The Physiology and Immunology of the Endocrine Testis

tì/ jì;:llilul s\ t: 1.q_ 81 LI Àt{Y

THE PHYSIOLOGY AND IMMUNOLOGY OF THE ENDOCRINE TESTIS

Simon MADDOCKS, B.Ag.Scl. (Hons)

A thesls submltted to the Unlversfty of Adelalde ln fulfl'lment of the requlrements for the degree of Doctor of PhllosoPhY

Department of Anlmal Scfences Walte Agrlcultural Research Instltute The UnlversitY of Adelalde

March 1987

lì,.,ì,r(,:,td r/ riÈ"1 Know'ledge and wlsdom far f rom belng oner. Have ofl tlmes no connexion. Knorledge dwells In heads replete with thoughts of other men; Wlsdom ln minds attentlve to thelr otvn' Know'ledger a rude unprof ltab'le massr The meré materials with whlch wlsdom bul'ldsr Tfl.|smoothldandsquarldandflttedtoftsplacer Does but encumber whom lt seems trenrlch' Knowledge ls proud that he has learnrd so much; Wisdom ls humb'le that he knows no more'

I{il'l lam CowPer ( 1731-1800) '_ .å Cowper Poetical Works TABLE OF CONTENTS f CONTENTS vii ABSTRACT x DECLARATION xl ACKNOt{LEDGEMENTS PREFACE xlff

PART I:

T CTIAPTER I: INTRODUCTION 2 1. 1. INTRODUCTION 2 L.2, THE PHYSIOLOGY OF THE ENDOCRINE TESTIS L.2,L. Introducti on 2 L.2.2. The Leydfg cells 7 r.2,2. I, Anatomy 7 L.2.2. 2. Relatiónship to blood vessels, lymph vessels and seminiferous tubul es L2 .2.2 3, Re]atfonshlp to other ce]ls in fnterstlt'lal tissue I5 I 18 ,212 4. Hormones produced by the Leydfg ce1ìs I 22 L .2.3 The Sertoll cells 24 T .2,3 I. AnatomY I .2.3 2. Relatlônshlp to the blood-testis barrier 29 I ,2,3 3. RelationshiP to other ce'lls 32 I 2.3 4. Hormones produced by the Serto'li cells 35 I .2.4 Mechanfs|n of transport of hormones out of and across cel'ls 38 L.2,4 .I.. Hormone secreti on 38 1.2,4.2. TransPort 4L L,2.5 . Cotpositìon and functlon of interstitial f'luid 45 L.2.6 . Conirol of endocrine activity in the testis 52 57 ,3 THE IMMUNOLOGY OF THE ENDOCRINE TESTIS I 57 T .3 Introducti on .r 58 ,3 r2 The Immune sYstem I 69 13 .3 Immunologica'lly privlleged sltes I 7L ,3 r4 The testis as án.fmmunologfcally privileged sÍte t 78 ..3 .5 TheorlesforthePrlvilegedstatusofthetestis I 84 I .4 THE PURPOSE OF THIS STUDY

BART II: 86 CTIAPTER 2: MATERIALS AND METHODS 87 2.L. REAGENTS 87 2.2. ANIMALS 88 2. 3 SURGICAL PROCEDURES 88 2, 3 r f . ThyroidectomY 9I 2' 3 t2.. Hypophysectomy 93 2. 3 13r Cryptorch I dectomY 93 2. 3 Efferent Duct Llgatlon .4, 94 2. 3 .5. Blood col lectlon if

2.3.5 Rats 94 .I. 95 2.3.5 SheeP .2. 97 2.3.6 . Lymph col lectlon 2.3.7 . Rat TransPl ants 97 2.3.7 .I. Under the kfdneY caPsule 97 2.3.7 Into the testfs 98 .2. 99 2.3.8 . Sheep Transplants lnto the testis 99 2.4. ISOLATION AND CULTURE OF RAT PITUITARY CELLS 2.4.L. Buffers and SolutÍons 99 'l IOI 2.4.2. Co1 agenase d f gestl on 2.4.3. Trypsfn dlgestlon t02 2.4.4. Tlssue culture 103 Routine method 103 2.4.4. I. r03 2.4.4. 2. LHRH chal'lenge

TESTS 104 2.5 . THYROID FUNCTION I04 Iod I ne accumu'l atf on 2.5 . 1. 104 2.5 . Rats l. I. 106 2.5 SheeP .L.2. 106 2.5 .2. TRH chal'lenge 108 2.6 . PITUITARY FUNCTION TEST - LHRH CHALLENGE 2,6 .I. Intravenous iniectlon 108 Intravenous lnfusion I08 2.6 .2. r08 2.6 -.2.2,,2,L. Va]idation of method Calculatfon of dose of fnfused LHRH r09 2.6 r09 2.6 .2.3. Intratesticular arterlal lnfuslon 110 2.7. HCG CTIALLENGE rl0 2.8. EDS ADMINISTRATION 1r0 2.9. SILICA ADMINISTRATION VOLUMES TIO 2.L0. MEASUREMENT OF ALBUMIN AND CT-EDTA DISTRIBUTION ttl z.LL. VASCULAR PERMEABILITY LL2 2.L2. LYMPH FLOW 113 2.T3. HORMONE ASSAYS Buffers and Solutions 113 2,L3,L. 113 2.L3.L,L. PhosPhate buffer L25 Blocker so]ution wlth I-thy roxi ne t14 2.L3.L.2. 114 2.L3 .L.3. TO-f ree serum 1r4 2.L3 .I.4. LÍquid scintfl'latlon system L-thYroxine radlofmmunoassaY T$ 2,r3 ,2. r16 2.r3 116 2,r3 L2l 2.L3 L23 2.L3 L24 2,L3 L25 2,L3 L27 2.13 Rat Prolactin (rPRL) .3.6. (oTSH) t28 2.L3 .3.7 . Ovlne Thyroid stlmul ating hormone ftf

r29 2.L4. HISTOLOGY 2.I4.I. Ff xatlves r29 2.L4.2. Tfssue Processlng L29 13I 2.L5 . IMIVIIJNOH I STOCTIEMISTRY 2.L5.L. Flxatlves 131 2.L5.2. Tlssue Processlng 133 2.L6. STATISTICAL ANALYSIS 135

GTIAPTER 3: THE RODENT TESTIS AS AN IMMUNOLOGICALLY PRTVILEGED SITE 136

L37 3.1. INTRODUCTION r37 3.2. CHOICE OF TISSUE GRAFTS

3.3, PITUITARY TRANSPLANTS 138 3..3. I, Experlmenta'l Procedures 139 139 3.3, I . l. Anlmal s 3.3. I.2. Col I agenase-df spersed cel'l s 139 3,3. I.3. TrYpsin-disPersed cel'ls r40 r40 3 .3. I.4. Ouarters of whole Pltultarfes 141 3.3, 2 . Results 3.3. 2 Col1 agenase-df spersed cell s 14t .I. r42 3 2 ,2. Trypsln-dlsPersed cel'ls '3. 145 3.3. 2 .3. Ouarters of who'le pituitaries 3.3.3. Discusslon 150 il 156 ip 3 .4. THYROID TRANSPI-ANTS tI Experf menta'l 157 ;l 3.4.1 . Procedure 3.4.r .1. Anfma'ls r57 3.4.1 .2. Survival of thyroid allografts in the testis rs7 3 .4.L .3. A comparlson of graft survlval ln the kidney and the testis 158 3.4.2. Resul ts 158 3.4.2.r. Survival of thyroid allografts in the testis 158 3 .4.2.2. A comparison of graft survfval fn the kldney and the testls 159 3.4.3 Dfscussion 165

3.5. EXPERIMENTAL MANIPULATIONS OF THE TESTIS AND THEIR EFFECTS ON ALLOGRAFT SURVIVAL r69 3.5. I. Efferent Duct Ligation 169 3.5. 1. I, Experimental Procedure 17I 3,5. I. 2. Results 17I 3.5, t.3. Dlscusslon r72 L72 3.5. 2' Cryptorch Íd i sm 3.5. 2. l. Experimenta'l Procedure 175 175 3 .5, 2.2, Results 3.5. 2.3 . Dlscusslon L76 3.5, 3. Human Chorionic GonadotroPhin L76 3.5. 3.1 . Mafntenance of lncreased lymph flow wlth hCG L77 3.5.3,r ,1, ExPerfmental Procedure L77 3.5.3.r .2. Resu'lts 178 3.5.3.r .3. Dfscusslon r80 l lv

3.5.3.2. Effect of lncreased lymph f'lorv on allograft surv lval 184 r84 2.L. Experf mental Procedure 3.5.2. r85 3.5.3. 2.2. Resul ts 187 3.5.3. 2.3. Df scusslon

3.6. IMPoRTANcEoFLEYDIGCELLSAND/oRMACRoPHAGESFoR 188 ALLOGRAFT SURVIVAL IN THE TESTIS r89 3.6.1. Experlmental Procedure 190 3.6.2. Resul ts 3.6.3. Discusslon 195

CHAPTER4:THEOVINETESTISASANIMMUNOLOGICALLY 200 PRIVILEGED SITE 20L 4.I. INTRODUCTION 202 4.2. EXPERIMENTAL PROCEDURE 202 4.2.L. Animal s 4.2.2. Thyrold transPl ants 203 4.2,3 . Pitultary transPl ants 204 206 4.2.4. Immunohl stochenl strY 206 4.2.5. Hormone assays 206 4,3, RESULTS 4.3. r. Thyrold transPl ants 206 206 l_ TRH challenge 4.3. r. 208 4 2 Iodine local lsatlon 13 .L. 208 4..3. 3 Hi sto'logY 'j r. 208 fr,| 4 .3 4 Plasma ãn¿ lymph hormone concentrations ì{ ,1. 2L2 4,.3.1. 5 P]asmahormoneconcentratÍonsfnweek]ysamples È 2L2 4.3 .2, Pitultary transPl ants 2L2 4.3 LHRH challenge ,2, L 2L2 4.3 .2, 2. Plasma and lymph hormone concentrations 215 4.3,2. 3. Immunohl stocheml strY 4.3.2. 4. plasrna hormone concèntrations in weekly samp'les 2t5 2r5 4.4. DISCUSSIoN

2L8 DTSCUSSION

PART III: 225 CTIAPTER 6: MATERIALS AND METHODS 226 6.1. REAGENTS 226 6.2. HEATING OF TESTES 226 6.3. PREGNENOLONE ADMINISTRATION t 227 I 6.4. TESTOSTERONE IMPLANTS I

¡ 227 6.5. CELL DISPERSION TECI{NIOUE

ì v

228 ASSAY 6.6. HORMONE 228 6.6.1 Testosterone radi oimmunoassay 230 6.7. PROTEIN ASSAY reagent BL 6.7 .Lr Proteln 231 6.7.2. Proteln assay LEVELS B4 6.8. MEASUREMENT OF SODIUM AND POTASSIUM STUDIES 234 6.9. ADMINISTRATION OF RADIOISOTOPES IN PHYSIOLOGICAL 6.9.1. 3H-v,lut". 234 6.9.2. 3H-Testosterone 235 14c-Mann 235 6.9.3 . ltol 125r-No.*u] 236 6.9.4. Rat Pl asma 6.9.5. 22Nu-sodium Chloride 236 237 6.T0. STATISTICAL ANALYSIS

238 REGION 239 7.L. INTRODUCTION 24L CANNULA 7 .2, THE PUSH-PULL 24L on 7.2,L, Introductl 242 7.2.2' Construction 245 Apparatus 7 ,2.3, 249 7.2.4. Usä of the Push-Pu11 cannula 249 IN THE RAT 7 .3. INVESTIGATIONS 25r 7.3.1. Experimental Procedure 'App'l in the testis 25L 7 .3.L, I. ication 25L 7 ,3.L, 2, Measurement of dilution Chofce of infusates 253 7 ,3 ,1, 3. 253 7 4. Protocol used .3 'Lr 254 7 ,3,2. Resu'lts Applicatfon ln the testls 254 7 .3.2, I. 254 7 .3.2' 2. Measurement of dllutlon of interstitfal flufd 258 7 .3.2.3. AnalYsls 258 7 .3.3. Di scussi on 263 7.4. CONCTUSIONS

268

269 8.1 INTRODUCTION 269 I r2r EXPERIMENTAL PROCEDURE anaesthesla and push-pull cannulatlon on I 2.L. Effect of 269 I testicularvenousconcentrationsoftestosterone ementatlon 270 i 8.2,2. Hypophysectomy with pregnenol one suppl GonadotroPhl n 27L 8,2,3. Himan Ôtrorionic 27L 8.2.4. Ethane Dimethane Sul phonate (EDS)

* vi

8.2.5. Heat treatment 272 8.2.6. Efferent duct I lgatfon 273 8.2.7. Testosterone lmPl ants 274 8.3. 275 8.3. r. 275 275 9.3.2. 278 8.3.3. 28L 8.3 .4. Ethane Dimethane Sulphonate (EDS) 8.3.5. Heat treatment 283 Efferent duct I igation 283 8.3.6. 2æ 8.3 .7 . Testosterone imP'l ants 289 8.4. DISCUSSTON

304 PUSH-PULL CANNULA 305 9.I. INTRODUCTION 305 MATERIALS AND METHODS 9.2, 306 9'2,L . Rams 9.2.2 . Boars 307 309 9.3 . RESULTS 309 9.4. DISCUSSION

PART TV: 315

316 TO.I. INTRODUCTION 3L7 EXPERIMENTAL PROCEDURES T0.2. 3L7 Animal s IO.2.I. 318 L0.2.2, CYtochemi strY L0.2.2,I, PreParation of tl ssue 3r8 Acld-phosphatase detection 318 I0.2.2.2. 32L L0.2,3. fmmunocYtochem Í strY I0.2.3.L, PreParatÍon of tissue 32r L0.2.3,2. Detect'ion of ce11 type-speclf ic antlgens 32r L0.2.4, Semlthin sectlons 323 I0.2.5. Paraffin sections 324 324 I0.3. RESULTS 328 I0.4. DISCUSSION

PART V: 333 CHAPTER IT: CONCLUSIONS

PART VI: 343 BTBLIOGRAPHY vif

ABSTRACT

The testfs has long enJoyed a reputatfon as an lmmunologfca'lly prlvlleged slte. This thesls re-examlnes the immune prfvlleged status of the testls, and 'likely mechanlsms fnvolved ln provfdlng thfs prlvileged status.

with The prlvileged status of the rodent testis was confirmed thyrold and pftuitary allografts. The effects of cryptorchidÍsmr efferent duct'ligationr and heat on the testis did not affect f'l ow after hcG i ntratesticul ar graft surv'ival . Increased lymph admlnlstration u,,as also unable to fnftlate an lmmune resPonse' speclflc destructfon of Leydig ce]ls by EDS prior to transplantation cells 'led to reJection of thyrold allografts, suggestfng that Leydfg maybelnvolvedlnimmunoprotectionintherattestls.

Toinvestigatetheroleof]ymPhinconveylnggraft-derived testisr hormones and possible immunosupPresslve factors from the

experiments were performed in sheep - a specles ln which testicular lymphatics are easlly accessed' Al'l prevlous studies on intratestlcu'lar graft survlval have been performed in rodents and relatedspecles.Thfsstudyalsoprovidedanopportunityto not re]ated lnvestlgate the lmmune status of the testls in a specfes to rodents. l/\lhile lntratestlcular thyrofd autografts survlved and concentrated fodlne four weeks after transp]antationr thyroid and pitultary allografts were promptly reJected ln thfs time' None of the respond grafts were abl e to secrete detectabl e I eve'ls of hormorìêSr or the lmmune to other hormona'l stlmul i. These studles suggested that vlii

superlor to that of the rodent testfs state of the ovlne testls 'ras was not and that the immune prlvl'leged status of the rodent testls necessarlly a general mammal lan characteristlc' two To allow comparlson of the lnterstltial environment of these speclesr interstltla'l fluld was sampled. A number of authors have are suggested that the hlgh levels of lntratesticular sterofds rodents' responsfb'le for the fmmune prlvllege afforded thfs organ ln Interstitfal fluid ln rodents has been col'lected by a method termed ls Drip-collectionr although the physlological state of such f'lufd so an open to questlon. This technfque has many shortcomings often a'lternative method was developed using the Push-pull cannula with thls employed in neurophyslo'logica] studies. Fluld co]lected by drfp cannula t{as dlfferent ln compositftion to that col'lected was the co]'lection. 0f most consequence for the studfes of thls thesls than lndfcation that intratestlcu'lar steroid levels are no greater testlcular venous b'lood leve'ls, suggestlng that previous values The possib'ly over-estimated actua'l concentrations by up to ten tfmes' of push-pul1 technlque tras also used to monltor the composltion lnterstltfal f'lufd after a variety of experfmental manlpulatlonsr including heat treatmentr efferent duct 1 igation, hcc lnjectfon' and The results of these hypophysectomy wlth pregnenolone supplementatlon. fnto studies suggested that Leydfg cells secrete as much testosterone of blood as they do into interstitia'l fluidr and that levels of testosterone in lnterstitla'l fluid may aríse more as a consequence and vascu'lar b'lood levelsr and are regulated by changes ln blood flow of exchange. A small study on rams and boars found sfmilar levels and no testosterone fn fnterstitlal fluid to those found fn the ratr otherreportssuggestthatramsandratshavedifferentsterofd lx profiles ln anY other resPects.

These studies suggested that the immune privlleged status of the rodent testls was un'likely to be malntafned by sterolds' At thfs stage the It was declded to examfne the ro'le of speclfic cell types wlthin testlcular interstitfum. Durlng a vfslt to Ffnland, a co-oPeratfve study was inltlated wlth Prof. M. Niemf and Dr P' Pollanen fn Turku' This to examlne the immunological cells of the rat and ram testls. study demonstrated slmilar'lymphocyte popu'latlons ln both specles' l,lhile the rat possessed numerous macrophages in the f nterstitlal regionr the ram was almost lackíng fn these ce]ls. such a variance cou'ld account for the difference ln the immune state of the testes of these two specíes.

The use of the testfs as a slte for endocrlne transP'lants rernalns an excltfng prospect. The vrork in thls thesis Provides a new approach for work fn this field with the indfcation that the lmmunologlcally privi'leged status of the rodent testis may not be a general mammalian

characteristic. Some new concepts on the possible mechanÍsms that afford an lmmunologÍcal'ly protected envlronment in the rodent testis are presented. X

DECLARATION

Thls thesls contalns no material whlch has been accepted fon the of award of any other degree or dlploma ln any Unfversfty. To the best my knotledge and belfef, the thesls contains no materlal prevlously publ lshed or wrftten by anothe¡ pepsorìr except where due reference ls made ln the te>Ê of the thesls.

I consent to thls thesls being made avallab'le for photocopylng and loan lf accepted for the award of the degree'

Sfmon Maddocks. x1

ACKNOt{LEDGEMENTS

It ls wfth great pleasure that I acknowledge the asslstance of all those who helped me wfth the studles presented ln thfs thesls'

Professor Brlan Setche'll fntroduced me to this fleld of research' and hls dedlcated supervlsion and enthusfastlc approach to science constant were key elements fn my completlng these studfes. Brian was a at source of inspfrationr and was always available to dfscuss anything anytime. His frfendshlpr guidance and assistance are highly valued' and slncerelY aPPreciated. wíthout ski'llful technical assfstance, many of the experiments in this study could not have been completed. I am most grateful to Mr efforts Shawn Sorerbuttsr Mr James Zupp and Mr Rex Connolty for thelr onmybeha]f.I.learntsomuchfromthlsta.lentedanddedicatedteam. I thank Drs John 0'l lverr John cormack and John Furness of the Fllnders Medfca'l centre for their involvement in these studies and for also the use of thefr facllities. John 0'livers valued frÍendship is

gratef u'l 'lY acknow'l edged. Professor Mfkko Niemi and Dr Pasl Pollanen of the Institute of f acil lties Bl ornedf ci ner Turku, Fl n'land made avall abl e thei r I aboratory

and excellant hospitality during a short visÍt to Fin'land' The fnvestigations on fmmunocompetant cells of the testls were inftÍated during this vfsitr but were hampered by industrlal actfon in the country at the tlme. That Prof. Nfemi and Dr Po'l'lanen were ab'le to

complete much of the work after my return to Australla is slncerely the appreclated. I am also grateful to them for al'lowlng me to lnc'lude work in this thesls. xif

Iwishtoacknowledgetheexpertasslstanceofthestaffofthe I am also farm and the englneering workshops at the waite Instltute' untlrfng efforts lndebted to the l'fbrarlans at the Instltute for thefr of in obtainlng rare and at tlmes obscure, manuscrfpts. Ms M' Morrls statistlca'l the Biometry sectlon gave lnva'luable assistance wfth the assessment of results. Ms C' C'lark My sincere thanks to Mr B.A. Pa'lk and his asslstant forthelrphotographlcservlce.TheydfdnIta]wayshavethebest of the materlal to work fromr but the finished product was a'lways hÍghest qual ltY. IammostgratefultotheAustra]ianWoo]Corporatlonforthe sustain my Post-graduate scholarshlp that allowed me to survive and famlly during these studles' for thelr Thanks to everyone in the Department of Anima'l Sciences provided a continued supportr encouragement and friendshlps. They ]lve.|yatmosphereinwhichtowork,andremainedagoodbandof on aPPropriate companions with whlch to escape to the Edinburgh Hotel occasslons of trlumPh or desPair'

support of my Final.ly, I am eternally grateful for the love and whlle I wife vicky and daughter carolyn. They went wlthout so muchr Thanks a'lso to attended to my studies and the wrftfng of this thesfs' and my parents lan and Diana for thefr continued support

encouragement. xffi

PREFACE

Aspects of the work présented in thls thesls have been reported el sewhere:

Abstracts (1985) Maddocks, S; Cormackr J and Setchell' BP Thefai.|ureofthyroldallograftsfntheovlnetestis. Proc. Aust. Soc. Reprod. Bfol. lZ : 5I'

Maddocksr S and Setchellr BP (1986) tt" of the lnterstitiat fluid of the rat testls' fV euroþean"onposftfon Testfs Workshop Mfniposters: L54'

Maddocks, s; Zuppr JL; Sowerbutts, S; and setchel'1, BP(1986) Testosterone l"ublr ln rat testlcular fnterstitfal fluld' Proc. Aust. Soc. Reprod. Bio'|. Jls : 58' P and Maddocksr S (1987) Pollanenr and rat Macrophagesr tyrpñ;tiãs- an¿ MHC II antlgen ln the ram the testl s. organs: in vII Wkshop. on Development and Functlon of the Reproductlve Press. Papers (1987) Maddocksr S; Sovrerbutts, SF and Setche'll' BP inJectlons of human chorÍonlc gonadotrophin on The effects of .ãpããt"¿ of vascular permeaLiiiit, ext'racellular fluld volume and the flow lymph in the testes of rats. Int. J. Androl.r in Press.

CHAPTER 1: INTRODUCTION IN5 íIIUTE 2 t,iBä,^,RY

I.1. INTRODUCTION

The testlsrr I lke the bralnr anterior chamber of the eye, and the hamster cheek pouch, is often cited as an fmmunologlcally privlleged site (Barker and Bi'llfngham, L9771. It protects tissue grafts from the fmmunologÍcal reJection that they would otherrfse suffer in other regions of the body. Such grafts are placed fnto the Ínterstitia'l region of the testis, and a number of authors have attempted to explain the immunologfca'lty privi'leged status of this region by factors present in the Ínterstitial milieau.

The work i n thi s thesf s re-examf r,res the immunol ogf cal I y prfvileged status of the testis. Both the physio'logy and the immune immunology of the testicular interstitium may contribute to the priviìeged status afforded thfs region, and the fo]loring revlew introduces these two aspects of the endocrine testis.

