotak e liveran dth ecarcas ebuffe rth eeffec to f intoaccoun tth eecolog yo fth especie ss o variationsi nfoo dsupply . Thusphotoperio d thatmeaningfu l combinationso fth erelevan t and temperature cuesar euse dt ocontro lth e environmental factorsca nb epresente d timingo freproduction ,bu tth enumbe ro f (Scott,1979) . Thegeographica l origino f spawnings afterth eonse to fbreedin g isa theexperimenta l population should alsob e functiono ffoo d availability (Baggerman, considered,fo rther ema yb esignifican t 1980;Wootton ,1977 ;Alle n& Wootton ,i n inter-population differences inth erespons e press). tocombination so fenvironmenta lfactors . Anydifference swoul db erelevan tt oth e Breedingi nth efemal e(î .aculeatu smake s predictiono fth eeffect so fenvironmenta l heavy demandsfo renerg y andmaterial s factorso nreproduction ,bu tma yals oyiel d becauseo fth ehig h cytoplasmicproductio ni n someinsigh t intoth ephysiologica lmechan thefor mo feggs ,whil ebreedin g inth emal e ismsthroug hwhic hth efactor shav ethei r makesheav ydemand so nenerg y becauseo fth e effects. complexreproductiv ebehaviour . Baggerman (1980)ha ssuggeste d thatchange si nsensit Thebioenergetic smode lo ffis hreproduct ivityt ophotoperio d duringth eannua l cycle ioncoul d forma valuabl e frameworkwithi n °*^L *aculeatu sdepen do nchange si na photo - whichth ephysiologica l analysesar esynth - reactivity threshold,th etimin go fwhic h esised. Themode l centres attentiono na n dependso na circadia nrhyth m insensitivit y environmental factortha ti sfrequentl yig tolight . Itwoul db einterestin gt okno wi f noredo rinadequatel y controlled inexperi therear eals ochange si nfeedin gmotivatio n ments,tha ti sth erat eo ffoo dconsumption . correlated withchange si nth ethreshold ,s o Giventh eenerg y andmateria l costso fre thatth eenerg ydemand so fsexua lmaturatio n production,foo d consumptionan dth esub aremet . Sucha correlatio n issuggeste db y sequentpartitionin g ofenerg yan dmaterial s theobservatio n thata tth een do fa breedin g betweenreproductiv e and^ion-reproductiv e season,th ematur e Q. aculeatusbecom ere activities representmajo rprocesse swhic h fractoryt olon gphotoperiod s (Baggerman, willb einfluence db yenvironmenta l factors 1980),an dals osho wa declin ei nth ea dli b andth ephysiologica lmechanism stha tmed foodconsumptio n (Fletcher,unpub) . iateth eeffect so fth efactors . InC L aculgatu^a relativel y endogenous References cycleo frecrudescenc e andregressio no fth e gonadsha sit stimin gmodulate d bytempera Allen,J.R.M. ,1980 . Theestimatio no fth e ture-dependent photoperiodic responses,bu t natural feedingrat eo fth ethree-spine d thequantit yo freproductio ni sprimaril y stickleback (Gasterosteusaculeatu sL. ) determinedb yfoo dsupply . (Pisces). Ph.D.Thesis ,Universit yo f Wales. Conclusion Allen,J.R.M .& R.J .Wootton ,1982 . The effecto fratio nan dtemperatur eo nth e Becauseo fth epotentia l complexitiesi n growtho fth ethree-spine d stickleback, therelationship s between environmentalfact Gasterosteus aculeatusL . J.Fis hBiol . orsan dreproduction ,bot h carefulexperi 20:409-422. mental design andstatistica l analysisar e Alm,G. ,1959 . Connectionbetwee nmaturity , required inth eelucidatio n ofthes erela sizean dag ei nfishes . Rep.Inst..Freshw . tionships. Manyo fth erelationship sar e Res. Drottningholm40:5-145 . size-dependent sucha sth erelationshi p Baggerman,B .1980 . Photoperiodic andendo between fecundity andsize ,s otha t adequate genouscontro lo fth eannua l reproductive techniques forcontrollin g forsize-depen cyclei nth eteleos t fishes. In:M.A .Al i dencemus tb eused . Theexperimenta l design (Ed.):Environmenta lphysiolog y offishes . should alsopermi tth edetectio nbot ho fth e Plenum,Ne wYork .p .533-567 . maineffect so fth eexperimenta l factorsan d Billard,R. ,1980 . Lespossibilitie sd e theirinteractions . Careful attentionmus t controled el areproductio n chezle s alsob epai dt oth eappropriat e levelo f poissonspa rmodulatio nde sfacteur sd e replication. Someexperiment sar edifficul t l'environnement etmanipulation shormon tointerpre tbecaus ewhil eth eexperimen ti s ales. Cah.Lab .Hydrobiol .Monterea u10 : essentially anested-desig nwit ha grou po f 11-32. fishi na tan kal lgive n thesam etreatment , Billard,R. ,C .Br y& C .Gillet ,1981 . thesubsequen t analysis treatseac hfis ha s Stress,environmen t andreproductio ni n anindependen t replicate,s otha tan y"tank " teleost fish. In:A.D .Pickerin g (Ed.): effectsar econfounde d with"treatment " Stressan dfish . AcademicPress ,London , effects. Such "tank"effect sma yb eimport p.185-208 . anti nspecie si nwhic h social interactions Borowsky,R.L. ,1978 . Social inhibitiono f sucha sdominanc ehierarchie sdevelop . maturation innatura lpopulation so f Xiphophorusvariatu s (Pisces:Poeciliidae) . 217 Science201:933-935 . MelànOgrâmmyisaeglefinu s (L.)i ncaptivity . Conover,D.O .& B.E .Kynard ,1981 . Environ J.Fish .Biol . 13:85-98. mental sexdetermination : interactiono f Kamler,E .& H .Zuromska ,1979 . Qualityo f temperature andgenotyp e ina fish . eggsan dproductio no fCoregonu s albula Science213:577-579 . (L.)i nthre eMasuria nLakes . Pol.Arch . Constantz,G.D. , 1979. Lifehistor ypattern s Hydobiol.26:595-623. ' of alivebearin g fishi ncontrastin gen Kapur,K. ,1981 . Spawning ofmajo r Indian vironments. Oecologia40:189-201 . carps- a review . Actahydochi mhydobiol . Dahlgren,B.T. ,1979 . Theeffect so fpopul 9:163-182. ationdensit y onfecundit y and fertility Kuznetsov,V.A .& N .Kh .Khalitov ,1978 . inth eguppy ,Poecili areticulat a (Peters). Alterations inth e fecundity andeg g J.Fis hBiol .15:71-92 . quality ofth eroach ,Rutilu srutilu si n Dahlgren,B.T. ,1980a . Theeffect so fthre e connectionwit hdifferen t feedingcondit different dietaryprotei n levelso nth e ions. J. Ichthyol.18:63-70 . fecundity inth eguppy ,Poecili areticulat a Matty,A.J .fe K.P .Lone ,1979 . Theeffec t (Peters). J. FishBiol . 16:83-987 ofandrogeni c steroids asdietar yaddit Dahlgren,B.T. ,1980b . The influenceo f iveso nth egrowt ho fcar p (Cyprinus threedietar y non-protein levelso nfecund caprio). Proc.Worl .Maricul .Soc .10 : ity andfertilit y inth egupp yPoecili a 735-745. reticulata. Oikos34:337-346 . Mehsin,K.A.A. , 1982. Someeffect so ffoo d Dahlgren,B.T. ,1980c . Influenceso fpopul supplyo nth eannua lreproductiv e cycleo f ationdensit y onreproductiv eoutpu ta t femalePhOxinu sphoxinu s (L.). Ph.D. food excessi nth eguppy ,Poecili a Thesis,Universit y ofWales . reticulata (Peters). Biol.Reprod .22 : Reisman,H.M .& T.J .Cade ,1967 . Physio 1047-1061. logical andbehavioura l aspectso frepro deVlaming ,V.L. ,1974 . Environmental and ductioni nbroo k stickleback,Culae a endocrine controlo fteleos t reproduction. incOnstans. Amer.Midi .Nat .77:257-295 . In:C.B .Schre k (Ed.):Contro l ofse xi n Reznick,D v& J.A .Endler ,1982 . Theimpac t fishes. VirginiaPolytechni c andStat e ofprédatio no nlif ehistor y evolutioni n University,Blacksburg ,VA .p .18-83 . Trinidadian guppies (Poeciliareticulata) . Elliott,J.M. ,1981 . Someaspect so ftherma l Evolution36:160-177 . stresso nfreshwate r teleosts. In:A.D . Robb,A.P. ,1982 . Histological observations Pickering (Ed.): Stress and fish. Academic onth ereproductiv ebiolog y ofth ehaddock , Press,London ,p .208-245 . Melanogrâmmusaeglefinu s (L.). J.Fis h Fagerlund,U.H.M. , D.A.Higg s& J.R .McBride , Biol.20:397-408 . 1979. Influenceo f feeding adie tcontain Scott,D.B.C. ,1979 . Environmental timing ing17a-methy ltestosteron eo r testosterone and thecontro lo freproductio n inteleos t attw oratio n levelso ngrowth ,appetit e fish. Symp.zool .Soc .Lond .44:105-132 . andfoo dconversio nefficienc yo funder - Shapiro,D.Y. , 1979. Socialbehaviou rgrou p yearling cohosalmo n (Oncorhynchuskisutch) . structure andth econtro lo f sexreversa l Proc.World .Symp .Finfis hNutr .Fis hFee d inhermaphroditi c fish. Adv.Stud yBehav . Tech.1:221-230 . 10:43-102. Gerking,S.D. , 1980. Fishreproductio nan d Silverman,H.I. ,1978 . Theeffect so fvisua l stress. In:M.A .Al i (Ed.): Environmental social stimulationupo nag ea tfirs tspawn physiology of fishes. Plenum,Ne wYork , ingi nth emout hbroodin g fishSarotherodo n p.569-587 . (Tilapia)mossambicu s (Peters). Anim. Glebe,B.D .& W.C .Leggett ,1981 . Latitudin Behav.26:1120-1125 . aldifference s inenerg y allocationan dus e Sohn,J.J. , 1977. Socially inducedinhib during thefreshwate rmigration so fAmeri itiono fgeneticall y determined maturation cansha d (Alosasapidissima )an dthei rlif e inth eplatyfish ,Xiphophoru smaculatus . history consequences. Can.J .Fish .Aquat . Science196:199-201 . Sei.38:806-820 . Stacey,N.E. ,A.F .Coo k& R.E .Peter ,1979 . Hirshfield,M.F. ,1980 . Anexperimenta l Spontaneous andgonadotropi n induced analysiso freproductiv eeffor t andcos ti n ovulation inth egol d fishCarassiu s theJapanes emedaka ,Oryzia slatipes . auratusL. :th eeffect so fexterna lfact Ecology61:282-292 . ors. J.Fis hBiol .15:349-362 . Hislop,J.R.G .& A.M .Shanks ,1981 . Recent Stearns,S.C. , 1980. Ane wvie wo flife - investigationso nth ereproductiv ebiolog y history evolution. Oikos35:266-281 . ofth ehaddoc kMelanogrammu saeglefinus ,o f Sundararaj,B.I. ,1978 . Environmentalregul thenorther nNort hSe aan dth eeffect so n ationo f annualreproductiv e cycles inth e fecundity ofinfectio nwit hth ecopepo d catfishHeteropneuste s fossilis. In:I . parasiteLernaeocerc abranchialis . J. Cons, Assemacher &D.S .Farne r (Eds.):Environ int.Explor .Mer .39:244-251 . mental endocrinology. Springer-Verlag, Hislop,J.R.G. ,A.P .Rob b& J.A .Gauld ,1978 . Berlin,p .26-27 . Observationso nth eeffect so ffeedin g Whitehead,C. ,N.R .Bromage ,J.R.M .Forste r levelo ngrowt h and reproduction inhaddock , & A.J.Matty ,1978 . Theeffect so falter - 218 ationsi nphotoperio d onovaria ndevelop mentan dspawnin g timei nth erainbo wtrou t (Salmogairdneri) . Ann.Biol .anim .Bioch . Biophys.18:1035-1043 . Wootton,R.J. ,1976 . Thebiolog yo fth e sticklebacks. Academic Press,London . Wootton,R.J. ,1977 . Effecto ffoo dlimit ationdurin gth ebreedin g seasono nth e size,bod y components andeg gproductio n of female sticklebacks (Gasterosteus aculeatus). J.Anim .Ecol .46:823-834 . Wootton,R.J. ,1979 . Energy costso feg g productionan denvironmenta l determinants of fecundity inteleos t fishes. Symp. zool.Soc .Lond .44:133-159 . Wootton,R.J. ,J.R.M .Alle n& S.J .Cole , 1980a. Energeticso fth eannua lrepro ductive cyclei nfemal esticklebacks , Gasterosteus aculeatusL . J.Fis hBiol . 17:387-394. Wootton,R.J. ,J.R.M .Alle n& S.J .Cole , 1980b. Effecto fbod yweigh tan dtempera tureo nth emaximu m daily food consumption ofGasterosteu s aculeatusL .an dPhoxinu s phoxirius (L.): selectinga nappropriat e model. J.Fis hBiol .17:695-705 . 219 CONTROLLEDREPRODUCTIO N INTH EROAC H (RUTILUSRUTILU S L.) A.D.Worthington ,N.A.A .Macfarlan e andK.W .Easton * Department ofLif e Sciences,Tren tPolytechnic ,Nottingham ,U.K . *AreaFisherie s Office,Severn-Tren tWate rAuthority ,Nottingham ,U . Summary use themt oregulat ereproductiv ecycles . For example,daylengt h plays themajo rrol e Timingo freproductio n inteleosts ,deter insynchronizin g reproduction insalmonids , mined principally by environmentalfactors , whereasbot hdaylengt h and temperaturema yb e generally ensures that favourable conditions important inman y cyprinids (deVlaming , 1972). areavailabl e for larvae. However infis h Hence,strategie s for controlling reproduction culture artificial advancement of spawningi s artificially,mus tb ebase d onnatura lregu avaluabl e technique,particularl y inextend latingmechanisms . Thus,tim eo f spawning ingth egrowin g season forfry . Manipulation canb e considerablymodifie d bymanipulatin g ofphotoperio d and temperatureha sbee nuse d photoperiod forrainbo wtrou t Salmogairdner i toaccelerat e gametogenesis and spawning in (Whitehead et al., 1978)an dwate r tempera- theroach ,a spar to f astud yo ncultur eo f .tur efo r carpCyprinu s carpio (Kossman, 1975). thisspecies . 48fis ho fmixe d sex (60-120g) This studyconcern s theroach ,a freshwate r were introduced toaquari a inlat eautumn . cyprinid oftemperat eregion swhic hha sbee n Experimental groupswer e subjected toa phot o rarely studied previously. Commercialdeman d -period accelerating attwic eth enatura l byangler s inBritai nha sprompte d theinvest rate (whichwa suse d forcontrols )fro mNov igationo fcultur e techniques includingin ember,wit hbot hgroup skep t in spring-like duced spawning,whic hha sbee n shown tob e water temperature (9-I6°c). Experimental straightforward inrip efis h (Worthingtone t groups spawned inearl yFebruary ,som e 13-16 al., inpress) . However,spawnin gma yb e weeksbefor e thenorma l spawning period (May advanced only afe wweek sb y thismetho d /June)o f localwil d populations. Onlyon e alone,an d inorde r toexten d thegrowin g control fish spawned,an d thenpoorl y inres seasono ffr y significantly,th eacceleratio n ponse tohypophysation . Experimental females ofgametogenesi s itself isrequired . Since showed asurg e inplasm a calcium (indicative photoperiod and temperaturebot h influence ofvitellogenesis )prio r tospawning ,bu tno t reproduction stronglv inroac h (Jafri,1980 ) thecontrols . Evidentlyphotoperio d isth e bothwer emanipulate d inthi sstud y ina n principal factor synchronizing reproduction attempt toaccelerat e gametogenesis andthere inth eroac h though temperatureha s asuppor by advance spawning. tiverole . Keywords:roach ,reproduction ,photoperiod , Materials andMethod s advanced spawning,gametogenesis . 48roac h ofmixe d sex (60-120g)fro m Introduction Severn-TrentWate rAuthority' s coarsefis h farmwer e introduced totw o 14001recirculat Themechanism s controlling reproduction in ing systems,eac hequippe dwit h anultra teleosts ensure that favourable conditions violet steriliser,heater ,settlin g tankan d (e.g.,temperatur e and food supply)ar epre biological filter. Each aquariumwa spro sentwhe n larvae emerge. Thusmos t species videdwit hartificia l spawning substrate. useregular ,predictabl e changes inth e All fishwer e individually identified bysub environment,daylengt h and temperaturefo r cutaneous injectiono falcia nblu edye ,the n example,t ocu ereproduction ; thesefactor s maintained under thefollowin gregimes : probably interactwit hendogenou srhythms . Thisprincipl ema yb e exploited byma ni n order tocontro l thereproductio n ofcaptiv e species. Environmental regulation ofrepro Advanced Natural ductionha sbee n frequently reviewed Oe.g.d e photoperiod photoperiod Vlaming, 1972;Billar d &Breton ,1978 ;Crim , 1982)an dha sbee n shown todiffe r according n 24 24 tospecies . Endogenous rhythmsar eimport Temperature °C 9-16 9-16 anti nsom efish ,e.g. , thecatfis h Photoperiod 8L:16Dt o16L:8 D Natural Heteropneustes fossilis (Sundararaje t al., 1978),whic h occupy environmentswit h little seasonalchang ei nligh tan d temperature. In temperate climes,seasona l changes inday - The advanced photoperiod beganwit h8L:16 D lengthparticularly ,an d temperature,becom e inlat eNovember ,acceleratin g thereaftera t more pronounced,an d fisho f theseregion s twice thenatura l rate to 16L:8D scheduled for lateFebruary . Naturalphotoperio d 220 Table. Reproductive events recorded from roach maintained under different photoperiod regimes (advanced: (S n=8, Q =16; natural: <5'=9,Q= 1^ . ADVANCED PHOTOPERIOD NATURAL PHOTOPERIOD —6, RELEASINLEASI G WITH RELEASING SPAWNING RELEASIN? G WITH RELEASING SPAWNING DATE OVULATED TUBERCLES SEMEN OVULATE.EASI D TUBERCLES SEMEN EGGS (N) (N) (N) EGGS (N) (N) (N) 5.2.82 0 7 3 • - 0 0 7,2 2 8 1 J 0 0 8.2 0 8 1 - 0 0 10.2 ] 8 7 V 0 2 11= 2 1 8 7 4 0 2 11.2 1 6 6 y 0 0 17.2 2R 1 2 - 0 3 1 19.2 ADMINISTERED CPE 7.5 - 12.5 MG KG-1 20,2 21.2 R= RESORBIN GFEMAL E *= POO RSPAWNIN G Figure. Plasma calcium values (x + s.e.) for roach maintained under the indicated photoperiod and water temperatures. *- 18 Ol Advanced photoperiod Natural photoperiod '•% 16 ° 14 I— OJ % 12 -t— O £ 10 20 ° 16 a. J 12 Advanced photoperiod $>•—»én—• Natural photoperiod ço—oef o—o o E E 3 m E Oct Nov Dec Jan Feb 221 corresponded tosun-ris e and sun-set (GMT). group,b y the spontaneousnatur e ofspawning . Temperatureswer emaintaine d above9°C . Thecontro l group didno t spawn spontaneously Photoperiodwa s adjustedweekly ,an d temper despite thefac t that temperature