1.2. THE PHYSIOLOGY OF THE ENDOCRTNE TESTIS

1.2.I. Introduction

Thetestisisanextreme]ycompleXorganproducfngthemale

gametes (spermatozoa) and the male sex hormones (androgens). In 1668 Regnier de Graaf showed convincingly that the testis consists of a series of e'longatedr convoluted tubules which produce Semen' These tubules are usually two ended (Clermont and Huckins, f96I) and open at (see both ends into the rete-testfs (Roosen-Runge, 1961) Figure I'I')' the The productlon of spermatozoa and their successful delivery from Flgure I.I. Diagram showing the arrangement of one of the seminiferous tubules and the rete testfs in the testfs of a rat (from

C]ermont and Hucki nsr f 961) 3

Tunica albuginea Epididymis

Ductulus Tubulus rectus e fferen s Rete testis

Cranial turns

Seminiferous tubule

Caudal turns 4

'largely male reproductfve tract ls dependent upon an adequate supp'ly of androgen - an endocrfne functfon of the testfs. This u,as demonstrated.in 1849 by Berthold in one of the first experlments in endocrfno'logy when he showed that regression of the secondary sexual characterfstlcs ln a castrated rooster cou'ld be prevented by ectopically re-implanting part of the testis. Because the seminÍferous tubules are cylindricaì structures, an interstitial space is formed between adjacent tubules (Figure L.2.). Blood vesse'ls, lymphatics and rìêFVêSr which do not penetrate the serniniferous tubulesr along wfth the hormone-producing Leydig cel'ls are usually found in this extracellular region. However, the specific composition and anatomy of this extracellular reglon does vary between species (see Fawcett et al, 1973). The whole structure is encased ln a tough capsule' The boundary wall of the semlniferous tubules consists main'ly of

myold cells and non-cellular materialr invo'lved in movement of the tubules. They are c'losely associated withr and probab'ly influence the 'lumen Serto'li cells which extend f rom the boundary Yrall to the of the tubule. The Serto'l i cells have contact with all other ceì'l types Ín the epithelium and the varfous germ cells are either sandwiched

between adJacent Serto'l i ce]ls or found embedded in the lumina'l surface of the sertoli cell cytoplasm. Germ cel'l development is fnfluenced by the pftuitary gonadotrophin FSHr and the testicular

androgen testosteroner both of which apPear to act through the Sertoli cells. The Serto'li cells secrete most if not all of the fìuid found within the tubular lumen (setchel'1, 1969)r ârìd can do so against considerab'le hydrostatic and diffusfonal gradients' Part of this secretfon fs now recognised as befng hormonal and the protein hormone InhibÍn has received much attentlon as a Sertoli cell product capable Figure 1.2. Cross-section of a testis from a mature rat. The fnterstltia'l space betueen the seminlferous tubules (ST) contains the Leydig cel'ls (L)r môcrophages (M) and blood vessels (B) and fs filled wlth extracellular lnterstltÍal fluld. The outer layer of the semlnlferous tubu'les (Þ conslsts of a layer of myofd cells surroundlng an lnner 'layer of epithe'lla'l cells. The inslde of the tubules ls lÍned by Sertoli cells (S) which enclose the spermatogenic cells (Sp). Llght micrograph X..1250. *: '{ \É' a t# I F \ t ç È ü la dg s,,i; {} ,l / t,

ç ,.1 *

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a a a tt* a j â,

I 6

the tubules of regu'lating pltuitary FSH release. The boundary wall of physlologically separates the lumlnal compartment from the - interstftlal tlssue by cetlutar barrlers that affect the free.exchange dlffers of water-soluble materlals, and as a consequence lumlna'l fluid markedly ln compositfon from fnterstitlal fluid' androgen The Leydig cells of the fnterstitium are responsible for productionandthlsprocessÍsinf]uencedbythepituitary may also gonadotrophin LH. Recent evidence suggests local regulation occur fnvolvfng factors present in testicular ínterstitial fluid from (sharpe and cooper, 1984; Rommerts et al, 1986)r possÍbly derived Sertoli cel'ls (Benahmed et alr I985a)' Inthe.lasttenyearstherehasbeenagro}ringinterestfnother celltypesoftheinterstitiumlandmacrophages,fibrob]asts' have been endothel f al cells, mesenchymal cel'ls and mast ce]ls and theÍr described. The importance of many of these other cell types role in the testis fs yet to be fully realised, although Ít is likely that they are involved in the endocrine activities of this organ' wfth LeydÍg Macrophages are frequent'ly found in close association Mi'ller et cells (connell and christensenr 1975; Wlng and Lfnr 1977i LeydÍg cells al, lgg3; Niernf et alr tgg6) and morphologlcal changes in are (caused by cryptorchidismr LH withdrawal, seasonal dysfunction) (see Bergh' concomltant with morphologfcal changes in the macrophages endothe]ia] ce].ls 1985; Gondos et a], 1980; Wing and Lin,1977). The linethebloodvesselsandthetymphaticsrâfìdtogetherwfth as possible mesenchymal cells and macrophages have been consldered

Leydlg cel'l Precursors. of these There rernalns much uncertainty as to the other functions been cells hovlever. some of them (eg. macrophages) have tradÍtfonally 7

recognlsedasfmportantcomponentsofthefmmunesystemandthe lmmuno]ogyofthelnterstitia]reglonwfllbediscussedinJater sectfons of this revlew (see I'3')'

L.2.2. The LeYdig cells

cells In 1850 Franz Leydig described the Presence of interstitial but did not situated between the semfnlferous tubules in the testis, were named after suggest any partÍcu'lar function for them. These cells Leydigby.laterworkersandinlg03BouinandAnce]provided cfrcumstantial evidence that they were the source of the male hormone' late 1920f s' Hovrever this proposal rernaÍned controversia'l until the Leydig cells modified and some workers continued to suggest that the (see 1984)' In and stored hormones produced f n the tubules Setche'l'l' the 1965, Christensen and Mason demonstrated directly that interstitlaltfssueproducedmuchmoretestosteronethanthe tubules could seminiferous tubules. Bell et al (r971) showed that the the convert'labelled sterold precursors fnto other steroids' however interstitial reglon, and particularlY the Leydig cells seemed to be steroids the on'ly ce]ls ln the testis able to synthesize androgenlc from cholestero'l or acetate.

1.2.2.I. AnatomY

polyhedra.l, and The mammalian LeydÍg ce1.l is a re]atively large, epithe'lioid cell. It is surrounded by a typlcal plasna membrane which is frequently folded with microvill i. It usually has a sÍngle' are not eccentrically ìocated nucleus, atthough binucleated cells I

vesicuìar' uncommon. The nucleus ls spherlcal or ovofd and dlstlnctly is and contains one to three'large nucleoll. The remafning chromatin present as granuìes dfstributed predominantly around the nuc'lear thickness mgnbrane gfving this mernbrane an appearence of exaggerated other ce'll of when seen wlth the light mlcroscope (Hookerr 1970)''No the fnterstitial tissue has a nucleus wlth these characterfsticsr usually making fdentificatfon re]atively easy. The cytoplasm is

abundant, with a promlnent amount of smooth endop'lagnlc reticulum' Thls is particularly so in gufnea pigs' opossumsr mice and boars' whfch There are also scattered patches of rough endoplas¡nfc reticulum slze interconnect with the smooth ER. The mitochondria are of moderate a'lthough and number and apPear fn the characteristlc lamellar formr not many are tubu'lar (christensenr 1975). Tubular crfstae are partlcularly dlstlnct in the human, guinea pfg or rat. The Golgi nucleus' complex ls well developed and often found at one pole of the # '.[ mature cell contains lipid globules of dlfferent I The cytoplagn of the sizeandnumberdependingonthespecies.TheyarepartÍcularly and mice' These abundant in the cat, and common in humansr guinea pigs Loisel g'lobules t{ere mentioned fn Leydigst origfnal descrfption and be in I (1903) guessed that the secretion of these cel'ls may lipfd are a'lso nature. Reinke crystal s, microtubu'les, and mlcrof il aments found ln the cytoplagn along vrith primary lysosomesr digestive (late vacuoles (secondary lysosomes) and resldual bodies secondary lysosomes). These resldual bodfes often take the form of lipofuscln granuìeswhenllpiddrop.|etsarepresent,butareusua]lyrarein which the cel'ls lack lfpid droplets such as rat or oPossum' ir specfes ln do Boar Leydig cells do not show lipid dropletsr however they Possess cells residual bodfes contafning an unusual reddish pigment' Leydig

I 9

um ln the range in size from about 10 um dlameter fn the human to 30 (boar) the boarr and constltute anything from 76 (ratl to 37l of i lnterstltlal vo'lume. The number of Leydlg ce'lls ln the boar ls extreme and they vlrtua'lly ffl'l the interstltla'l tlssue' Most seasonal breeders a'lso show Leydlg célls of dlfferlng cells appearance dependlng on the season. In such animals, Leydfg appear to transform from an Itundffferentiatedrr interstitial cell iust before the breeding season, into an active Leydig cell with abundant and possibly smooth ER, possessing mitochondria with tubular crfstae, period of a number of medium sized lipfd droplets. Following the activity,theyFê$Fessrand.losethespecificLeydigcell characteristics ( Wing and Lin, L977; Hochereau de Reviers and Lincol nr I978). most Two generatlons of Leydig cells have been described for

1 been found fn the il mammals studiedr although three popu'lations have 'tl, 1969) and pig (van t'Ì human (Manclni et a'|, 1965; Pe]l iniemi and Nieml, The ffrst Straaten and wensingr 1978; van vorstenbosch et aJ' 1984)' is termed the ltfetalrrgeneration because this is when they ffrst for appear. Their functfon is to secrete the androgens responsib'le different'iation of the male reproductive tract but not the regression independently of of the paramesonephric Mue'llerian ducts. They function period' gonadotrophins and regress at birth or in the early post-nata'l the extent of regression depending on the species. within a few months after birth, the interstftial tlssue of the human testis becomes essentially devofd of Leydlg cells and remafns in thfs condltion until puberty. 0n the other hand' there fs t throughout childhood I I apparentlylitt.lelnvo]utionpostnatallyintheguÍneapigorinthe i number mouse. The rat undergoes some decrease in Leydfg cell

Ì 10

whfch postnatallyr however there remalns a PoPulatfon of Leydlg cells fn continue to secrete lon, but measurable amounts of testosterone ¡1 plasma. Thfs appanently lnltiates neura'l changes lmportant for pfg' a subsequent development of male behaviour. In the human and the are second populatlon of Leydfg cells has been described whlch gonadotrophln dependent, transientr and present fn the perfnata'l period. The fina] generatfon in all species aPPear to be lnitlated post-natallyatthetimeofpuberty.Theydlfferentiatefrom rradu'lttr interstitlal cells to aquf re the characterstlcs of the LeydÍg ce]l. Exact'ly whÍch ce]'ls are the precursors for thi s popul ation has (Manclni et yet to be determined. Fibroblast-l ike or mesenchyma'l cel'ls a'|, 1965; Kerr and sharpe, 1985)r macrophages (c1egg and MacMil'lan' I965a)r âfìd endothellal cells (Lawsr 1985) have been suggested as cells Leydfg cell pFêcuFSoFSr as have dedifferentfated feta'l Leydig in the testls (Prlncer 1984). Proliferatfon of those d remaining r,t+ products takes place I organe'lles involved fn manufacturing secretory (mitochondriar smooth ER, Golgi apparatus) with nuclear changes and an (see Burgos fncrease in the microvfl'li processes on the cell surface of et alr 1970; Chr{stensen, 1975). During maturatfonr the number Leydig cells and their slze fncreases (Knorr et alr 1970; Tapanainen (Ketelslegers a'l' et alr 1984) along with LH receptor numbers et occur to 1978). Mitosfs of fully differentfated Leydig cel'ls may also a'lesser extent and chrlstensen and Peacock (1980) and Amat et a'l is (1986) have shovrn that Leydig cell division and differentiation

sti'l 'l possl bl e I n mature rats. Leydig ce]ls occur as clusters fn the triangular I In most specÍes ï intertubular spacer ârìd as strands between closely opposed tubules ti 1973¡ (see Burgos et a'1, 1970; Fawcett et a'l , I97Oi Fawcett et al '

! 1I

the chrlstensenr 1975; Neaves, 1975). In the sexual'ly mature anlmal' fncluding plasna membrane of the Leydfg celt has many sPeclalisations that are of Junctfonal compl€Xêsr ProJectlons and surface lndentatlons 1975)' both the smooth and coated variety (conne'll and chrfstensenr Surroundlng cluste¡:s of Leydlg çells is a basa'l lamlna of varfab'le widthanddlsPosltlonthataPPearstofunctionasasupportlng structure in conJunctlon with collagen fÍbres located around the the Leydig cells and basal lamlna. These collagen flbres are few in loop out rat and are usually found ln bund'les of several ffbres' whfch (clark' over the Leydig cells and extend into the fluid space 1975)' and Much of the avallable lnformation on Leydig celt function cells metabolfgn has been derlved from fn vltro studies using isolated 'laboratory ls ln culture. Because the interstltlal tlssue of the rat the scarcely attatched to the semfnlferous tubulesr lts fsolatlon in plece of past was accomplished by sfmply grasping the tubules in a testicular tlssue and pulllng them out. when the tubules were removed (chrlstensen Masonr 1965)' the web of Ínterstltial tlssue rernafned and decapsu'lated More recentìy techniques have been developed where the to testis ls incubated with digestive enzymes such as co]lagenase assist ln the separation of tubules (Payne et al' L982i Shaw et al' 1979). The population of fnterstitial cells obtained ln this manner Percoll can then be further purfffed by passing the cells down a gradÍent (Browning et al, I98I). These procedures yleld a reasonab'ly purepopulatlonofLeydÍgcellsforlnvitrostudies.Howeverrsuch studies remaln llmited tanporalty to relatively short fntervalsr (see use carefully regulated cu'lture systems Brownlng r despite the of ì I et a'|, 1983). Molenaar et al (1983) examined three dlfferent fsolation I procedures used to obtaln Leydig ce]ls and found the steroidogenlc

! l T2 activity of fsolated cells varfed between different methods of Íso]ation. This seemed partly attributable to the amount of ce]l (1984) have damage caused durlng lsolatfon. Aqui:lano and Dufau recently used a combinatlon of centrlfugal e'lutriation and centrffugation on Metrfzamfde gradlents to procure reportedly 100% pure Leydf g cel'l PreParati ons. Isolatlon studies have led to reports of heterogenelty of et lsolated Leydig ce'lls (Payne et a'lr 1980¡ Chen et a't, I98I; Cooke al, 1981) with two popu'lations being found in the adult rat testis and c'lassfffed as LH responsive or LH unresponsive (see sharpe, L982',' and Dufau However¡ thfs subJect remalns controverslal with Aquilano (1984) reporting the active Leydig cell populatfon to functlonally consist of only one populatlon of Leydig cells wfth comparab'le LH receptor numbersr steroldogenic activity and susceptibll ity to gonadotroPhic desensitizati on.

L.2.2.2. Re1ationship to blood vessels' lymph vessels and semlniferous tubu'les

The relationship between the wa'lls of the tubulesr the Leydig cells, and the blood and lymph vessels varies amongst different in species. In 1973, Fawcett et al studied the intertubular'lymphatics 14 specÍes. The abundance of Leydig cells, the amount of intertubular the connective tissue and the location and degree of development of of lymphatics allowed these authors to defÍne three maÍn categories interstftial tissue organisation. The folloving descriptfons' and Figure L.3., are from Fawcett (1973) who increased this classification by the to four groups. The ffrst group (Figure 1.3.a) is characterfsed Figure I.3. Diagram shc¡rvlng the varíatÍon in the anatomy of the interstitial tissue in severa'l species. (a) Guinea Pfg, shoving Leydig cells clustered around blood vesse'ls' wÍth the whole groups of cells comp'lete'ly surrounded by

'l 'lymphatic endothel ia'l cel s and f I oati ng i n a sÍ nusoid' the contents of which a'lso bathe the walls of the seminiferous tubules. (b) Rat, simllar to the GuÍnea Pig except that the groups of Leydig ce'l'ls are surrounded by

an f ncomp'lete I ayer of endothel ia'l ce'lls. (c) Ram, shovl ing the Leydlg cells either in groups near a capillary or in separate clusters embedded in loose connectÍve tissue which also contains tymph vessels^and other blood vessels. (d) Boar, shoning the Ínterstitial sPace crammed with c'losely packed Leydig cells with a few small blood and lymph vessels (from Fawcettr 1973). I3

I ('v(lr(, a. ( t'lls @ Paflelal o endollìehurn

Lynrplraftc Srnusord

o c Elood vessel Myord layer o Visceral endolhel¡um ø o

b. o o

Lymph sPace g

o o o o o

c. s

Lynìphat¡c o \ vessel Loose c0nnecl¡ve tissue

Leydtg cells 14

the gulnea pig, ln whlch the Leydlg cel'ls comprlse a s¡nall fractfon of testf cular volume, and occur ln clusters closely aPp]led to b'lood vessels. The greater part of the lnterstltlum fs occuPfed by extensfve lymphatic sfnusolds of irregular out'line. These are bounded by a rrvfsceralrr (or.lnterstltlal; Clarkr 1975) layer of attenuated endothelium coverfng the vessels and thelr assoclated Leydlg ce'll c'lustersr and a ltparletal rr (or peritubul ar; C'lark' 1975) I ayer of endothel fum closely appl ied to the myoid layer of the semfniferous tubules. The endothelÍum of the'lymphatic sinusoids in thfs space fs generally continuous. Narrow sheets of collagen bounded by two layers of endothelium attach the vascular-endocrfne comPlexes to the tubules' In this specles thereforer Leydlg ce]ls are lnterposed between the walls of the blood vessels and the endothelfum of the lymphatic sf nusoids. In the second grouP (Figure I.3.b)r characterised by the rat and llìousêr lnterstitfal organfsatlon is baslcally the same as in the first over group. However the visceral layer of endothelium Ís dfscontinuous wide areas so that Leydig cells are directly bathed in lymph.

The maJorlty of I arger mammôl sr incl udf ng the bul l, Fâlllr

elephant, monkey and man, have a very different lnterstitial organfsatfon (Flgure 1.3.c). In these sPecÍes, Leydlg cel'ls do not have such an obvious association wlth blood vessels, occuring fn c'lusters of varying slzer scattered fn an oedematous loose connectlve tlssue which ls drafned by consPfcuous lymph vessels located centrally or eccentrfca'lly in each intertubular area' The fourth group (Flgure 1.3.d) includes the dornestlc boarr warthog, zebra and naked mole rat. closely packed Leydfg cel'ls occupy large lntertubular spaces and comprise uP to50l of the volume of the I5 testls. There ls very llttle lnterstltlal connectlve tlssue ln these specfes, and.smaìI lymphatlc vessels are infrequently encountered' Gfven that the Leydlg ceì'l fs an lmportant source of androgen fn the testfsr these specfes dlfferences ln interstitlal organlsation the have obvlous impl fcatlons for how androgen ls partltloned between (1973) vascu'lar and fntratesticu'lar clrcu'latlon. Fawcett et al hypothesized that fn the rodents (Groups I and 2), movement of testosterone from the Leydig cells could be envisioned as a release of androgens into the blood caPfllarfes and lnto the protein-rich extracellular fluids that move from the blood vascu'lar system into the lymphatic sfnusofds surroundlng the seminiferous tubules. These authors suggested that the'lymphatic route of testosterone transport might be the prlmary source of androgens for the seminiferous tubules fn these anfmals. In the larger mamma'ls of Group 3' blood capfllarles are in much closer contact with the tubules and therefore may directly supply testoterone to the tubules. Lymphatlcs fn these specfes are believed to be more fnvolved in return of extracel'lu'lar flufd to the general clrculatlonr than fn dlstrfbution of testosterone' The large provlde mass of Leydlg ce]ls in Group 4 ls probably able to dÍrectly testosterone to the tubules without the need for any Índfrect system (connell such as the lymphatlcs or b'lood vascular system and connell' Ig77). The high density of Leydfg cells in these species probab'ly relates to the production of other hormones such as oestrogens and

pheromones (eg. rrboar taintn' 5c-androst-16-en-3-one)'

!.2.2.3. Relatfonship to other cells ln fnterstitial tissue

WhiletheLeydigcelìiswidelyrecognisedasthemqjorcelltype 16 of the interstitlal compartment of the mature testfs (Nfernf and Ikonen, Lg73l I ô rìurnber of other connectlve tissue cells have been reported ln thfs reglon lnc]udlng flbroblasts¡ macrophagêsr ßâst cells (1979) and'lymphocytes (chrlstensen, 1975). Ewlng et al and Niemi et al (1986) have reported that testfcular macroPhages ln the rat may (1985) estlmated constitute up to 25% of interstitial cel'ls and Bergh been one macrophage per four Leydig cells. Macrophages have frequently found ln close assoclatfon wfth Leydig cetls (connell and chrlstensenr and L975¡ Wlng and Lfnr L977i Miller et al, 1983; Nlenri et alr 1986) morphological changes ln Leydlg ce]ls (caused by gonadotrophin withdrawl, cryptorchidf sm or seasonal dysfunction) are concommltant wlth morphologfca'l changes in the macrophages (see wlng and Lln'L977;

Gondos et al, I98O; Bergh, 1985). The fmportance of this assoclatlon is unclear although Bergh (1985) indicated that the morphology of the two cell types ls corre'lated such that a functfonal inter-relationshfp ls tikely. Indeedr macrophage assocÍation wfth Leydig cells leads to consfderable contaminatlon of Leydfg cell preparationsr and simf'larities fn functlon between the two ce]l types has led Nleml et ltunwarrantedrr al (1986) to suggest that thfs may ln fact account for suggestions of Leydfg cell heterogeneity (see cooke et al, l98l)' Mllewlch et a'l (1982) found peritoneal macrophages to be sterofdogenic, and Molenaar et al (1984) have shown macrophages to al possess common surface antfgens wfth Leydfg cel'ls. Wahlstrom et

(1983) and Hovatta et al (1986) have found macrophages rhich contaln r FSH demonstrable by fmmunohlstochernfstry in the human testls suggestfng that these cells may also respond to gonadotrophins' Mi'ller et al (1983) found that portfons of Leydig cells may be endocytosed by are macrophagesr supportfng the suggestion that testfcular macrophages T7

fnvolved fn Leydlg celt functfon. Yee and Hutson (1985) demonstrated that condltioned medlum from.cultures of testicular macrophages stfmulated testosterone productlon when added to Leydig cells ln vftro. On the other hand, Niernl et al (1986) observed macrophages ln prepubertal anlmals whlch may Ímply other functlons such as lmmunologfcal roles, for these macrophages. Most of these studfes have

been carrfed out wlth rats and the situation in other species is yet to be futly fnvestigated. Leydfg cells have been found fn the boundary tlssue of the seminfferous tubu'les ln guinea pfgs (Fawcett et al, 1970) and man (Fawcett and Burgos, 1960) in association wfth the myold cel'ls and fibroblasts of thls tissue. These Leydlg cells may be lmportant fn Leydlg cell - Sertoll ce1l interactfons and Bergh (L982'1983) has

shown that perltubular Leydfg cells fn the rat testfs change in slze accordlng to the stage of the spermatogenic cyc'le in the adJacent seminlferous tubule. Macrophages have also been reported ln this region of the rat testis (Ross, 1967). ThÍs perÍtubular region is closely bounded by the peritubular or parleta'l endothelfum' and Leydig cel'ls of the Ínterstltium which are not necessarily incorporated fn the boundary tissuêr but whfch lfe adJacent to the seminiferous tubules are in close association wlth this endothelium (Clark, 1975).

Thls may be of consequence Ín affecting distribution of hormones produced by these celts. It has a'lso been suggested that newly dffferentiated Leydig cells may arise from peritubular cells and subsequently 'leave the lamlna proprla to gain access to the fnterstitlal tissue (Kerr et al, 1985). Durlng pubertal maturatlon'

new Leydig cells apparently dlfferentiate from perftubular Ínterstftlal cells (chrfstensenr 1975¡ de Kretser, L967i Fawcett' t8

also the Lg73¡ van Straaten and wensing, 1978)r ônd thls reglon fs slte of development of numerous Leydig cel]s 4 weeks after EDS treatment (Kerr et al' 1985). Mastce.|lsareafreece.ll-typeusua]lyfoundlnconnectlve tfssues, and thelr Presence has been conffrmed ln fnterstltlal testlcular and epididymal tissues, as well as the testls medlastlnum 1980; and tunfca albuginea (Hermo and Laltir 1978; Nfsta'l et al' (1984) have shown Nykanem, 1980; Maseki et alr 198I). Nistal et a'l those that mast cells of the testis and epldidymfs are sfmf'lar to lncreases found in other connectfve tissues. The number of mast cells in infancyr decreases ln childhoodr and lncreases agafn at puberty' a'll During adulthoodr mast cel'l numbers progressfvely decline in et al testicular and epfdldymal connective tlssuesr although Masekl patients wlth (198I) reported an lncrease ln numbers (Mastocytosis) in in the idiopathfc male infertlllty. The fncrease in mast cell numbers connective normal indivfdual correlates wfth developnent of testicular tfssesr and Mast cell degranulatlon and heparin release is (see 1984)' At the eoncommÍtant with co]lagen synthesis Nistal et a'l' occursr begfnnlng of pubertyr prol iferatlon of active fibroblasts 'level and Leydf g cel I co.tncl di ng w f th i ncreased gonadotrophi n s differentiatfon. The lncrease of Mast ce]l numbers during thls tfme given their may be of importance for developlng Leydig cells Ínevitable associatfon fn the fnterstftlum'

L.2.2.4. Hormones produced by the Leydfg cells

ThattheLeydigcelllsthepredominantsourceofandrogeninthe review' However a mammallan testis has a'lready been addressed in this I9 varfety of sterolds are syntheslsed ln the testfs from efther cholesterol, formed elsewhere ln the body and transported to the testls fn blood; or acetate, derlved from the blood or formed as (Setchel'lr acetyl-coenzyme A durfng metabollsm of g'lucose 1978)' Testosterone fs qúantftatively the most lmportant sterold secreted by the testls, and produced by the'Leydlg cells. That a number of other steroids can be measured in the testis ls not surprÍsing when one examines the pathways that a'llow cho'lesterol to be transformed to testosterone (see Ffgure 1.4.). Setchell (1978) has reviewed the endocrlno]ogy of the testisr and androstenedione' androstenedlol' dehyd roe pf and rosteFofìêr 17 cr- hyd roxy p rogest@Fofì€r

17c-hydroxyPregnenolone, pregnenolone' progesteroner 5c¡-dihydrotestosteroner androstanediols, and a varlety of l6-unsaturated Crn sterolds have been found ln the venous b'lood of the testes of a number of specÍes. There is great variation amongst and Ín mamma'ls in terms of which steroids are secreted by the testisr what concentrations. However it fs fair to say that most androgens appear to be produced ln the testiscular interstitium, Primarily by the Leydig ce11s. Prepubertal and fetal Leydig cel1s appear to produce dlfferent (1981) products compared to the adult Leydig cell. Hsueh et al have

reported i sol ated cel I s of the prepuberta'l rat testi s to secrete androsterone and Sa-androstene-3ar L7 þ- rdi ol whereas testosterone was the major product in adult cells (Hsuehr 1980). Likewise' the Leydig cell ln the sexually mature animal is responsible for testicular oestrogen production whereas the sertoll cell performs this functfon ln the immature animal (Payne and valladares, 1980). This has obvious must fmp'ì lcations for studies on lsolated ce'lls sf nce age differences Figure 1.4. Outl ine of the steroid biosynthetic pathways within the testis (from de Kretser, 1984). 20

Acc tatc

I + Cholcstcrol

CH¡ cH3 + I I co CHtco CH.

CH¡ CH¡

Pregnenolone Progestcrone

CH¡ CH CHt I I co H-C cl CH -OH OH -oH --oll CHs CH¡ CH¡

HO o o

I 7a-hydroxypregncnolonc I 7æhydroxyprogesterone I ?c-hydroxy, 20o{ihydro Progcsterone

o o CH¡ CH¡

CH¡ CHr

HO o HO Dchydrocpia ndrosteronc Androstencd ione Oestrone

CHtOH cH! CH¡

HO HO

Androstcncdiol Tcstos¡crone Ocstradiol-l7P 2T

be carefully consldered before any concluslons are drawn about cell functi on.

Apart from the androgenlc products, lt fs now evldent that the Leydig cet'l synthesizes other bioactlve substances that are maf n'ly involved fn the paracrfne and autocrine regulation of tesicular function. Ear'ly studles by Nlemi and Kormano (1965) demonstrated a posslble role for Qxytocfn fn medfating contractlìfty of the seminlferous tubulesr and since then Oxytocln and Vassopressfn have been found in smaìl amounts f n testicular materia'l from ratsr men ârìd bulls (wathesr 1984) together with Neurophysin suggestfng local biosynthesfs. Recentìy Guldenaar and Pfckering (1985) have demonstrated lmmunocytochemical locallsatfon of oxytocln in rat LeydÍg cellsr although they found no evldence of vasopressln or neurophysfn' Synthesls of prostaglandins has also been demonstrated fn the testls, although their preclse effects and role in testicular tissue remain to be clearly deflned. Haour et al (1979) found an increased secretion of prostag'landfn Fro and prostaglandin Ezby rat Leydlg cells, assocfated wlth the down-regulatlon of LH-hCG receptors after

hCG adminÍstratlon. Carpenter et al (I978) investigated prostaglandin production by rat testls tfssue, and found PGFZ,. was prÍmarlly synthesised by tubulesr whlle fnterstitial cel'ls (predominantly Leydig cells) had a greater net synthesis of PGE than PGF2a. A number of reports indicate that prostaglandfns can direct'ly lnhfbit LH-fnduced steroidogenesfs ln dlspersed rat Leydlg cells (see Salram' 1979) whfch suggests that prostaglandlns may have an autocrlne/Paracrlne control function over steroldogenesfs. Eltls et al (1975) have proposed an lntegrated mechanism of synthesfs of prostag'landins and testosterone 22

(see Ffgure I.5.), and suggest that a number of alterations seen ln the testls after heatrcryptorchldfsm, or radlation may relate to prostaglandln medlated effects involvlng cAMP. p-endorphin production in rat Leydfg cells has been demonstrated (Shaha et al, 1984) and there fs some evidence that it may facllftate testosterone secretion either directly as an autocrfne effectr or indi rectly by way of Serto'l i cel1-produced or myof d ceìl-produced

f ntermediates (Bardin et al, 1984). A number of other Pof'CI-derived peptides have also been located in Leydig cells (Margioris et alr 1983) and opiate receptors found fn the sertoli cel]s by Fabbri et al (1985) suggest that these cells are the targets for testicular opfates. The exact ro'les for these products rernain to be e'lucldated.

Howeverr ACTH and MSHfs have been shovln to stimu'late growth and cAMP accumulatlon in Sertoll cells and p-Endorphinr possibly urith another testlcular opiater inhf blts Seto'lf ceì1 proì iferation and ABP secretion (see Bardln et al, 1984). parmentier et al (1983) found renin-like immunoreactivlty in the testfsr and Pandey et al (1984) have found sfmi'lar activlty in purified Leydig ce'l'l PreParations.

L.2.3. Sertol i ce]l s

In 1g65r Enrico Sertoli descrfbed columnar cells with cytoplasnic processes extending from the bassnent membrane to the lumen of the seminiferous tubules, and envelopfng the nelghbouring germ ce'l'ls to provide physical support and rrnursingrr functfon. Von Ebner (1871' I8S8) recognfsed that the ce'lls of the semfniferous tubules were always found in speclflc rrstagesrr or assoclatlons, and that the Flgure 1.5. Proposed scheme sholing the apparent routes for the

synthesis of malona'ldehyde' Prostaglandin Et and Prostaglandfn Fror and testosterone synthesfs from pregnenolone. Related mechanlsms ln both pathways involve

peroxÍdatlon of l lpf ds via elther NADPI-I or ascorblc acf d (ADP). See Ellls et al (1975) for further detai'ls. CH i =O Lipid {-Lipid* Glucose NADP o2 NADPH - to -

o2 t ¡ OH

Choleslerol-

/c-AMP Stimuloles\ o 6 p pst Inhibir Esterose o. \ / Testosterone o E ( øo, Choleslerol o c- FA co0H E I o C- PUFA t PhosPholiPose ãJFA+ I c0, c-PO4-X o2 o Dihorne-y-linolenic ocid -o E (l) Alkdine r (mernhonous) = cooH cooH LYsosome Stobilizers' Lobilizers, Heot c-AMP ocid Coff eine c-GMP I 2- Hydroxy-8,1O-hepûodecodienoic Theophyl line Vrt. A + Epinephrine AcetYlcholtne OH o Noreornephine LH H H Vit. E Testosteone + cooH* Cortisol LiPrd Peroxides ç H 0 o OH 0. TBA Molonoldehyde ":V 4RSH I 2RS-SR OH 'ç N cooH cooH (¡)

OH H H H PGE PGFro TBA Chromogen I 24

developmental stages of a spermatogenlc cycle are arranged spatlally fn the form of a wave. However he fncorrectly concluded that the germ the cel'ls deve'loped from the cytoplasm of the Sertolf cells' and that (see spermatlds degenerated to provlde the llquld part of the semen Setchelt, 1984). The true orlgin of the spermatozoa and the concept of a symbiotic relationshfp between Sertoli ce]ìs and the developing germ ( ce]l s was deduced by Merkel ( 1871) I Serto'l i ( 1878) and Renson 1882) ' whi'le germ cells continualìy divide, dlfferentiate and are released Ínto the tubular lumen to eventually pass out of the testisr the Sertoli cells are a stab'le populatlon of cel]s' whfch do not dlvide in the adu'lt animal (steinberger and steinberger, 197I; orthr I982t 1984). The importance of Sertol i cel'ls ln regu'lating and maintainlng theprocessofspermatogenesfshasbeenemphasisedbynumerous investigatorsr although thelr precise functions and sfgniflcance for

the devel opi ng germ cel I s are stil'l not f ul'ly understood.

L.2.3.I. AnatomY

the The sertoll cell rests upon the llmfting basement membrane of recognised semenlferous tubule. The basa'l portion of Sertoli celìs are fn tubular cross section as polygona'l âFêâsr easfly distingulshed by (Burgos et al' their shape and nuclear and cytopìagnlc characterfstlcs

1970). The nucleus is infolded ln all specles studfed' being elaborate'ly

'lobulated fn some (Fawcett, 1975). It often lacks both the flbrous in lamina that refnforces the inner surface of the nuclear envelope of many cell typesr and the karyosome and perfpheral clumps types' heterochromatin found ln the nuclei of most other somatic celì 25

Speclallsed Junctlonal comp'lexes are formed between neighbourlng Sertoll cellsr wlth oppossed membranes apProaching to wlthin 20 À ln some areas (Dym and Fawcettr 1970).. These Junctlonal compìexes dlvlde frad'lumlna'ltt the germlnal eplthet lum f nto rrbasaln and compartments' and provfde a structural basls for the malntenance of a dffferent mllfeu in each of these compartments whfch may be crucfa'l durfng specfffc stages of cell develoPment. The cytoplasrn of the Serto'l i cells contalns ìong mf tochondria'

Whlle randomly orÍentated fn the basal cytoplasm, they tend to be orientated parallel to the ce]l axfs fn the supranuclear columnar portion of the cel'l (Fawcett' 1975). Agaln unlike other cell typesr the Golgl apparatus appears to consfst of mu]tlPle separate Golgi e'lqnents scattered throughout the basal cytoplasm and occassional'ly fn the supranuclear region.

Numerous membrane-'lfmfted dense bodfes are found fn the 'lysosomesr cytop'lasmr and fnclude Prlmary autophaglc and heterophagic vacuoles, and deposits of lipochrome pfgment (Fawcett, 1975). In 'lysosomal addltlon to norma'l housekeeplng activltles f n a cel'l' the Sertoli cells digest those germ celts that normally degenerate durfng spermatogenesisr along 11lth lobu'les of resldual sPermatld cytoplasm left behfnd fn the release of spermatozoa (Black, L97L¡ Cìegg and McMitlan' 1965b¡ LacY' L962).

Rough and smooth endoplagnlc reticulum is found in the sertoli cell cytoplasmr with rough or granular retfculum Predominantly in the basal reglon. This granular form ls usually tubu'lar' and whl'le the populatlon of attatched ribosomes is large fn the monkey, it ls relatively sparse ln other species (Dym, 1973¡ Fawcett' 1975)' The

smooth endop'lagnfc reticulum ls general'ly more abundant than the 26

roughr but agaln Fawcett (1975) descrfbes slgniflcant specles dffferencesr t{fth rumfnants possesslng more extenslve quantitles than the laboratory rat. The ram Sertoll cell contalns aggregates of snooth retlculum ln the basa'l cytoplasrn often closely assocfated wlth llpfd.

Loca'llsatf on al so occurs f n cytoP'lasm f mmedlately surroundlng developlng acrosomes of assocfated spermatlds. Crystall ine lnc1uslons

have also been descrlbed in the Serto'l i ce]lr although they are se]dom found ln laboratory and domestlc anlmals. Lipld content ls highly variable between speciesr ln both total

amount and size of droplets. It is general'ly loca'lised near the base of cells and aPpears to vary with the stage of the sPermatogenlc cycle fn some species (Lacyr 1967). After heatr X-irradlation or oestogen treatment whlch al'l cause germ ceì'l degeneratlon, the amount of lfpfd lncreasesr ôod Lacy (1967) has speculated that phagocytosis by Sertoll 'l cel I s of germ cell resldual bodf es may contribute to thl s I pld pool I which in turn might be utillzed by Sertoli cel'ls for sterofd synthesfs. The abundance of srnooth endoplas¡nfc reticulum suggests that Sertolf cells may have the capaclty for sterold biosynthesls

(SteÍnberger and Steinberger, L977r - At certain stages of the spermatogenic cycler mlcrotubules are abundant in the Serto'l i cell cytop'lasm. While consldered to be maf n1y cytoskeletal e'lements ProvÍding suPport for the columnar Portlons of the ce'lì, they may also be actively fnvolved ln movements of cytoPlasm lnvolved ín dlsplacÍng late spermatids (Russellr 1980).

Unl ike the Leydlg ceì'l popu'lationr whlch is prfmarlly establ ished ln the adult anfma'l at puberty; the mltotlc actlvity of the Sertoli cel'l fs most pronounced fn the fetal and early postnatal Perlod (Orthr 1984). In the rat, Serto'li cell proliferation ls maximal on day 20 27

postconceptlon (Orth, 1982) and falls steadlly after parturltlon,

(Clermont and ,l ceaslng between the twelth and flfteenth day after birth Perey, 1957; Hf'lscher and Makoskl, 1968; Orthr L982,1984). Indeed,

the number of Sertoll cel'ls per testls apPears to rqnaln unchanged after the second postnatal week, desplte some evldence of DNA synthesfs fn nondfvidfng Sertoli ceìls (Stelnberger and Stefnbergerr

L977 ) . Investigations on Sertoll celìs fn vivo, or usfng whole testis tissue fn vitro are extremely dffffcult because of the ceìlular heterogenelty of the tÍssue. WhÍ1e the germ celì populatlon of the tubules can be dep'leted by various experfmental methods already mentloned, they do not eliminate other cell types (peritubular cel'ls' fnterstltlal cells). Thus likð tnose studylng the Leydig cellr

numerous workers have attempted to iso'late and culture Sertoll ce'lls (see ü fn vltro. These attempts have been of variable success irü i Steinberger and Stelnberger, L977), due maÍnly to the difficulty ln separatlng the Sertoli ce'lls from other associated cell types. That the mitotic process ceases two weeks after birth Ín these cells has also restrlcted the establlshment of pure lfnes and the propagatlon of cells ln vltro, although Mather (1980) has established a clonal ce'll lfne (TMO) and a tumour cell'llne (TR-ST) with Sertoll celì characterfstfcs (Mather et al t L982). In the maJorlty of studies, Sertol f cel'ls have been obtafned from the prepubertal rat testls (Dorrfngton et al t L975; Welsh and Wiebe, L975¡ Frltz et al t L976i Lacroix et al t L977; Wflson and Grfswold, L979i Ílrfght et al, l98I¡

Klsslnger et a'l t L9823 Le Gac and de Kretserr L98\ Sharpe et al, I Lg82). A few lnvestlgators have also used SertolÍ cells from lmmature plgs (Vazeille and Chevaller, L979i Perrard et a'|, 1985)' immature r 28

buil (Francls et alr 1981; Vfglerl et aI,1985; Hayesr 1986) and (Lee l{hf'le the culture of Sertolf cells ,l immature monkey et alr 1983). i alr fso.lated from mature testes has been reported (rat: Stelnberger et Lee L975i DeMartino et al, 1977; Cameron and Merkwatd' 1981; monkey: et al, 1983) the contamlnatlon of such PreParatlons wlth germ cells been obtalned remaf ns a problem. trlhile much useful informatlon has fn from such studiesr it rernains to be shown that such cells behave methods of the same manner as those in the adult animal. Isolation

some success uti'lize a double enzyme digestlon of mfnced testls tissue, wlth collagenase and trypsfn (welsh and wiebe, L975¡ Dorrlngton et a'|, L975) and produce cell preparatlons free of Leydig (1984) ce]l and fibroblast contamination. Recently Tung et a'l have described an improved lsolatlon procedure using hyaluronldase whlch yie'lds Sertol f ce]1 preparatlons of less than 0'3% perf tubular cell

contaml nati on. ü are of obvious ,.¡ These problems of non-sertoll cel'l contamlnants obtafned concern when it comes to lnterpretlng data on cell products from in vltro studles. However a further compl icatlon arfses from the process of culture itself. sertolf cells grown on culture plates in defined medium loose their columnar form and adopt a flattened

morphology (suarez-ouian et al, 1984). Thls lmp'lies important cell-cell relationships as I shall discuss shortly' However ft has perform been of concern that cells in such a state may not contlnue to Dym and the same functions that columnar cells perform ln vfvo. workers have recently cultured sertolf ce]ls on a reconstituted such msnbrane extract in bicameral culture chambers' and in t basement I I (suarez-oufan al' cu'ltures the columnar form of the cells persists et I 1986)' 1984; Hadley et a'1, 1985; Byers et a'lr 1986¡ DJakiew et a'lr

? 29

Relationship to the Btood-testfs Barrfer 1 L.2.3.2.

A number of substances of wfdely varylng mo]ecular size Íntroduced into the blood stream rapidly appear fn testicu'lar lymph' but not in the fluid collected from the cannu'lated rete testis (Waites and setchelt, 1969; Setchell, I97ü. Such studies defined the existence of a blood-testis barrierr anôlagous to the b'lood-braÍn barrier dfscovered several decades prevfousìy. However unl ike the blood-brain barrÍer which has been found to reside in cell-cell junctlons ln the waì'ls of cerebra'l caplllaries (Reese and Karnovsky, Lg67), the blood-testls barrier Ís found Principal'ly in the walls of the semlniferous tubu'les. The divlsf on of the Sertoli cel'l epithel f um into basa'l and adlumf na'l compartments has a'lready been addressed in

,.'I il this revfew. It fs the specfalised JunctÍons between pairs of Sertoli rfr! I cells above the spermatogonla, but below the spermatocytes that allows this divisfonr and it is these junctiona'l complexes that are the prlnclple component of the btood-testls barrier (see Setchell' 1978)' These sites of restrlcted permeabil lty have been deflned by studÍes using electron opaque markers (see Fawcett, 1975). They are symmetrical specfallzations of adioining Sertoli cells' confined to the basal third of the epithetium (see Figure I.6.). Just above the spermatogonfar overarchlng lateral processes of the Sertoli cel'ls meet. In these Junctions subsurface cisternae and bundles of fllaments develop, and the opposÍng membranes (normally søne 150 - 200 A apart) 'ii{ approach to wfthin 20 Â to form the gap Junctions. Multiple sÍtes of ï I obliteration of the Íntercel'lular cleft are apparent, and Ít is at fi these sftes that penetrating electron-opaque markers abruptly stop

! Flgure I.6. Drawing l'llustratlng the occludlng junctions between

Sertol i cel'ls. These dfvlde the seminf ferous eplthel lum lnto basa'l and adìuminal compartmentsr and form the prfnclple component of the blood-testfs barrfer (adapted from Fawcettr 1975). 30

ADUN,IINAt COifARIl¡ENf

Sertoli- Sertoli iunction

¡ I 31

(Fawcett, 1975). In the lmmature testisr occludlng Junctfons are absentr and while typfcal gaP junctions are coffilllorìr they gradually dlsappear (Gl'luIa et al, Lg76l. The develoPment of the sPeclalfsed

Junctlons'between Sertol l Ce]ls would seem 'to be under hormonal control slnce the blood-testls barrier does not aPpear unti'l the onset of spermatogenesfs at Puberty (Kormanor 1967a). The unlque cell-cêll a'l Junctf ons between sertol f ce]1s are an effective lntra-epithel f component of the blood-testls barrler. However, the Sertoli cel'l has lmportant lnteractf ons with other cel'l typesr and the epithe'liof d cells ln the peritubu'lar contractile layer of the seminiferous tubules

are noYl thought to provfde an adventitialr a'lbeit incomplete' component to the b'lood-testls barrfer. This is particularly so ln rodentsr although in larger mamma]s lt may be relatively unfmpo¡tantr and Fawcett (1975) suggests that in prf matesr inc]udir$ Ílilfìr the extenslve gaps ln the multlple layers of adventltfal cells mean that the blood-testls barrler depends exclusfvely on the Sertolf cell

j unctf on s. The divfsion of the Sertoli ce]l epftheìlum by the Junctfonal complexes ls of maJor consequence for fluid partitfonfng. Substances reaching the base of the epithelium from the b'lood have more or less dfrect access to cells in the basal compartment. However the occludÍng ceìl Junctfons mean that substances must pass through the Sertoli cytoplasm to reach germ cel]s in the adluminal comPartment' Llkewlse the ftuld of the tubular lumen must be derlved from materfal passed selectively throughr or synthesised by the sertol i cel'l (setche'll, 1969). A conspicuous feature of the composftlon of the f'luld of the seminfferous tubu'le ls a high content of potassÍum (40 mM in the rat; Setchell and Waltes, 1975)r ôrd Muffly et aì (1985) conclude that 32

Sertoll cells are responsible for this hfgh concentratfon and that the pumP' plasna membrane of these cells contains an active potassium

L.2.3.3. Rel atf onshi p to other cel I s

In the intact testis, the mesenchymal cells (peritubular myofd cells and fibroblasts) in the boundary tlssue of the seminfferous tubu'les are adJacent to the epithe'lial-type Sertol I cells' separated by a basal lamfna (Fawcett, 1975) with which only 3% of the sertolÍ cell is ln contact (weber et al, 1983). These two ce'll types have been ÎlârìfìêFr observed to interact specffically wlth each other in a comp'lêX both in vivo and in vitro (Tung and FrÍtz, 1986)' Peritubular myoÍd cel'ls celts of rodents are thought to be derived from fibroblast-like ln the fetal gonada'l interstitium under the influence of adJacent s and Sertol i Sertol Í ce]l s (Bressl er and Ross, 1973 ) . Peritubul ar ce]l ce]]slnco-cu]turefnteractinavarietyofwaysresultingfn proteln' pro'longed mutual survfval in a medlum containing no added survfve in under condltlons ln whfch nelther cel1 type would otherwlse monoculture (Tung and Frltz, 1980). Peritubular ce1ls in co-culture Protein wfth Sertol i cells sustafn the production of Androgen Bindfng (ABP, a recognfsed marker for Sertolf cell function; Tung and Fritz' ce]l 1980; Hutson and stocco, 1981) r âfìd the addÍtion of perÍtubu'lar conditÍoned medlum to sertoli cells in culture lncreases ABP and Transferrln formation (Skinner and Frftzr 1985a)' Indeedr perltubular paracrfne factor cells are known to secrete a 70 kDa non-mitogenlcr cu'ltured termed P-Mod-s which maxfmally stimulates ABP productlon ln sertoli cells (skinner and Fr1t2,1986). This indfcates that a protein secreted by perltubu'lar cells may be involved f n mesenchyma'l- 33 epithe'lfal cell lnteractlons between perftubular and sertoll cells. Ihese two cell types also co-operate to form components of the extracellular matrfx (ECM) and basal lamlna of the tubular boundary ìayer. Cultured peritubular cells secrete collagen and flbronectln' and sertolf cells fn monoculture produce type IV collagen and'lamlnln (Tung et al, 1984; Tung et alr 1985; Skinner et al, 1985). Pollanen et al (1985) have also demonstrated laminin and type IV collagen immunohÍstochemically in myoid cell layersr and lamfnin fn Serto'll ceìls of both normal and pathologfca'l human testes. The finding by

Hermo and Lalli (1978) of mast cells and monocytes as regular components of the walls of seminiferous tubules may relate to co]lagen synthesis fn thls region. Proteoglycans constitute important

components of the ECM¡ and are thought to pìay an essential role ln

ECM depositlon. Skinner and Frltz (1985b) have demonstrated that cu'ltured Sertolf cells synthesize and secrete proteoglycans whfch contafn both chondroftln and heparfn glycosamlnoglycan chains (GAG chains)r while cultured peritubular cel'ls produce Proteoglycans of higher molecular mass containfng chondroitln GAG but not heparin GAG

chai ns. As culture and partlcular'ly co-cu'lture systems are further

developed and lmproved, the physlological signfficance of much of thls

work will become c'learer. l{hi'le the physioìogfcal role of perftubular ce]lsr collagen fibrils and other ECM components fn the boundary wall ln I lmitlng the passage of cells and macrorno'lecules and preventf ng the penetration of blood vessels fs recognlsed (Dym and Fawcett' 1970)' they also p'lay an important role in shfelding the immunologically-foreign haplofd germ cel'ls from Ímmune surveillance'

Hermo and Lalli (1978) reported leukocytes in the the'limfting 34

membrane of the sernlnlferous tubules ln human testlcu'lar blopsles' which whfle found subJacent to Sertoll and germ cells |n contact wfth the basal lamlnar were never found ln the semfnfferous eplthellum. By far the greatest contact between Sertolf cells and other cell types fnvolves the germ cellsr and Weber et al (f983) report 849á of the sertoll cell to be ln contact wlth germ cells. Russell (1980) has revlewed the known interactfons between Sertolf cells and germ cells. Sertoll cells show conflguratlonal relatlonships and speclallsed contacts wlth developfng germ cells. Degnosome-lÍke contacts functlon as attachment devices to maintafn the fntegrfty of the semfniferous epithellum, and at the same time ensure that germ cel]s are transported fn an orderly fashion towards the tubular lumen by vlrtue of conflguratlona'l changes of the Sertoll cel1. Gap Junctlons are

assocf ated wfth desmosomællke Junctlons and are I fke'ly avenues for transport of srnal'l mo]ecular wef ght substances from the sertol f cel'l to the germ ce1ì populatlon. E'longated spermatlds aPPear to be he'ld withfn the deep recesses of the Sertoll ce'll by ectoplasnfc speclallzatlons, the dfssolutlon of which appears important for sperm

re]easer and seems to allov excess spermatf d cytoplasm to be rernoved from the spermatld head. Sertoll cells phagocytose these complexes just prlor to sPerm release. whfle the sertoli celt ls obvlously actively engaged fn the

spermatogenlc process, the lnabllfty of lnvestigators to culture germ cells rsnains hlghly suggestive that the Sertoli cell provides a mf'lieu essentlal for germ cell dffferentlatfonr and lndicates hor much

more work is to be done ln thf s area. Hovreverr co-cu'lture of Sertol I cells with germ cells has been partlcularly successful (Zlparo et al' L982i Galdlerf et al, 1983, I9B4; Tres and Kierzenbaum' 1983) ln 35

studylng such lnteractions. As mlght be expected fn such a close relatlonshfpr germ cells alsO aPPear capable of signalling to Serto'l I cells, and Ílelsh et al (1985) have reported stfmulatfon of Sertoll cell adenylate cyclase ln vftro by germ cells.