surpassed aturesrecorde d daily. thespawnin g threshold of 14°C (K.Easton , Blood sampleswer e taken from thecauda l pers. coram.). However,on e controlfemal e vasculature at4 the n3 weekl y intervals. did ovulate and spawn sparingly afterhypo Plasmawa sanalyse d byfluorometri c titration physation,thoug h thehatchin g ratewa sver y for total calcium (indicative ofvitellogenin , poor. Thisma yhav ebee ndu e topoo reg g Elliot et al., 1979). During blood sampling, quality or fertilization. Overall,i tseem s fishwer e examined for the developmento f thatwhils tdaylengt h isth emajo renviron tubercles,spermiat-ion ,o rovulation . To mental factor synchronizing theprespawnin g check the stateo fgona dmaturit y allfis h and spawning stages of thereproductiv e werehypophysise d with carp pituitary extract cycle of theroach ,temperatur eplay sa (CPE)give na s3 I/ Pinjection s (12.5mgkg -' modulatory role. Unfortunately, incomplete over24h) . temperature controlprevent s further inter pretationo fthi saspect . It isconsidere d Results thata perio d of shortdaylengt h and low temperature isprobabl y required,becaus e Datao nphotoperiod , temperature andplasm a roachkep tunde r long dayan d 16°Cdurin g the calciumar e shown. Precise temperaturematch prespawning phase showgona d regression ingo fth e two groupswa sno tpossibl ewit h (Worthington et al.,unpubl.) . Thus,shor t the available facilities and thever y severe daylength and lowtemperatur ema yb e stim winter. On 29Januar y females inth e ulatory asi nanothe r cyprinidMirogre x "advanced"grou pwer e observed tob ever y terrae-sanctae (Yarone t al., 1980)i nwhic h rotund andnea r spawning. Subsequentrepro hightemperature sbloc kvitellogenesis ,al ductiveevent s are summarized inth etable . though thisspecie s isa winter-spawner . The advanced fish spawned spontaneously on Iti snoteworth y that allfis h inth econ four occasionsbeginnin g 7February . One trolgrou p.mature dwithi n onewee k ofeac h female fromeac hregim eovulate d and spawned other,whe n thephotoperio dwa s 14.5L:9.5D afterhypophysation . Both of these spawnings (corresponding tomid-May ,th e start ofth e gaveviabl eegg s althoughhatchin gwa sbette r natural spawning season). Thisgrou psyn inexperimenta l (70-80%)tha ncontro lfis h chrony isessentia li na specie swhic h (30-40%). Overall,viabilit y of larvaewa s spawnsannuall y inshoals ,an d indicatesth e excellent. Marked changes inplasm a calcium precision of themechanis m controllingrepro were confined toth efemale sunde r advanced duction. However,becaus e theaquari awer e photoperiod,whic h increased greatlybetwee n arranged inparalle l ina recirculatin g 8 and 29Januar y (P< 0.001) . Thendurin g system,i ti spossibl e thatpheromone sre spawning,calciu mfel l sharply (P< 0.001) . leased from earlymaturin g fish induced No suchchange swer e observed incontrols . spawningbehaviou r andmaturatio n infis hi n Prespawning calcium levels inmale swer econ other tanks,a ssuggeste d by evidencefro m sistently lower thani nfemales . Levelsi n thegoldfis h (Partridge et al., 1976). allgroup sdecline dmarkedl y (P< 0.05 ) bet The increases inplasm acalciu m infemale s ween 18an d 23February . duet ovitellogenesi s arelowe r thanthos e characteristic of salmonids,presumabl y Discussion because these specieshav emuc h larger,mor e yolkyeggs ,requirin g ahighe r levelo f Thegrou pmaintaine d under thecompresse d vitellogenesis. Despite thedifference s in naturalphotoperio d spawned 13t o 16week s absolutevalues ,th egona dpattern s are before alocal ,wil d population,whic hspa similar tothos e seen inth erainbo w trout wnedbetwee n 19Ma y and 9Jun e (Worthington subjected tocompresse d photoperiod cycles et al.,unpubl.) . Similar studies onannuall y (Whitehead et al., 1978). Incontrast ,th e spawning teleosts showtha t spawning canb e peak seen inth ecompresse d cycle roachgrou p advanced 11week s incoh osalmo nOncorhynchu s ismor e acute compared toth econtro lgroup , kisutch (Macquarrie et al., 1978)an d 12 and alsoa wil d population,wher eplasm a weeks inrainbo w trout,Salm ogairdner i calciumfo r threemonth s prior tospawnin g (Whitehead et al., 1978). However,th efis h was elevated (Worthington etal. ,unpubl.) . inth epresen t studywer e onlymanipulate d This indicates thatvitellogenesi s during the for 10weeks ,a soppose d toth e3 3month sfo r prespawning periodwa s accelerated undera cohosalmo nan d 6month s forrainbo wtrout . regime of increasing daylength and temperature. Iti slikel y that theprespawnin g period Further experiments areneede d inorde rt o offersth egreates t potential for advancing definemor eprecisel y theenvironmenta l spawning incyprinids ,becaus e gonad activity requirements forreproductio n inroach . Never islo wo rretarde d overwinte r (Billard& -theless,applicatio n of the environmental Breton, 1978). regimedescribe dwoul d facilitatea 30-40 % It isclea r thatal lo fth e environmental extensiono fth egrowin g seasono f0 +roach . requirements for completegametogenesi san d spawningwer e fulfilled inth eexperimenta l 222 Acknowledgement ADWan dNAA M aregratefu l toth eSevern - TrentWate rAuthorit y forfundin gthi s project. References Billard,R . &B .Breton , 1978. Rhythmso f reproduction inteleos t fish. In:J.E . Thorpe (Ed.): Rhythmic activity offishes . Acad.Pres ,London ,pp .31-53 . Crim,L.W. , 1982. Environmentalmodulatio n ofannua l and dailyrhythm s associated withreproductio n inteleos t fishes. Can. J.Fish .Aquat .Sei .39:17-21 . Elliott,J.A.K. ,N.R .Bromag e& C .Whitehead , 1979. Effects ofoestradiol-17 ßo nseru m calcium andvitellogeni n levels inrainbo w trout. J.Endocrinol .83:54-55 . Jafri,S.I.H. , 1980. Thecontro lo frepro duction in theroach . Ph.D.thesis . University ofLiverpool . Kossman,H. , 1975. Reproduction experiments oncar p (Cyprinus carpio). EIFAC25:122 - 126. Macquarrie,D.W. , J.R. Markert &W.E .Van - stone, 1978. Photoperiod induced off season spawning of coho salmon (Oncorhyncuskisutch) • Ann.Biol .anim . Bioch.Biophys . 18:1051-1058. Partridge,B.L. ,N.R .Lile y &N.E. Stacy , 1976. Therol eo fpheromone s inth e sexualbehaviou r of thegoldfish . Anim. Behav.24:291-299 . Sundararaj,B.I. ,P .Nat h& V .Jeet ,1978 . Roleo fcircadia n and circannualrhythm s inth eregulatio n ofovaria n cyclesi n fishes: acatfis hmodel . In:P.J . Gaillard andH.H .Boe r (Ed.): Comparative Endocrinology. Elsevier/North Holland, Amsterdam, pp.137-140 . deVlaming ,V.L. ,1972 . Environmentalcon trolo fteleos t reproductive cycles:a brief review. J..Fis hBiol .4:131-140 . Whitehead,C , N.R. Bromage,J.R.M .Forste r & A.J.Matty , 1978. The effects ofalter ations inphotoperio d onovaria ndevelop ment and spawning time inth erainbo w trout (Salmogairdneri) . Ann.Biol .anim . Bioch.Biophys . 18:1035-1043. Worthington,A.D. ,N.A.A .Macfarlan e& K.W . Easton. The induced spawning ofroach , Rutilus rutilusL. ,wit hth e antioestrogens clomiphene and tamoxifen. J.Fis hBiol , (inpress ). Yaron,Z. ,M .Coco s &H . Salzer,1980 . Effects oftemperatur e andphotoperio d on ovarianrecrudescenc e inth ecyprini d fish Mirogrex terrae-sanctae. J. FishBiol . 16:371-382. 223 THESENSITIVIT YO FREPRODUCTIO N INFISHE ST OSTRESSFU L ENVIRONMENTALCONDITION S Shelby D.Gerkin g Arizona State University,Tempe ,Arizona ,U.S.A . 85287 Abstract Cyrinodontidae,wa schose n asth eexperiment al animal because it laysegg s nearlyever y Temperature,salinit y and acidity havea day fora sustaine d periodof"tim e (14week s pronounced influenceo n reproduction of the inon eexperiment) , it issmal l and resistant desert pupfish,Cyprinodo n n.nevadensis . todisease ,an d itsegg sca n becollecte d and Reproductive performancewa sevaluate d using cultured easily. Thisspecie s isadapte d to dual criteriao feg g production andeg g watero f high temperature,hig h salinityan d viability. Someobservation s wereals o high alkalinity which must betake n into madeo negg sdurin g theirmaturatio n inth e account whencomparin g itsresponse swit h ovary. Testswer eperforme d under conditions other species. Twoothe r cyprinodonts,th e thatwer eoptima l for reproduction aswel l as flagfish,Jordanell afloridae ,an d thesheeps - sublethal environments above and below the headminnow ,Cyprinodo n variegatus,an da optimum. Oogenesis proved tob esensitiv et o •membe ro f the family Cyprinidae, theblunt - stress and underlay the reduced production noseminnow ,Pimephale spromelas , havebee n and viabilityo fegg s that wasobserve d under used formos to f the reproductive studies stressful conditions. The reproductive similar tothos e reported here (Craig& Baksi , tolerance limits (50$o f peak performance)fo r 1977; Hansene t al., 1977; Mount, 1973). thethre estressfu l factorsemploye d were considerably narrower than the lethal limits Oogenesiseffec t for Larvaean dadults . Testsdesigne d torevea l physiological com Temperaturestres s pensationo f thereproductiv eproces st o temperature and pHstres swer enegative . Theoogenesi s.effec t isth enegativ eeffec t Evidently the reproductive tolerance limits of astressfu l environmento n somestag eo f are fixed, i.e. theycanno t beshifte db y development while theeg g is inth eovary . prolonged exposure tostressfu l environments. We first recognized thiseffec t wheneg gvia Keywords: fish reproduction,stres so n bilitywa scompare d at two temperaturere reproduction,oogenesis ,eg g production, gimes: (1)succes so f hatchingo f eggs laid reproductiveperformance ,temperature ,ph , by females at28 C (optimum temperature)an d salinity . incubated abovean d below thattemperature , and (2)succes so f hatchingo fegg s laidan d Introduction incubated at thesam e temperature, i.e.no t transferred toanothe r temperature. The Thispape r will review thewor k inm ylabo latter regimesubject s theeg g toa constan t ratoryo n theeffect so f temperature, temperature inth eovar y aswel l asdurin g salinity and acidityo n reproduction inth e incubation. Thisexperimenta l designmor e desert pupfish,Cyprinodo n n.nevadensis . closely resembles thesituatio n innatur e Departures fromoptimu m conditions for these than the firstwhic h isdesigne d totes tth e threefactor scaus ea reduce d reproductive developmental toleranceo fegg s all laid ata performance asmeasure d byeg g production uniform temperature. The two typeso fexperi andeg g viability. Under thesecondition s mentsyielde d strikingly different results the fish isconsidere d tob eunde rstress . both abovean d below theoptimu m temperature. Wehav ecalle d itreproductiv e stressbecaus e Forexample ,a t22 C the formerexperimen t gave theothe r vital functions areaffecte d little, 75$o f anorma l hatchwhil e the latter gave ifa t all,an d thecircumstance sunde r which 1$; about thesam edisparit ywa s founda t itoccur sd ono t lead toprematur edeath . 34C. Temperature stresswa sevidentl y res Thestresse s thatw ehav eapplie d aresystem ponsible for thedisparity ,causin g some ic innature ,affectin g thephysiolog yo f the change inth eeg gwhil e itdevelope d inth e wholeanimal , incontras t tospecifi c poisons ovary. This viewwa sstrengthene d when weob that actdirectl yo n thedevelopin g eggo r served that acclimated females laid numerous sperm. eggs (5egg sg"'day _1)a t32 Cbu t hatching The term reproduction isuse d ina restric success wasonl y 15$. Manyegg s reached ted senset o includeoogenesis ,eg gpro maturity, therefore,bu tonl y a fewpossesse d duction andeg g viability. Somemateria lo n thequalitie s required for hatching. Small, larval survival tests is included tosho w fragileegg swit h softchorion s and littleo r that theearlie r stages inth e reproductive noyol k wereobserved . Mean yolk diameter processar emor e sensitive tostres s than is was reduced to 1.02a t 32C from 1.08m ma t theorganis m which hatches from theegg . 20C. The relatively higheg g production Thepupfish ,a membe r of thefamil y coupled withpoo r quality at32 Cha sbee n 224 observed several timesamon g thevariet yo f vitellogenin,a yol k precursor, (2)transfe r experiments on temperatureeffect s thatw e of vitellogenin from blood toyol kspheres , haveperforme d (Shrode& Gerking ,1977 ; (3)enhancemen to f vitellogenin transferb y Gerkinge t al., 1979). pituitary gonadotropin and (4)fusio no f Investigators working inOntario ,Canada , yolk spheres. Thus, theblockag eo foogene onsmallmout h bass,Micropteru sdoLomieui , sisdu e toaci d stressma y because d bya n havebee n among the foremost advocateso f inhibitiono f thesynthesi so f vitellogenin the influenceo f temperatureo n reproductive inth e livero r toa depresse d secretiono f success innature . Examining thepopula gonadotropin whichenhance s thevitellogeni n tiondynamic so f thebass ,Christi e & transfer. Regier (1973)explaine d year toyea rvaria Atresia,o reg g resorption,wa sobserve d tions inrecruitmen t by theunpredictabl e byRub ye t al. (1977) invaryin g amountsa t occurrenceso f lowsumme r air temperatures. all pH levels including thecontrol . We Twoeffect swer e noted: desertion ofnest s alsoobserve d asimila r response inth epup - bymale swhe n the temperature fell below 14C fishovary . Atp H8. 3 (control)ther ewer e during spawning and poor growtho fyoung-of - 343oocyte so f which 18wer emature ; atp H the-year at low temperatures resulting in 5.5oocyte s numbered 269wit h 12mature ;an d abnormal overwinter survival. These ideas atp H 5.0,25 5oocyte san d 8mature . The were laterexpande d upon byShute r (1980). percentageo fmatur eoocyte s judged tob e Forney (1972)fel t that recruitment wasde abnormal were: 12$a tp H8.3 ,24 $a tp H 5.5 pendent upon theaverag e temperaturedurin g and 60$a tp H5.0 , Presumably theseabnor theJun espawnin g season. Theseauthor sd o mal eggsar e resorbed. Ball (1960)fel ttha t notentertai n thepossibilit y of adirec t environmental stresswa s theprincipa l cause influenceo f temperatureo n thegonads . We of atresia,althoug h heobserve d thephago suggested (Shrode &Gerking , 1977)tha t the cytic resorptiono fegg s inovarie so fnorma l temperatureexperienc eo f femalesa tcriti fish aswell . Thiscorrespond s withobser cal timesdurin g eggmaturatio n mightex vationso n thepupfis h and the flagfish. An plain someo f theorde ro fmagnitud ediffer outstandingexampl eo f atresia innatur ei s ences inyea r class strength innatura l provided byBeamis h (1974)wh oobserve d mat populations. This pointo f view isbacke d ureegg s inth eovarie so f acid-stressed up byKay a (1973a)wh odemonstrate d that whitesuckers ,Catostomu scommersoni ,bu t temperature hasa direc t actiono n thego theseegg swer e notdeposited . nads independento f pituitary and gonadal hormones. Kaya (1973b)als oconclude d that Eggproductio n gonad regression after thespawnin g period wasmor e rapid at high temperatures (24-25 The numbero fegg s spawned isa reflectio n compared with 10.5C)regardles so f thephoto - of thenumbe r that successfully progress period (4an d 15hour so f light). Therela through the final stageso foogenesis . Since tive importanceo f photoperiod and tempera eggproductio n can bestudie d without kill ture,whe n applied together,o n teleostre ing theanimal s and preparing theovarie s production isno tentirel y clear (seerevie w formicroscopi c analysis,thi smeasur eo fre byd eVlaming , 1975), but the facttha t productiveoutpu twa suse dmos toften . An temperature hassom e influenceo ngameto - aquarium divider betweenmale san d female genesis isno tdisputed . pupfish isremove donc ea da y for2 1days ; courting behavior ensures;th efemale ,foll pHstres s owed by themale ,dive s intoa blac kyar n mopo n thebotto m of thetank ;an don eo r Theoogenesi seffec t isals oeviden t inacid - moreegg s are laid individually and fertili stressed fish. Rubye t al. (1977)classi zedo n thethreads . Theadhesiv eegg sca n fied seven stageso foogenesi s inth eflag - beremove d from the threadswit h tweezers fish,subjecte d the fish toa grade d series and transferred to incubatingdishes . of acidities ranging fromp H6. 