I.2.3.4. Hormones Produced by Sertoli cells

The possibf'lity that the seminfferous tubules mfght Possess sterofdogenic functions was suggested by the occurrence of tumours be'lieved to orfginate from Serto'li cells' which secreted oestrogens (Huggins and Mouìder, 1945). The abundance of gnooth endop'lagnic reticulum fn the Serto'lf cells, simflar to other steroid produclng

cellsr made thls suggestion plausible on a morphological basfs (Chrlstensen and Fawcett, 1961). HouJeverr there has been some controversy over what hormones are actually produced. Whlle Chrfstensen and Mason (1965) found slgnlflcant androgen biosynthesls by lsolated semfniferous tubules in vltro, it seems likely that these preparatfons were contamfnated by interstitial cells. Cooke et al Íg72') lncubated fnterstitlal tfssue in vitro and found the content of testosterone fncreased with tfmer whereas the amount of testosterone fn fsolated tubules decreased durlng incubation or was unchanged' Howeverr de Jong et al (1974) found the oestradiol content of

separated tubules increased during incubation, whlle that ln interstitial tlssue did not change. Sertoll cells cultured from the testes of prepubertal rats formed appreciable amounts of oestradiol-I7p and oestrone when testosteroner

I one 19-hyd roxytestosteForì€¡ an d rostened f one or 19- hydroxyan d rostened

were added to the medlum. However thÍs activfty was greatest in ce'lls 36

from 5 day old ratsr and had fallen to undetectable'levels ln cells cultured from 30 to 40 day old rats (Armstrong et alr L975¡ Dorrfngton and Armstrongr 1975¡ Dorrfngton et al, L976). l{hf'le the tubules may have the capaclty for convertlng progesterone and pregnenolone to a number of compounds lncluding both testosterone and androstenedfone, and other compounds not formed by the fnterstltial tlssuêr they do not appear able to synthesise androgens from cholesterolr which remalns a function of the lnterstftial tissue and prlmarfly the Leydfg cells ( Setche'l'l ' 1978) . Oestrogen production by the Sertoli cell is now thought to be important ln regulating Leydlg cell activfty. A dfrect effect of oestadfol on testosterone synthesls in the testis has been

demonstrated by a number of I aboratorfesr and oestrogen recePtors are found fn lnterstftlal ce]ls (see Stelnberger and Steinberger, L977). The abllity of androgens and oestrogens to dlfferentially lnfluence LH

and FSH secretion has a'lso been reported (Stelnberger and Chowdhurryr L974¡ Steinberger and Chovdhurry' L977). Howeverr the hormone produced by Sertoli cells which probably has the most chequered hlstory of all reproductive hormones fs lnhibln. Whfte the existence of fnhibin was postu'lated over 60 years ago' it has only recently been lsolated and characterfsed. Its name was coined by McOultagh Ã93Ð for a non-steroÍdal substancer rìolv known to be a glycoprotefn, that feeds back on the anterior pftultary to speclfically decrease the secretlon of FSH (see revlews by Setchell, 1980; Steinbergerr 1983; de Jong and Robertson' 1985). By its

inhÍbiting effects on FSH secretionr lt can alter spermatogenesfs (see de Jong et al, 1978) and white thfs probabty remains the major role for fnhfbin in the male¡ it may exert other effects wlthin the testis' 37

Franchfmont et al (198f) found a dose-dependant lnhÍbltory effect on the dfvlslon of germ cells (see also Demoulln et al, 1981).

Nagendrahath et al (1g82l, report that dfhydrotestosterone lnhlblts the rèlease of lnhibln by Serto'l f cel'lsr and Hurkatdl et al (f9&t) suggest that testosterone concentratfons may regulate lnhibln levels' Inhibin has recently been shovrn to be composed of an lnter'linked (1986) c- and p- subunit, and Vale et a'l (f986) and Ling et al have shoyn that dlmers of the p-subunlt can form (which compl icates any

immunoassays of this hormone uthere the antlsera is directed agafnst the p-subunit)r êrìd that these dlmers are potent stlmulators of FSH synthesfs and secretíonr whlch now raises the possibility of duaì control of FSH from within the gonads (Tsonls and Sharpe' 1986)' Just

how this dfmer lnteracts at the level of the pltuitary with LHRH remains to be determfned. There has a'lso been consfderable lnterest expressed recently in paracrfne substances t.,lthfn the testis (Sharpe' 1986; Tahkar 1986) and I have already touched on some known interactfons between LeydÍg cells and semlnfferous tubules. However a number of factors are novr being 'located ln the testis that are either unique to this or$ôrìr or sfmllar to substances found elsewhere in the body but whlch possess specffic paracrine roles fn the testis. Sharpe (19S4) has reviewed the known intragonadal hormones, and gonadal LHRH ls the hormone with the most extensÍve bibllography. There is evfdence for the Sertoli cell orfgln of a testlcular LHRI{-'|fke peptide (sharpe et alr 1981) and it likely acts as a communlcator between Sertoll and Leydig ceìls to control intratestlcular testosterone leve'ls (Sharpe et al t L982) ' Whlle testosterone p'lays a central ro'le in drlving spermatogenesf s' there ls grovrfng evldence for the productfon and actlon of peptlde growth 38

factors whlch may medlate the gonadotrophic and steroldal effects' and Felg et al (1983) have lsolated one such factor from Sertoli cells. Recently Benhamed et al (1985b) have reported FSH regulatfon of Leydig celt function vla at least two types of Sertòll ce'll generated protefnsr and Perrard-Saperl et al (1986) report a dlrect Leydlg cell regulation of Serto]l cell function highlightlng yet agaln the ultÍmate importance of co-culture and in vfvo studfesr and the consideration of paracrfne control and fnteractions.

L.2.4. Mechanlsm of transport of hormones out of and across cells

A number of dlfferent cel'ls fn the testis produce a varlety of substances that act wlthln the testis and e'lsewhere fn the body general'ly, necessitatlng the secretfon and transport of these products fn a manner yJhfch al'lotvs their action to occur fn the right Place and at the rlght time. Such processes must a'lso be carefully regulated' Just as the mechanisms of action of steroid and Protefn hormones fn target ce'lls are different (see Mainwarfng, L977¡ Catt et alr 1980) so thefr production and mechanfgn of transport dlffer. 0f the testicu'lar steroids, testosterone ls by far the most studied'

I.2.4.I. Hormone Secretion

Cho'lesterol is produced by the Leydlg celJ srnooth endoplagnlc retfculumr passes to the mitochondrla for sfde-chafn cleavage and conversion to pregnenolone, which then returns to the smooth endoplas¡nfc reticulum for conversion to testosterone (see Ffgure (see 1.7.). How thÍs then leaves the cell is unknown Chrlstensenr Figure 1.7. The spatlal organisation of the testosterone biosynthetic apparatus Ín a Leydlg cel1. Cholestero'l (CtlOL) in the metabollcally active pool is derived from de novo 'l synthesi sr cho'lestero'l esters i n f pid dropl etsr or i n blood plasna. Cholesterol probably is transported from the blood plasma into the Leydig ce'I1 by lipoprotein (LP),

which may bind to Leydfg cell membrane receptors.

Cholesterol bÍosynthesis takes place fn the cytoplasm and endoplasmic reticulum. Cholesterol bindÍng proteins (CBP)

may exÍst to transport cholesterol to the mitochondria for conversion to pregnenolone. Pregneno'lone binding protelns (PBP) are then thought to return the pregnenolone to the

endoplasmic reticulum for conversion to testosterone. How this then leaves the cel'l is unknown, although binding protefns may be involved (from Ewing and ZirkÍn, 1983). 39

CHOL

I MEVALONATE f ACETATE I HMG-COA \ NE

CHOL / /\ ESTERS / / ACTIVE \ CHOL I CHOL I \ POOL I LIP IO \ / DROPLET -> -- -/ I CHOL ENOOPLASMIC RETICULUM

6 PREG + C PREG - PBP MITOCHONDRION

\ .-.__, CELL MEMERANE

T- BINDING PROTEINS 40

1975). Sterofds and thelr substrates are so]uble, and cannot be local lsed by conventional technfques such as autoradlography. The

expected mode of secretlon depends on whether the n€ryly syntheslsed testosterone ls released lnto the cytoPlasrn outsfde the tubules of the

srnooth retlculum; lnto the hydrophoblc portlon of the retlculum

membrane itse'lf, or into the cavlty of the gnooth reticu'lum. Christensen (1975) dlscusses the fmpt lcatfons of each of these

possible mechanismsr on the mode of testosterone Secretion. Simple dfffusfon fs unl ikely to occurr and it ls more than I ikely that a carrier proteln is lnvolvedr possfbly fn assocfation wlth the Golgi bodfes. Whatever the mode of secretfonr testosterone eventualìy

emerges from the Leydfg cell lnto the extracelìular spâc€r possibly

bound to a carrler protein (which would increase its solubllity)' protef n hormone secretlon by testicu'lar cells f s unl ike'ly to be any different to the mechanÍsn employed by other protefn secretfng cells. Studles of the pancreas (Jamfeson and Palade, 1967a'b) demonstrated that after proteln synthesls on the rough endoplagnÍc retlculumr the product passes pronptly fnto the cavlty of the endoplagnlc reticulum and ls then transported ln solutfon to the perlphery of the Go'lgi comPlex. There small membrane vesfcles bud off the retlculum and carry the protein into the Golgf reglon where they are fused wlth condensf ng vacuoles. t¡later withdrawal from the vacuoles concentrates the protefn thereln. The resulting secretory granule fs transported to the surface of the cell where its content is released lnto extracellular space (Christensen, 1975). 41

L.2.4.2. Transport

Hormones secreted into the lnterstitlal tissue leave the testls by one of three routes : by entering a caplllaryr by passlng lnto a lymph vesse'lr oF by traversing the myold cell layer to enter a semfnfferous tubule. As dlscussed earlier, the great specles varfatfon ln interstitiaì anatomy probably affects the relative partitlonlng of testosterone between lymph and blood. In those species possesslng ìarge lymphatlc slnusoids (rodents)r the 'lymphatic route is envisaged as the prfmary source of androgen supplled for the sqnfniferous tubulesr whfle those anfmals wíth more c'learly defined lymphatlc vessels (sheep, humans) have blood caplllarfes ln much c'loser contact wfth the tubules whfch may dlrectly suppty testosterone (Fawcett et alr 1973). While androgens are found in the lymph of all speciesr ôrìd testlcular ìymph contafns much more testosterone than testlcular arterlal blood (Llndnerr 1963, L967, 1969) blood fs the maJor route of transport for testosterone from the testls to the rest of the body. This fs prlmari'ly because of the hundred-fold dlfference ln flow rate (more than 1000 ml of b'lood per hour compared wlth about I0 ml of lymph per hour; Setchell, 1978). Blood ln the deferentfal veln however contalns more testosterone than arterial bloodr and blood from arteries in the spermatic cord may contafn more testosterone than arterfal blood elsewhere ln the body because of vefn to artery transfer (Free and Jaffer 1975; Free and Til'lson' 1975). A sfgniflcant ProPortion of testosterone fn blood ls bound to plasma protefn, lncludfng albumin and cortlcosteroid-blndlng globul fn (Eik-Nes et al, I954i Eik-Nesr 1970). A sex hormone-bfndlng globulln (SHBG) dÍstlnct from cortlsol-binding globul fn (CBG) is present in the 42

plasma of several specfes fncludlrì$ mêrìr but fs not found fn the rat or boar (Corvaì and Bardlnr 1973). The afflnfties of physfologlca'lìy fmportant sterolds for CBG or SHBG are two to three orders of magnltude higher than those for albumln. Horever albumfn ls fmportant rffreerl in determfnlng the magnftude of the non-protefn bound or fractlon of sterold ln plasna (slfterf et a'|, L982). It ls generally held that only the free fractlon ls available to target tissues' and that the level of SHBG and CBG regulate the free hormone concentration with SHBG or CBG-bound hormone formlng a pool of readily avallable hormone that wlll not be degraded by the liver. Howeverr Silterl et a'l (1982) suggest that plasma sterold bindlng protefn may p'lay an essential role fn the uptake of sterold by target cells, slnce lntracellular CBG has been found in a number of rat tlssues' Pardridge and Landaw (1985) have recently modelled testosterone transport ln the brain and predfct steady-state concentratlons of lntracellu'lar free hormone to change ln paraìlel more closely to changes in the concentratlon of bound hormone rather than free horrnoner as measured in vftro (see Figure I.8.). Free (lg77l suggests that venous-arteria'l transfer would also be affected by hormone blndlng proteins' aìthough the specfffc fnteraction of capfllary endothetial ce'lls and hormone bfndfng protelns must be consldered (Pardrldge and Landlaw' 1985). Lymph is an fmportant route for the secretlon of conJugated steroids and some protelns. In pigs and horsesr there ls appreclably

more testosteroner dehydroeplandrosterone and total unconJugated sterolds ln'lymph than ln venous blood (Setchell and Coxr 1982; Setchell et a'1, 1983). Galfl et al (1981) found that more than 70% of radlofodlnated albumln fnJected dlrectly fnto the testfs of rams was cleared in testicular lymPh. Siml'lar resu'lts have been found ln rats Figure 1.8. Steady-state mode'l of testosterone transport through the

braf n caplllary wa1'l and f nto brain ce1ls. Pools of globulin-boundr albumin-bound and free ìigand ln the systemic clrculatlon are denoted by GLorALo, and LFo, and pools of gtobul ln-boundr albumin-bound, and transportable ligand ln the brain capillary are GLrAL and Lt respectívely. Pools of free and cytop'lasmic-bound

testosterone in brain celìs are given bY Lm and Pl- respectively. Varfous rate constants are denoted by Kl-g' wfth the rate constant of hormone metabolism designated This model does not adhere to the restrictions of Kmet .. the free hormone hypothesis and allows for enhanced

transport of hormone from the p'lasna protein-bound pool fnto the tissue extravascular space. This process is bel feved to occur via an endothel ial inhibition of I igand blnding to the plasma proteÍn without the protein crossing the endothel ial wa'll. The model predicts that the steady-state concentration of intracellular free hormone changes ln parallel more closely to changes in the concentratfon of p'lasma protein-bound hormone as measured fn vftro and not the free hormone as measured in vitro (from Pardridge and Landaw' f985). 43

o L F

K6 K Ã

K3 K7

K I Kq K met \ K1 K2

T 44

(Setchell and Zuppt personal communlcatlon). The protefn hormone Inhibin ls also found ln higher concentratlons in testlcular lymph than fn venous b'lood (see Hudson et alr L979). The blood testls barrfer restrfcts the entry of many but not a'll substances to the semfniferous tubules, and in general tlpfd solublllty appears the maln factor determlnlng the rate of entry (Setchell 1978). ParvInen et a'l (t97O) found exogenous cholestero'l penetrated poorly lnto seminlferous tubulesr nhile progesterone, pregnenolone and testosterone readfly passed the blood-testis barrler. Ilhfle testosterone is found in tubular and rete testis fluidr fts concentration fs much less than that of the internal spermatfc veflìr and appears to enter the tubule by facflitated dfffusfonr fnvolvlng a saturable carrler (setchell et al t L978i Main and setchellr 1978). Retentlon and dlstrlbutlon of androgen wfthin the tubules and epldldymls seems to lnvolve a speciflc bindfng proteln ln some species. Several mammal ian specfes have been shown to contaln a speciffc Androgen-blndÍng protein (ABP) whfch 1 Íkely causes accumulatlon of androgen ln the tubules (Hanson et al' 1975)' This proteln ls produced by the Sertolf cells and enters the epfdidymfs along wlth other testlcular secretions (Danzo et al, L974). Secretlon

of ABP ls stimulated by FSH and androgen (Frltz et al, L975; Ritzen et a'l, Lg77r. In sheepr protefns genera'lly penetrate slowly through the blood-testfs barrfer (Setchell et al' 1969) although fodinated ovlne

LH, FSH and GH entered ram rete testis ftuid more rapidly than lodlnated albumln or Prolactin (Setchellr L9741. In the ratr gonadotrophins penetrated through the walìs of the semfnfferous tubules but dfd so very slovly (Setchell et alr 1976)' 45

L.2.5. Composltfon and functlon of lnterstitlal flufd

ht.

By now ft should be apparent that the extracelìular or rfinterstltialrr space ln the testis fs crltlcally important for this organs continued normal endocrfne functlon. The fluld ln thfs regfon not only bathes the prlncfpal cells of the testfs but performs the vftal role of providlng an avenue for communicatlon both between ce'lls wlthin the testls, and between the testfs and the rest of the body' Interstitfal flufd ls a'lways assumed to be formed by filtratfon of a fluid of 'low protein concentration at the arteria'l end of the testfcu'lar capillarles and resorptlon of most of thÍs flufd but not the protein at the venous end of the capitlaries. The proteln and the rest of the fluid leaves the testls as'lymPh. while direct evfdence for thls process has been demonstrated for other tlssues (see Yoffey and Courtlce, I97O) we can only assume that the system ls simflar in E I the testls, where regulatlon of tlssue pressure by contractlon of the capsule may be of consfderable slgnlffcance (Setchell, 1986).

For some !êaFS oovtr lt has been recognised that all substances entering or leaving the tubules and probabìy the Leydfg cells' must pass through the lnterstltial fluid. The close assoclation of some Leydig cells wfth the wal'ls of capillarles may mean that they have a dfrect associatlon with blood plasma. Indeedr the ffndlng that the concentration of testosterone rlas higher in the spermatlc veln blood than ln lymph mlght suggest a closer assoclatlon of Leydig cells with blood vessels than with lymphatics. Mclntosh (1969) has shown by electron mlcroscopy that Leydlg cells may be fntlmately assoclated with eftherr but that there are more blood capfllarfes than lymph capÍllaries (see also Yoffey and CourtÍce, 1970). Likewfse, certafn 46

sertolf cell products may be directly secreted into the tubular lumen. Howeverr the fluld ln the lnterstltial reglon rernafns an fmportant

avenue for cellular communlcation, and thus a potentfally usefuì windov on what ls happenfng wlthfn the testls. Not surprlslnglyt a

number of attempts have been made to monltor the fnterstitial regfon. practlcally all fn vfvo studles have relied on measuring fluids draining the testÍs (blood, lymph, rete testfs flufd), although as may be appreciated by now, the composition of these f'lulds does not necessarily reflect the compositÍon of fluids withfn the testis' Lymphatic vessels Ín the spermatic cord are closely associated with blood v€sselsr Particularly veins, and there fs a strong possibility that transfer of materia'l can occur between the varfous flufds in the cord (setche'l'lr 1986). This ls partlcularly so for compounds such as testosterone whose productfon and Prlmary actlons both take place in the testis itself. The ffrst real attempt at obtaÍning fnterstitial dr$ ).î fluid lras reported by Pande et al in 1966' using a method that mfght be termed rrDrip Col'lectionrr. Thls method Ínvolves

. . . rr ( remov i ng the testes f rom ratsr ônd maki ng_ a) sma'l l inclsfon at a èuitable slte through the tunlca albuginea avoidlng lnJury to btood vessels and semfniferous tubules. The orgãnr werê then placed in a test tube wlth a srnall perforãtion at the bottom and contafning a few glass.beads. Ìhe testes were suspended above the glass beads which prevented blockfng of the perforatfon. The test tube was put insfde a conica'l bottom centrlfuge tube fnto whlch the fluÍd from the testes trlckled dorn through the perfor¡tion. The operatfon was carrled out at a temperature of 2 -C under sterile conditions.rr .. .

The collected fluld was centrifuged and the ce]l free supernatant taken for estlmatlons. While no mentfon fs made in the PaPer as to how presenting simllar data for the human t long coìlectlon contfnuedr fn I I testlsr Pande et al (1967) report co'llection for 15-17 hours. Since I these reportsr a number of researchers have used thfs technlque or r 47

varlatfons of lt ( eg. centrifugatlon of the whole apparatus for 15-20 mfn at 54g instead of gravity collection for 15-17 hours) to obtaln fnterstitfal fluld for analysis, or for addition to ln vltro cultures (see Hagenas et alr 1978; Sharper L979; Sharpe and cooper, 1983;

sharpe et al, 1983; Turner et al, 1984). However the reported composition of fluid obtafned by this method ls somewhat unusua'l for a (1966' fluid supposedly formed by b'lood filtration. While Pande et al (see 1967) discuss thefr resu'lts fn comparison to blood serum Figures L.9.t1.I0.), they make no attempt to explain any of the dlfferences found. In terms of actual ratios the followfng differences can be

hight ighted 3

Ratio of IFlSerum I 2 Substance measured Rat Human Rats * hcc3 LlT Protei n 2.3/r Ll2 nl dehydrogenase 3.s/r 400/l lÈ Lactic Gl ucose-6-phos Phatase 2/r 3/L ti' G'lucose-6-phosPhate dehydrogenase 1000/ I 5000/ I Acid phosphatase 65/L LI2/I Ascorbic acid L5/L 30/L Ll7 L/3.s Gl ucose " Gl ycogen s/r 7/t Total Lipids r/3.s L/3 165/L52 L83/L46 L4O/L4L I Sodium lon Potasslum fon 7 /5.6 7.7/5.r 3/L Chloride ion L25/t15 13 9/ lrs

r) Pande et alr 1966 Ð Pande et a'l,1967 3) Sharper 1979

A number of the enzymes listed are usually recognised as fntracellular enZ!ft€sr and together with the high potasslum levels are

somewhat suggestive of cellular damage and contaminatfon of samples' Apart from the potential prob'lems of the human tfssue used by Pande I I befng post-mortem; that the technfque of isolating an organ from its i oC blood supply and collectlng fluid from it for 17 hours at2-4 tfght

! Figure 1.9. BÍochemica'l composítion of drip-collected rat testis ÍnterstitÍa'l fluid presented by Pande et al (1966).

i d lË I

Ì 4B

T^Ilt,tl I. Prolcilr, (i¡¡rlxrl¡.vrlr¡rto ¡rrrrl Vitnnritr ('o¡trtitt¡el¡t¡ of Rnt Ttrstix l'h¡irl.

(,'r¡r¡st.i I ttt rrt s Ttxti¡ lluirl Scrr¡ trr

7.04 7.4 ¡rl t ti Totll ¡rrott'itr (g,/lf)0 ttrl) 5.31 ( {.xl- t¡.lr{) ( r.:r - s.5 ) f ,:rtrtic rlt'hJ'rì rr)g('¡tr:lde ( rr tr i t x/rrr g .007 .tll8 (.0:17-.():19) ¡rrotein/nri n) ( .rlfÞ .10) Glucose-(i-¡rlros¡rlrttrtrt' (R:rcker 1.88 1.00 urriLs/I00 rul) (.0-J.5) (.0-3.0)

G lueosc-6-plrot¡rlrn tc rlclt¡'rì rogettnse 0tì7.5 .0 (units/rnl) (95Þ1025 ) (i5.5 .0 Âcirl lthosphntasc (rrrgP/l{ì0 rrrl/lr) (59.9 -7l.ll ) (i{.(ì Âlk¡rlint' ¡rltos¡tlrttl:tst' (rrrgP/10{) rrrl,/hr) 61.r (5f).8 -(i{.5 ) ((i3.1 -rifr.:t ) I [yalrt rottirltrst' (t¡rritÈ/I (r(ì tt¡l) .0 .0 9.0 .0 C¡r rbo¡¡ie nlrlr.T(ìrrtsc ( rrrrits/nrl) (9.0-:.0) Gl¡rcose (nrg/I00 ntì) 9.0 63.7 ( 8.5 -10.3 ) (5(i.6 -(i0.r) ) Gl¡'cogcrr 17.2 3.15 (r5.0 -1e.6 ) (2.5-4.0) f,actic ncid 800.0 1.37 (70(Leo0) ( r.r5- r.7 ) tSialic aciil 15.0 35.0 Ascorbic ncid " 10.25 .69 ( e.6%r0.87) ( .60- .6e)

l Data l¡nst'il otr n s¡nglc' cstin¡ntiort.

TÀnt,T: lf. Li¡rirìs :rrrrl Hlrtlrol.vlr* of R¡rt Tcslir 1'lrrirl'

Corrstittlelrt¡ Te¡ti¡ flr¡irl Sr.rt¡¡rt Totrrl li¡rirls (nrg,/l{)0 tnl) l::0.:ì I 405.¿ (¡ijr¡.(i -lTl.l{) (.100.:¿l--r0ô.1 0) Free storols ll.l6 28.5 ( 10.2.r- ¡3.3i) ( !6.: - 3{.6 ) Sterol ctrters 37.79 50.91 ( 30.14- 3fl.5o) ( 50.37- cl.3{) Phos¡rholi¡rirìs 15.65 198.2 ( r3.02- 18.r2) (1e6.8 -119.6 ) 46.09 Choli¡rc (/o of ¡rlrospholi¡idx) 30.81 ( 3:1.2f'r 3{1.93) ( 41.31- 49.20) Srritro 3.(i4 8.99 ( 3.ol- 4.:ls) ( 7.e - 8.26) Ethnnolnnrirre 10.10 94.1 2 ( $.se- ¡0.97) ( 23.03- s6.14) Sodir¡nr (rrtEq/l) ¡ 65.1 7 I 59.r7 ( I fi3.:17-l 70.75) (146.0 -153.r2) Pot¡uqsil¡m 0.s 5.6 ( 5.4 - 7.!t ) ( 5.2- 5.e) Cl¡lt¡ritlc l .0 I15.0 (u0.9 -120.3 ) (lr9.r -118.9 ) ooa C¡¡leiunr 5.3 ( r.91- 9.4e) ( 4.1 - 5.7) ot eo Dienrbo¡¡ntc (nrMrul) l3.l { I ( 20.07- e5.õe) ( e.o - 14.07) ì s0.0 80.0 I Zirrc (ag,/100 rnl) ( r8.0 - 93.0 ) ( 75.0 - 85.0 ) I

Ì Figure 1.10. BiochemÍcal composition of drip-co'llected human testis

Í nterstiti al f'l u i d presented by Pande et al (L967 ) . 49

Trnr.u l. Protein, carbohydral.e and vitamin cons[ituente of human teel.¡s fluid Serum Oons[ituents Testis lluid pl{' 7.0 7.5 3.71 I ? .18 Total protein (6927 .45) (g/lo0 ml) (3 .60 -3 . ?7) l,nctic dehydroßenrsc 0 .251 0 . 0007 (U/mC protein,/min) (0.243-0.259) (0 .000- 0.0007) 6.3 2.3 G I ucose-6-phospha [ase (r 0-3.0) (Racker unit/100 ml) (3.0-e.0) 5250 0.0 G lucose-6-phospha tc dchyd rogcnase (0.0-o.0) (U/ml) (4950-5550) 0.o Acid phosphntase 1L2.4 (o.H.o) (mg P/100 ml/hr) (l 03 .6-1 16 . 8) 19 .23 4.5 Alkaline phosphatase (2.7-5.41 (mg P/100 ml/hr) (16 .7-20 . 5) 0.0 0.0 Hvaluronidase (0.o-o.0) iu/roo mtl (0.0-0.0) anhydrase 0.0 0.0 Carbonic (0.0-0.0) (0.0-0.0) 1U/100 ml) 2l .l 73 .0 Clucose (71.0-74.0) (mg1100 ml) (2O.4-22.5) 20.2 3.0 Glycogen (2.5-3 .25) (mg/100 ml) (19.6-21.0) 46.8 Lactic acid 600.00 (42 . 8-4e .6) (mgl100 ml) (544-63e) 30.9 0 .314 Ascolbic acid (0 .146-0 .482) (m¡r/100 ml) (28.0-34.0)

r Determined bv BDH indicator paper. t Mean, with ránge in Parentheses.

Tesr,B 2. Lipids and electrolytes of human testis fluid

Constituents Testis fluid Semm Total lipids (mg/100 ml) 28L .32 656 .25 Free sterols (mg/100 mlì 42 .11 57 .36 Sterol ester¡ (mS1100 ml) ?9 .23 13? .15 Phospholipids (mg/100 ml) 19 .93 246 .55 Sodium (mEq /l) 183 .t2 146.33 I)olassium (mEqI) 7 .76 5.t2 Ohloride (mEq /l) 139 .65 115.37 (mEq/l) 3 .7A 5.71 Calcium r llica¡'bonate (mEq /l) 28 .67 23.44 (25 .44-30.36) (22.61-25.93) Zinc (¡gl100 ml) ?0 130 (55- 80) (125-133)

. Mcan f

be rrunphysf ologicalrf was never addressed. The low g'lucose levels' and the hfgh g'lycogen and'lactate Ievels mfght also be fndfcatlve of anaerobfc metabol fsm.

In I970r Tuck et al empl oyed a mlcropuncture technfque to suck up interstltia'l f'luld from between the tubules. Thls technlque requlred the rqnoval of at least part of the tunfca albuglnea whfch may have dlsturbed fluld formatfon and removaì. The reported potassium level in

samples collected was about 50% hlgher than blood plasnar whereas 'lymph Setchell (1970) found potassf um 'levels f n spermatic cord to be

equa'l to b'lood plasrna. Whf le equit ibration of potassium levels ln the lymph collected by Setchell may have already occurred with b'lood fn the spermatfc cord, the suggestfon of cell damage and contamination of

samples by Tuckrs technique remains possÍble. Small volumes were also

a drawback with the technlque. Interstltla'l testosterone concentrations have been reported by a

number of workers using the Drip collection methodr and Comhafre and

Vermeu]en (1976) were also ab'le to measure testosterone ln samples coJ'lected by mlcropuncture (see Table I.I.). The currently accepted

hypothesf s that arose f rom such resu'lts, and ls well ref'lected in the table, ls that lnterstitial ftuid contaÍns aPpreclably hfgher concentrations of testosterone than testicular venous blood (or perlpheral blood). However¡ what ls also aPparent Ín thfs table' and yet never discussed ls that the faster the method of recovery of fluid (centrifugation / mlcropuncture vs gravlty ) the lower the level of testosterone. This must surely ralse further questlons about the usefulness or vatidity of methods of collection. 0f further consfderation are the detailed studfes of testicular

lymph carrfed out several years ago in the ram. Llndner (1963' 1969)' the of the ratr Tab.le l.l. The testosterone concentratlons ln lnterstltla'l fluid and venous blood from testls and fn perfphera'l blooà plasma. Values are glven in ng/mlr-with standard errors of the means and had been the numbers of observatlbns. The values lndicated as LHRH-A were from testes whlch lnJected dlrect'ly wfth I ng of an agonfst of LHRH (see original paPer for details).

Bl ood Author Techn lque Interstltf a'l f'luld P'lasma Testfcul ar veln Perl Pheral

+ (I7) Comhal re and Mlcropuncture ¡59 27 Vermeul enr 1976 (10) r.6 0.7 (10) Hagenas et al, 1978 Centrl fugatl on L37 + 25 1 (26) 28 + 5.3 (10) r.2 0.1 (8) Turner et a'l t 1984, 1985 Centr I fu gatl on 73 + 5 ! (4) 2.7 0.3 (4) Sharpe et a'|, 1983 Gravlty Control 3L5 + 29 ! il il 590 + 20 (8) 99+20 (8)

(8) + 20 (8) ll LHRH-A 800 + 30 I75 (¡ H 52

tr{allace and Lascelles (1964) and Setchell et al (1967) reported testfcu'lar lymph fn the ram to be sfmilar fn composftlon to blood plasma from the lnternal spermatic vefn. Honever testosterone levels were approxlmatel y 7M of those fn b'lood. Even allowlng for lymph/vascular transfer and equi'libratlonr it is extremely dffflcult to reconclle the reported high concentrations of testosterone fn interstitial f'luid with lot{er levels fn testicular lymph and venous b'lood.

It is no'longer sufficient to suggest that substances need only be detected in Ínterstitial fluid and that knovledge of sPeciflc levels fs not all that important. Knorving the actua'l comPosftion of interstitial fluid ls lmportant if we are to understand fntercelluìar

communf catlon and the physf o]ogf cal control of testlcu'lar function'

Much more work fs needed ln thfs ôF€âr and techniques must be improved if any advances are to be made. It is a reglon of prlme lmportance ln paracrÍne regulatfon and communfcatlon (Sharper 1986). That the majorlty of infertfle men present wlth normal or near normal gonadotrophin levels and normal or varylng degrees of subnormal unable numbers of sperm (de Kretser and Kerrr 1983) and that we remafn to provfde suitable therapy ln most cases ref]ects our Poor understandlng of the control of sPermatogenesls. Much of this fnformatfon ls probably to be found in the fnterstltla'l regfon' and consequently in fnterstitlal fluld.

L.2.6. Control of endocrlne activlty in the testls

The endocrfne functlon of the testis has hÍstorically been seen 53

to be controlted by the anterfor pltuitaryr slnce testicular development and functfon are so clearly dependent on the gonadotrophlc hormones LH and FSH. However lt ls becomlng increaslngly evfdent that the day-to-day functfons of the testis are largely under local control (sharpe, 1983r 1986). r have discussed the role of testicular b'lood vessels and lymphatlcs in conveying substances to and from the cells in this organ. Horveverr that these systans can be regulated by the testis is of immense physÍological fmportancer and of prfme consideratÍon when discussfng the mechanfsns of control of endocrfne

fu ncti on.

The capillarfes of the testls are unusua1 compared wlth those of other endocrÍne tissues fn the rat, in that they appear to be unfenestrated (wolff and Merker, 1966). They also shovr an extraordinary sensltfvlty to the toxfc effects of cadmfum salts

(Setche'l'l and WaÍtesr 1970; Aokf and Hofferr 1978). However thef r

norma'l permeabillty wouìd appear to be sfmilar to that of other capillarfes fn the body as determined by measurements of their permeability-surface area product (PS) for CTEDTA' Sodiumr Vitamin BtZ (Bustamante and Setche'llr t98I) and Albumln (Setchell et alr l9B4). The permeability of testicular blood vessels to a'lbumln can be markedly increased if the anima'l ls injected with human chorionfc Gonadotrophin (hCG) between I and 24 hours beforehand (Setchell and Sharpe, t98I; Sowerbutts et a'|, 1986). The Permeabllity returns to

normal between 36 and 48 hours after the hCG. The increase ln permeabil ity is accompanÍed by an I ncreased vo'lume of interstitia'l fluld withfn the testls and by fncreased'levels of testosterone in testfcular venous blood which peak inltially at 2 hoursr wfth further peaks so far recorded at 16 and72 hours after hCG (Risbrldger et al' 54

1981; Hodgson and de Kretser, L982,1984; Hodgson et al, 1983;

Sowerbutts et al, 1986). However testicu'lar permeability fs normal at the tfme of the 72 hour testosterone peak suggestlng that dlfferent mechanlsfns may be lnvOlved ln these two effects. The Permeability lncrease does not fnvolve androgens' prostaglandinsr hlstamlne or bradykfnfnr but fs medlated by S-hydroxytryptamlne whfch may arlse from the abundant mast cells found fn the vicinity of the artery on the surface of the testls (Sowerbutts et al' 1986). HCG is known to bind to LH receptors on Leydfg ceìls and thfs ls the likely stÍmulus for fncreased testosterone productfon by thÍs treatment. However' Bergh et al (1986) and Wfdmark et al (1986) have produced evidence for the fnvolvement of leukocytes in the Permeabil ity resPonse to hCG whfch supports the above proposltfon that hCG has more than one mode of actfon. These effects are of consequence physiological'ly because LH fs reported to have ldentlcal effects to hCG on capi'l'lary permeability and interstitial flufd volume changesr and is probably normally fnvolved fn regulating the tonlc control of testicular blood vessel permeabÍ'ltly (see ShaFper 1983). The testicular LHRH-Ifke substance thought to be secreted by the Sertoli cells appears to not only regu'late testosterone secretion, but also to modulate interstltial flufd formatlon and the results of Sharpe and Fraser (1980) and Sharpe et al (1982) rafse the posslbllity that the de'lay in actlon of LH and

hCG may in part represent the tlme taken for substances to fnduce

intratesticu'l ar secretion of LHRFI. Just as these changes ln btood vessel permeabflÍty can dramatica'l1y affect the endocrine actlvity of the testls, so too can the rate of blood flor through these vessels. The vascu'lar system can control the entry or egress of substances into and out of the testis 55

fn two ways. For those substances for whlch there are concentratlon gradients across the vasculaF wallr movement can be regulated by varying the rate of elther passive or facllltated dlffuslon' If thfs dlffuslon ls not llmfted, then transport can be regulated by changlng blood flow (Setchell, 1986). The findfng in the asPermatogenfc testls of hfgher than norma'l concentratlons sf testosterone in the testls and 'levels fn testÍcular venous blood, but lower than normal in the peri pheral cf rcul ati on i s probab'ly due to a reducti on I n testicu'l ar (Setchel'l blood florv in proportlon to the reductfon ln testf s weight

and Gal ilr 1983). Testlcular b'lood f'lour is lnfluenced by a variety of factors lncludfng posture' temperature and the common vasoactfve (see substances such as prostaglandfns and neurotransrnltters Setche'll,

L97Ot 1978; Free, Lg77). It ls unl ikely howeverr that the potential abÍtity of LH to lncrease testicular blood flonr as discussed earlierr 'lag-time ls of any physlologlcal signfflcance sfnce the for the response ls so large, and the resulting change of such small magnftude (Freer 1977¡ Setchell' 1978). Indeedr the enhanced secretlon of testosterone by the ram testis fn response to a spontaneous pulse of LH (Laurle and Setchel'I, 1978) must be due either to a raPfd change fn permeabillty of testicu'lar blood vessels or the walls of the Leydlg cells, or an equally rapid change ln the rate of testosterone (1985) synthesls by these cells (see setchel'|, 1986). Damber et al

have found the lower secretfon of testosterone from the abdominal testls after hCG is malnly due to reduced blood flow and not to any dlsabillty of the Leydfg cells of abdominal testes to produce testosterone. It remains'llkely that any effects of LH on testicular blood flow are indirect effectsr probably medfated by testicular factors produced fn response to LH (such as the LHRFI-Iike factors 56

descrfbed by Sharpe and co-workers)r ônd the suggestfon that some product of fnterstitial cel'ls (Leydig cel'ls, Macrophages) is lnvolved ln regulatlng the nelghbourfng blood supply (ll,flllamsr 1949) requlres further I nvestlgatlon. A blood-testls barrier fs probably lnvoìved fn the control of endocrine actlvfty in the testls. Whfle I have already dlscussed the maJor b'lood-testis barrier fn the seminiferous tubules; Kormano (I967b) has demonstrated that at pubertyr the penetratlon of certafn dyes fnto the fnterstitial fluid is reducedr and some may no'longer penetrate at al'1. Thls lmplf es scrne barrier at the level of the vascular endothelfum. The barrfer does not appear to restrfct the (Waitesr Waites passage of most protefns fnto Ínterstitial flufd 1977"

and Gladwell, LgïÐ unllke the blood-braln barrlerr êrd the substances that lt ls lntended to exclude rsnaln to be ldentlfied' It ls of lnterest that the establishment of this barrfer co-lncides wfth an increase ln the activity of alka]fne phosphataser an enzyme considered to be important ln transporting substances across capfllary wal'ls (Kormano, 1967b). After pubertyr the testfcular blood vessels are

second only to the capl'llaries of the braln ln thelr level of alkaline phosphatase. while the enzyme ls found ln the endothe'llal cells of a'll other blood vessels ln the testlsr virtual'ly no actlvlty is found in interstitial cells or the tubules (Setchell, 1986)' fn contrast' the high enzyme gamma-g'lutamyl transpeptidase whfch is also present in concentratlons in brain capitlariesr ls abundant fn the endothellal cells of the artery on the surface of the testis and fn the spermatic cord and fn testlcular arteriolesr but not in testlcuìar capil'larfes (Nleml and Setchellr 1986). In the testls, as fn other tlssuesr this As emphasised enzyme ls probably associated wfth amino acfd transport. 57

by Sharpe (1983), the varlous blood-testls barrlers ln the testls (vascular endothe'llumr myold cells, tight-Junctions of the Sertolf cells) wlll not only selectfvely prevent certafn substances from enterfng the testls, but may also act to prevent others from leavfng the testfs. These various barrlers allow for the creation of unlque microenvf ronments wfthf n the tubu'les, and f n the f nterstitlal spôcer and are probably essential for 'loca'l lnteractf on between the varÍous ce'll types and compartments wf thin the testis.

1.3. THE IMlvluNOLOGY 0F THE ENDOCRINE TESTIS

I.3.I. Introduction

The fields of reproduction and immunoìogy have historlca'l1y been classifled as separate biologfcal dlsclpllnes. A connection between the two was ffrst reported in 1898 when Ca'lzolarÍ observed that the thymus of rabbits castrated before sexual maturlty was larger than that of controls. However ft took some 70 years before the slgniflcance of thfs observatÍon became apparent and lt fs only recently that greater emphasls has been placed on interactlons betveen the reproductlve and fmmune systøns. It is nour recognlsed that reproductive-lmmunologlcal lnteractions are common in mamma'ls' They appear to be hormonally regulated and the hormones fnvo'lved origfnate from the thymusr the hypothaìamlc-pltuitary unit and the gonads' A varlety of c'linfcal and experlmental evidence supports the hypothesis that gonadal sterofds regu'late fmmune functlon (see Grossman, 1985 for a recent review) and the research fnto the mechanfgns fnvolved ln maternal acceptance of the immunologica]ly forefgn foetus is typlcal 58 of work fn whlch the actfons of gonadal hormones have been fmplfcated (see Jacoby et al, 1984). White a sfgnificant amount of work has concentrated on the regulatlon of the thymus and othe¡: frunmune canponents by the male gonads, there has also been consfderable lnterest expressed fn aspects of immune function wlthin the gonads, and as outlined in the introduction to this reviewr the immunology of the endocrfne testis has proved especlally interestlng. Along with the braln' anterior chamber of the eye, and the hamster cheek pouch' the testis has ìong enjoyed a reputation as a peculÍar1y hospitable site for tfssue grafts (Barker and Billingham, L977). These sites consistently rtprotecttl transplants from the process of fmmunologlcal reJectlon usually encountered e'lsewhere ln the body (Bi'll lngham and Silvers, 1965; Merrlll, 1965) and have been termed Immunologically Privileged Sltes. Before elaborating on the methods of like'ly protection afforded a graft ln such a siter ft fs necessary to review some baslc concepts of

transpl antatf on immunol ogy.

L.3.2. The Immune System

The fmmune system lncorporates the ìymphatlc and vascular systems of the body. The most fmportant cel I s I n the lmmune system are Lymphocytes. These are basical'ly metabolically qufescent cells which recirculate continuously around the bodyr passing out of the blood fnto the lymphatic clrculatlon within the lymph node and other organlsed lymphoid tfssue. They are broadly classifled lnto two main

categori es (i) T-lymphocytes : derived from the thymus 59

fii) B-lymphocytes : derlved from bone marrou ln adult

mammal s whlch dlffer ln thefr developrnentat backgroundr ILfe spôor and functlons. They are not dfstlngulshable morphologlcally, but dfstlnctfve surface markers can be detected by lmmunofluorescence. wfth Lymphoid tlssue is a term used to lnclude these cells, together the prfmary lymphoid organs where they are produced (thymus and bone (lymph marrov, ln adult mammals)r and the secondary tymphofd organs nodes, part of the spleenr tonsi'ls, Peyers patches ln the lntestine) where lymphocytes come fnto contact wfth antfgen and make an immune

response (Cunnlnghamr 1978). Foreign antigens enter the cfrculatlng pool and localise ln the to lymphold tfssue. There they stlmulate a proportlon of lymphocytes (see prollferate and differentiate fnto rreffectorrr cells Flgure 1.11.). These effector ce'lls can be antfbody producfng cells ln the case of B-lymphocytesr or a varlety of antlgen sensltive and regulatory cells ln the case of T-'lymphocytes. Assoclated with effector cell production fs the generation of Memory cells which ioin encountered the immune system library as a reference to previously antigens. Antfgens stimu'l ating B-lymphocyte prol iferation induce antlbody forming cel'ls that aPPear ln the stlmulated lymphold tlssue wlthÍn a fery days. These cells are usually plagna cel]s, or basophlllic lymphofd mononuclear cel'ls of various slzes' and each produces an antlbody of sfngle speciflcfty and of on'ly one Ís Ímmunoglobulfn class (Yoffey and courtlce,1970). Thls response calledtheHumora]ImmuneResponse.Theregulatorycellsof Helper T-tymphocyte orfgfn are extrerne'ly diverse ln thelr function' T-cells fnteract wfth B-lymphocytes to stimu'late antlbody production Figure I.II. Genera'l pattern of cellular events in an immune response (from CunnÍngham, 1978). 60

Antigen + accessory cel 'ls (macrophager hel per T cells)

stimu]ation

Precursor Precursor B-cel I s T-cel'l s

Pro'l iferation and Differentiation

Effector B-cel'ls Eff""."JT-cel 1 s Antlbody fo rm I ng Cytotoxf c ceìl s cel ls Sensitlzed T-ce'l I s Regul ator cel'ls ( he'l Per and suppressor) 6I

and Suppressor T-cel'ls depress antfbody productlon. Cytotoxlc T-cells seek out forelgn cells and lnteract wfth macrophages to promote destructlon of these forêlgn.cells. These protectlve functlons are referred to as the Cell-Medlated ResPqnse. That T;lymphocytes rather than B-lymphocytes or macrophages- are prlmarfly responbfble for graft reJection has been demonstrated in a number of ways. Nude mlce and 'lack fail rats (genetlcal ly def f cf ent i n T-ce'l1s through of a thymus) to reJect skfn grafts, and rodents experfmenta'l1y deprived of T-lymphocytes (thymectomlsedr f rradi ated and bone-marrovl-reconstltuted anfmals) also fafl to reJect transplants (Tf'lney et al, l98I; Klndred'

1979).

When an antfgen ls lntroduced fnto the body for the flrst tlme' there ls a condltloning of the fmmune cel'ls or thelr Pre-cursors that extends over a number of weeks, and may result in]ow levels of cfrculatfng antibody. The events occurrfng ln this latent Period are described as the Primary resPonse. In the case of a response agaínst a tfssue graft whfch results in reJectfont the graft ls safd to have suffered Ffrst-set reJectfon. ff a second exposure to the same antigen or tfssue occurs weeks or months aftenrards, the memory cel'ls of the

fmmune system generate a rapld productÍon of antlbody or cytotoxic

cells, with fmmune response befng much quÍcker than in the primary response. Thfs fs called the secondary lêspofìser and ln the case of reJected tlssue grafts fs referred to as second-set reJectfon.

Histocompatibllity molecu'les are a class of membrane glycoproteins which are Present on the surface of eukaryotic ce]ls and are coded for by a set of genes known as the MaJor Hfstocompatibility 62

Compìex (l'frlO). Hlstocompatiblllty molecules belong to two typesr oo€ expressed on the surface of all cells, and the other exclusfvely represented on cells of the fmmune system. Those exPressed on the surface of all cells dlffer from lndividua'l to lndlvldua'l and thelr genetic dlversity provldes blo'logfcal unfqueness for every subJect fn a normaì population. When organs or tissues are transplanted from one lndlvidual to another bearlng a dffferent MHC codlng' they are recognlsed as antlgenlcally forelgn by the host and a reJectlon response fs fnitlated. Tab'le 1.2. deflnes the standard terminology used fn transplantatlon ímmuno'logy. Autografts and Isografts wilì not dfffer from the host ln lvllc coding and wil'l therefore not fnduce an immune response. Allografts howeverr while matching the I'tlC gene loci' wlll not possess the hosts codfng pattern and will like'ly fnitiate an lmmune response. Xenografts usually suffer reJection because thelr l'vl|C wll'l not only dfffer in terms of coding pattern ' but also gene 'locatfon. Xenografts nfll also often suffer from physiological incompatibll ity which affects survival.