7 to4.5 ,an d examined theovarie s histologically after2 0 Temperature stress dayso fexposure . Thenumbe ro fearl y stages (Stages0-1 ) increased,o r "piledup" , Eggproductio n exhibited agrade d responset o and thenumbe ro f the latestage s (Stage6 ) temperature stressabov ean d below theopti were reduced ascompare d with thesam e mum. Thenumbe ro fegg swer ecounte d daily stageso f development inth econtrol s atp H at 10temperature s ranging from 18t o36 Ca t 6.7. Theoocyte s apparently havedifficult y twodegre e intervals. They wereexpresse da s progressing through thematuratio nprocess . eggspe rspawning ,egg sg~lday~ 1 andpercen Primary vitellogenesi soccur sa tStag e4 , tageo f thetota l numbero fday s (21)o n and theauthor s felt that vitellogenesi san d which spawningoccurred . All threecriteri a RNAprotei n synthesis leading toyol k forma gave thesam e result,an donl yegg s g^day-1 tionwer e both inhibited byaci dstress . are reported here. Theoptimu m temperature Stage 4correspond s toth ebeginnin go f true was30 Cwit h aneg gproductio no f8 egg sg -' vitellogenesi s inteleos t fish (Wallace, day-1, and 50$o f this valuewa sachieve da t 1981),whic h involves the followingevents : 24an d 32C. Since theegg sproduce d at32 C (1)sequesterin go f hepatically derived wereo fpoo r quality (see above), thetoler - 225 ance Limits for reproduction werearbitrari viewo f thefac t that the LC5Q of larval pup lyestablishe d at50 $o f peak egg production fishwa sp H4.7 , it isclea r that reproduct andeg g viability. These limitswer eob ion respondsmor equickl y andmor eseverel y served at2 4an d 30C, farnarrowe r than the toaci d stress thand oothe r vital functions. lethal limitso f 2an d 44C. Upon comparing This result issupporte d byCrai g &Baks i this narrow bando f favorable temperatures (1977)usin g the flagfish and byMoun t (1973) withcondition s at the locality fromwhic h with the fathead minnow. Flagfish reproduct the fishwer ecollected , Saratoga Springs in ion, forexample ,wa s halved atabou t pH 6.0 Death Valley,Californi a (Deacon, 1968), from thecontro l level atp H6.7 ,whil eth e successful spawning,judge d on thebasi so f lethal point wasp H4.5 . Thepupfis h is temperature alone,extend s fromApri l toNo adapted to live inmor ealkalin ewate r than vember. Rarely aretemperature s exceeding theseothe r species,bu t thedifferenc ebe theuppe r limit for reproductionexperien tween the levelo f pHcausin g reproductive ced during thisperiod . The upper limitfo r stress and that resulting indeat h isth e reproduction inth epupfis h was fhehighes t same inever y species tested inthi sfashion . known for teleostsunti l Smith &Chernof f (1981)reporte d thatCyprinodo n span d Acclimation Gambusiacf .seniIi s "...appear toconsti tutebreedin g populations"a t 39.2-43.8Ci n Reproduction ison eo f themos tcomple x a thermal stream near SanDiego ,Chihuahua , vital functions, involvingman y elements Mexico. .whichmus tmes h together inorde r toproduc e viableegg s insufficien t quantity,suc ha s Salinit ystres s thebrai n-p ituitary-adrenal-gona dcomplex , neurological mechanismsan dme ioti cchromo Theeuryhalin e cyprinodonts have thehigh somal re-arrangements. Iffis h reproduction est tolerance tosalinit y of any teleost adapts toa stressfu l environment,o rcompen group. Forexample , thesheepshea d minnow sates for thephysiologica l disturbance it survives inwate ru p to 142p.p.t . (Simpson suffers,b yproducin g anorma l numbero f & Gunter, 1956). The reproductive salinity viableeggs , then thespectru mo fenviron tolerance limitso f thepupfis h proved tob e mentswoul d bebroadene dove r whichsuccess broad, ranging from0. 5 to2 0p.p.t . Re ful reproduction^could beachieved . This productive performance dropped rapidly above responsewoul d havebot h theoretical and and below these levels. The lethal toler practical significance; survival valuewoul d ance range,however , iseve n broader,exten beenhance d and physical-chemical habitat ding from 0.1 to5 3p.p.t . (Lee& Gerking , alterations would not bea sdetrimenta l as 1980). Thepupfis hobviousl y isabl e tore would otherwise be indicated. produce successfullyove ronl y a fractiono f this range. Kinne (1964)state s that this Temperature isth ecas e for several organisms,bu tlittl e work on fishes hasbee n done forcompariso n Wehav e tested theabilit yo f pupfish re with these results. Kinne& Kinn e (1962) production toadap t to temperatures thatre found that successful hatchingoccurre d in sult insubnorma l eggproductio n andviabili C.maculariu su p to7 0p.p.t . incontras t to ty. Fishwer eexpose d fora perio d of time the2 0p.p.t .observe d inou rexperiments . toa nenvironmen tknow n toreduc e therepro Of the littleadditiona l related information ductiveoutput ; eggproductio n and viability inth e literature,ther e issom edebat eab weremeasured ; and thisoutpu t wascompare d out theeffec to f lowsalinit yo n reproduct with thato f fish which did not have theben ion inth eeuryhalin emullets ,Mug iIcepha - efito f suchexposure , i.e. fishwhic h were lusan d M.cap ito . Abraham et al. (1966, • transferred immediately from normal tosub 1967)clai m that vitellogenesi s is inhibited lethal conditions. The reversewa sdon e infres hwater ,bu tEckste m &Eylat h (1970) also. Fish acclimated toth esubletha len report that reproduction isno t disturbed if vironment were transferred and tested imme thesespecie s are transferred tofres hwate r diately under normalconditions . as fryan d are reared tomaturit y inthi s Sincew eha d noguidanc e about how longre environment . productive acclimation totemperatur emigh t take,a nentir e generation frommatur e female pHstres s tomatur e femalewa sexpose d toth esam e temperature (Gerkinge t al., 1979). Thus, Pupfish reproduction is severely inhibited oogenesis, theegg ,th e larvaan d finally the when thep H is loweredo n lya modes t amount sexually matureadul t had thesam etempera from the levelo f itsha b itat,p H8.3 . Egg tureexperience . Broodswer e reared attw o reproduction was halveda tp H 7.0 (8.75egg s sublethal temperatures,2 4an d 32C,an da t g-^ay-1 atp H8.3 ; 3.86 eggsg~'day -^ at the28 Coptimum . Eggproductio n wasmeasure d pH7.0) ,an d reproductive output dropped at the rearing temperature and alsoa tth e steadily top H5. 0 atwh i ch level virtually temperature towhic h theyha d not beenex noegg swer e laid. Eggv iabilitywa sredu - posed fora whol e generation. Thus the24 C ced tohal f thecontro lv aluea tabou tp H fishwer espawne d at that temperaturean da t 7.0,an d noegg shatche d below pH6.0 . In 28an d 32C; the32 C fish at24 ,2 8an d32C ; 226 the2 8fis h at24 ,2 8an d 32C. Noadapta landlocked habitats. Israel J. Zool.18 : tionwa sobserved . Forexample , the32 C 155-172. acclimated fish laid thesam enumbe ro fegg s Abraham,M. ,A .Youshou w &N .Blanc ,1967 . at itsaccustome d temperature (ca 1.4egg s Inductionexperimentall e de lapont eche z g~1day~1)a sdi d those reared at2 4an d28C , MugiI cap it ocon fi ne nea u douce. Cr. and thishel d for the fish acclimated at2 4 hebd.Séanc .Acad . Sei.Paris .265:818-821 . and 28also . All groups laid nearly 9egg s Beamish,R.J. , 1974. Growth and survivalo f g~'day~1 at28 Cwhethe r they had been reared whitesucker s(Catosfomu scommersoni ) at that temperatureo r not. Egg viability ina nacidifie d lake. J.Fish .Res . testswer e not helpful in interpretingacc Board Can.31:49-54 . limation experiments,sinc e rearingegg sa t Christie,.W.J . &H.A . Regier, 1973. Tem 32Cwa sdisadvantageou s nomatte r what the peraturea sa majo r factor influencing previous thermal historyo f the femalesha d reproductivesucces so f fish -tw oex been. amples. Rapp.e tProces-verbeau x de Réunions. Cons. Intern,pou r I'explor.d e Acidit y laMer .164:208-218 . Craig,G.R . &W.F .Baksi , 1977. Theeffect s Reproductive adaptation top Hwa sattempt of depressed pHo n flagfish reproduction, ed byexposin g several pairso f fish fortw o growth and survival. WaterRes.11:621-626 . successive three-week periods top H 8.3 Deacon, J.E., 1968. Ecological studieso f (control)an d top H6.3 ,a subletha l condi aquatic habitats inDeat h Valley National tionwhic h leads to loweg gproductio nan d Monument with special reference toSarato viability. Reproductiveoutpu twa smeasure d gaSprings . Final Report under contract during thesecon d three-week period. Thena 14-10-0434-1989,U.S .Fores tService . reciprocal transfer wasmad ean d afive-wee k deVlaming ,V.L. , 1972. Environmentalcon measurement of reproductiveoutpu t wasre trolo f teleost reproductive cycles. J. peated oneac h group (Gerking &Lee , 1982). Fish Biol. 4:131-140. ' Ifcompensatio n hadoccurre d inth ep H 6.3 Eckstein,B .& U .Eylath , 1970. Theoccur group, itseg gproductio n should havebee n rencean d biosynthesis invitr oo f 11-ket- greater than thato f thegrou p transferred otestosterone inovaria n tissueo f the from thecontro l pHwhic h did not haveth e mullet,Mug iI cap ito ,derive d from two benefito fprio rexposur e toth e lowpH . biotypes. Gen.Comp .Endocrin .14:396-403 . This did notoccur ,however . Thereproduct Forney, J.L., 1972. Biology andmanagemen t iveoutput so fth e twogroup swer e notstat of smallmouth bass inOneid a Lake,Ne w istically distinguishable (pH6. 3 =5.2 7 - York. N.Y.Fis h andGam eJ . 19:132-154. 4.12; transferred from pH8. 3 to6. 3 =4.7 7 Gerking,S.D . &R.M . Lee, 1982. Survival 1 - 4.06 eggs g~'day" ). When thep H 6.3 and reproductiono f thedeser tpupfis h group was transferred toth econtro l pH,a n (Cyprinodon n.nevadensis )afte racclima unexpected resultoccurred . Egg production tion toaci d water. J. Exp.Zool . did not recover to thecontro l level asi t (accepted for publication). hadwhe n the fishwer e released from tempera turestress ,bu t remained at theleve l Gerking,S.D. , R. Lee& J.B . Shrode,1979 . characteristico f thesubletha lexposure . Effectso f generation-long temperature Control egg production wasabou t 8.0 eggsg -^ acclimation on reproductiveperformanc eo f day-'',bu t thegrou p transferred from pH 6.3 thedeser t pupfish,Cyprinodo n n.neva to8. 3 was5. 5 eggs g~1day~1. This suggests densis. Physiol.Zool .52:113-121 . thatpossibl y somedamag e toth ereproduct Hansen,D.J. , S.C. Schimmel &J .Forester , iveproces s had been produced by theexpo 1973. Arochlor 1254 inegg so fsheeps - sure to thesubletha l pH. Thisoutcom ere head minnows: effecto n fertilization peated the resulto f asimilar ,earLie rex success and survival ofembryo s andfry . periment,s ow ear e reasonably certain that 27thAnn .Conf .S .E .Assn .Gam e andFis h theobservatio nwa s nota n anomaly. There Commissioners, p.420-426 . isalway s thepossibility ,o f course,tha t Kaya,CM. , 1973a. Effectso f temperature the five-week period was not sufficient for on responseso f thegonad so f greensun - recovery totak eplace . Thus,unde r the fish (Lepomiscyanellus )t otreatmen twit h Iimitation so fou r experiments pupfish repro carp pitu itarie san d testosteronepro - duction did not adapt toa nexposur e tosub prionate. J.Fish .Res .Boar d Can.30 : lethal pH,an dwa sdisturbe d tosuc ha 905-912. degree that recovery tonorma l wasno tevi Kaya,CM. ,1973b . Effectso f temperature dent when the fishwer e released fromaci d and photoperiod on seasonal regressiono f stress. gonadso f green sunfish,Lepomi scyanellus . Copeia 1973:369-373. Kinne,0. , 1964. Theeffect so f temperature and salinity onmarin ean d brackish water References animals II. Salinity and temperaturecom binations. Oceanogr.mar .Biol .Ann .Rev . Abraham,M. ,N .Blan c &A .Youshouw ,1966 . 2:281-339. Oogenesis infiv especie so f greymullet s Kinne,0 .& E.M . Kinne, 1962. Rateso f (Teleostei,Mugilidae )fro m natural and 227 development ofembryo so f acyprinodon t fishexpose d todifferen t temperature- salinity-oxygencombinations . Can.J . Zool.40:231-253 . Lee,R.M . &S.D . Gerking, 1980. Reproduct iveperformanc eo f thedeser tpupfis h (Cyprinodonn .nevadensis )i n relation to salinity. Env.Biol .Fish .5:375-378 . Mount,D.I. , 1973. Chroniceffec to f lowp H on fatheadminno w survival,growt han d reproduction. WaterRes .7:987-993 . Ruby,5.M. , J.Acze l &G.R . Craig,1977 . Theeffect so f depressed pHo noogenesi s inflagfish ,Jordanell a floridae. Water Res. 11:757-762. Shrode,J.B . &S.D . Gerking, 1977. Effects ofconstan t and fluctuating temperatures on reproductiveperformanc eo f adeser t pupfish,Cyprinodo nn .nevadens is . Phy siol Zool.50:378-389 . Simpson,D.G . &G .Gunter , 1956. Noteso n habitats, systematic characters,an d Life historieso f Texas saltwater Cyprinodon- tes. TulaneStud .Zool .4:115-134 . Smith,M.L . &B .Chernoff , 1981. Breeding populationso fcyprinodontoi d fishes ina thermal stream. Copeia 1981:701-702. Wallace,R.A. , 1981. Cellular and Dynamic aspectso foocyt e growth inteleosts . Am.Zool .21:325-343 . Added references Shuter,B.J. ,J.A .MacLean ,F.E.J .Fr y& H.A.Regier , 1980. Stochastic simulation of temperatureeffect so n firstyea rsur vivalo f smallmouth bass. Trans.Am . Fish.Soc . 60:53-61. Ball,J.N. , 1960. Reproduction infemal e bony fishes. Symp. Zool.Soc .London . 1:105-137. 228 THE ROLES OF LIGHTAN D TEMPERATURE IN THE REPRODUCTIVE CYCLES OF THREE BITTERLING SPECIES; RHODEUS OCELLATUS OCELLATUS,ACHEILOGNATU S TABIRA AND PSEUDOPERILAMPUS TYPUS Isao Hanyu, Kiyoshi Asahina*and Akio Shimizu Department of Fisheries,Th e University ofTokyo ,Bunkyo ,Tokyo ,Japa n Summary 1955;Nish i &Takano , 1979). We have selected 3 bitterling species which In Rhodeus ocellatus ocellatus which exhibit different spawning types: Rhodeus continues cyclic spawning from spring to ocellatus ocellatus. spring to summer; summer,th egonada l maturation was induced by Acheilognathus tabira. spring; Pseudck- raising temperature above 10 SC in early perilampus tvpus.autumn .Thi s paper reports spring. Contrastedly,th e gonadal- regression on the environmental regulation of the at the termination of spawning season was reproductive cycle in these species clarified effected solely by shortening the daylength by a series of experiments. In these below 13L in early autum, when the natural experiments,adul t fish in different phases temperature was well above 20 CC. If the of their annual reproductive cycle were regressed fish were reared under a photo- subjected to various photoperiod and period over 14L,the y could be brought soon temperature regimes,whic h were similar to into recrudescence and oviposition could be their natural environmental conditions. started in4 weeks . In Acheiloanathus tabira,a spring- spawn- Rhodeus ocellatus ocellatus er, the lower critical temperature for maturation was about 13"C ,whil e the gonadal It has been reported earlier that the involution, unlike in _B_. ocellatus. was spawning period of this fish extends from caused by temperature rising beyond 25 °C in lateMarc h tomid-Septembe r (Asahina et al., early summer. However, ifth e fish kept 1980). Male and female GSIs (gonad weight x spawning under moderate temperatures, they 100/ bodyweight )depic t a sudden rise, a were found to develop photoperiodism, which peak inMa y and/or a plateau, and then an resembled that of _R_. ocellatus. in a few abrupt decline.Alon g with yolk accumulation months. During autumn, the gonadal recru in the ovary inMarch ,th efemal e ovipositor descence proceeded only slowly owing to the grows to about the anal fin height and then shortening daylength. continues cyclic elongation throughout the On the other hand, in Pseudoperilampus breeding season.Towar d the season's end,th e tvpus which is an autumn-spawner,th e gonadal ovipositor dwindles,concurren twit h ovarian maturation was brought about by the involution. In the prespawning season, the photoperiod shortening below 13L. Moreover, testicular lobules contain cysts filled with the gonadal regression was induced by the spermatogonia and spermatocytes. The testis temperature falling below about 15BC. undergoes shrinkage and spermatogenesis stops These results are compared with well abruptly at the season's end. studied cases of other cyprinids and possible rules in the environmental regulation of Initiating factor of spawning season teleostean reproductive cycle are discussed. Keywords: reproductive cycle, bitterling, The bitterling collected in mid-January photoperiodism,critica l temperature, recru were exposed to several combinations of descence. temperatures (10,1 6 or 22 °C) and photo- periods (7,9 , 12o r 16L — L/D cycle in 24 Introduction hr period). They were fed mainly with tubifex worms. Freshwater mussels as spawning beds Among cyprinids,th e bitterling group is were also placed in the aquaria. characterized by its various types of Within 4week s of rearing, GSIs increased spawning periods,i.e. , spring-,autumn- ,an d rapidly at both 16an d 22 °C, regardless of spring to summer-spawning types, respec photoperiod, and some females started cyclic tively. Therefore, they are ideal for oviposition. GSIs showed no significant comparative studies on the environmental change at 10°C. After raising temperature to factors involved in the annual reproductive 13°C,however ,a rapid increase of GSI and cycle of teleost. Until now, however, very initiation of oviposition were induced inal l few experimental works have been conducted on the photoperiod groups. the bitter!ings (Verhoeven & van Oordt, Apparently,th e gonadal maturation in the * Present address:Colleg e of Agriculture and Veterinary Medicine,Niho n University, Setagaya,Tokyo ,Japa n 229 % 4 Female 1.5 -o 15L/25°C Male •o 14L 3^ •* 13L «- Ü2L 1.0 • CO 2 .111_ CO CD o 0.5 '77 Sep. Oct. Nov. '77 Sep. Oct. Nov. Fig. 1.Effect so fvariou sphotoperiod s onGS Io fRhodeu socellatu socellatu sdurin g late- and post-spawning periods.I ntw ogroups ,photoperio dwer echange d onth e36t hda y (arrows).Temperatur ewa skep ta t 25°C.Symbol san dbars ,mea n± S.E.M . prespawning period dependso nth etemperatur e different conditionsa teithe r 15o r25° Can d rising above 10°C . Increasing daylengths with or without mussel. Photoperiod was during this period seem to have little fixed at16L .I nth eabsenc eo f mussel, the effect. fish soon stopped oviposition cycle,althoug h high levels "of GSI were maintained. In Terminating factoro fspawnin g season Septembero r5 month s later,th e photoperiod was shifted tonatura l daylengths. Fish collected inlat e August were used. In responset oth e shift of photoperiod, About halfo f them had regressing gonads. GSIs decreased rapidlyi neithe ro fth e2 5° C Experimental fish were first divided into 5 groups, with or without mussel. This groupswit h different photoperiods; 11, 12, demonstrated that the development of 13, 14o r15L .Th etemperatur ewa smaintaine d photoperiodism was not ascribable to at 25°C. continuationo fspawnin g cycles.I naddition , Withina spa no f5 weeks ,ovipositio n cycle when thetemperatur ewa s changed to 15°C , resumed in both the 14 and 15L groups, some females kept spawning for2 mor emonths , whereasn oovipositio n occurred and female indicating delayed manifestation of GSIsdecrease d significantly inth eremainin g photoperiodism.O nth eothe r hand, spawning groups. However,b yfollowin g lengthening of continued fora duratio n ofon e month, when photoperiods from 11an d12 Lt o1 5 and 14L, the temperaturewa sraise d from 15t o 25°C . respectively,ovaria n recrudescencewa s soon This indicated that there was insufficient inducedan dovipositio n commenced eventually. developmento fphotoperiodis m at15°C . GSIs increased markedly. In contrast, Therefore, the temperature can affect neither oviposition nor GSI increase was considerably both the development andth e observed inth egrou p kepta t13 L throughout manifestation of photoperiodism. The the experiment (Fig. 1.). developmento fphotoperiodis m ispresume d to These results indicate that the be induced, however, by some innate photoperiodism as affecting the gonadal mechanism,probabl y related tomaintenanc eo f activity develops during the course of full gonadal maturation, since it takes- breeding period,an d manifests itself when place under regular,favorabl econditions . the natural daylengh approachesth e critical level between 13 and 14L. This phenomenon Declineo fphotoperiodis m undoubtedly confirms that the natural spawning period ofthi s species terminatess o The responsiveness tophotoperio d seems to abruptly in September, when the water be lost beforeth efollowin g spring. Possible temperature isstil l warm. involvement of temperature in this phenomenonwa sinvestigated . Regressed fish Developmento fphotoperiodis m caught inNovembe r were kepto n the regimes of5 , 10 or 15° C at 10L for 9 weeks. From similar experiments conducted monthly, Subsequently, each groupwa sdivide d into 2 this photoperiodismwa sfoun d to develop in subgroups,1 0o r14 La t15°C , to be reared July.T oidentif y factors responsiblefo r its for another5 weeks . development,bitterlin g having just started At the termination of rearing, gonadal spawning were brought in April under 4 recrudescence was most advanced, but photoperiodic response was.leas t clear in the gonadal histology changed little in 22 °C 15t o 15°C subgroups. Even the subgroup kept groups (8,1 2an d 15L). The decrease in GSI at 10L throughout the experiment had a was less at 26°C than at30°C , marked increase in GSI. In the 5t o 15°C and These results indicate that the termination 10 to 15°C subgroups, the photoperiodism of the spawning season in this species is persisted rather distinctly. caused by the temperature rising beyond the Additional experiments showed that the upper critical level of about 25 °C. On the recrudescence under short daylength was other hand, it was also found that the completed in 2 to 3 months at higher photoperiodism could develop in this species. temperatures. Itappear s likely, in nature, Thus, fish kept under 22°C-15L for 2 months the photoperiodism becomes weak enough or commencing from May responded to shortening lost by mid-winter so that the rising photoperiod (to 11L)an d gonadal regression temperature in early spring takes over the occurred rapidly. Itma y be possible for this control ofmaturation . The importance of this species to terminate spawning in this way in finding has to be clarified by further some cool habitats. investigation. Factors involved in recrudescence Acheilognathus tabira Regressed fish with oocytes at Judging from the ovipositor elongation and peri-nucleolus stage in early September were presence of ovulated eggs inth e ovary, the maintained on regimes of 22°C^8L, -11L, -15L spawning period of this fish extends from or 27°C-15L. After 6weeks ,a slight increase late April to late June (Shimizu & Hanyu, in GSI and concomitant formation of both 1981). Gonads remain involuted during high yolk vesicles and spermatocytes were observed summer months, when most oocytes are at in all the 22 Cgroups , whereas hardly any peri-nucleolus stage and male germ cells are gonadal change occurred in the 27 °C group. all spermatogonia. In the first maturational After another 4weeks ,however ,onl y the 22 phase starting in September, formation of °CT15L group revealed high gonadal activity yolk vesicles and spermatocytes occurs, with sharply increased GSI. The remaining accompanying slow increase of GSI and egg groups did not undergo any further changes. diameter. In the second maturational phase It is therefore concluded that, the 'first starting in March or early April, phase of gonadal recrudescence is induced by accumulation of yolk globules and active the lowering temperature in autumn, but the spermatogenesis proceed with concomitant second phase of recrudescence does not occur rapid increase of GSI and egg diameter. because of shortening daylenghs of this season. The latter situation isver y similar Initiating factor of spawning season to that of_R_ .ocellatu s inautumn . The bitterling maintained in an outdoor Pseudoperilampust.ypu s pond were used. In late February, experi mental fish were brought under conditions This bitterling species spawns inautumn .A with combinations of temperatures (8, 13 or detailed study of its annual reproductive 16°C)an d photoperiods (11 or 15L). After 60 cycle is prevented by difficulties in days, GSIs of both sexes were greatly sampling except for the larval schooling increased in the 16°Cgroups , regardless of stage in spring. photoperiod. This increase was associated with active spermatogenesis in males and Initiating factor of spawning season heavy accumulation ofyol k in females. Some females had ovulated eggs. Under 13 °C, male Larvae collected inMa ywer e reared in an GSIs increased considerably, but females outdoor pond and brought up to the adult size GSIs showed no significant increase. GSI bymid-summer . In late August, experimental changed scarcely in the 8°Cgroups . fish were transferred to artificial Thus the rising temperature is considered conditions of temperatures (18,2 5 or 28 °C) to be the only initiating factor of spawning combined with photoperiods (12, 13, 14 or season in this species,a s also observed in 15L). JL ocellatus. The critical temperature is After onemonth ,mal e and female GSIs were almost 13°C, although a slight difference markedly increased in the 12L groups, exists between sexes. regardless of temperature,wherea s they were hardly augmented in both the 14 and 15L Terminating factor of spawning season groups. Under 13L-18°C , only part of the females showed an increase in GSI, although Fish continuing cyclic oviposition in late mostmales showed an uniform increase. May were exposed to several combinations of This species is, therefore, a short day temperatures (22,2 6 or 30 °C) and photo- fish which matures in response to the periods (8, 12 or 15L). During 3 weeks' shortening of daylength below 13L. exposure,bot hmal e and female GSIs decreased greatly in 30 °C groups, while GSIs and Terminating factor of spawning season Fish were first made to start their asmentione d earlier. In the latter,afte ra spawning cycle underlO L*-19°Ci nOctober , and strong suppression by high temperature theni nearl y November, they were divided during summer,_ th e f irst phase of into4 group so nregime so f1 O La t8 ,12 , 15 recrudescence proceeds under lowering or 19°C. After about6 weeks ,mal ean dfemal e temperature regardlesso f photoperiod. This GSIswer e reduced inth e8 an d1 2° C groups. instance suggests thatth e refractorinessha s Parto ffis h showed decrease inGS Ii nth e 15 some causal relation to high temperatures, °Cgroup .A t19°C , females continued cyclic especially in fish whos e upper critical spawning and high levels of GSI were temperaturefo rmaturatio n is relativelylow . maintained. Thus,th eterminatio n of spawning season Lastly mention should be made of the canb eattribute d toth etemperatur e lowering gonadal responsiveness to long below about 15°C. photoperiod-warm temperature in late summer and autumn .Th eresponsivenes s seems to be Discussion rather common in the spring-spawning cyprinids, although it does not play a Itha softe n been pointed out that both positive role under short daylength present photoperiod andtemperatur ear eimportan t in in autumn.I tcan ,however ,b e utilized for regulating reproductive cycles of some artificial control of"ou to fseason " sexual teleosts inhabiting the temperate zone maturation inthes e fishes, and should be (Sundararaj& Vasal, 1976; Peter & Crim, •searchedfo ri nfishe s other than cyprinids. 1979).Th egonada l activityo fth ebitter!in g is also affected significantly by photoperiod and temperature, but in a References different way.I nthi s group,unlik e inothe r cyprinid fishes so far reported, thetw o Asahina,K, ,I .Iwashita ,I .Hany u &T .Hibi - factorsar eno tsimultaneousl y effective,on e ya, 1980..Annua lreproductiv e cycleo fa dominates over theothe r insom e phases and bitterllng,Rhodeu socellatu socellatus . vice versa inth eremainin g phases of their Bull.Japan .Soc .Sex .Fish .46 :299-30 5 annual reproductive cycles. deVlaming ,V.L. ,1975 .Effect so fphotoperi As described above, Rhodeus ocellatus, od and temperature ongonada lactivit yi n spawning from spring to summer, matures thecyprini d teleost,Notemigonu s crvsoleu- depending solelyo nth erisin g temperaturei n cas.Biol .Bull .148 :402-415 . early spring and undergoes regression Harrington,R.W.Jr. ,1957 .Sexua lphotoperio - responding exclusively to the shortening dicity of thecyprini d fish,Notropi sbifr e daylengh in early autumn. Spring^spawning natus (Cope),i nrelatio n toth ephase so f Acheilognathus tabira behaves in the itsannua lreproductiv e cycle.J . Exp.Zool . corresponding manner inspring , and cand o 135: 529-553. so againfo rth eterminatio n ofit s spawning Nishi,K .& K .Takano ,1979 .Effect so fpho period, provided this lasts longer under cool toperiod and temperature onth eovar yo f temperatures. Autumn-spawning Psudo- thebitterling ,Rhodeu s ocellatusocellatus . perilampus tvpusmature s inrespons e toth e Bull.Fac .Fish .Hokkaid o Univ.30 :63-73 . shortening daylength in early autumn and Peter,R.E .& L.W .Crim ,1979 .Reproductiv e regresses depending on the lowering endocrinology offishes :gonada l cyclesan d temperature inlat e autumn. gonadotropin inteleosts .Ann .Rev .Physiol . Similar alternation of dominant factors 41: 323-335. during annual reproductive cycle may take Shimizu,A .& I .Hanyu ,1981 .Annua lrepro placei nspecie s other thanth e bitter!ings. ductivecycl eo fa spring-spawnin gbitter - Thus,th egoldfish , Carassius auratus. has ling,Acheilognathu s tabira.Bull .Japan . been reported to fully mature in winter Soc. Sei.Fish .47 :333-339 . depending onlyo nth e temperature (Yamamoto Sundararaj,B.I .& S .Vasal ,1976 .Photope et al., 1966).W ehav e succeeded in inducing riod and temperature controli nth eregu the goldfish to remature and spawn during lationo freproductio n inth efemal ecat autumnb yexposin g themt o long photoperiod fishHeteropneuste sfossi l1« .J .Fish . combined with warm temperature which is only Res. Board Can.33 :959-973 . slightly higherfo rth eseaso n (Hanyue tal. , Verhoeven,B .& G.J .va nOordt ,1955 .Th ein unpublished). fluenceo f lightan d temperature onth e sexualcycl eo f thebitterling ,Rhodeu s The occurrence of postspawning amarus.Proc .Akad . Sei.Amst .C58:624-634 . refractoriness has been indicated in Yamamoto,K. ,Y .Nagaham a &F .Yamazaki ,1966 . cyprinids sucha sRhodeu s amarus (Verboeven& Ametho d toinduc eartificia lspawnin go f van Oordt, 1955) and Notropis bifrenatus goldfishal lthroug h theyear .Bull .Japan . (Harrington, 1957), although deVlamin g Soc. Sei.Fish .32 :977-983 . (1975)ha srecentl y reported that Notemigonus r.rysoleucas shows no such refractoriness. Interestingly,J. ,ocellatu s neither exhibits refractoriness while A.tabira clearly does, 232 INVESTIGATIONS INTO THE IMPORTANCE OF DAYLENGTH ON THE PHOTOPERIODIC CONTROL OF REPRODUCTION IN THE FEMALE RAINBOW TROUT N. Bromage, C. Whitehead, J. Elliott, *B. Breton, A.Matty Fish Culture Unit, Biological Sciences Department, University of Aston, Birmingham UK *INRA, Universite de Rennes-Beaulieu 35031 Rennes, Cedex, France Summary Thus, in the present work the nature of the photoperiodic cues involved in reproductive Exposure of female rainbow trout to photo- development in female salmonids, and also periods of constant length produced a simil the neuro-endocrine mechanisms by which ar sequence of changes of gonadotropin, this control is achieved, are further invest oestradiol 17B and vitellogenin as seen under igated by studying the effects of light cycles natural seasonal cycles. Constant long of constant length on the timing of spawning days or long days until June 21 followed by and the levels of gonadotropin, oestradiol- short days significantly advanced spawning, 1 7B and calcium as an index of vitellogenin whereas 12L: 12D produced similar timings in the rainbow trout. to the controls. These results confirm that a seasonally-changing daylength is unnecess Materials and methods ary for reproductive development in the Five groups of 3 yr-old rainbow trout, of trout and support a circadian or circannual Danish origin with a natural spawning time mechanism of photosensitivity with differing of Jan-Feb were maintained in light-proof light cues at different times of the annual 8001 tanks, each with a single 40-watt fluor cycle (Photoperiod, Spawning, Trout, escent tube (200 lux at the water surface) Endocrine Control). controlled by time clocks adjusted weekly Introduction and exposed to the following light cycles: (1) Simulated normal seasonal light cycle It is now well recognised that the repro (Control); (2) Normal seasonal (increasing) ductive cycle of salmonid fish is controlled light cycle until 21 June followed by constant by modifications in levels of hormones 16-hour light: 8-hour dark (1 6L:8D); from the hypothalamic-pituitary-gonadal (3) Constant 12L: 1 2D throughout the year; axis induced by cues from the external en (4) Constant 16L: 8D (Long day); vironment in particular seasonal changes in (5) Constant 16L: 8D until the summer sol daylength (Billard et al, 1978). Thus, stice (June 21) followed by 8L: 16D (Long- under conditions of constant temperature Short). and rate of feeding, spawning of rainbow trout can be advanced or delayed by accel The water temperature was constant at erating or slowing down respectively the 9 C with a dissolved oxygen of 100% satura seasonal changes in daylength into shorter tion in the effluent and a pH of 6.6. The or longer periods of time than the one year fish were fed at a level of 0.5% body weight/ of the natural cycle (Bromage et al, 1982a). day with BP Nutrition Mainstream diet. It has been suggested that the increasing and All fish were blood sampled at the start of decreasing components of a natural seasonal the experiment in Feb and each month there cycle may be required for appropriate re after. Methods for sampling and subse productive development in fish (Erikson & quent assay of gonadotropin, oestradiol and Lundquist, 1980). However, in other calcium are described in Bromage et al animals and plants on which studies of light (1982a). Fish were also examined each measurement have been conducted, repro month for signs of sexual maturation. ductive responses occur to absolute changes Spawning was said to have occurred when in daylength as a result of mechanisms in ripe eggs were expelled after gentle hand volving either circadian or circannual pressure on the abdomen, i.e. stripping. phases of photosensitivity or 'hour glass' Differences between means were tested measurement and do not require the pro statistically by either a Student's t-test or gressive seasonal changes in daylength an F-test if the variances were dissimilar. which are seen in natural cycles (Follett & Davies , 1975). Whether such mechanisms operate in salmonid fish is not known. 