Unmatched graft antigens (a'llografts, xenografts) reaching the organlsed tymPhatlc tlssue of the host witì trigger a number of processes (for a detal'led descrfptlon see Hayry et al' 1984) of which

T-lymphocyte blast formation and divfsion, and the assoclated productlon of effector T-cells ls of most consequence for graft reJectlon. A number of dlfferent types of T-cells are involved fn graft reJectlon, and they can be dffferentlated accordlng to mqnbrane expression of partlcular phenotypes. In mlcer one classfflcatlon nLyn system dlstfngufshes lymphocytes by the phenotypte. T-lymphocytes expressfng the Ly -Lt+2 phenotype are dfrectly stfmulated by antfgens such as tfssue grafts and cells bearfng tumour antigens or infected Table 1.2. Terms used ln Transplantatlon Immunology

Immu nogenetic Preflx Mean I ng Nouns AdJ ectlves (+ synonyms) (+ synonyms) re'l atl onsh I p

Se] sel Auto- Sel f Autograft Autogenelc f to f ( autochthonous) ( auto'logous) Dif l ndlvf dua'ls but Iso- Same Isograft Isogenelc ferent ( syngenelc ) genetica'l ly ldentlcal

Same specles but Allo- 0ther Al 1 ograft A'll ogenelc ( homograft) genetlcal'ly dlfferent specles Xeno- Forelgn Xenograft Xenogenelc Dlfferent ( heterograft) ( heterol ogous)

(¡or 64

wlth vfruses. These T-cells prolfferate and differentfate to produce the Cytotoxfc or Cytolytlc ìymphocytes whfch have the abillty to cause I lysfs of forelgn cells. The mechanfsm of thls dlrect cytotoxlclty ls poorly understoodr but ft ls speclfic for the cells that caused

fmmunf zatf onr and nearby ce'llular elements are not affected. C'lose cell-cell contact is required (Fawcett' 1986). Helper T-ceì1s express a dffferent phenotyper the Ly +Lt-Zt and enhance the development of cyto'lytlc T-cells. Transp'lantatlon antigefìs pêF sêr are poor

fmmunogens wlthfn a species (Batchelor et al' 1978) and Lafferty (1980) and Bach (1980) suggest that co-presentation ls required wfth a 'lymphoreticul metabol ical ly actf ve ar cel'l . In ordl nary Parenchymal

organ transpl ants these cell s woul d f ncl ude bl ood mononuc'lear cel'l s

and bone-marrow derfved npassengerrr leukocytes (Steinmuller, L967 i

Guttman and Lindqvist, 1969), but not graft parenchymal cells. Support for the co-presenter model comes from a number of authors who have I I demonstrated that prfor culture of tissue makes it possfble to transplant skfnr thyroidr parathyrold and pancreatlc lslets of

Langerhans across allogenelc or even xenogenelc barrfers wfthout

fmmunosuppressfon. This ls suggested to be due to the reduction of

immunogenic capacfty through the loss of highly lmmunogenfc Passenger cells ln culture (see Lafferty et alt L975; Lafferty and Woolnough, 1977i Soltlnger et alr 1977i Lacy et al, 1979). The lnteractfon of an Antigen Presentfng Cell (APC) and a Helper T-cell appears central to the whole rejectiorì procêsSr as descrfbed by

Mason (1983, Ffgure L.Lz.r. In thls model macrophages / dendrltic cells are envlsaged as lfkely antfgen Presentfng celIs. Macrophages have the abillty to take partfculate matter into their cytop'lasm and to degrade the ingested substances with hydrolytic enzymes. This Figure L.L2. The generation of effector mechanisms of allograft rejectfon (from Masonr 1983).

ftl Itr I

t I I

I 65

o^tt1c^ þr¿s¿ntrn5 -c¿tt (nøc¡ Antlgen recognition lLl by T, cells hcl¡cr Tcelt

Prol iferation and differentiation of Th vr¡th prodwtion of lymphokines

Act ion of lle TRF I L2- I.1A F lymphokines on effector cel I recu r sors NK l'4ó E P celÌ T. AR celt interacting wlth P APC

'\& rLt I

Generatlon of effector tvq led acl- mechan I sms 4ct IYCted 11ø NK Tc A rc I

lLl, lnterleukln I lL2, interleukln 2 l'tAF, macrophage actlvatlng factor TRF, T cell rcPlaclng frctor NK cel I , natural kl I l¿r ccl I Tcp, cytotoxlc T cell Precursor Tc, cytotoxlc T cell Hø, macroPhagc q't5r,s ADc¿ APC, antlgen Prcrentlng cel I AFC, antlbody formlng cell ADCC, entlbody dependcnt ccll-rnedlated toxlcltY Ì^ hclpcr f ccll

r 66

phagocytfc functlon a'l'lows them to destroy bacterla, dead cells and debrfs from inJury and they are important fn malntafnfng and repafrlng tfssues, and fn defence of the body from fnvadlng mlcro-organlsns' They exfst as elther free macrophages, whfch are motlle and wander through the ground substance of connective tissue' or as ffxed

macrophages which are sessile, and stretch out a1ong bundles of

collagen fibres. It is no1rr wldely accepted that both forms are different functional phases fn the life history of cells of the same 'lineage, and al'l orfginate from precursors in the bone marrow (Fawcett, 1986). Macrophages actively engaged fn engulfing antlgen, whfch seem to retain a sma'll amount of antÍgen bound to thefr surface they present fn a concentrated form to lymphocytes. However' to be

fmmunogenfc such antigen must be presented by macrophages ln assocfatfon wfth thelr surface lFlC mo]ecules (the Ia molecu]e fn the ii il mouse)r ôrìd only macrophages with the Ia molecules are caPable of r,'i 'lymphoid 'lymph I co-operating wlth T-lymphocytes. In tlssue such as nodes, dendritfc cel'ls are likely candidates for antlgen presenting cells. They are very sfmflar to macrophages fn many waysr but are not phagocytlc (Fawcett, 1986).

The Efferent phase of the fmmune response fnvolves the destructfon of the graft by the effector lymphocytes. While sensltfzed T-cells are an essential ffrst weaPon in graft rejectfonr an exceedlngly compìex and slzable prollferative resPonse takes place in the graft itse'lf which is associated with that occurring ln the lymphoretlcular system of the recfpfent. In the $raftr the I I 'lymphold prol iferatÍng ce]'ls are mostly blast cells or lymphocytes' i a'lthough some macrophages would a'lso appear to Prollferate ln sftu'

3 67

and the overall lnftammatory response ls now recognfsed as an fmportant part of al'lograft reJection. The assoclatlon of antigen and T-lymphocytes wlth an antf gen-presenting macroPhage/dendrltlc cel l

causes the APC to release the lymphoklne Interleukfn-I (IL-I). Thls stlmu'lates the Helper T-ce'l'l to proì lferate and dlfferentlate and 'lymphokines. appears to fnduce secretlon of a variety of other Interleukln-2 ÃL-Ð causes proliferatlon of cytotoxlc effector cel'ls, and activated natural killer cells (Nem'lander et al, 1983). Accordfng to Carpentier (19S3) t IL-Z ls now seen as a maJor component trfggerÍng

the lmmune response ln allografts. Indeed' many of the

immunosuppresslve drugs used fn modern organ transplantatfon

programmes to prevent allogeneic graft reJection operate to prevent

IL-2 re'lease (see Wang et a'l , L982't. Macrophage Actfvatf ng Factor probably stlmulates both the macrophage component of the ce'llu'lar

i i lnffttrate, and secretlon of plasnfnogen activator - a neutral q protease known to be assocfated wlth macrophage effector function I (cohn, L978¡ Nathan et a'|, 1980). HelPer T-cells are also lnvolved fn the B-lymphocyte generated Antlbody Dependant Cet'lu'lar Cytotoxf xity (ADCC) associated with the lnflammatory response (see Figure L'L2"

and Hayry et al, 1984). The inflammatory ìymphocytes, monocytes and

macrophages dlsplay large numbers of ldlotyplc receptors to donor lfrlC antigens, and have a hlgh number of IgG receptors (Roberts and Hayry'

L9763 Hayry et al, 1984). Assocfated wlth thfs generation of fnflammatfon is the accumulatlon of large numbers of thrombocytes fn the graft which may contribute 1n partr to the vascu'lar changes observed fn an organ graft vascular tree (Hanto et a'|, L982)' It ls of interest that an fnf]aÍrnatory eplsode ls often a'lso seen I in autograftsr although ft only suPerflclalty resembles that seen fn

I 68

an allograft. The response ls a'lways much gnaller, and dlstlnctly lacks blast celIs (Hayny et al,1984).

Flna'ltyr the concept of transplantatlon Tolerance requfres mention. Immunotolerance fs a state of fmmuno'logfc fnactlvlty that ls speciffc ln regard to antfgens or cells which, ln normal anfmals would

lnduce an lmmune resPonse. Cultured allografts that have been depleted of passenger leukocytesr câPFt recognlsable antfgen and are Promptly reJected ff reclPlent anima'ls are challenged with donor leukocytes at the tlme of transplantatfon. Howeverr anfmals carryfng al'lografts for a prolonged perlod (>= 100 days) become progressively more reslstant to challenge with donor cells (Zitron et al, 198I) and Donohoe et al (1983) have demonstrated that thyrold graft adaptÍon to the host results from the develoPment of tolerance fn the reclpient. As dlscussed by Lafferty et al (1983)' the lnductlon of tolerance for 'leakage tlssue grafts is 'lf ke'ly to develop as a result of the slon of free antlgen lnto the immune system of the reciPient. Several

mechanlsms have been proposed that account for fnductfon and

mafntenance of tolerance by such a methodr resuìtlng ln elther preventlon of T-he'lper cell actfvation, or the predornf nant actfvatlon of T-suppressor cells (see Gotze and Motar l98I). It appears that to]erance ls the result of an actfvely malntained balance between help and suppression and requfres the continuous Presence of lovl leve'ls of antigen and antibodies.

Transplantatlon lmmunology ls a rapidly advanclng field ln whlch controversey ls wfdespreadr and changes in hypotheses of speclflc ¡ I mechanlgns of action occur frequently. The above revievl fs an 69

ln extrqnely slmplfstlc representatfon of the mechanlgns fnvo'lved al'lograft reJection. Horyever ft provfdes a sufffcfent foundation on whfch to proceed wlth,a dfscusslon of Immunologfcaìly Prlvlleged

Si tes.

I.3.3. Immunologically Privi'leged Sites

The immunologfca'lly privf'leged status of the brain' anterior been chamber of the eyer and the hamster cheek pouch has primarily attrÍbuted to their lack of lymphatic drainage (Head et a'lr 1983a)' wlthout lymphatlc drafnage there is an effective b'lockade fn the Barker and afferent lmmune Fêsponser and this lÚas clariffed by are Biltingham (1968) when they demonstrated that if skln lslands raised on vascular pedlcles wlth thelr lymphatic dralnage severed' they then become effective privlleged sites' this The testls however, beccrnes a unique'ly privileged site fn context, because it is known to possess very effectlve lymphatic drainage (demonstrated as early as 1830 by Panlzza)r involvfng 'lymph primarf 1y the i'l lac and renal nodes (Ti'lney, I97t; Mccullough' The drafnage of L975¡ Whftmore and Gittes, L976¡, Head et al, I983b). Fawcett et al the human testis is shown fn Figure 1.I3. Furthermore, (1969)andFawcetteta.I(1973)havedernonstratedthatthetesticular interstltlal connective tlssue fs'laced wlth a comp'lex fine lymphatic network,asulasdlscussedearllerfnthisreview.Thus,withsucha lymphatlc connection, other factors must contrfbute to the lmmunologlcal privilege afforded the testis' and numerous investigators have attempted to define the mechanisrn(s) involved' Obviously, lf some speclfic process can be Figure 1.13. Diagramatic representation of the lymph nodes which

co'l'lect the lymph of the human testic'le and scrotum (f rom DonÍni and Battezatir L9721. 70

lumbo-aortic

common iliac

--'--:.:----

I I I

I iexternal iliac

I I I I

I l--t t---. Ç:í ,,--. í i.; superf icial

I inguinal it ñ ,¡ 'ç I I I

I I 7t fdentifled fn the testls that provldes local fmmunosuppresslonr wlthout the need to admfnfster fmmunosuppressfve drugs whÍch supPress all fmmune functlonsr the consequences for organ t¡ansplantatfon are lmmense.

1.3.4. The Testis as an Immunologlcally Prfvileged Site

The use of the testfs fn transplantatlon studies has a long history and in I77I John Hunter descrfbed the transfer of rfthe testes of a cock Ínto the be'lly of a henrr (see Forbesr L947r. However studfes on the transfer of other tissues fnto the testis aPpear to have orfgfnated early thfs century. In 1935r Gardner and Hlll publlshed work on the persfstence of pituftary grafts fn the testls of the ltìouser and reported many investigators of the tfme to have found the testis to be a favourable slte for ovarian transplants. From these early reports unfolds a fascinatlng tale of fnvestigatfon fnto the

immune privÍ'leged status of the testis' the history of which was revfened by Barker and BlllÍngham In L977. In 1949, Greene successfu'l'ly used the testicle in mice' rats and hamsters as a transplant slte for Brown-Pearce tumors of rabbit orlgfn. However, he

observed that on'ly SO-LOOZI of reciplents grew tumors comPared wlth

100% when the same tumor was transplanted to the anterior chamber of the eye. After perfods varyfng from several days to more than a month, the lntratestfcular tumor grafts regressed and thereafter reslstance to refnnoculatlon of this tumor was noted. Lymphatlc extenslon and metastasfs did not occur. Aron et al (1957) also reported successful aìlo-transplantation of pitultaryr thyroidr ôrìd kfdney tlssue to the guinea plg testfsr and Medawar and Russell (1958) used adrenal 72

iso-grafts ln mfce testes. More recently, Dib-Kurl et at (1975) found that parathyrofd allografts sustafned normal serum ca'lcium levels ln outbred parathyrof dectomlsed rats for ìonger.'than 3 .monthsr f f pìaced

f n the testis. Intramuscu'lar imp'lants in such anf mals hovever fal'led to achieve even transient normocalcaernia, presumably because of early

rej ect i on. The role of hÍstocompatibÍllty was addressed by NaJi and Barker

(1976) when they studfed the fate of parathyrold allografts in parathyroidectomlsed fnbred rats. Survlval of fntratesticular grafts transplanted across hfstocompatibi'lity barriers exceeded by many weeks (L2-L6 that observed for allogenefc parathyrofds ln subdermal pockets days). However, hfstocompatibility Ylas a significant factor since only

20% of grafts in the testes of incompatfbile hosts survived for 150

days, while 62% of grafts ln the testes of a more compatible host survfved for this time. Human parathyroid xenografts were reJected so rapidty that normocalcaqnla tras never seen after Íntratestfcular transplantatlon in rats. Further evídence that the testis is frequently effective, but l¡cgn¿lete.u privileged for other tissue allografts comes from the work of Whltmore and Gfttes (1975) who found that intratesticular skin allografts fn rats were not all protected Ín thfs envfronment wlth 9 out of 29 grafts shorfng hlstologfcal evÍdence of reJection after 26 days. The findings of Ferguson and Scothorne (lg77 arb)r Bobzfen et al (1983) and Selawry and Whlttington (1984) also support the suggestion that the testls Provides effectfve but Íncomplete immunological protectlon. As seen in Table 1.3.r a number of other reports also ffnd variability ln graft survival tlmes even

when the genetlc relatfonshlp of the host and donor fs constant (autografts and lsografts). Table 1.3. Summary of lfterature reports of lntra-testlcular transplants.

d Reference Tl ssue/Graft Used Donor/Recl pl ent Graft lo An ima] s Rel atf onsh i p Du ratf on Success Used Rate

Medawar and Adrena'l Isograft Termlnated at 75 Mice 14 days Russel t (1958) 'a'l 100 Rat D i b-Ku rf et Pa rathy rol d Autografts > 3 months ( 1975 ) Aì 1 ografts > 3 months 100 Rat Xenografts 25days->6 90 Gulnea Plg -> Rat months Rabblt -> Rat

80 Rat Nagi and Barker (1976) Parathy rol d A1 I ografts > 118 days

85 Rat Whltmore and Glttes Parathyrof d Autografts Terminated at 6 weeks ( 1979) A'l l ografts Termlnated at 80 Rat 6 weeks r00 Rat Head et a'l (1983a) Pa rathy rol d I sografts > 400 days Al 1 ografts > 25 days 50 Rat > 200 days 25 Rat

Xenografts Ave. 30 days 100 Rat -> Mouse Bobz i en et a'l ( 1983 ) Pancreatf c Is'lets (¡{

cont/. . . ø Reference Tlssue/Graft Used Donor/Recl plent Graft m An f ma] s Rel ati onsh I p Du rati on Success Used Rate

Is'lets I sografts > 90 days 75 Gufnea Plg Ferguson and Pancreatic Scothorne (1977arb) Al 1 ografts up to 82 daYs 75 Gulnea Pfg 100 Gulnea Pig Skl n Autografts 56 days Plg A1 1 ografts L4/2I/28/47 days L00/80/L5/0 Guinea

85 Mice H i'l 'l and Gardner Pl tu f tary Isografts Term'lnated at 3 weeks ( 1936 ) Aron a'l (1957) Anterior PftultarY Homografts Unknow n 100 Gulnea Pig et 80 Gulnea Pig Thyrol d Homografts (> I week) Plg 0vary Homografts Not stated Gulnea (1940) Papill ary Tumour (8240) Homografts No take Nil Rabb i t Greene Ac'lnar Type Tumour (T36) - Prlmary tumour Homografts No take Nfl Rabb i t - Lymphatlc extenslon Homografts No take Nfl Rabb i t - Splenlc metastasls Homografts No take Nfl Rabb i t Mlce Greene (1949) Brown Pearce Tumour Xenografts Varfabl e. L2 50-I00 Rabbit -> days to more 50-100 Rabbit -> Hamster than 2 months 50-100 Rabbit -> Rat Pig Nil Rabbit -> G. -J 5

cont/. . . d Tlssue/Graft Used Donor/Recfplent Graft lo An imal s Reference Re1 atl onsh f p Du ratl on Success Used Rate

( Wflms Tumour Autografts Termlnated at r00 Wistar Rat WaJsman et a'l 1980) 14 days A'l ì ografts Termlnated at 0 Lew ls Rat 21 days A'l'lografts + Testosterone/ Termlnated at 60/80 Lewls Rat 0estradl ol 21 days 70/50 Flscher Rat A'l'lografts Termlnated at 100 Fischer Rat 2I days

Renal Cel'l Adenocarcfnoma Autografts Termlnated at 0 Mice 14 days A1'lografts Termlnated at 0 Mice 21 days A1ìografts + Termlnated at 0/0 Mice Testosterone/ 2l days Oestradl ol

Neurob'lastoma Autografts Termlnated at 90 Mice 14 days A1 I ografts Termlnated at 40 Mice 2l days (¡{

cont/ ... o Reference Tlssue/Graft Used Donor/Recl plent Graft lo An ima'l s Rel atl onsh i p Du ratl on Success Used Rate

Wajsman et al (1980) Neurobl astoma A'll ografts + Termlnated at 40/60 Mice Testosterone/ 2I days 0estradi ol Rat Akimaru et a'l (1981) Insul i noma Al 1 ografts - normal > 50 days 6 - dlabetlc 20-30 days 15 Mlce Hogan (1981) Teratocarci nomas Isografts Unknown Not Speclfled rrCells contlnue to grow and gfve rlse to tumoursrf

Rat Canivenc (1936) Pl acenta Isografts Termlnated at 100 5 days Rat Takewakl (1956) Vagi na Isografts Termlnated at r00 I month Isografts Termlnated at 100 Rat Takewakl (1957) 7 weeks Rat Takewakf (1958) Vaglna + OvarY Isografts Termlnated at 100 { 2 months o\

cont/... o Reference Tlssue/Graft Used Donor/Recf plent Graft /o Anfmal s Rel atl onsh f p Du ratl on Success Used Rate

Mice Bl'll f ngton (1965) Morul a A'll ografts 20 Termlnated at 62 Mice Bl astocyst A'l l ografts 15 days Ectop'lacental cones A1'lografts 94 Mf ce Mice Klrby (1963) 81 astocyt Al I ografts - scrotal testes > 14 days 90 - cryptorchid > 14 days 94 testes

{ aq1, q+ln aq+ u! uJP oq1, 6u¡eq qu¡t t Jo+ralJa Jo luaJa¡la 's!+sol oq+ +Ptl+ asodo¡d a^llcP [[l+s s¡ uels'ts c¡1eqduÁ[ aqî. ]o lxJP +ueJo]]P uP ]o acus+slxo pup .a[qec! ldde 1ou 1sn[ s! apelcotq ounu¡ul! +uoJo]]P so++19 pue aJotrr]ltlil 'apou qduÁ¡ gec¡6oI otl+ +Pql po+seô6ns eneq (9¿6I) /(¡l'ro¡¡un po¡ sc¡leqdur(¡ luale¡¡e c¡1eur6e.rqde¡pqns ouo +seo[ +P o+ s!+so+ at{+ }o a6eu¡elp c¡leqdur([ aq+ +prll puno¡ q+oq puP 's+PJ u! pup (I¿6T¡ ÁauL}I po!pn+s os[P o^Pt'l (5¿6Ï¡ q6noILnCct/{ 'Ja^a'ioH'(896I) uPq+ Á'roaq1 oLqlsnP1d alou e '{¡ute+Jo3 ILossnu PuP JP','rPpari'ì¡ ]o lPtl+ o+ paLlpJ luan[]]o cllPqduÁ¡ spn l.r .apou qdur,(¡ Áup es¡s¡supJ+ u! s!1¡so+ aq+ pêpJo¡¡e eq Á1uo Je[nc!+so+ oq+ qclqil u! sLellllue osot{+ peltodel satcuo+slsuo3u! ps+ou oq+ Jo] plnor,l e6e1¡n¡ld acur', '1"![JPa clcPJotl+ oq+ o1 Á11ca'r¡p pessed pue lled ul +unocc. p[no"t s!t{I 'lcnp uloJ] slossa^ cl+er{duÁ[ ,opou qd'rr{¡ p asJo^sueJ+ o+ poIle] s]lsa+ aq+ pellodal 1ase6u3 uI aq1l .palpnxs s+PJ S9 Jo 6T u! lPql '696T qdur(1 6u¡u¡e'tp 'saJloulllua3 6T Jo ssocxs ul Jo epou ue e1¡dsap rpelce[e't lsoJeou aq+ 01 Áer'rq1ed c¡1eqduÁ[ +uaJa]]e sdl+ oq+ .leeu /(¡¡ec¡do1oq1'to Á11d,ro.rd aJor,r s[!pl +eJ +[npp ;o po'þtls /(aq1 ueqn (€¿6I¡ ueq6ul[llg palue¡dsue.t1 s1¡elôot[e u!ìs +Ptl+ a+eLd oos¡ Áaup¡¡ aq1 pup JaxJpg r{q peouallpr{c sen,(loeql slqr'(r atll pue ue6'to aql uaa''qoq ¡'o [a^oI ot]+ +P epou qdtuÁ¡ 6u¡uleJp 1.saJeau uP asnPcâq +uo!c!]op s! s!+so+ otl+ Áenqled cllerldu¡Ál 6uo¡ Á[tensnun ]o ¡oa6eulPJpc!1.PqdruÁ[aq+./(Joaq+sltllo+ôu¡p.toccy.856Iu1[Lossnu se',r e6e¡¡lr¡.rd slr{+ Jo} uo¡1eueldxe puP JP,.rPpoh¡ ,(q pesodo.td ,(t[p!+!u! aql 's!+sol oq+ pap¡nold eôe1¡n¡ld eunuutl pollo Á¡1uenbe.r, +sot¡l ^q arl+ o+ papêou set't Áloaq1 atll Jo suo!+Eclpu! [P+uol¡¡!Jodxa lloddns aq+ c¡leqdurÁ[ oq+ ocuo ' Äau p rpet,eJlsuoulop sP' sllsel Jo'IJon+au

solJoaql 'S'€'I s!lsa+ otll Jo sn+P+s pe6el¡r¡'ld arl+ JoJ

8L 79

ma'lfunctfoning. Thfs fmplfes that antlgenlc material can reach the response-lnitiating 'lymph node, but for some reason the effector cel'ls are not producedr or do not retur'n to the graft s!te' " Ferguson and Scothorne (1977 arb) demonstrated that whfle first-set fntratesticular grafts rarely fnduce systemfc lmmunlty ln the host, second-set intratesticular grafts were usually rejected fn the typlcal rapid second-set fashf onr succußìbing to a preexistf ng systemic lmmunlty generated by ffrst-set skin grafts. This suggests that both arms of the fmmune system can be actlvated under the right condftions. Reports of high leve'ls of gonadal infiltration by leukaemlc T-ce'lJs in cases of acute ìymphoblastlc'leukaernfa wlth prfmary testlcular relapse (Reid and Marsdenr 1980; Eden et al' 1978;

Tfedmann et a'l , Ig82) also lend support to this suggestion sfnce the

efferent immune resPonse must be rractiverr to allow such lnflltratlon' It seqns fn effectr that the fmmunotogical'ly prfmed male host cannot be regarded as possesslng any degree of prfvflege in the testis (in relation to tissue from the donor against whfch lt ls prlmed)' where as the unprÍmed host does have thÍs prfvi'lege' and rernains rrunprlmedrr after flrst-set lntratestlcular transplantatlon' Thfs

lmpl ies that factors in the testfs, or between the testis and any given dralning lymph node somehow suppress the recognftfon of testicular-derfved antigerìsr and thus prevent lnltiation of the lmmune

response. The studfes of Tllney (197I), McCullough (1975) and Whitmore and Gittes (1976) report the investfgation of 'lymphatfc connectlons of the testisr and demonstrate that the lymphatics aPparent'ly comblne to enter a lymph node Yúithout connectfng to other organs or vasculature' This suggests that very lfttle happens fn the afferent lymphatic 80

vessels leavlng the testls whlch cou'ld prevent antlgen recognitlon ln the tymph node. That the given nodes a'lso recefve lymph from other sltes not consldered fnrnunologfcally prlvlleggd'(eg. scrotum) meang that they are probabty not speclffcally involved in conferrfng the

fmmune privileged status on the testls. Likewfser the testfcular route of exposfng the host to antigen is not in itself immunosuppresslve as 'lack fsr apparentlYr the anterior chamber of the eye where the of lymphatic dralnage results fn exPosure of the hosts lmmune system to graft antfgens by the vasculâF Foutêr with subsequent systemic suppression (Kaplan and streilen, 1978; Whittum et a'l , 1982¡ Strei'lein et al, 1980). Indeedr Parental straln lymphocytes inJected lnto the testes of F, hybrfd males fnduce graft-versus-host reactions fn the drainlng 'lymph nodesr and intratesticular innoculations with a'llogenelc lymphold cel'ls result in norma'l systemic antfbody resPonses (Head et alr I983c).