233 Results or at least not a decreasing or short photo- In the control fish, spawning occurred in period after the summer solstice. The late Jan, at the same time as similar lowest peak level attained and the one of groups of fish maintained in outside tanks the shortest duration occurred in the fish under ambient conditions. In contrast, on long days until June 21, followed by a fish exposed to long days until the 21 June direct switch to short days. In all treat followed by short days as well as those ments the levels of oestradiol were falling under constant long days spawned in Oct and towards basal values before the beginning Nov respectively, some 14 and 10 weeks of the increases in gonadotropin which earlier than the controls. The two groups occurred at the time of spawning. offish under 12L: 1 2D and a normal season Calcium (see Fig 3): Basal levels of cal al cycle until 21 June + long days spawned cium were found until May in the two in early and late Jan respectively. groups of fish under 16L: 8D, until July- Under all regimes a similar profile of Aug under 12L: 12D, and Sept under the changes of gonadotropin, oestradiol 17B increasing + long regime. After these and calcium was shown although the timing dates significant increases in serum levels and duration of these changes was necess occurred (p <0.001 ) up to peak heights arily modified by the differences in day- which under each regime usually preceded length to produce the clear differences in the time of spawning by 2-3 weeks. spawning time. These are considered in more detail below:- Discussion It is clear from the present results that Gonadotropin (see Fig 1): The lowest or spawning occurs in the rainbow trout ex basal levels of this hormone were shown at posed to light regimes of constant length the beginning of the experiment in Feb and and that seasonally-changing daylength is during the mid-cycle of each of the experi not essential for the cueing and modulation mental and control groups. In April/May of reproductive development. This con there were increases in gonadotropin which firms other studies (Bromage et al, 1982b) in all groups except those under the in - from this laboratory and similar results in creasing + long regime were significantly other species of teleost (Baggerman, 1972; different from basal values(p< 0,05) These Sundararaj & Vasal, 1976). increases lasted for 2-4 months and in each case was followed by a reduction to basal The finding that the time of spawning is values which occurred more quickly under unrelated to the total number of hours of the long to short regime. The return to light or dark received by the different low levels during mid-cycle in all photo- groups of fish strongly indicates the pres period treatments was then followed by ence of a circadian or circannual-based much more rapid and significant (p <0.01) mechanism for photoperiodic response increases in gonadotropin and in all cases rather than the 'hour-glass' seen in some initiation of these spikes of activity occurr animals (Lees, 1972). Circadian phases ed before the time of spawning, although of photosensitivity have also been demon levels continued to rise after completion of strated in the stickleback (Baggerman, this process. 1972) and catfish (Sundararaj & Vasal, 1976) although in these species environ Oestradiol-1 7B (see Fig 2): Under all mental factors other than light are of photoperiod regimes low serum oestradiol similar or greater importance in the cue values were maintained from Feb to June, ing of development. although during this period there were modest changes in some of the cycles. Bet The earlier spawning times of fish ex ween June and Aug more significant increas posed to longer days earlier in the year es were shown (p< 0.001) and these suggest that the trout is a 'long-day animal', occurred earlier in fish which had been ex although the further acceleration of spawn posed to longer light regimes earlier in the ing by the switching of fish from long to year, i.e., 12L: 12D, Long-Short and 16L: short days at the time of the summer sol 8D. Peaks in serum levels of this hormone stice provide evidence of an additional were subsequently shown in the Autumn short-day requirement later in the cycle. (Sept-Dec) and they were at their highest It is probable that the extended period of in those fish which had received longer days egg development in salmonids, when com- 234 Fig, 1 Changes in serum gonadotropin (mean + SEM, n=6) under the five photoperiod regimes. Oestradiol 17ß ipg/ml) 26000 r- - Increase + hold at 16L8D from June 21st - Control - 16L:8D •12L12D - 16L8D until June 21st then8L:16D Time of Spawning Fig. 2 Changes in serum oestradiol-17 B (mean + SEM, n=6) under the five photoperiod regimes. Increase + hold at 16L8D from June 21st Control 16L8D 12L12D 16L8D until June 21st then 8L:16D f Time of Spawning _L _L _L_ _L Fig. 3 Changes in serum calcium (mean + SEM, n=6) under the five photoperiod regimes. 235 pared with other fish, requires more than References one photoperiod cue during the annual cycle. Baggerman, B.,-1972. Photoperiodic The failure of either constant long or short responses in the stickleback and their days to arrest development in the trout control by a daily rhythm of photosens (Bromage et al, 1982b) argues against this itivity. Gen.Comp. Endo. Supp 3:466-476. hypothesis, although there is evidence in Billard, R., B. Breton, A.Fostier, other animals for movement of phases of B.Jalabert & C.Weil, 1978. Endocrine photosensitivity in the absence of expected control of theteleost reproductive cycle environmental triggers (Gwinner, 1973). and its relation to external factors: Sal- It is also possible that the egg and develop monid and Cyprinid models. In P.Gaill ing follicle have a programmed life and that ard & H.Boer (Ed): Comparative Endocrin after this point has been reached, feedback ology. Elsevier Press, Amsterdam. mechanisms may provide the signal for Bromage, N., C. Whitehead & B.Breton, final oocyte maturation and spawning. 1982a. Relationships beb/veen serum Turning now to the neuroendocrine media levels of gonadotropin, oestradiol 17B tion of the photoperiodic response, one sees and vitellogenin in the control of ovarian a similar sequence of changes of gonado development in the rainbow trout. Gen. tropin, oestradiol-1 7B and vitellogenin Com. Endo. 47: 366-376. under all regimes. Such profiles have al Bromage, N., C. Whitehead & J.Elliott, so been reported for fish on seasonally- 1982b. Daylength requirements and the changing light cycles (Bromage et al, 1982a). control of reproduction in the female The first measurable change under all re rainbow trout. Gen.Comp.Endo.46: 391 . gimes was an increase (early in the cycle) Eriksson, L. & H. Lundqvist, 1980. of gonadotropin which probably initiates sub Photoperiod entrains ripening by its sequent alterations in oestradiol (Bromage differential effect in salmon. Naturwiss et al 1982a). The apparent failure of the 67: 202. „ different photoperiod regimes to affect the Follett, B. & D. Davies, 1975. Photo- timing of onset of this early change in periodicity and the neuroendocrine control gonadotropin is not unexpected as the experi of reproduction in birds. Symp.Zool. ment was not begun until Feb. However, Soc.Lond., 35: 199-224. there were differences in duration of this Gwinner, E., 1973. Circannual rhythms in increase which was shortened in fish which birds: their interaction with circadian were moved from long to short days in June. rhythms and environmental photoperiod. Furthermore, the curtailment of gonado J.Repro.Fert.Sup. 19: 51-65. tropin secretion by exposure to short days Lees, A., 1973. Photoperiodic time was paralleled by reductions in the peak measurement in the aphid Megoura oestradiol level subsequently achieved in viciae. J.Ins.Phys. 19:2279-2316. the same group of fish. In contrast levels Sundararaj, B. &S. Vasal, 1976. Photo in fish on longer days (16L: 8D or 12L: 1 2D) period and temperature control in the after June 21 were higher and maintained regulation of reproduction in the female for longer periods of time. Subsequently, catfish. J. Fish. Res. Bd. Can. 33:959- gonadotropin levels under all regimes were 973. reduced in mid-cycle when oestradiol levels were rapidly increasing. The timing of the second and much larger increase in gonadotropin was clearly mod ified by photoperiod manipulation. Such increases which are thought to be concerned with the control of final oocyte maturation and ovulation, have also been reported by other workers (Billard et al, 1978). In all regimes oestradiol had reached basal levels at this time. The maintenance of high levels of gonadotropin after spawning, al though probably due to the marked reduction This work was supported by a NERC in steroid feedback which occurs after ovula Grant to N. Bromage. tion, may also be important in the control of early egg development for the next cycle. 236 INFLUENCEO FTEMPERATUR E ONGONA DDEVELOPMEN T INA STRONGLYPHOTOPERIODI CSPECIES , GASTmOSTEUSACULEATU SL . BerthaBaggerma n Department ofZoology ,Universit y ofGroningen ,P.O.Bo x 14,975 1 NN HAREN,Th eNetherland s Gonadmaturatio n in thesticklebac k is By repeating theexperimen t offig. 1a t mainly dependent onth ephotoperiod ,bu ti s different timesbetwee n late summeran d modulated by temperature ina t least twodif spring,i twa s found that therang eo fth e ferentways ,a sshow nbelow . photo-induciblephas e gradually extends into Gonad recrudescencebegin s inlat esumme r theearlie rhour s of theday;fig.2 .Thi s andphas e 1(spermproduction;oocyt egrowt h figure thus explains thedecreas e ofth e up tovacuolisation)i s completedb yDecember . photoreactivity thresholdbetwee n latesumme r Development ofphas e 2(Leydigcell develop and spring. ment andnes tbuilding;furthe r oocytegrowt h Bykeepin g fish(hatched inJune)fro mAugus t and ovulation)begins inJan./Febr.an d iscom under aconstan tphotoperiod(8L-16D )a ttw o pleted aroundmid-April,whe nbreedin gbegins . different temperatures,15°an d 20°C,i twa s Itwa s found that development ofphas e2 found thatth erang eo f thephoto-inducibl e canb e initiated onlywhe nth ephotoperio d phaseunde r 20°di dno t extend asfa rint o isabl e toovercom e thephotoreactivit y theearlie rhour s of theda ya shappene dun threshold.Thisthreshol d ishig h inSeptem - der 15°,thelatte r equalling theextensio n ber(whenphas e 2ca nb e initiated onlyb y occurringunde rnatura l conditions (fig.2). photoperiods longer than 14L-10D)and declines tover y lowi nJan./Febr.(whe n phase 2ca nb e rangeo f initiatedb yphotoperiod s as shorta s photo-inducible c 9L-15D).As shownbelow,th eheigh t ofth e phase: 5\ threshold dependso nth erang e ofth ephoto - •>81 induciblephase ,whic h isdetermine d bya daily rhythmo fphotosensitivity . nat.cond. /MM Figure 1show s therespons e of fish(caught >8L-16D innatur e inOct./Nov.)whe nexpose d toa se t Spn"/ of skeletonphotoperiods(II-VI)i nwhic hth e 24 total amount of lightwa s8 hours,divide d at.cond. intoa mai n lightperio d of6 hours,plu sa ' )8L-16D twohou r lightpuls ea tvariou s times inth e April 14,1977 darkperiod.I tca nb e seentha tth efis hdi d -//- 10 12 14 16 notrespond,o ronl yver y little toligh t hours after "light-on"(a tOhrs ) between6-1 2 hrs(I-IIl),butwer equit ere sponsivebetwee n 12-19hrs(IV-V),wit h apea k Fig.2.Extensiono f therang eo f thephoto - between 14-16hrs.Th eperio dbetwee n 12-19 induciblephas eunde r different external hrs isth ephoto-inducibl ephase .Figur e1 conditionsbetwee nautum nan d spring. thus explainswh y inOct./Nov .developmen t ofphas e 2ca nb e initiated onlyb yphoto Theconclusion s istha t the lowertempera periods longer than 12L-12D,becauseonl yi n tureso fwinte r favour theextensio no fth e thosecase s thephoto-inducibl e phasei s photo-inducible phase intoth eearlie rhour s exposed tolight . of theday,allowin gphas e 2t o startit s development inJan./Febr.,whe nth enatura l means of4exps . day lengths arestil lver y short.Fromtha t in Oct/No v T=20"C time(as otherexperiment s showed)therat eo f itsdevelopmen t isfavoure db ybot h longer photoperiodsan dhighe r temperatures. References. Baggerman,B.1972.Photoperiodicresponse s in the stickleback and theircontro lb y adail y rhythmo fphotosensitivity.Gen.Comp.Endocri nol.Suppl.3,466-476 . Baggerman,B.1980.Photoperiodican d endogenous control of theannua l reproductive cyclei n teleost fishes.In:EnvironmentalPhysiolog yo f Fishes.Ed.M.A.Ali.533-567 .Plenu mPubl.Corp. , NewYork . 12 18 daily light/dark cycles Fig. 1.Determinationo f therang eo fth e photo-inducible phaseb ymean s ofa serie s of skeletonphotoperiods . 237 EFFECTS OF PHOTOPERIOD AND TEMPERATURE REPRODUCTION OF MALE THREE-SPINED STICKLEBACK DURING WINTER B. Borg,M . Reschke,Th . van Veen Department of Zoology,Universit y of Lund,Swede n Photoperiods have amarked , though season- Initially the testes contained spermatozoa and temperature-dependent influence on the together with spermatids and spermatocytes, reproduction of the three-spined stickleback or spermatozoa almost alone.A quiescent Gasterosteus aculeatus L. (Baggerman,1957 , spermatogenesis with few cells other than Borg, 1982]. Spermatogenesis isactiv e from spermatozoa dominated inL D 16:8 20 and in the end of the breeding season in late the low temperature groups,bu t was not summer to late autumn.Th e kidney of the found in LD 8:16 20°.Mos t fish in LD 8:16 male hypertrophies in the breeding season 20 displayed a vigorous spermatogenesis and secretes a "glue"tha t is used in the with large numbers of spermatogonia and also building of the nest.Thi s hypertrophy is other stages.Thi s condition was not found androgendependent. •i n any other group,includin g the initial In order to study environmental effects on controls. androgen-production and spermatogenesis,an d Both LD 16:8group s had significantly. their relation to each other,stickleback s higher second proximal renal tubuleepi - were subjected to photoperiods ofLight : thelia (KEH)tha n both LD 8:16 groups.Th e Dark (LD) 16:8o r 8:16 h and 20°o r 9° Cfo r KEH in LD 8:16 20°wa s also significantly 30 days in December-January. The activities lower than in LD 8:16 9 ,an d lower than in of 3/3-hydroxysteroid dehydrogenase (3p-HSD) the initial controls. and glucose-6-phosphate dehydrogenase Androgen-stinjulation was most marked in (G-6-PD), two enzymes necessary for produc the LD 16:8 20 C group,an d least inL D tion ofactiv e sex steroids were investi 8:16 20 C.A n active spermatogenesis was gated histochemically. Also the histology of only present in the latter group. Thus, testes and kidney were studied. there was a negative correlation between 3/5-HSDreaction s were carried out for 3h spermatogenesis and steroid-production. at 37 C in phosphatebuffer with NAD and These effects are probably due to androgen- nitrobluetetrazolium (NBT)added .Epiandro - inhibition of spermatogenesis,whic h has sterone,firs t dissolved in dimethylform - previously been demonstrated at/after the amide served as substrate.G-6-P D incuba breeding season in this species (Borg, 1981). tions were carried out for 15mi n at 37 in Trisbuffer,wit h NADP,NB T and polyvinyl- alcoholeadded . Glucose-6-phosphate was used References as a substrate. In both types of reactionsa blue formazan stain was found in thecyto rman,B . 