Recent evidence that sfgniffcant events may take p'lace at the slte of exposure rather than fn the regfonal tymph node ln certain grafting sltuatlons fs of signfficance fn this regard. Pederson and Morrls (1970) emP'loyed heterotoplc sheep kidney alìografts with chronic fistulas ln the rena'l lymphatics to prevent draining antigen or ce'lls from reachlng the regional 'lymph nodes. Neverthelessr the lymph soon contained stlmulated lymphocytesr and the graft became infiltrated with tymphold cells and was promPtìy reJected' Ascher et al (1981) have shown that cytotoxlc Precursor cells recruited to

sponge matrix allograftsr undergo pro'l iferatlon and maturatlon to itself. cytotoxfc effector cells '',Íthin the graft sfte All the evldence suggests that lmmune privi'lege fn the testfs

depends on loca'l factors f nterferlng wfth these Processes to inf'luence 81 the lnltfatlon and development of the reJection resPonse' Although benefícial for graft réclplents, the sltuatlon may be detrfmenta'l for 'fmmune survelllance and may'exP'laf n why the testls' llke the b¡'aln' ls (see a maJor slte of recurrent tumours ln male leukaemlc patients díscussion earlier, and Baum et al t 19791. In the earller part of thfs revlew, I dfscussed the existance of a blood-testis barrfer around the se¡niniferous tubules. Thls barrler is capable of excludfng from the and lumen of these tubules many substances normalìy present in blood of lymph, including antÍbodies, and at the same time preventlng escape autoantigenic materfal assocfated with maturlng components of the germinal eplthelfum (testlcular antfgens). l{hile numerous studfes on auto-immunity in the male have stressed the importance of this barrÍer (see Tung, I98O for a detaf'led revlew on thls topic)r it ls unlikely that lt plays any dlrect ro]e ln mltÍgating host reactlvity to L977) Since such f ntratestf cu'lar allografts (Barker and Bil'lf nghamr ' grafts are lnvarlably placed in the interstitial region of the testfs' this is where attentfon must be focused. A'lthough the testicular caplllarfes and venules are unfenestratedr they do allovr diffuslon of fn immunog'lobu'lfn G (Manclnf et al, 1965). As already discussed detailr the lnterstitiat space contalns extensfve lymphatics and islands of blood vessels surrounded by Leydig cellsr macrophagêS¡ llìâst cells and plasma cel'ls. Resfdent macrophages have been isolated from the perfused testis (Tung et al t L979)r ôrìd the Leydig ce]l-macrophage ratlo ls about 4:1. Circulatlng lymphocytes do reach the testfcular lnterstltlum, and from there they enter the tymphatlcs and can be (Morris' recovered by cannulae placed fnto afferent lymphatlc vessels 1968). Thusr humoral antfbody and recfrculatfng lymphocytes attendant space on a systemlc immune resPonse can readlly reach the lnterstitial 82 of the testls (Tungr 1980).

Head et al (1983c) revfewed several possibilftles for local suppresslveagents.lnthe'testfcular.mf.|lei¡.Themostcomnonlyclted candldate ls the Leydlg ce]'tr whfch pnoduces sterolds ln the fnterstftial tissue. There is substantial evldence that a number of androgens are lmmunosuppressfve (Clemens et al t 1979; Roubínlan et a'l'

Lg77) a'lbeit i n extreme'ly hf gh concentratf onsr and the reportedly high levels of testosterone produced by Leydlg cells ln the testis (certalnly hfgher than levels of this hormone seen e'lsewhere fn the body) have led to speculation on thefr role as local supPressors of the fmmune response. However, androgen fmmunosuppression is a contioversfal subJect (see Cohnt L979)r ârìd other factors need conslderatlon. Leydfg ce]ls produce other products such as Progesterone and Prostaglandinsr both of which can also be demonstrated to be lmmunosuppressive. A'lso, reports that Leydig cells wlll spontaneously adhere to lymphocytes and nonspeclffca'lly suppress prol lferatfon of mltogen-stlmulated and alloantigen-stlmulated ìymphocytes fn vftro (Born and }lerkerle, 1982) may be of consequence' It remafns posslble thereforer that suppresslon of the fmmune response by local hormones ls a feaslb'le explanation for toca'l immunological prlvilege and Head et a'l (1983c) suggest that thfs mechanlsm may also explain both the increased number of infections and the somewhat prolonged graft survival noted by Frenkel (196I) in the adrenal

g1 and.

The germ cell popu'lation ftself is another cadldate for provldfng (1982) the lmmune prfvl'lege of the testis. Shearer and Hurtenbach describe the depressed response of human and mouse lymphocytes to

mitogens in vitro in the Presence of spermatozoa or their Precursors' 83

t¡lhen mlce are lnJected f ntravenously with syngenefc sperm¡ T-cell responses to both a'l'logenelc lymphocytes and hapten-modlffed self are lmpalredr and Hurtenbach and Shearer (1982) found the 'latter to be controlted by an induced T-suppressor cell. It is of interest ln this regard then, that Anderson et a] (1984) could not detect any lûlC transplantatfon antigens on post-mefotic germ cells. The lower temperature of the testis has also been proposed as a possfble factor conveylng immune prfvilege (see Bobzlen et a'lr 1983) although Selawry and tr|hittlngton (1984) reported that the lower temperature of the testis may in fact be detrimental for the survival and functfon of some grafts. Semfnat p'lasna from several species has been shown to suppress the in vitro stfmulatfon of T and B lymphocytes (Stltes and Erickson, L975; Prakash et al , L976i Lord et al, L9773 Marcus et a'l t 1978i

Anderson and Tarter, L982¡ Peluso et al' 1986). Tarter and Alexander (1984) have reported semÍnal plasna samples of Prfmarf'ly prostatlc' vesfcular, or epidfdymal/testicular origln as u{ell as samples from vasectomÍsed men to al'l contain comP'lement-f nhibiting activity' although epidÍdymal fluld contafned nearly twlce the amount as other

Sources. That samples from vasectomlsed men showed the same leve'l of inhÍbition suggests that secretion comes from the prostate and ssnlnal vesicles, as well as from the testis and epidfdymis. However the testicular contribution may be fmportant fn maintaining the immunologfca'l barrler provided by the Sertoli cells for the developfng germ celìs, and Tarter and Alexander (1984) cfte evldence of a high mo1ecular welght antl-haemolytf c factor secreted by Sertol I ceìls ln tissue culture. Secretfon of such factors fnto the fnterstitia'l regfon might be of consequence for fmmune function in this regfon. 84

Final'ly, the evldence that culture of tlssue prior to transplantation wl'l'l greatly lncrease the chances of graft survival is of lnterest. Lafferty (1980) and Hart and Fàb¡"e (1981) have demonstrated that Passenger ìeukocytes, and partlcularly dendritic cells with abundant Ia antfgen, contrfbute signfffcantly to the immunogenÍcity of allografts. The process of cu]ture rernoves such cells, and dimlnishes the effect. Thus, whlle sorne sPeclffc process might operate local'ly in the testis to provide inununosuppression' one might envf sage the testis as providing a culture]Íke environment where the graduat replacement of donor passenger cells by host ceìls would sfgnlfÍcantty weaken the capaclty of a graft to evoke a reJection response. This might in fact allow induction of tolerance in

the hosts Ímmune systemr wlth the continued presence of low amounts of donor antlgen.

1.4. THE PURPOSE OF THIS STUDY

The aim of the present study was to reexamÍne the immunologically-privfleged status of the testis. InitÍally' work was carrled out in rats to confirm the privileged status of the rodent testfs. Once a routlne transplantation protocol was establishedr various aspects of testicular functlon were modlffed experfmentally to

examlne those aspects of potential importance for allograft survival in this sfte. All previous work in thfs area has utilised rodents and related specfes. These animals have a testfcular interstltial anatomy that is different to that of larger mamma'ls. To allovl lnvestlgatÍon of the mammals' lmmune state of a testls more representatlve of the larger 85 thisstudyrÚasextendedtoincludethesheepasanexperfmental mode'|.

Follovring these studfes on graft survfval' a number of of experfments were conducted (prlmarily in rats) with the aim elucidating factors Ín the interstitial milieu of the testis that might contrfbute to the immune privilege afforded thfs region' to be Existing techniques for samp'l lng interstf tia'l ftuid were found generaìly unsatisfactory, and potentially unphysiologica]r ôfìd a new technique involving the use of a Push-Pull cannula ldas tested' A cannula was number of experÍments were performed where the push-pul1 i n whÍch norma'l used to samp'l e i nterstitf a'l fl ui d f rom rat testes functlon was aìtered by varlous experimental manfpulatÍons' The these technfque was also tested in sheep and pigs. The results of studles have led to the development of a new hypothesfs concernfng physfologfcal aspects of hormone secretlon by the endocrfne testis'

in The opportunlty to collaborate with Professor Mikko Nlemi Turkur Finlandr at this time allowed a study of the immunocompetent cells ln the testis of the rat and the ram. ThÍs revealed potentia'lly important aspects of Ímmune function fn the testis that might contribute to an lmmunologically prÍvlleged environment' and the present study concludes with a new proposal as to why the rodent

testi s i s an immuno'logf ca1 1y priv f I eged si te'

CI-IAPTER 2: MATERIALS AND METHODS 87

2. t REAGENTS

LHRH, TRH,, l--Thyroxlne'. Human Chorlonfc Gqnadotrophln and Testosterone were obtained from sfgma chemlcal co.r st. Louisr

Missourir U.S.A. TRFI for inJectÍon vÚas obtalned from Roche Dlagnostlca 3H-Testosterone, l25l-l-fhyroxine, (protirel in). Radlolabelled Sodf um 13tIo¿ide I25Iodid", and Sodfrr (InJection BP) were purchased from were P¡nersham (Australia) Pty Ltdr Sydneyr N.S.|r|. Qther materlals obtafned as follovrs: Heparin from Commonwealth Serum LaboratorÍes' Melbourne, vÍctorfa; sodium pentobarbitone (Nembuta'l, Abbott) from

ceva chemfca'ls Australia pty. Ltd.r Hornsby, N.S.tll.t Polyethelene and EngÍ neeri ngr po'lyv i nyl ch1 orl de cannul ae tubi ng f rom Dura'l P'l astlcs and Dural, N.S.W.. A'l'l so]vents used were analytfcal grade. Tlssue cu'lture medÍa were obtaf ned from Gibco Laboratoriesr Grand Is'land' New Yorkr and from the commonwealth serum Laboratoriesr Parkvil'ler Melbourne, VictorÍa.

2.2 ANIMALS

Rats were of the Porton strafn bred fn the Adelaide unÍversity central anlmal house and were not Ínbred; sheep used were of the Merino strain, bred on the unlversftyrs Mortlock experiment statlon

and were also not inbred. 8B

2.3. SURGICAL PROCEDURES

2.3.L. ThYroldectomY

Rat Thepairedthyroldglandsaresmallpinkorgansroneoneither sideofthetracheaJustbe]owthe.|arynx.Theyareconnectedacross the ventral aspect of the trachea by a thin band of tissuêr the isthmus.Becausetheminuteparathyrofdglandsareembeddedfnthe anterfor part of each thyroid gland and cannot be satisfactorÍly a removed from them, the re¡noval of the thyroid is technlca'lly

thy ro-parathY roi dectomY.

Each anÍmal was anaesthetised with an intraperltoneal lnjection of pentobarbitone (60 mg/kg bw; Nembutal, Abbott) and lald on its back was wfth its head toward the investigator. A midtine skin incision madealongthelengthoftheneckfromltsbasetojustbelowthe point of the lovler jaw. The subcutaneous fat and connective tissue

were cleared and the salivary gìands separated and retracted latera]ly.Thetwohalvesofthesternohyoidmusclewerealso for this separated and retracted laterally, using a paperc'lip reformed aspect purpose. The thyrohyoid muscle runnfng a'long the dorso-lateral dfssection and of each lobe was separated from each gland with blunt then cut retracted aìong with the sternohyold muscle. The fsthmus was the in the mldllne and the connective tissue between each g'land and gland away from trachea carefully cleared by repeatedly pushing the edge of the the trachea. Extreme cautfon is needed near the dorsal Severfng of gland slnce ft runs along the recurrent laryngeal nerve' respiratory thÍs nerve can cause paralysfs of the larynx leadlng to B9

Each gland was impalrment whlch can be fata'l (waynforth, I98O). finally severed from lts vascular connectlons anterforly and posterforlY and removed.

SheeP Thethyrofdofthesheepisareddish-brownco]ourandis end of the sltuated on the]ateral and ventral surfaces of the cranial lobes trachea (May, 1964). As fn the rat, it consists of two lateral unitedatthefrcauda.|endsbythefibrousorg.|andularfsthmuswhlch lobes passes across the ventral surface of the trachea. The lateral or eleventh extend from the ffrst or second tracheal ring to the tenth rfngandmeasure4to5cmlongandltol.5cmwfde.Theyaresmooth, to the elongate and oval fn outllne. The gland is loosely attached sternocephallcr trachea and ls covered'latera'lly and ventra'l1y by the omohyoldandsternothyrofdmusc]es.Onepairofparathyroidsare and are sftuated on the deep face of each lobe near the caudal border also lnevltably removed durÍng thyrofdectomy. Hoveverr rumfnants the thyrold possess a paÍr of external parathyrofds found cranfa'lly to tota.| gland, and thus thyroldectomy fn sheep does not also fnvolve parathyroÍdectomY, as in the rat' Forthyroldectomy,eachanima]wasfastedfor4Shoursbefore Sur$er}randkeptoffwaterfor24hoursbeforesurgerytoreduce because durfng flufdlty of rumen contents. ThÍs is fmportant in sheep' head is the operation the anlmal ls placed on lts back and the rumen contents to sometlmes posftloned lower than the rumenr câusing of thfs flow back up the oesophagus durlng anaesthesia' The llkellhood Anaesthesfa can be greatly reduced by the above preventative measures' pentobarbltone (60 mg/kg was lnduced by an lntravenous lnJection of 90

admfnistered as bw) into the Jugular and malntafned by pentobarbltone veln' To insert this needed through a cannula in the lateral saphenous laterally' cannulai a skin tnclslon was made fn one of the r:ea¡:.legsi abovethetarsalJointatthedfsta]endofthetlbia.B]unt veÍn dÍssectfon through the fascia revea'led the latera'l saphenous (recurrent tarsal vein, see May, 1964). A section of this vefn v{as threads p'laced cleaned of connective tissue on all sides and two silk proximal to the around the vessel. The first thread t{as securely tÍed proposedpointofinsertion,agnaì.lcutwasmadeÍnthewallofthe (0'D' I'Smm' I'D' vein above the first tle and a polyethylene cannu'la 1.0mm)fntroducedlntotheveinforadlstanceof30-40cm.The veln and the enc'losed second thread was then tied securely around the cannula in position' cannula above the fnsertlon pofntr to secure the Amid-ventra]lnclsfonYJasmadefntheanterforpartoftheneck facia startlng Just behfnd the crlcoid carti'lage. The subcutaneous coverfng the gland was separated by blunt dfssection and the muscles The thyroid fn were separated at the mldline and retracted laterally' btood supplied from the sheep is exceptlonally we1'l vascularised, wfth pairedposteriorandanterlorthyroidarterieswhlcharisefromthe carotidartery.Venousdralnagelsmalnlythroughthepairedanterfor thyroldveinswhlchJointheJugularvefn.Each.lobewascarefully and the trachea' and separated from the surroundfng connectlve tissue both lobes and the all b'lood vessels lfgated prfor to rernoval of isthmus.Mfnorbleedfngwascontrolledwithgauzeswabs.Before c]oslngtheskinlnclsionrretractorsandstlabst{ereremovedand posltions. As Ín the ratr no musc.les alloned to return to their normal suturing of the muscles Ylas required' 91

2.3.2. HypoPhYsectomY

The pltultary gland i,s a smaJt plnk organ found at the base of the brain. It is 'located in a srnall bony fossa, the sella turcica' which is in the sphenoid bone of the craníum. The pftuftary fs in attached to the floor of the thfrd ventricle by a thin stalk and is close relationship with the optic chiagna'

Rats

Rats were hypophysectomlsed by Mr J.L. Zupp using the intra-aura'l to method of Gay (1967). The specfal stereotaxic apparatus requfred perform this operation successfully was obtained from the Stoeltfng

Companyr Chicago, UsA. Each anlma] was anaesthetfsed by an intraperitoneal inJectÍon of Avertin (0.8 m1/I00 g bw)r which fs no longer available commercia'lly but can be constituted from a solutfon of trlbromoethanol and amylene hydrate as described by Dyer et a'l (1981). Thls relatlve'ly short acting anaesthetfc was found preferable to pentobarbÍtone. The anfmal was p'laced on its ventral surface wÍth the its tall away from the operator. The cartllage at the base of externa'l audftary meatus was cut wÍth a pafr of scissors' the animals teeth pìaced lnto the tooth holder, and the ear bars lnserted into the containing ears. An 18G thin-wa'lled needle mounted on a 10 ml syringe 2-3 nls of physiologfcal saline was inserted into the hole fn the ear 50 to bars. The needle ,,,as pushed along the ear canal at an ang'le of the horizontal fn a dorsal direction untfl it touched the media'l wall of the periotic capsule where the bone is particularly thÍn' The upwards needle tÚas then forced through the bone with the bevel facfng damagfng it' so that the pofnt slid under the trfgemfnal nerve wlthout 92

The needle was then pushed fonvard a predetermfned dfstance (establlshed ln sacrificed animals of the same slze' with the brain

removed) so that the.bevel 'could be turned down to be sltuated directly over the pituitaryr un¿". the sella turclca. The gland was then slowly aspirated fnto the water-filled syrÍnge and carefu'lly vltaìly examfned to ensure comp'lete removal. Post-operative care fs Anlmals important to ensure a 'low mortality rate from hypophysectomy. so]utfon were kept warm after the operation, and given a 5% dextrose Thls was ad I ibitum to prevent development of fatal hypogtycaefiria' (Pfizer Pty routfne'ly combÍned with a Terramycfn solution Agrfcare

Ltdr West Ryde, NSW) to reduce infections' To obtafn tissue for transplantatfon puFposesr donor animals were sacrÍficed using an overdose of chloroform. The skfn on top of the tissue and skull was rqnoved, and the skul'l scraped free of connective the skull fat. uslng a denta'l drlll with a round bone burrr the top of was scoredrcut and llfted off wfth the dura. MaintaÍnlng steri'lity' The area the braln t+as removed to allovl access to the sella turcica' sella was was carefully cleaned and uslng sterÍle flne forcepsr the and llfted to expose the pitultary. This was then carefulìy removed (Gibco' placed into sterile Dulbeccors Phosphate Buffered Saline Grand

Island, New York) ready for transplantatfon'

Sheep sheep' The pituitary is an frregu'lar conical shaped structure ln It fs slight'ly reddish-brown and fs relatlvely larger in sheep than fn other domestic anlmals (May, 1964). Pltuftarfes were removed for transplantation from sacrlficed Merino males castrated soon after and the top of the bi rth (wethers). These anfmal s were decapitated, 93

sterl]ity was skull removed with an electrlc band-saw. As ln rats' carefu]lymaintafned.ThebraÍnwasrqnovedtoexposethese]la The pitultArY turclca whlch was carefully 1f fted rr1Íth sterile forceps' wasthencarefu.|lyremovedandp.lacedlntosterfleDu]becco|sPBS ready for transPlantatfon'

2.3 .3. CrYPtorch i dectomY

(see 2'3'I') Each animal was anaesthetised wlth pentobarbitone Ínvestigator' A srnall and laid on its back with the tail toYards the vfsua'lise each medlan incision was made fn the skln of scrotum, to each testis testis fn its muscular sac. Each sac was cut opefìr and abdominal cavity' The pushed up through the ingufnal opening into the ingulnal canal on each sfde was then sutured closed'

2.3 .4. Ef f erent Duct L i gati on

Totietheefferentductsfnratsreachanimaìwasanaesthetised withpentobarbitone(see2.3.1.)and]afdonitsbackwiththetal] through an abdomlnal towards the investÍgator. A testls was exposed the testis by inclsion, and the head of the epidfdymis lifted from carefu]lydlssectingtheJoiningconnectivetÍssueonthe]atera.l one sfde to side. This a'llowed the epidfdymis to be ro'lled over to exPosetheefferentducts.Theseweredissectedfreefromthe (4/0 sflk' surrounding fat and occluded by tying one lfgature ducts to the testisr cyanamid, Austral ia) dista'lly along the efferent andanotherllgaturearoundtheductsveryclosetothetestis.The testiswasthenreturnedtothescrotum,theabdomina]incfsfon 94

closed, and the anfma'l allotved to recover'

2.3 .5. Bl ood Col I ectl on

2.3.5.L. Rats

vefn ín Peripherat blood samples were taken from the iugular non-recovery anÍmals' recovery anfmals and the posterlor vena cava in with pentobarbitone For Jugular samples, the animal was anaesthetised (see 2.3.I.) and'laid on its back with its head towards the the jugular vefn investÍgator. The base of the neck YJas opened and exposedthroughthepectoralmuscle.A2IGneed]eattatchedtoalm] syringewasfntroducedfntotheveinrandb]oodwfthdrawnslowly. Afterremova]oftheneedlertheskfnlncfslonwassutured.In il after anaesthesia was induced wfth t,,. non-recovery anlmalsr )l taf'l towards pentobarbftone¡ the animal was lald on its back with its wall the investigator. A V-cut was made through the skin and abdomina'l across startfng at the base of the abdomen and proceeding dfagonally the thorax' The flap each side to end up at the dorso-lateral edges of entÍre gut ref'lected' of skin u{as moved onto the chest wall and the (near the leve'l of the The vena cava *,as entered at the wÍdest pofnt kidneys)usinga2IGneed]eattatchedtoa2mlsyringe,andb]ood withdrawnslowly.Inbothsituationsrbloodsamplesu,eretransferred toheparinfsedEppendorftubesrandplagnaseparatedbycentrffugation in a Beckman Mfcrofuge. t Testicularvenousb.loodsampìeswerecollectedfromthevelnson I I exposlng the testfsr usually via a i the surface of the testls. After the testlcular scrota] incisfon, the pofnt was ldentified at which

! 95

separatfng agafn to vefns merged on the surface of the testls prfor to vein was form the pampÍnfform plexus. using a 27G need'le, the largest puncturedatthepolntofminfmunbranchfng.TheneedlevJasremoved, Dade Diagnostics' and heparÍnlsed mfcrohaematocrlt tubes (capfllets, puerto Rfco) were held against the resu'ltant pool of blood florlng col'lected from each from the vein. In most côsêsr ten such tubes were to a plastic testfs. The contents of these tubes were then transferred plasna a Beckman Eppendorf tube and centrifuged to separate the in

Microfuge. oC In all cases, plasna was stored at'20 untÍ1 assayed'

2.3 .5.2. SheeP

from the Peripheral b'lood samples ïrere taken ln consclous anfmals } 10m1 it{ using a I9G needle attached to a ),t Jugular by venfpuncture, ¡! required syrÍnge. In those experiments where repetitive samplÍng was an indwelling fn consclous animalsr the jugular r,as cannulated with prÍor to polyethylene cannula (I.0. I.00 mm, o.D. 1.5 mm) on the day were samplfng. In anaesthetlsed anÍma'ls, perlpheral blood samples takenfromtheposterlorvenacavavfaacannu.laposltionedina

'lateral saphenous vein as described i n 2'3'L' animals Testicular venous blood was obtafned from anaesthetised (I'D' 0'8 mmr 0'D' fn which an fndweltlng polyvfny'lchloride cannula spermatlc veln (see I.2 mm) was posltioned fn the ipsl'lateral lnterna'l Flgure 2.L.). wlth 0'9% l After drawfng a blood sample the cannu'lae were flushed I sterlle salfne contaÍnlng heparin (50 I.U./ml). Blood samples were collected fnto I0 ml heparÍnized centrffuge tubes and usually

r Figure 2.I. Lymphatics draining the testis fn the ram. The animal was kil'led 15 minutes after receivÍng an injection of Índian ink into the testis. The course of the'lymph (black) from the testis to the 'lumbar 'lymph node (1.N.) Ís shown' The intestines have been removed. The deep inguina'l node (D.I.N.) remained unstained (from LÍndnerr 1963). A. Exposure of testlcular Iymphatics in the spermatic cord. Lr lymph vessels containfng indian Ínk; PL' pampiniform plexus drafned of blood; CR' cremaster muscle; T.V.r tunica vagÍnalis' reflected; VAS, vas rd rq i deferens, retracted; X' level at which lymphatics and internal spermatic veÍns u,ere cannulated; I.R., external Ínguinal ring. B. Testicu'lar lymphatics in inguÍnal canal and abdomen. ING.r inguina'l canalr exposed; VASr vas deferens 'lymph curvlng around ureter (UR); L.N., lumbar node; V.C., posterfor vena cava; H.L., lumbar haemoìymph

nodes.