1957.A n experimental study on plasm of interstitial cells.Th e intensity the timing of breeding and migration in of reactions was arbitrarily,an d blindly, the three-spined stickleback (Gasterosteus classified as 0,+ , ++ or+++ . aculeatus L.). Arch. Néerl. Zool. 12:105 - The most intense reactions of both enzymes TÏT. were found in LD 16:8 20 C, the only group Borg, B. 1981.Effect s ofmethy ltestosteron e where also red breeding colours and repro on spermatogenesis and secondary sexual ductive behaviour were observed.Th e low characters in the three-spined stickleback temperature groups were intermediate,an d (Gasterosteus aculeatus L.). Gen.Comp . the LD 8:16 20°grou p had significantly Endocrinol.44 :177-180 . fainter reactions of both enzymes than all Borg,B . 1982.Extraretina l photoreception other experimental groups. involved in photoperiodic effects onre production inmal e three-spined stickle back,Gasterosteu s aculeatus.Gen .Comp . Endocrinol.47 :84-87 . 238 REPRODUCTIVEBEHAVIO RAN DENDOCRIN EACTIVIT YO FTH EPUFFER , FUGU(= SPHÉROÏDES) NIPHOBLES, OCCURRINGO NTH ECOAS TO FSAD OISLAN DI NTH EJAPA NSE A Y.Honma , A.Chib a SadoMarin eBiologica lStation ,Niigat aUniversity ,Niigat a950-21 , and BiologicalLaboratory ,Nippo nDenta lUniversity ,Niigat aFaculty ,Niigat a951 , Japan Summary Thepuffer , Fugu( = SpheroidesJ nipho andMa yjus tprio r toth esexua lmatura bles, isa ver ycommo nspecie so ffis h tion. Duringa perio d fromJun et o along thecoas to fJapan . Itspeculia r October,th eactivit y isdecline d toa spawningbehavio r linkedwit h themoo no r rathermil do rquiescen tcondition .Histol - tidalphase si swel lknow no nth ePacifi c gicalchange si nth ethyroi dglan dcoincid coast.Sinc ether e isa grea tdifferenc e edroughl ywit h themorphometrica l changes inth ewate r levelan d itsamplitud ebe inth eTS Hcells . TheACT Hcell-interrena l tweenth ePacifi c coastan d theJapa nSe a axisals oshowe da seasona lchange ,bu t coast,i ti sneede d toelucidat eth e nots odirec tcorrelatio n }.s seenbetwee n reproductiveactivit yo f thispuffe r thisaxi san dgonada lmaturation . occurringo nth eJapa nSe acoast . This studywa s thereforedesigne d tokno wstil l Hour Number of spawning unclearproble mo f thisspecie so nth e i Wirt», JapanSe asid ewit hspecia l regard to maturationan dendocrin eactivity . La Full On thebasi so fgonosomati cinde xan d +o histological examinationo fbot hsexes ,i t isestimate d thatth epea ko fmaturatio n isearl yJun ei nth emal ean d lateJun ei n thefemale . Thespawnin goccurre dalmos t everyevenin gdurin ga perio do ffift y minutesbefor e and'afte rsunse ti nth e New breeding seasonlastin gfro mearl yJun e tolat eJul y (fig.1) . Theresul tobtaine d S o may intimate thatther e isno ts oclear-cu t o correlationbetwee nspawnin gan d lunar periodicity onth ecoas to fSad oIsland . Thisphenomeno nma yb eowin g tomino r spring tidean ddail yhig htide . TheGT Hcell si nth eadenohypophysi s -O showeda hyperfunctiona l figuresuc ha s hypertrophy anddegranulatio nwit hvariou s gradeo fvacuolizatio n in thepea ko fth e gonadalmaturatio nan d spawning. TheA F stainablemateria ldemonstrate d inth e )= preoptico-hypophyseal systemwa sver y abundant inApri lan dMay ,bu t itbecam e scanty inJune ,suggestin ga nacut ereleas e ofneurohypophysea l hormone. Toward the postspawning season,a gradua l increase -fr±- inth emateria lo f thissyste mi sseen . Sunset Thecel lo f thenucleu s lateralis tuberis showedonl ya faintdilatio nprio rt oth e fig. 1. Observation ofpuffer' sspawnin g peako fgonada lmaturation . Seasonal on thecoas to fSad o Island,wit h timeo f change inth ethyroi dglan d isdistinc tan d full (t)an d low (J),tide ,duratio nan d itshighes tactivit yi srecognize d inApri l number (frequency)o fspawnings . 239 SEXUAL STIMULI ASSOCIATED WITH INCREASED GONADOTROPIN AND MILT LEVELS INGOLDFIS H Ann L. Kyle, N.E. Staoey, and R.E. Peter Department of Zoology, University of Alberta,Edmonton ,Alberta ,T6 G 2E9,Canad a In many male mammals and birds, exposure to In contrast, males that did not court or spawn had sexualstimul i results inacut e elevations of circulating GtH and milt levels not different from isolated fish. luteinizing hormone (LH) and testosterone (Tl This suggests that male sexual activity and elevations Previous work in our laboratory showed that asimila r in milt and GtH are concurrent events which may phenomenon exists in the male goldfish (Carassius share acommo n activational mechanism inth ebrain . auratus). Males placed with stimulus pairs of In summary, the performance of male sexual spawning goldfish had "Con A II" or "maturational" behaviour is correlated with the rapid elevation of gonadotropin (GtH) concentrations and expressible GtH concentrations and expressible milt volumes, milt volumes that were significantly elevated as which may act as a positive feedback system to compared to males kept in all-male groups; maintain sexualreadiness . stimulatory effects on GtH and milt persisted for at least 2 and 24 h, respectively, at 14°C (Kyle et al., Fig._ L Milt volumes, GtH concentrations after 2 h of 1979). Inth e present experiment, we determined the stimulation, and GSIs (mean ± S.E.) of male goldfish sensory cues needed to evoke these rapid that were isolated from (I), in contact with (C), or physiological changes. separated (S) from either a heterosexual (Ç) or homosexual (0) pair of spawning fish. GtH values Methods were combinedt o yieldthre e groups: I,C ,an dS . Experimental males were initially hand-stripped MILT VOLUME (ul) GtH (ng/ml) of milt and either isolated in 65 litre glass aquaria at 20°C, placed in contact with a pair of spawning Cçc*cfc)* goldfish, or separated from the pair with a 60 perforated, transparent partition. This spawning pair consisted of amatur e male and either a prostaglandin 50 F2alpha (PGHnjected female or male; PG treatment induces qualitatively normal female sexual behaviour 40 in both male and female goldfish (Stacey, 1981). [1 After 2 h,th e experimental males were anesthetized, stripped of milt Died, and killed for determination of 30 the gonadosorhatic index (GSI). Expressed milt was r- aspirated into micropipettes, centrifuged, and the 20 \Wt\ volume of milt and packed sperm measured;th e ratio of these volumes gave the percentage of seminal 10 plasma ineac hsample . i C S c S n . Results ; of stimulation Males placed in.contact ' with either a hetero sexual or homosexual (where a PG-treated male Fig. 2. Milt volumes, GtH concentrations after 2 h of played the female role) spawning pair showed stimulation, and GSIs (mean + S.E.) of isolated, significantly increased GtH and milt levels over those sexually inactive, and sexually active male goldfish. values for isolated fish. In contrast, no increases Those values underlined by the same bar are not occurred if contact with the spawning pair was significantly different at p<0.05. prevented, even though visual and chemical cues MILT VOLUME (u.1) could be perceived (Fig. 1). The percentage of I I Isolated seminal plasma and GSIs were not different among treatment groups. Some or the males placed in contact with [;:I;L| Sexually inactive spawning fish failedt o court or spawn during the 2 h test When the data for the combined "contact" ![•: I Sexually active group were divided into sexually active and inactive categories and compared with the isolated controls, the sexually active males had significantly higher GtH andmil t levels than either the inactive or isolated fish (Fig. 2). Discussion [%fÎ While the sight, sound, or smell of a female can cause rapid, transient elevations of LHan d T in some ia] male mammals and birds, visual and chemical stimuli alone from a spawning female goldfish, either Hours of stimulation PG-treated (present study) or ovulated (unpublished results), djd not elevate GtH or milt levels in the male. References These elevations only occurred in males that were placed in contact with a spawning pair (even when Kyle, A.L., N.E. Stacey, R. Billard, and R.E. Peter. that pair contained a PG-treated male playing the 1979. Amèr. Zool, 19:Abstr . 554. female role) andwer e sexually active during the test Stacey, N.E. 1981. Amer..Zool. 21: 305-316. 240 CHANGES IN PLASMA LEVELS OF GONADOTROPIN AND THYROID HORMONES DURING SPAWNING IN THE WHITE SUCKER (CATOSTOMUS COMMERSONI). Duncan S. MacKenzie and N.E. Stacey Department of Zoology, University of Alberta, Edmonton, Alberta, CANADA, T6G 2E9. Summary - Prespawning ia. 1981 MALES (mean + s.e.,N) Plasma gonadotropin and thyroid hormone levels were - measured in white suckers during their spawning migration. Gonadotropin levels were low in both males i-*2 na osa and females prior to spawning, and increased to peak na rrit- levels in spermiated males and ovulated females. Levels O) then declined in spent fish returning to the lake. c Thyroxine levels were highest in prespawning animals, Spawning and tended to decline over spawning, with highest jj 35 1 levels and diurnal variations found in males. Triiodothyronine levels were constant in both sexes. O 30 25 1 Introduction J, 20- The white sucker {Catostomus commersoni) is abundant 15 and widely distributed in North American lakes and rivers. In the spring of 1981 and 1982, adult suckers 10 - were captured during their spawning migration at a 5 number of sites along a small stream draining into Lac 17 £j_ 23 59 Ste. Anne, Alberta. Blood samples were taken from the caudal vasculature of each fish within 15 minutes of 0300090 0 1500210 0 Total netting. Sex and gonadal condition were determined by TIME ovulated females absent external morphology and (in 1981) oocytes were aspirated from unovulated females and checked for Prespawning 1b. 198 1 FEMALES germinal vesicle migration (GVM) and breakdown (mean + s.e.,N) (GVBD). Blood from each fish was assayed for gonadotropin (GtH), total triiodothyronine (T,), and total thyroxine (T4). Blood GtH was measured using a jaü caio MBL2 OS I CaU radioimmunoassay for carp gonadotropin. T3 and T4 were also measured by RIA. 15 - Preovulatory 10 Results and Discussion - 5 . Gonadotropin _J±tU ttlâl rr26 1-131 1—1109 GtH levels in spermiated male suckers were uniformly E 15 - GVM low prior to the appearance of ovulated females, at °> io|- which time levels increased dramatically and remained high throughout spawning with no significant diurnal I variation (fig. 1 a). Spermiating males on the spawning I- JÎL Jl M ia i n grounds in 1982 also had significantly higher GtH levels C5 than spent males caught returning to the lake (means ± Ovulated m s.e.= 11.3 ± 1.0 vs. 4.4 ± 0.3). High GtH levels in spawning males may in part be a response to ovulated --Fl females; on May 12, 1981, GtH levels of spermiating males caught-in the presence of ovulated females (23.4 fcjpj l2ä hâ ± 3.1) were significantly higher than those of Spent spermiating males caught in an area devoid of females. 15 (4.4 ±1.1) GtH levels in females were correlated with 10 rn ovulation. Levels were lowest in prespawning fish, and remained low throughout the spawning season in preovulatory fish (fig. 1b). GtH increased significantly in ji. Ù LCL fish with GVM and GVBD, reaching peak levels in 0300 0900 1500210 0 Total ovulated fish and decreasing with completion of oviposition and return to the lake (levels in returning TIME fish= 5.2 ± 0.5). No consistent diurnal variation in Fig. 1. Gonadotropin levels in male (1 a ) and female (1 b ) plasma GtH was seen. suckers at four different times of day during the 1981 spawning season. Thyroid Hormones spawning activity and may therefore reflect increased metabolic demands at these times. There was no T3 levels in both sexes showed very little variation of apparent diurnal variation in T4 levels in female suckers. any kind during spawning. Mean T3 levels ranged from In general, T, levels in females were lower than those in 1.8 to 5.0 ng/ml in both males and females. There were males, and tended to decline with reproductive stage significant diurnal variations in T4 levels in male suckers. (highest = 5.0 ± 0.3 in prespawning females declining In prespawning animals, highest levels were found at gradually to a low of 2.1 ± 0.4 in spent females). No 0300 hrs (10. 2 ± 1.6 ng/ml) and lowest at 1500 hrs observable changes in T4 levels were seen associated (4.3 ± 0.6). In spawning animals, T4 levels were elevated with the initiation or cessation of female spawning at 1500 hrs (1 1.0 + 1.2), but means ranged from 5.9 to activity, although higher levels at the start of spawning 6.7 ng/ml at all other times. These peaks in T, levels support a role for thyroid hormones in ovarian correspond to times of greatest migratory and maturation. 241 MANIPULATION OFSPAWNIN GACTIVIT Y INRAINBO WTROU TB YLIGH TPROGRAM S M. Pohl,R . Schmidt,W .Holt z Institut fürTierzuch t undHaustiergeneti k Göttingen,West-German y The spawning in rainbow trout coincides Itwa spossibl e toreduc eth e spawningin with shortday so fwinter . terval of males and females from 12 months The purpose ofthi sexperimen twa st oen to 9 or6 month s (Fig. 1an d 2). Infemale s hance the frequency of spawning by way of the period between the first and the last artificial lightprograms . fish spawning was reduced from 14t o8. 5 to Eightymal e and 60femal e troutwer eevenl y 6 weeks, respectively. The output ofsper m and randomly allotted to8 fis htank slocate d and eggs wasnormal ,an d therewa sno tmuc h in a dark basement and supplied with well difference between groups (Table 1).Egg s water of constant quality and temperature obtained from the9 an d 6month s groupwer e (10± 1°C).Tw otank s eachwer e subjectedt o slightly smallerbu t thequalit y ofbot hmal e an artificial light year of 12,9 , 6o r3 and female germ cellswa snormal . months, respectively. All fish were anaes The 3 months group started to spawnver y thetized and checked for mature germcell s earlyan d the spawning seasonlaste d foral at two-week intervals,durin g peak periods most sixmonth s regardless ofth e continuing females were checked weekly. Quantity and lightprogra m (Fig. 1an d 2). The individual quality of germ cells,sper m motility,eg g response was erratican d unpredictable.Th e sizean dhatchin g ratewer e determined. eggs were smaller,bu tbot hmal e and female germ cellswer e functionally normal (Table1) . Figr J^:Proportio n of milters spawning inrespons e toth e respective light program OCT NOV DEC JAN DATE OF STRIPPING A O I 9 MO .- a y g 6 MO Z 50 ,--r | 3 MO A Lengtho f Sperm/male Weight of10 0 Eyed eggs Hatching artif.yea r (X109) rate(% ) greenegg s (g) eyed eggs (g) (%) (mo) 12 336 7.3 10.6 87.9 84.0 9 399 6.4 11.9 88.1 84.0 6 308 6.0 10.5 93.5 91.8 3 335 5.4 8.3 94.2 91.6 242 LONGPHOTOPERIOD SSTIMULAT EGONA DDEVELOPMEN T INRAINBO WTROU T 0.Skarphédinsson ,A .P .Scot t andV .J .By e MAFF,Directorat e ofFisherie sResearch ,Fisherie sLaboratory , Pakefield Road,Lowestoft ,Suffol kNR3 3OHT ,U.K . Summary Experimentswit hvariou s photoperiods under-yearlings. However,o nth e 19h an d revealed that longconstan t daylengths 20h photoperiods ,growt hwa s consistently induced precociousmaturatio n inmal eunder - less thano nS N (p< 0.01 ,student' s t- yearling rainbowtrout . Ovariandevelopmen t test). The stimulatory influence oflon g intw oyea rol d female troutwa sals ostimu daylength onprecociou smal ematurit y is lated by similardaylength ,wit h ovulation thusno t primarilydependen t on fastgrowt h andplasm a levels of testosterone showing rate. cycleso fapproximatel y 6months . Experi Ovariandevelopmen t in2 yea rol d female ments inprogres s ona commercia l fishfar m troutwa s alsostimulate d by constant 18L : haveals odemonstrate d that longphoto - 6D . Ovulation andplasm a levelso ftesto periodsca nb euse d toadvanc e spawningu p sterone showed a6 mont h cycle and afe w to sixmonth s and thusensur ea suppl yo f fishproduce dviabl eegg s three timeswithi n out-of-season eggs. 12months . Inth estrai nunde r test,whic hnormall y Materials andmethod s maturesDecember-January/ ,spawnin g was slightlydelaye d (January-February 1982)b y 1.Th e influence ofdaylengt h onprecociou s the 18L: 6 Dphotoperiod . One third of maturationwa s investigated intw oexperi these fishovulate d againi nJul y 1982,an d ments. In the first,under-yearlin g trout plasma testosterone levels indicatetha t werekep t at 13.5°Co nseve ndifferen t over75 %o f the fishwil lhav e ovulatedb y photoperiods: 9h ,1 4h , 19h ,natura l(N) , September. Ovulationoccure d shortly after simulated natural (SN)an dchangin g4 t o testosterone levelsexceede d 200ng/ml . 20h and 20t o4 h (1h/week) . Whenth e Testosterone levels incontro l fish inJul y increasing anddecreasin gdaylengt h camet o were around 10ng/ml . anend ,the ywer e continued atconstan t 20h and4 h ,respectively . Inth esecon d Discussion experiment,onl y9 h ,1 4h , 19h and SNwer e used. The stimulatory effect of longphoto periods runscontrar y toth ecurren t 2. Ina preliminar y experiment,a smal lnum hypothesis that shortday s are required to bero fmatur e female troutwer ekep t ona accelerate gonadalmaturatio n inrainbo w constant 18h daylength . trout. A reproductive cycle of6 month s durationunde r constant longda y conditions 3.I na curren t experiment ona commercia l has alsobee n reported inbroo ktrout , fish farm,starte d inJun e 1981,tw oyea r stickleback andda ban d iti spossibl etha t old female trout (n= 42 )hav ebee nkep to n under thesecondition s twogametogeni c aconstan t 18h photoperio d (naturalda y cycles arerunnin gconcurrentl y andou to f lightextende d byartificia l light)a ta phase. constant 10°C. Control fishar emaintaine d Wehav edemonstrate d that longconstan t undernatura l illumination. photoperiods canb e applied torainbo wtrou t toobtai novulatio na t6 mont h intervals. Results The apparatus required ischea pan d easy to operate,offerin g asimple ,commerciall y Precociousmaturatio n inmale swa sonl y applicablemetho d forobtainin ggoo d inducedb yconstan t longda ylengt h quality,out-of-seaso n eggs. (Table 1). When the increasing photoperiod reached 20L: 4D after 16week sn ofis hha d spermiated. However,afte r 6week smain Table 1. %precociou smale s ondifferen t tained onconstan t 20h da y length,87 %o f photoperiods themale swer e running. Fertilization rates inexces so f90 %wer eobtaine d using the 9 h 14h 19h Inc.Dec .S N N milt from these fish. Earlymaturatio n insalmonid s isfre Expt.1 5.8 15.795. 086. 720. 1 15.010. 0 quently associated with rapid growth ofth e Expt.2 7.7 17.683. 8 - - 16.7 - 243 INFLUENCE OF SALTWATER SPAWNINGAN D STRESSO NQUALIT Y OF SEXPRODUCT SO F CHUMSALMO N (ONCORHYNCHUSKETA ) Joachim Stoss,Ul fH.M .Fagerlun d WestVancouve r Laboratory,Fisherie sResearc hBranch ,Dept .o fFisherie s andOceans ,416 0 MarineDrive ,Wes tVancouver ,B.C .V7 V 1N6 Canada. Summary Chum salmon enterfres hwate r (FW)fo r spawning ofteni na nadvance d stageo r after completion of sexualmaturation . Therefore,holdin g ofbroodstoc ki nse a water (SW)appear s tob ea feasibl e Salt water Fresh water management option for the enhancement of X i SO X ± SD P chum salmon. However,capture d fish maturing in SWhav ebot h poor gamete Fertility % 55.2 i 40.0 93.6 i 7.1 0.02 fertility and high prespawning mortality. Egg diameter (cm/20 ova) 13.8 t 0.5 14.4 t 0.5 0.02 Toidentif y theunderlyin g causesw e Ovarian fluid/ measured characteristics of sexproduct s ovarian mass (%) 5.0 t 2.2 5.8 t 2.9 NS ofchu m salmonhel d inF Wan d SWan d also Ovarian fluid pH 8.7 i 0.2 8.8 i 0.1 MS investigated theeffec t of stresso n Ovarian fluid pOsm 394 t 44 311 i 51 0.001 plasma Cortisolconcentrations . Ovarian fluid Na*(mM) 176 t 23 141 i 24 0.001 Maturing salmoncaugh t at seawer e Ovarian fluid K* (mM) 5.3 i 3.5 3.2 i 0.5 NS tranferred totank swit h FWo rS W Ovarian fluid Ca4"1 (mM) 2.6 S 0.7 1.5 i 0.6 0.001 (28%0 salinity). Stresswa s applied toon eS Wgrou p by lowering thewate r Ovarian fluid Mg'*'' (mM) 2.9 i 1.0 1.1 i 0.3 0.01 leveli nth e tanks from 80t o1 5c mfo r onehou r daily over8 days , starting two daysprio r to thefirs t recorded ovulation. Blood samples forcortisol-RI A seminal plasma,perhap s reflecting secretion takenfro m SWfis hafte r 6day s ofstres s ofbivalen t ionsvi ath eintestine . Sperm treatment were compared with unstressed fertility rates*'inS Wan d FWfis hwer e controls and pre-stressvalues . similar (SW:38. 2± 39.9%,FW:53. 3± 29.8%), Femaleswer e spawned within twoday so f butmotilit y (score0-5 )wa s lowi nS W(0. 9 ovulation. Gamete fertility wasmeasure d ± 1.1)i ncontras t toF Wfis h (3.2± 1.2) . by crossing with pooled gametes ofth e Motility wascorrelate d with fertility inS W opposite sexcollecte d from fisho fth e males (r= 0.53,P < 0.01). same stock returned to theriver . Plasma Cortisol levels instresse d and Inth eF W (n= 19),SW-unstresse d (n= unstressed fishwer e similar (pooled data: 55)an d SW-stressed (n= 42)fish , females 28.7 ± 14.3,n = 26 ,male s 14.8± prespawning mortalitywa s26% ,27 %an d 8.8,n = 33)bu tsignificantl y different 40%,respectively . Fish inal lgroup s fromvalue smeasure d 6day s earlier (11.4± reached sexualmaturit ywithi n 7days . 8.3,n = 9an d 8.0± 3.5 ,n = 14 , Amongmale s whichwer eno t sacrificed for respectively). semencollectio n 100%mortalit y was The following conclusionsma y bedrawn : reached 10day s earlier inS W thani nF W High fertility (> 80%)occure d in42 %o f SW- fish. Gamete characteristics of stressed maturing female chum salmon. Poor fertility and unstressed fishi nS Wwer esimilar . was directly correlated withosmoregulator y Therefore,value s from thesegroup swer e malfunction. Thebreakdow ni n pooled forcompariso nwit hF Wfish . osmoregulationma y becorrelate d withth e Females: Significant differences between degeneration of osmoregulatory tissues which SW (n= 31-32)an dF W (n= 7-8)group s occurs during sexualmaturatio n inPacifi c were found (see Table). salmon (Williamse t al., 1977). Our data InS Wfemale s fertility was negatively suggest thatth eresultin g highosmoti c correlated withpOs m (r= -0.68,P < . pressure ofth e ovarian fluid is the cause 0.01),Na +( r= -0.47,P < 0.02)an dK +( r ofdecrease d fertility of females heldi n = -0.42,P < 0.02). SW. Males: (no.o fobservation s SW:24-34 , Stress,a sapplied ,di d not affect plasma FW:4-6 ,x ± SD). Nopur emil twa s Cortisolconcentration s or gamete obtained from SW-males since large amounts characteristics. of liquid,accumulatin g in the posterior intestine,contaminate d the samples. This References was confirmed byhig hconcentration s of Williams,P.V. , et al. 1977. Investigation Mg"1-1"(SW :63. 5± 20. 3mM ,FW : 1.52 ± 0.55 of prespawning mortality of197 3Horsefl y mM,P < 0.001 )an d Ca++(SW : 1.53 ± 0.75 River sockeye salmon. Int.Pac .Salmo n mM,FW :0.8 4 ± 0.52mM ,P < 0.01)i n Fish.Comm .Progr .Rep . 37:3 7pp . 244 CONCLUSION 245 BASICAN DAPPLIE DRESEARC HO NFIS HREPRODUCTION :A BALANCE ? (Finaladdres s onbehal fo fth eOrganizin g Committee) E.A.Huisma n Department forFis hCultur e and InlandFisheries ,Wageningen ,Th eNetherl'and s Mr.Honorar yPresident ,Ladie s andGentlemen , plexmod e ofaction san d interactions ofre DearFriends , leasing factors, precursors," hormone s and pheromones,I ofte nwondered ,how ,fo rgood Firsto fal l Ishoul d liket oconfes stha t ness sake, we succeeded in getting these iti sbot ha pleasur e and anhonour ,a swel l creatures reproduced. And thiswa smor ees asa stres s tob eth e last speaker ina lon g pecially the caseafte rtha tmos tcomprehen rowo fsuc hdistinguishe d speakers.Th emor e sive and excellent reviewo fDr .R .Billar d so, when Iscrutinize d the titleo fm ycon (InstitutNationa l del aRecherch eAgronomi tribution: Is therea balanc ebetwee nbasi c que, Jouy-en-Josas, France), our honorary and applied research infis h reproduction? President,o nth epresen t status inth econ We have had,ladie s and gentlemen,a won trolo f reproductiono f fish inaquaculture , derfuleven there . where he elaborated on the fact that,al The fact that some 90 contributionswer e though hypophysation procedures are widely submitted tothi s conference,an d thatabou t and successfully practised toinduc ereprod 150 scientists participated, representing uctioni nfish ,thi sma y representth eadmin notles stha n3 0nations ,clearl y illustrates istration of awron g hormone in the wrong the enormous interest in this particular dosea tth ewron gtime . field:Fis hReproductiv e Physiology. The idea as well as the conceptfo rthi s I think weal lagree ,tha tth eartificia l symposium originated atth ePalmpon tMeetin g induction of reproduction infis hb yinjec in 1977i nFranc e and ourcolleagu eProf.Dr . tion of hormonal substances canb e regarded P.G.J.W.va nOord t (University ofUtrecht , as shooting witha gu no na mosquit o andw e TheNetherlands )advocate d todevot e afutur e aredesperatel y An need ofmor eaccurat ean d meeting toa nintegratio no fbasi c and applied dependable methods in order to guaranteea aspects of fish reproductive physiology. I safe and continuous supplyo fyoun g fisho f take it,tha tth einteres t ofal lo fyo ui n different species foraquacultura lpurposes . coming to andparticipatin g inthi smeetin g is a sign that this combinationha sworke d Quite an important part of fish culture quitewel lthroughou t thisparticula rweek . still relieso nth e supplyo ffr yo rfinger - Coming to thetopi co fthi s shortaddres s lings from the wild, which of course can - is there a balance? -I mus t admit that fluctuate quite considerably. while attending thismeetin g Ihav ewondere d - In South-East Asia thecultur eo fmilk - howman ytype so fbalance s there couldbe . fish (Chanos chanos)no t only provides for Of one I am sure,becaus e that hasbee n animportan t contribution toth ehuma ndiet , revealed to usalread ydurin g the reception but also offers hundreds of thousands of at "KasteelGroeneveld " last Tuesday; the jobs in ruralareas .However ,thi saquacul organizing committee facesa deficit ;a nega turalenterpris e totally depends oncatc ho f tivebalance . milkfish fry in the wild and from yeart o Speaking now infinancia l terms itma yb e year this catchma y fluctuate regionallyb y added that thetotalize d financial inputi n a factor10 . thismeetin g amountst o- an d Ihop etha ti n -Th e outputvolum eo fee l inJapa ndroppe d the meantime all of you are familiar with considerably in the years '70 and thiswa s the Dutch currency -abou t 1millio n Dutch correlatedwit h astron g decline inth ecatc h Guilders (U.S.$ 350.000.-).Takin g intoac ofelvers .Thi s independable supply isrespons count that in any agricultural projectth e ible for the facttha t sometimesprice s for rentability reaches hardly 10%,th e trout elvers go skyhigh and reach afe whundred s production of, for instance,Franc e hast o ofU.S .dollar spe rk go felvers . increase next yearwit h some 1-2millio nk g -Althoug h thesituatio nstart s toimprove , inorde rt oreac hanothe r typeo fbalance . during the last decade only somewhat more We may also look fora balanc e inou row n than half ofth eavailabl e fishpond areai n field of research on reproduction and look Central Africa was inful loperation ,whic h for benefits ofthi smeeting . Indoin g soI wasmainl y due toinadequat e availabilityo f shallopenl yadmi ttha tI hav e learned alo t youngfish . inthi smeeting . Thisaddres sstarte d witha confessio nan d The important roleo f fishreproductio ni n I dolik et omak eon emore .I ,myself ,a ma n aquaculture canb e illustrated bymentionin g aquaculturist fromprofessio n and inlisten justtw o"historical "facts . ingt oth e speakerswh odemonstrate d thecom 246 -Afte r Remy and Gehin rediscovered the Ladies angentlemen ,i nlookin gbac kI lik e artificial reproduction of fish and imple topropos e another title forthi saddress .I mented this in fish farming activities in should like toomi tth equestionmark ;I eve n the Elsace in 1843,th e culture of trout, should liket oomi t ado ta tth een d inorde r pike and other species became awel lesta to ensure that this dialogue is not ended blished activity. but can be continued inth enex tmeetin ga t -Afte r the introduction ofth eso-calle d St.John's ,Canad a in1987 . "hypophysation" in fish culture during the In this respectw e canal lb egratefu lt o years '50 the farming of forinstanc echi - the Canadian delegation, which informed us nesian herbivorous fish speciesbecam ecom that they arewillin g toorganiz e afollow - monus ethroughou t theworld . up tothi smeetin g in5 year stime . Withthes epoint s inmin d Ishoul d liket o At the endo fthi s symposium Iwoul d like make afe wremarks . toexpres sman y thankst oou r sessionchair Isther ea balance ?Ye s andNo ! men and chairladies who so ably conducted Yes, because,whe n looking at thepaper s theirsessions . both ofth ePaimpon tMeetin g and thisevent , Our sincere thanks go to all those,wh o we learn that 80%,respectivel y 60%o fth e contributedb ygivin g lectures,b y submitting papers are dealing with species of which posters,b yparticipatin g inth ediscussion s either the farming isa nestablishe denter - andb yprovidin g company. .pris eo rwhic h representpromisin g candidates We like toacknowledg e gratefully thein foraquaculture . terest that theMiniste ro fAgricultur ean d No, because of a lot oflabou r inputob Fisheries took inthi seven tan dmaterialize d viously goes into the research of species insuc ha super b receptiona t"Kastee lGroene - (mainly Salmonids), which from anaquacul - veld". tural point of view do not form too much Ladies andGentlemen ,thi seven t couldno t difficulties in reproductive management on have takenplac ewithou tth eenormou seffort s fishfarms . of both Dr.H.J.Th . Goos/(Universit y0 fUt There is certainly astron gnee d forfur recht,Th eNetherlands )an dDr .C.J.J .Richte r ther research inreproductiv emanagemen t for (StateAgricultur eUniversit y ofWageningen , example on milkfish, on eel,o nmullet ,o n The Netherlands). Since we do haveelecte d Indian carps, on Latin-American Characids an honorary chairman in the person of and onquit e afe wAsia nan dAfrica nSiganid s Dr.R .Billard ,I lik et orecommen dDr .Goo s andGroupers . and Dr. Richter to be nominated honorary secretaries retro-actively fromAugus t2nd , Basic research and applied researchsome 1982. times do stand apart in thesens etha tth e On behalf ofth eparticipant s Ials owan t former confines itself toth equestio n"ho w to thank the studentsbot h fromTh eNether it is controlled" and the latter's interest lands and from abroad,no tonl y forcontri is in "how it can be governed toobtai na butingt oth epresentatio n ofillustrations , tooli nth eenhencemen t ofaquaculture" . butals obecaus e theirpresenc e and interest In respect to this I like to recall isth ebes tguarante e forth e continuationo f Dr.R .Billard ,wh o said inhi s introductory this importantwork . lecture: "The typeo fknowledg e required in I liket orecal lth eoutstandin g cooperation reproductive physiology of fish depends on ofth edirectio nan d thestaf f ofth eInter thetyp eo faquaculture" . nationalAgricultura l Centre (IAC)b yprovid I completely agreewit hhi man d Ia msur eh e ing us with this nice ambience for our will agree with a slight alterationo fth e meeting. sentence,base d onth eabov ementione dpoints : It is a pleasure to be suretha t Ispea k "Thetyp e ofaquacultur e depends onth eknow on behalf ofal lparticipant s inaddressin g ledge available in reproductive physiology our sincere appreciation for all the work offish" .Onl y then fishreproductiv emanage done and still to be doneb yMrs .Ad ava n ment finds asoun dbasis . Ingen, Mrs.Elle nva nd eWeterin g andMrs . This laysa clai mbot ho napplie d andbasi c LiesbethHotke . researchers ofwhic h Ihop ew ear eal laware . Personally I found aga p inthi smeeting , Dear participants, let us finally thank especially in thediscussions .Th e question each other for coming here, let us thank "how itca nb egoverne d and used inaquacul each other for our most enjoyable company ture praxis"ha s not been put.forwar d too and, looking forward to our next meeting, often. letu sdeclar e I am suretha t infutur e events likethi s "This Symposium isclosed" . wewil l increasingly learn-eachother' s research aimsan d applicationsbette r andi n doing sob e able toclos e thisgap . 247 LISTO FPARTICIPANT S International Symposiumo nReproductiv ePhysiolog y ofFis h BELGIUM A.L.Kyle Department ofZoolog y H.J.Koch University ofAlbert a Zoological Institute Edmonton,Alb .T6 G2E 9 University ofLeuve n Naamsestraat5 9 D.S.MacKenzie B-3000 Leuven Department ofZoolog y University ofAlbert a F.P.Ollevier Edmonton,Alb .T6 G2E 9 Zoological Institute University ofLeuve n R.E.Peter Naamsestraat5 9 Department ofZoolog y B-3000Leuve n University ofAlbert a Edmonton,Alb .T6 G2E 9 CANADA N.E.Stacey J.P.Chang Department ofZoolog y Department ofZoolog y University ofAlbert a University ofAlbert a Edmonton,Alb .T6 G2E 9 Edmonton,Alb .T6 G2E 9 K.G.Waiwood A.F.Cook WestVancouve rLaborator y TheKing' s College 4160Marin eDriv e 10766-97Stree t WestVancouver ,Br.Columbi aV7 V 1N6 Edmonton,Alb .T5 H2M 1 M.Wiegand L.W.Crim MemorialUniversit y ofNewfoundlan d MemorialUniversit y ofNewfoundlan d Marine Sciences"ResearchLaborator y MarineScience sResearc hLaborator y St.John's,Newfdl .A1 C5S 7 St.John's,Newfdl .A1 C5S 7 CENTRALAFRICA NREPUBLI C E.M.Donaldson WestVancouve rLaborator y J.A.L.Janssen 4160Marin eDriv e Stationd ePiscicultur e del aLandji a WestVancouver ,Br.Columbi aV7 V 1N6 c/oPNU D B.P.87 2 L.M.B.Garcia Bangui Department ofZoolog y University ofAlbert a DENMARK Edmonton,Alb .T6 G2E 9 I.Boetius B.J.Harvey DenmarksFiskeri-o gHavunders^gelse r University ofVictori a Charlottenlund Slot Department ofBiolog y 2910-Charlottenlund P.O.Box 1700 Victoria,Br.Columbi aV8 W2Y 2 J.Boetius Denmarks Fiskeri-ogHavunders^gelse r D.R.Idler Charlottenlund Slot MemorialUniversit y ofNewfoundlan d 2910-Charlottenlund MarineScience sLaborator y St.John's,Newfdl .A1 C5S 7 F.Minck Waterquality Institute G.vande rKraa k Aquaculture Department WestVancouve rLaborator y 11,Ager nAll e 4160Marin eDriv e DK-2970HvSrshol m WestVancouver ,Br.Columbi aV7 V 1N6 248 EGYPT FRANCE H.vanWeer d K.Alagarswami Ilacob v Laboratoire dePhysiologi e desPoisson s P.O.Box2 I.N.R.A. Cairo 78350Jouy-en-Josa s N.Zonneveld E,Bedier Ilacob v France-Aquaculture P.O.Box2 StationDeva-Su d duCNEX O Cairo 34250Palavas-les-Flot s FEDERALREPUBLI CO FGERMAN Y R.Billard Laboratoire dePhysiologi ede sPoisson s H.Bayrle I.N.R.A. BayerischeLandesanstal t fürFischere i 78350Jouy-en-Josa s Weilheimerstrasse8 a 8130Starnber g B.Breton Laboratoire dePhysiologi e desPoisson s S.Enteneuer Avenue duGénéra lLecler c Sonderforschungsbereich9 4"Meeresforschung " 35042Renne s Bundesstrasse 55 2000Hambur g 13 J.Bruslé Laboratoire BiologieMarin e V.Hilge Université dePerpigna n Institut fürKüsten -un d Binnenfischerei Avenue deVilleneuv e derBF A 66Perpigna n Wulfsdorferweg 207Ahrensburg/De nHols t S.Brusl é LaboratoireBiologi eMarin e W.Holtz Université dePerpigna n Institut fürTierzuch tun dHaustiergeneti k Avenued eVilleneuv e derUniversitä t Göttingen 66Perpigna n Albrecht-Thaerweg1 3400Göttinge n E.Burzawa-Gerard MuséumNationa l d'Histoire Naturelle R.Reinboth Laboratoire dePhysiologi e Institut fürZoologi e derJohanne s Gutenberg 7,ru eCuvie r Universität 75231 Paris Cedex0 5 Saarstrasse 21,Postfac h 3980 6500Main z P.Chambolle Laboratoire deBiologi eMarin e FINLAND Université deBordeau x Avenue desFaculté s S.Lindgren 33405Talenc e Department ofPhysiologica l Zoology University ofHelsink i D.Coves SF-0100Helsink i 10 CentreOcéanologiqu e deBretagn e B.P.33 7 Nakari 29273Bres t Department ofPhysiologica l Zoology University ofHelsink i N.Devauchelle SF-0100Helsink i 10 CentreOcéanologiqu e deBretagn e B.P.33 7 S.Pesonen 29273Bres t Department ofPhysiologica l Zoology University ofHelsink i S.Dufour SF-0100Helsink i 10 MuséumNationa l d'Histoire Naturelle Laboratoire dePhysiologi e A.Soivio 7,ru eCuvie r Department ofPhysiologica l Zoology 75231 ParisCede x0 5 University ofHelsink i SF-0100Helsink i 10 Y.Fontaine MuséumNationa l d'HistoireNaturell e Laboratoire dePhysiologi e 7,ru eCuvie r 75231 Paris Cedex0 5 249 A.Fostier M.G.Plouidi Laboratoire dePhysiologi e desPoisson s Laboratoire dePhysiologi e desPoisson s Avenue duGénéra lLecler c I.N.R.A. 35042Renne s 78350Jouy-en-Josa s B.Jalabert P.Prunet Laboratoire dePhysiologi e desPoisson s Laboratoire dePhysiologi e desPoisson s Avenue duGénéra lLecler c I.N.R.A. 35402Renne s Campus deBeaulie u 35042Renne s O.Kah Arraché deRecherche s CNRS B.Querat Laboratoire deBiologi eAnimal e Laboratoire dePhysiologi e Généralee t Avenuede sFaculté s Comparée 33405Talenc e 7,ru eCuvie r 75005Pari s I.A.Khan Laboratoire dePhysiologi eGénéral ee t P.Reinaud Comparée Laboratoired ePhysiologi e desPoisson s 7,ru eCuvie r I.N.R.A. 75231Pari s Cedex0 5 Campus deBeaulie u 35042Renne s P.deKimp e CentreTechniqu e ForestierTropica l P.Zaborski 45B 13Avenu ed el aBell eGabriell e Centred eCytologi eExpérimental e 94130Nogent/Marn e C.N.R.S. 67 rue M.Giinsbourg J.Leloup-Hatey 94200 Ivry-sur-Seine Laboratoire dePhysiologi e Généralee t Comparée Y.Zohar 7,ru eCuvie r Laboratoire dePhysiologi e desPoisson s 75231 ParisCede x0 5 Campus deBeaulie u 35042Renne s M.H.Magri Laboratoire dePhysiologi e desPoisson s GABON I.N.R.A. 78350Jouy-en-Josa s A.Pham EcoleNational e desEau xe tForêt s J.Marcel B.P.396 0 Laboratoire dePhysiologi e desPoisson s Libreville I.N.R.A. 78350Jouy-en-Josa s HONGKON G F.leMen n Wen-young Tseng Institut deBiologi eAnimal e Marine ScienceLaborator y Universitéd eBordeau x TheChines eUniversit y ofHon gKon g Avenue desFaculté s ShatinN.T . 33405Talenc e INDIA J.NunezRodrigue z Laboratoire deBiologi eAnimal e H.Singh universitéd eBordeau x FishEndocrinolog y Laboratory Avenuede sFaculté s Department ofZoolog y 33405Talenc e BanarasHind uUniversit y Varanasi- 22100 5 M.Olivereau Laboratoire dePhysiologi e T.P.Singh Institut Océanographique FishEndocrinolog y Laboratory 195,ru eSaint-Jaque s Department ofZoolog y 75005Pari s BanarasHind uUniversit y Varanasi- 22100 5 250 B.I.Sundararaj I.Hanyu Departmento fZoolog y Universityo fToky o University ofDelh i Laboratory ofFis hPhysiolog y Delhi 110007 Bunkyo-ku Tokyo 113 ISRAEL M.Mori saw a M.Abraham OceanResearc h Institute Department ofZoolog y University ofToky o TheHebre wUniversit y Minami-daiNakano-k u Jerusalem Tokyo 164 S.Rothbard S.I.Ohtake Fishan dAquacultur eResearc hStatio n Departmento fBiolog y DOR NihonUniversit y School ofMedicin e D.N.HofHacarme l 30-820 Oyaguchi,Itabash i Tokyo 173 Z.Yaron Tel-AvivUniversit y H.Urasaki Departmento fZoolog y Departmento fBiolog y TelAvi v NihonUniversit y School ofMedicin e Oyaguchi,Itabash i ITALY Tokyo 173 D.Casciotti K.Yamauchi Institute ofAnima lProductio n Department ofBiolog y University ofPerugi a Faculty ofFisherie s ' 0160.0Perugi a Hokkaido University 3-1-1 Minatocho L.Colombo Hakodate04 1 University ofPadu a Instituteo fAnima lBiolog y KUWAIT ViaLOreda n1 0 35100Padu a R.Abu-Hakima Kuwait Institute forScientifi cResearc h C.Simontacchi Mariculture andFisherie s Department Institute ofVeterinar y Physiology P.O.Box 1638 University ofPerugi a Salmiya 01600Perugi a THENETHERLAND S IVORYCOAS T B.Baggerman S.Hem Zoological Laboratory Centre deRecherche s Océanographiques Kerklaan3 0 ORSTOM 9717H R Groningen B.P.V. 18 Abidjan H.Diederik ZoologicalLaborator y JAPAN Sectionfo rComparativ e Endocrinology P.O.Box 80058 T.Abe 3508T BUtrech t Department ofBiolog y NihonUniversit y Schoolo fMedicin e E.H.Eding Oyaguchi,Itabash i Department ofFis h Culture andInlan d Tokyo 173 Fisheries Marijkeweg 40 A.Hara 6709P GWageninge n Department ofBiochemistr y HokkaidoUniversit y Schoolo fMedicin e E.Egberts Kita 15,Nish i 7,Kita-k u Department ofExperimenta lAnima lMorpholog y Sapporo06 0 andCel lBiolog y Marijkeweg 40 6709P GWageninge n 251 G.E.Fahraeus-vanRe e C.J.J.Richter ZoologicalLaborator y Department ofFis hCultur e andInlan d P.O.Box8005 8 Fisheries 3584C HUtrech t Marijkeweg4 0 6709P GWageninge n " J.Th.Gielen ZoologicalLaborator y M.Siemelink Section forComparativ e Endocrinology Euroconsultb v Padualaan8 P.O.Box2 2 3564 CH Utrecht 6800A KArnhe m H.J.Th.Goos M.Terlou ZoologicalLaborator y ZoologicalLaborator y Section forComparativ e Endocrinology Sectionfo rComparativ e Endocrinology Padualaan8 Padualaan8 3584C HUtrech t 3584C HUtrech t H.Hogendoorn L.P.M.Timmermans Organization forImprovemen t ofInlan d Department ofExperimenta lAnima lMorpholog y FisheriesOV B andCel lBiolog y Karperweg 8-10 Marijkeweg 40 8221R BLelysta d 6709P GWageninge n E.A.Huisman J.A.J.Verreth Department ofFis hCultur ean dInlan d Department ofFis h Culturean dInlan d Fisheries Fisheries Marijkeweg 40 Marijkeweg4. 0 6709P BWageninge n 6709P GWageninge n R.vande nHur k S.vanVlie t ZoologicalLaborator y Euroconsultb v Section forComparativ e Endocrinology P.O.Box2 2 Padualaan8 6800A KArnhe m 3584C HUtrech t NORWAY A.vanIngen-Bouwman s Departmento fFis hCultur ean dInlan d D.Aasjord Fisheries University ofTroms ^ Marijkeweg 40 Institute ofFisherie s 6709 PG Wageningen B.P.48 8 9000-11Tromsj S J. G. D.Lambert Zoological Laboratory K.G.Forberg Section forComparativ e Endocrinology University ofTroms ^ Padualaan8 Instituteo fFisherie s 3584C HUtrech t P.O.Box308 3Gulen g 9001Tromsj S M.A.M.Machiels Department ofFis hCultur ean dInlan d A.Kolbeinshavn Fisheries University ofTroms ^ Marijkeweg 40 Institute ofFisherie s 6709P GWageninge n P.O.Box79 0 9001Tromss S W.B.vanMuiswinke l Departmento fExperimenta lAnima lMorpholog y T.Refstie andCel lBiolog y Research Station forSalmonid s Marijkeweg 40 N-6600SunndalsjSr a 6709P GWageninge n PEOPLE'SREPUBLI CO FCHIN A P.G.W.J.vanOord t ZoologicalLaborator y LinHao-Re n Section forComparativ e Endocrinology Department ofBiolog y Padualaan8 Zhongshan(Sun-Yet-Sen)Universit y 3584C HUtrech t Guangshou,Guangdon g Province 252 THEPHILIPPINE S TAIWAN J.V.Juario N-H.Chao-Liao AquacultureDepartmen t SoutheastAsia n TungkangMarin eLaborator y Fisheries Development Center TaiwanFisherie s Institute P.O.Box25 6 Tungkang,Pingtun g 916 IloiloCit y UNITEDKINGDO M C.M.Kuo ICLARM R.Alderson P.O.Box150 1 DevelopmentManage r Makati,Metr oManil a MarineHarves tLtd . Lochailort M.S.Marte Inverness-Shire PH38 4LZ,Scotlan d Aquaculture Department SoutheastAsia n Fisheries Development Center J.N.Ball P.O.Box25 6 Department ofZoolog y IloiloCit y University ofSheffiel d Sheffield S102T N POLAND S.M.Baynes K.Bieniarz MAFF Academy ofAgricultur e Directorate ofFisherie s Research Department of Ichthyobiology andFisherie s FisheriesLaborator y ul.I.Ambrozowa6 Lowestoft,Suffol kNR3 30H T 30-149Krako w N.R.Bromage PORTUGAL FishCultur eUni t Department ofBiologica l Sciences M.A.Ramos GostaGree n Ministerio daAgricultur a ePesca s BirminghamB 47E T INIP Avenidad eBrasili a P.Copeland 1400Lisbo a BruneiUniversit y Department ofApplie dBiolog y SOUTHAFRIC A Uxbridge,Mddx .UB 83P H H.J.Schoonbee J.A.K.Elliott ResearchUni tfo rFis hBiolog y University ofAsto n RandAfrikaan s University Department ofBiologica l Sciences P.O.Box52 4 Fish CultureUni t Johannesburg 2000 Birmingham B47E T SWEDEN J.Finlay UnileverResearc h B.Borg ColworthLaborator y Department ofBiolog y GreyhopeRoa d University ofLun d AberdeenAB 9 2JA,Scotlan d Helgonavagen3 22362Lun d R.Johnstone MarineLaborator y SWITSERLAND P.O.Box10 1 VictoriaRoa d J.Weibel AberdeenAB 9 8DB,Scotlan d OptigalS A 2,Rout ed'Oro n D.E.Kime CasePostal e 324 Department ofZoolog y CH-1010Lausann e TheUniversit y Sheffield S102T N N.MacFarlane Departmento fLif e Sciences TrentPolytechni c NottinghamNG 1 4BV 253 A.J.Matty M.P.Schreibman TheUniversit y ofAsto n Department ofBiolog y Department ofBiologica l Sciences CityUniversit y ofNe wYor k GostaGree n BedfortAvenu ean dAvenu eH BirminghamB4 7E T Brooklyn,Ne wYor k 11210 R.Roberts K.Selman B.P.NutritionU.K .Limite d Department ofAnatom y Wincham,Minse lWork s Collegeo fMedicin s Northwich,Cheshir eCW 96D F University ofFlorid a BoxJ-23 5 A.P.Scott Gainsville,Florid a3261 0 Directorate of.Fisherie sResearc h FisheriesLaborator y W.L.Shelton Lowestoft,Suffol kNR3 3OH T Department of Fisheries and Allied Aqua- cultures C.J.Secombes Auburn University University ofAberdee n Auburn, Alabama 36849 Department ofZoolog y TillydroneAvenu e P.Thomas AberdeenAB 9 2TN,Scotlan d University ofTexa s &Austi n Marine Science Institute 0.Skarphedinsson PortAransas ,Texa s 78373 FisheriesLaborator y Pakefield R.A.Wallace Lowestoft,Suffol kNR3 3OH T OakRidg eNationa lLaboratorie s P.O.BoxY J.P.Sumpter OakRidge ,Tennessee3783 0 Department ofBiochemistr y BruneiUniversit y U.S.S.R. Uxbridge,Middx .UB 83P H I.A.Barannikova P.West Physiological Institute FinfishHatcher y Leningrad University ScottishSe aFarm sLtd . Leningrad 199164 South Shian,Conne l ArgyllPA3 7 1SB,Scotlan d R.J.Wootton Department ofZoolog y University ofWale s Aberystwyth,Dyfe d SY233D A A.D.Worthington Departmento fLif e Sciences TrentPolytechni c NottinghamNG 1 4BV U.S.A. G.V.Callard BiologicalScienc eCente r BostonUniversit y 2 CummingtonStree t Boston,Massachusett s 02215 S.D.Gerking Department ofZoolog y ArizonaStat eUniversit y Tempe,Arizon a8528 1 J.Pudney BiologicalScienc eCente r BostonUniversit y 2 CummingtonStree t Boston,Massachusett s 02215 AUTHOR INDEX Abe, Takeyuki 204 Fahraeus-van Ree,G.E . 53 Abu-Hakima,R . 190 Fontaine, Y.A. 52 Abul-Ellah, K. 190 Forberg, K.G. 197 Akatso, S. 190 Fostier, A. 14, 164 Al-Shoushani,M . 190 Gerking, Shelby D. 224 Arnold, CR. Ill Gielen, J.Th. 53, 54, 55 Asahina, Kiyoshi 229 Goos, H.J.Th. 54, 55,57 ,5 9 Baggerman, Bertha 237 Groningen, J.J.M,va n 80 Barannikova, I.A. 49 Halpern-Sebold, L. 59 Baynes, S.M. 103, 128 Hanyu, Isao 229 Bayrle, H. 129 Hara,A . 158 Belvedere, P.C. 84 Hardy, A. 112 Bentivegna, F. 84 Harvey, Brian 123 Bieniarz, K. 196 Hilge,V . 27 Billard, R. 1, 50, 134, 192 Hirai,H . 158 Boëtius, I. 174 Holtz,W . 242 Boëtius, J. 174 Honma, Y. 23 9 Bogomolnaya,,Alissa 49 Huisman, E.A. 246 / Boiti, C. 110 Hunter, George A. 78, 177 Borg, B. 23 8 Hurk, R.vande n 69, 99 Breton, B. 14, 50, 233 Idler, D.R. 4 Brichon, G. 155 Jalabert, B. 164 Bromage, N. 23 3 Janssen, J.A.L. 195 Brown, N.J. Ill Johnstone, R. 79 Bruslé, S. 193 Kah, 0. 56 Bukovskaya, O.S. 49 Kerdelhué,B . 52 Burzawa-Gerard,Elisabet h 19 Kime,D.E . 95 Bye, V.J. 243 Kleine Staarman, G.H.J. 195 Callard, G.V. 40, 109 Kleijne, J.A.F.W. 53 Canick, Jacob 109 Kuo, Ching-ming 181 Casciotti, D. 110 Kyle,An n L. 240 Chambolle, P. 56, 58 Lace, Z.M. 28 Chang, John P. 51 Laird, L.M. 81 Chao,Nai-Haie n 132 Lambert, J.G.D. 57,69 ,99 , 107 Chiba, A. 239 Lamour, F. 155 Colombo, L. 84 Leloup-Hatey, J. 112 Covens,M . 159 Leuven, S.E.W. 147 Crim,L.W . 23, 26, 50 Lin Ding, 199 Datta, K.K. 200 Lin Hao-ren 199 Devauchelle,N . 155 MacFarlane, N.A.A. 220 Diederik, H. 107 MacKenzie, Duncan S. 241 Dikshith, T.S.S. 200 Marconato, A. 84 Donaldson, Edward M. 78,114 ,177 , Margolis-Kazan, H. 59 205 Matty, A. 23 3 Dubois,M.P . 56 Menn, F. le 198 Dubourg, P. 56, 58 Mol Moncourt-de Bruyn,Mariëll e G.A. Dufour, S. 52 de 54 Dye, Helen M. 177 Morisawa, M. 131 Easton, K.W. 220 Morisawa, S. 131 Eding, E.H. 147, 195 Ohtake, Sin-Iti 204 Efimova, N.A. 49 Okuno,M . 131 Egberts, E. 80 Olivereau, M. 58 Elliott, J. 233 Ollevier, F. 159 El-Zahr, C. 190 Oordt, P.G.W.J,va n 44, 53,54 , 55, Epler, P. 196 57 Evans, D.M. 23 Panis, C.J. 53 Fagerlund, Ulf H.M. 244 Parmentier, H.K. 80 255 Pasqualini, C. 52 Peter, R.E. 30, 50,51 , 240 Plouidy, M.G. 134 Pohl,M . 242 Priede, I.G. 8i Prunet, P. 24 Pudney, Jeffrey 109 Querat, B. 112 Raizada, R.B. 200 Refstie, T. 73 Reschke,M . 23 8 Richter, C.J.J. 147, 195 Rothbard, Hamutal 201 Rothbard, S. 201 Schmidt, R. 242 Schoonbee, H.J. 202 Schreibman, M.D. 59 Scott,A.P . 103, 128, 243 Secombes, C.J. 81 Selman, K. 151, 161 Sesso,Antoni o 25 'Shelton,Willia m L. 82 Shimizu, Akio 22 9 Simontacchi, C. 110 Singh, H. 60 Singh, H.N. 203 Singh, I.J. 203 Singh, T.P. 60, 203 Skarphédinsson, O. 243 Stacey, N.E. 240, 241 Stephan, G. 155 Stoss,Joachi m 114, 254 Thomas, P. Ill Timmermans, L.P.M. 80 Urasaki, Hiroshi 204 Val-Sella,Maur a V. 25 Van der Kraak, Glen 177, 205 Veen, Th. van 238 Waiwood, K.G. 206 Wallace, R.A. 151, 161 Weil, C. 26 Whitehead, C. 23 3 Wiegand, Murray D. 136 Wilms, Petra G. 57 Wootton, R.J. 210 Worthington,A.D . 220 Wijst, J.G.M,va n der 147 Yamamoto, K. 185 Yamauchi K. 185 Yaron, Z. 27 Youngson, A.P. 79 Zaborski, P. 64 Zandwijk, M.L 57 Zenkevich, H.A. 28 Zohar, Y. 14 256