I {r

r 96

HL.

B v ¡

UR.

NG VAS A l_ A ú ,7- r

VAS

T.V

v 97

centrifuged fmmediate'ly. If there was an appreciable tlme lag between The plasna collection and centrlfugatlon the samp'les were kept on ice' required separated after centrffugatfon were stored at -20oC, until

for assaY.

2.3 .6. LYmPh Col I ecti on

in which Testicular lymph was obtained from anaesthetised animals apolyvinylchloridecannu]a(I.D.0.Bmm,o.D.I.2mm)wasposltioned (Llndner' L967¡ in a lymphatic vessel in the spermatic cord L963t to have this Covie et a.l , L964; see Flgure 2.L.1. It was lmportant and cannula 'loaded wÍth heparinised sa] f ne at the time of insertion' to ensure that lymph f'lowed freely after insertion' i 2.3.7. Rat TransPl ants

2.3.7.L. Under the KidneY caPsule

(see and Animals were anaesthetised wlth pentobarbftone 2'3'1') the tissue shaved on the flank over the kidney destfned to recelve by graft. An lncisfon was made fn the f'lank and the kÍdney exposed pair of gently iso]atfng it from the surroundÍng fat. using a sharp made in the kfdney forceps and maintaining sterilÍtY, a smalt hole was graft was capsule and with the aid of fine toothed forceps' the tissue damage the pl aced beneath the capsul e. Great care uras taken not to kidneyltse]f.Theorganwasthenreturnedtotheabdomenand carefully reposftioned in the fat cover' The fncisfon was then

cl osed. 98

2.3.7 .2 Into the Testi s

Animals were anaesthetised with pentobarbitone (see 2.3.I) and shaved on the abdomen. A mfdlfne incfsfon was made fn the abdomen, and the testÍs gent'ly pushed up from the scrotum fnto the abdomen' to be exposed through the lncfsion.

Whole tissue grafts

when transp'lanting a píece of tissuer a 25G needle l{as used to puncture the tunica and ffne toothed forceps used to Ínsert the tfssue graft into the testicular parenchyma. lllith this technique' suturfng of the tunica was rare'lY needed.

Isol ated cel I s If the graft consisted of Íso'lated ce'lls, these were loaded into a I ml syringe fn a srnall volume of carrÍer bufferr ôrd usfng a scalp-vefn needle setr the tunica ¡1as punctured and the needle of the scalp-vefn set pushed into the testicular parenchyma and the cells gently expelled. The sca'lp-veln needle was then wlthdrawn. As with

whole tfssue grafts, suturing of the tunfca I,as rarely required.

After transp'lantation, the testis was carefully returned to the scrotum ensurlng that the spermatfc cord was not twisted in the process. The abdomfnal fnclslon was then c'losed' 99

2.3.8. Transplants into the Ram Testis

The ovfne testis contafns much more connective tÍssue than the rodent testfs and tfssue grafts cannot be simply pushed Ínto the testlcu'lar parenchyma. consequently, a dlfferent method for transplantation had to be developed. The tlssue to be transp'lanted was ¡roxf mate'ly I mm3. The testis was part'ly exposed through a small incfsion fn the scrotum and a pursestrÍngsutureofapproxfmate]ylcmdfameterplacedinthe tunica in a region devofo'of any obvious blood vessels. The diced tissue was then loaded fnto a l2-gauge stafnless steel cannu'la whÍch the was pushed Ínto the testfs well clear of the rete testisr through centre of the purse-string suture. A trocar was then used gent'ly to expeì the tfssue into the testfs. As the cannu'la tras removed from the testfs, the purse-string suture was pulled closed. The scrotal Íncfsfon was then sutured, and the anfmals returned to thefr pens after recovery.

2.4. ISOLATION AND CULTURE OF RAT PITUITARY CELLS

2.4.1. Buffers and Solutíons

Hepes buffer

137 mM NaCl

5mM KCl

0.7 mM NarHPOO

1 I ethanesu'l fonlc 25 mM n-2- hydroxYethYl P Peraz ne acld (HEPES)r PH 7 2 100

l0 mM G'lucose

uM CaOl 360 2

Du'l beccos modi f ied Eagl es medl um (DMEM)

Thisbuffert{asobtainedfnpowderedformrspecified suitab'le for tf ssue cu'lture, f rom Gibco Laboratories' Grand Is'land' the New York, and reconstituted accordfng to the directions of supp'l i er.

(KRB/BSA) Krebbs Ringer Blcarbonate so]ution with Albumln

Solutlon l. 0.154 M NaCl Solution 2. 0.154 M KCl Solution 3. 0.II M CaClt Solutfon 4. 0.154 M KH2P04 Solution 5. 0.I54 M M9SOO.7H,O [So]utions I to 5 prepared as stocks of 5X glven concentratÍonl Solution6.o.I54MNaHCO,(gassedforlhourwfthc02)

Krebbs Ringer Bicarbonate (KRB) 100 Parts solutÍon I 4 Parts solutlon 2 3 Parts solution 3 I Part so'lutlon 4 t part solution 5 2I Parts solution 6 Gassed for I0 minutes wfth 5 % CO2/95 1¿ Oz ¡-UTË

A.R Y

To this KRB solutfon was added

14 mM Glucose 3 mg/ml Bovine serum albumin

Compìete amfno acid supplement (Eagler 1959)

++ ++ KRB/BSA (Ca _lrlg free)

Prepared as for KRB/BSA above¡ with solutions 3 and 5 omitted.

2.4.2. Co]lagenase df gestÍon (after vale et al ' L972)

Anterfor pltuitary glands were obtaíned as detailed in 2'3'2' and rÍnsed several tÍmes fn Hepes buffer. Pituitary quarters were fncubated Ín Hepes buffer containing 3jã BSA, O.I% hyaluronidase and

0.35% collagenase, at 37oC for 45 mlnutes wíth continual stirring'

Fragments of pitultary were drawn fn and out of a sllfconised Pasteur pipette every I0 minutes durfng this incubatÍon. The ÍncubatÍon

mÍxbure was then centrifuged at 475 g for 2 mÍnutes, the ce'llular

pellet resuspended in Hepes buffer containing 0.25% V iokase and stirred for another I0 mlnutes. Followfng a second centrifugatlon'

DMEM medf um contai ni ng l0% pe'|1 eted ce] 1 s tl,ere resuspended f n steri'l e

horse sêFUlrìr 2.57o feta'l ca'lf serumr 0.1 mM glutamÍne and 1ã Glbco non-essentfal amfno acfds. Celìs were washed 6 times fn sterfle medfum by repeated vortexing and centrifugatlon. After the flnal washr cells were suspended in a smal'l volume of sterlle DMEM for transplantatfon ro2

as descrl bed i n 2.3.7 .2.

2.4.3. Trypsin dfgestÍon (after Hopkins and Farquar' 1973)

Anterfor pituitary g'lands were obtafned as descrlbed in 2¿3'2' and each anterior lobe clean'ly chopped with a razor b'lade into 40-50 rectangular tÍssue blocks. Blocks were p'laced into KRB/BSA at room temperature durfng collection and finally washed once wfth KRB/BSA' A'll b'locks were then pooled into an Erlenmyer flask with 5 m] KRB/BSA oC, containing 12.5% trypsin and 20 ug/m1 DNAase at 25 and transferred oC to a shaking water bath for fncubation at 37 for 15 mlnutes' After trypsín lncubatlonr the solution was transferred to a conical centrifuge tube, the supernatant removed after settl ing of tissue contents (about I minute) and 5 ml of pre-warmed KRB/BSA containing I mg/m1 soya bean trypsfn ÍnhÍbftor added. Thfs was then incubated for 5 oC. mÍnutes at 37 Th" supernatant was again decanted, and the tÍssue washed with Ca++/Mg** free KRB/BSA contaÍning 2 mM EDTA and fncubated oC. for 5 minutes at 37 After decanting thfs so'lution, 4 ml of

Cu++/Mg++ free KRB/BSA containfng I mM EDTA and I ug/ml neuramfndase was added. The solution was transferred to another Erlenmyer flask and oC incubated Ín the shakÍng water bath at 37 for 15 minutes' The tfssue was then transferred to a conÍcal centrffuge tube and washed 3 times wfth 20 ml of Ca++/Mg** f."" KRB/BSA. Rfgorous pipetting of the mi¡ture with a f'lame-pol ished Pasteur pipette was then used to dfsperse the b'locks of tfssue. Flnal'ly' the cell suspension was spun

through BSA by layering 5 ml of 4% BSA below the cell suspensfon' The

tube was centrifuged at 70 g for 10 minutes to produce a cell pel'let relatlvely free of debrisr which was then resuspended ln a sma'll 103

vol ume of DMEM for transp'lantation as descrlbed 7n 2.3.7 .2.

2.4.4. Ti ssue Cul ture

2.4.4.L. Routf ne method

Following fsolatîon of pftuitary cells as described in 2.4'3' abover 2.5 X 105 cells were placed into each of 4 wel'ls on a tlssue culture plate (costar, cambridger Mass., usA). 0.5 nrl of DMEM containing 5% horse sêFUllìr 2.5% fetal calf serum and Ij[ non-essential amÍno acids was then added to each well. Cells were maintained under oC. an atmosphere of S% COZ in air ln an f ncubator at 37 Cu'lture medlum was changed every second day during lncubatÍon.

2.4.4.2. LHRH Chal 1 enge

Flve days after p'l ati ng out of i sol ated pf tuitary ce]l s, thei r functional capaclty lras assessed by providfng a stimulus of LHRH. Cells were washed severa] times wlth medium as detaÍ'led f n 2'4'4'L' and then finally gfven serum-free medÍum contaÍnfng only I% non-essential amlno acf ds. In each test for a 4 well cu'lture' 2 we]'ls received only serum-free medÍum and 2 welJs recelved serum-free medium

contaÍning 10 nM LHRH. Cells were Íncubated for 6 hours wÍth these so'lutfonsr after which the medium tras sampled from around the cel'ls oC and stored at -20 until assayed for LH. Cu'ltures were termfnated at this point. r04

2.5. THYROID FUNCTION TEST

A varfety of tests exlst to assess the functlon of the thyroíd g'landr as seen in Flgure 2.2. Three methods were found to be of use fn the studies in this thesis. Firstly, in some experlments lt was useful to monftor plasna levels of T4 and TSH, and these methods are detailed in 2.I3.2. and 2.13.3.7. In other experÍments¡ it was possible to

provide a TRH bolus to stÍnrulate TSH secretion by the anfma'lsr ot{n pitultary whÍch Ín turn should stimulate To secretfon by the graft' Thfs could then be monÍtored in plasma samples. It was not possfble to dlrectly chaìlenge the grafts wÍth oTSH because this was obtained from vÍvo' the uSA and quarantfne regulatlons would not permit lts use fn have such The TRH on the other hand was a synthetÍc ana'log and dld not restrictlons on its use. Flnally, fn most experiments the abilfty of a graft to accumulate radioactÍve fodfne was assessed.

2.5.1. IodÍ ne Accumul atf on

2.5 . l. l. Rats

Twenty-fourhourspriortograftremovalrFâtsweregivenan 125rodln". intraperÍtoneal Ínjectlon of I.0 uçi (37 kBq) Followlng removal of the testis or kidney containing the graftr and the contralateral ungrafted oF$ânr testes were placed fnto Bouins fixatfve and kidneys into buffered Formalin (see 2.8.). Uptake of lodine in all tissue samples was monitored in a gamma counter (Riagamma I271,

LKB-ülal I ac, Ff nl and). Figure 2.2. Physiologic leve'l of thyroid functf on tests (f rom

Zellmann, 1974). 10s

I PHYSIOLOGIC LEVEL OF CER EBRUM \) THYROID FUNCTION TESTS

US TE STS F

ITA <--- TSH Suppression

H +---- Rodioimmunoossoy

Rodiooctive Uptoke Perchlorote Dischorge \({t TSH Stimulotion @ ( ------Bio-ossoy -lq LYMPHOCYTE T3 PBI BEI TBG TqI T+D T¡ TBG

FREE Tq T¡ É FREE T+ ond Ts

PERTPHERAL /ãrurn Rerlex frs \^ @ )*-¿;tL------þnines 106

2.5.I.2. SheeP

Iodine localisation by thyroíd grafts ln sheep testes was assessed Ínitially.by gamma photography in the Dept. of RadÍology at the F'linders MedÍcal Centrer Bedford Parkr South Australia' To aid in LzslodÍne. photography, 13lto¿tne was used fnstead of Each anfmal was 131r-injectab'le gf ven an intravenous bo1us of I mci sol ution (funershamr Australia) 48 hours prÍor to graft remova'l' FolIoving castration, testes were placed on ice and transferred to the Ffinders Medfcal Centre for gamma photography. Upon return to the laboratory' each testis was serfal'ly cut lnto I cm3 pieces which were p'laced f nto Bouins fixative and counted fn a gamma counter'

2.5.2. TRI-I Chal l enge

sheep were fÍtted wÍth indwe'l1ing polyethylene jugular catheters

(I.0 mm o.dr I.5 mm i.d.). A bolus fnjection of 200 ug TRH was (0'9% admfnÍstered lntravenously (i.v.) in 2 ml physlo'logical saline NaCl) to all anfmals. This dose was selected from a dose-respose trial (TSH) as being ab'le to induce satlsfactory ThyroÍd Stimulating Hormone (see secretion and associated Thyroxine (TO) re'lease in normal rams Figure 2.3.). Bleedlng commenced two hours prior to TRH adminfstration at 20 min fntervals and continued for 3 hours after TRH admfnÍstratÍon at I0 minute intervals for the ffrst hour, and every 15 mÍnutes for the next two hours. Further samples were collected at 30 minute fntervals starting 7 hours after TRH admlnistratÍon and contlnuing for another 2 hours. Samples u,ere centrifuged and plasna tras stored at

-zOoC untÍl assaYed for TSH and TO. Figure 2.3. Levels of Thyroxine in peripheral p'lasna of rams after an (open i ntravenous I njection of 2m] physi ol ogica] sal i ne

dfamonds) or 200 ug TRH in 2 ml sa]ine (solid diamonds)' t40

TRH r20 + ^ E 100 u| vc BO ç Ì- o E 60 .n o -o. 40

H o\j 0 360 480 600 - r20 0 1.20 240 Time (min) 108

2.6. PITUITARY FUNCTION TEST - LHRH CI-IALLENGE

2,6.I Intravenous lnJectlon

Pituftary function ln sheep l{as assessed by monitoring the response of the pituÍtary to a bolus injectfon of LHRH. To ensure that the donor pltuitaries were functíoning normaì'ly prior to transplantatfon, donor wethers y¡ere prePared with lndweltlng Jugu'lar catheters as descrf bed i n 2.3.5.2. and challenged wlth an f ntravenous bolus of LHRH (5ng/kg body weight) lnjected lnto the uncannulated jugu'far vein. Blood samples were taken 20 min prÍor to the LHRH being admlnisteredr and continued for two hours after admfnfstratlon at I0 min intervals to monitor LH secretion.

2.6.2. Intravenous infuslon

2.6.2.I. Va'l idation of method

Prior to transp]antation¡ Fôltrs that were to be used as graft recipients were lnfused with LHRH via a cannula in the lateral of saphenous veln in the hind leg (see 2.3,L.) to check that an amount provide LHRH whichr if fnfused into the testlcular artery to sufficfent local concentrations of LHRH to stimu'late gonadotrophln secretfon by viable intratestÍcular pituÍtary transplantsr would not be sufficient ln the perfpheral cfrcu'lation to stÍmulate the recfpient vefn animalsr own pftuitary into secreting gonadotrophfns' The Jugu'lar in these anfmals was cannulated with an indwellfng PE catheter as descrlbed 1n 2.3.5.2. Samples were collected 20 min prf or to 109

corflnencsnent of the lnfusion, and then contlnued at I0 mlnute fntervals for two hours. The infuslon ftself was mafntafned for one hou r.

2.6.2.2. Calculation of dose of infused LHRH

Intravenous fnJectlon of LHRH at 5 ng/kg body weight (bw) produces a physfologÍcal pulse of LH in wethers (DtOcchio et a'l' 1982) and the calculated half-life for LHRH ln sheep ranges from 5 to 10 mlnutes (Swf ft and Crighton, Lg7Ð. Hwever for the Ínfusion of LHRH into the testisr côlculations were based on double the normal intravenous dose of LHRH (increased to 10n9/kg bw) to ensure that sufffcfent hormone reached the grafts. Assumlng an extracel'lular fluld volume of 15% bw, an i.v. bo]us injectlon of 10ng LHRH/kg bw into the jugular of a 60kg anlmal wou'ld produce concentrations at the level of the pltuitary ln the order of 70 pglml. To produce a sfmilar (see concentration Ín a testis with an assumed blood flow of 30 ml/mln Setchell and Brooks, 19S7) would require an LHRH ínfusfon of 2 ng/ntn into the testlcular artery and thÍs rate was chosen for infusfng grafted testes. The total amount of LHRH infused directly into the testlcular artery was I20 ng, or about 2 nglkg bw. This Ís hardly sufficfent to stfmulate the recipfentrs own pftuitaryr even if gfven as a slngle bolus, and was expected to be quite Íneffectfve in thÍs regard when infused over I hour.

2.6.2.3. Intratesticul ar arterf al f nfusion

To examf ne graft function before the testf s was F€lfiovêdr an r10

expanded catheter (PE, nomlnal I.D. at ffner end 0.2mmr o.D. 0.5mm) was lnserted into the testÍcular artery on the surface of the testÍs'

LHRH (2A ng/nl) was infused through thfs catheter at the rate of 0'I ml/min (fÍnaì dose of 2 ng/nin as calculated in 2.6.2'2') 'for one hour to stimulate gonadotrophfn secretion by the grafts'

2.7. HCG CTIALLENGE

50 HCG was adnrfnÍstered to rats as a subcutaneous injection of (pH í. u. hCG (Sigma, St. Louis) in 0.2 m] 0.0I tvl phosphate buffer

7 .2).

2.8. EDS ADMINISTRATION

Ethy'lene dimethane su]phonate (EDS) was klndly provf ded by Dr

F.F.G. Rommerts of Erasmus UnfversÍty, Rotterdam, The Netherlands' EDS

(30 mg/m1) was dlsso'lved Ín dimethyl sulphoxide water (1:3' vol/vol)

and adminfstered via intraperitonea'l fnJection (I00 mg/kg bw)'

2.9. SILICA ADMTNISTRATION

Sllica (SiO2, Sigma, PartÍcle sfze 1-5 u;5 mg in 0'2 ml physiologlcal sa] ine) was administered by Íntratesticular injectfon

usf ng a 25G needle and I m1 syringe.

VOLUMES 2.I0. MEASUREMENT OF ALBUMIN AND CT-EDTA DISTRIBUTION

one,hour 5tc.-labelled ethylene dlamine tetra-acetfc acid tIl

(r25r-nsR) fsPacesr (5Ìc.-EotR) und 125I-labelled human serum albumln prevlous'ly by in the testfs yrere measured usfng the method described with setche]1 and sharpe (1981). Animals were anaesthetised pentobarbitone sodfum (Nembuta'|, Abbottr 60 mg/kg lntraperltoneally)' was inserted A polyvinylchloride catheter (0'8mm o'd" 0'5 mm i'd') intooneJugularveinrandfora]buminsPacedeterminationsluCiof For I25r-nSn (Amersham, Australia) was 'injected through the catheter' Cr-EDTAsPacemeasurementsrbothureterst{ereseveredpriorto SIç¡-EDTA the injectÍon of 5 uçi of (Amersham, Austra'l ia) through and a blood samp'le catheter. one hour later, both testes were removed posterfor vena cava (see co]'lected with a syringe and needle from the 2.3.5.).Radioactivityinplasmaandfsolatedtestes¡,asmeasuredfn uslng different settings to a gamma spectrometer (LKB Wa]'lacr Finland) discrfminatebetweeenthetwodifferentfsotopesused.The u1lg was ca]culated distributÍon vo'lume or ltspacert for each marker in by dfviding the radfoactivity fn the tissue in dlsintegratlons/mlnute in per gram by the radfoactivity in the b'lood plasna

di si ntegrations/mi nute per uì p'lasma'

2.LL. VASCULAR PERMEABILITY

Vascu]arpermeabilltywasca.lcu.latedusfngtheformulaPS=K. Valb-final (Amtorp, 1980) where Valb-fina'l Ís the final equflfbrlum (reached hours after volume of dfstributfon of albumin 6 125r-hsA, hour cr-EDTA admfnistration of but found to equal the t K the rate constant spâcêr Setchell and Sharpe' 1981; see 2'I0')r fs from the slope of of penetration of a'lbumin fnto the testfs determlned ) and Valb-I and the llne'log" ( I - (Val¡-l hou./Vc.-EDTA) hour TT2

VC.-fOtR are the volumes of distrlbution of these markers one hour after thefr admfnistratfon. In calculatfng the rate constant K' Ínitial vu.,o values (time = 0.mfn) were assumed to be equÍvalent to (198I) testÍs b'lood volume as described by Setchell and Sharpe and their values of 10 ullg before hCG admínistration and 15 ullg after

hcG administration were used in calculatÍons. In the equation described by Amtorp (1980) for calculation of vascular permeabflity' the logarf thms were calcu]ated to base e, a'lthough thf s was not specified by the origÍna1 author. Unfortunately' in prevfous pubtÍcatíons (see setche'll et a'l , Ig84; Setchell and Tupp' t9B7) values were calcu'lated with base I0 logarithms. As a consêQUêrìc€r the absolute values for permeabÍ'lity-surface area products previously published are too]ow by a factor of 2.303r but this does not affect the val iditY of changes rePorted. il r¡¿ ,r 2.T2. LYMPFI FLOIII

Lymph flow was calculated from the formulu 9- = 0'693 the lymph f'low f n u'l /g/nin' Va'lb-f Va.lb-f inal / Tr, where 0a is Ína'l is the final equi'lÍbrfum volume in ul/g o1 distribution of the albumin (assumed to equal the I hour Cr-EDTA space as dÍscussed in 2'1I') and from Tttz 1s the half-time of clearance of directly-iniected albumin L25I-hSA the testis. For measurements of clearance rate from testes in-vivo, radioactivity was monitored using a directlona'l gamma counter with a ratemeter (Nuc'lear chicagor Illinois, u.s.A.) and a chart recorder (Rlkadenkl, Tokyo, Japan) fot'lowing the injection dÍrect'ly into the testis of I uCi in 50 u'l physio'logica'l saì ine, through a 30G

needl e.

I IT3

ASSAYS 4! 2.L3. HORMONE

2.L3.1. Buf f ers and So'l uti ons

2.L3.!.L. PhosPhate buffers

(PBS) (A) O.O5 M, PH 7.5

g Na,HPOO (anhYdrous) ! I.42

0.37 g EDTA t, 1.00 g NaNt

8.I8 g NaCl

adJusted to PH 7.5 with IN HCt water made up to I ìftre uslng double dlstilled

(gel PBS) (B) O.O5 M, PH 7.5 , O.26 gelatin

solutlon' To the PBS, added gelatfn to get O'2% NaOH adjusted PH to 7.5 wlth IN

(KPBS) (c) O.O7 M K2HPO4/KH2PO4, PH 7'4

I i 9.35 g KZHPO4 I ¡ t, 1.78 g KH2P04 water made up to I lltre uslng double distflled

I 114

L25 2.I3 .L .2. Bl ocker sol uti on w i th I-thyroxl ne

tl acld (ANSA) 0.0333 g B-Anil f no-naphthalene-sul phonfc

I.3320 g Thlmerosal (Merthi ol ate) (T4FS) 3.25 ml T* free serum (specif 40 - 60 uci/ug To) O.25 ul "tt-ro ic activity: made up to 100 ml wfth KPBS

2.I3.L.3. TO-f ree serum

r25r-To give 30 To a pool of serumr suffÍci"nt was added to - samptes were taken for 4O,OOO CPM/m'|. After mixfng wel'lr two I.t counting.2gcharcoa]/I5m]serumwasthenaddedandtheso]ution left stlrrlng overnfght at room temperature. Fo]'lowfng incubatÍon' the the solution was centrifuged for 30 mins at 2'OOO rpm to renrove mÍns' ü and then centrifuged at 20t000 rpm for 30 ,.1 bulk of the charcoal, Thfsprocesswasrepeatedunti.|thecountsÍnthesupernatantserum were less than L% of those added'

2.L3.I.4. Liquid sclntfllation system

( A) Sci nti'll ation f'l u I d 4.0 g PPO (2,5-diPhenY'loxazole) (5-phenyloxazole) ) g POPOP (2,21-P-Phenylenebis I 0.4 I dissolved in I lftre of toluene

(B) Countfng of aqueous sYstems T I

I AfteraddfngtheaqueoussampleandsclntÍllationf]ufdlthe on a poìyethylene vfals were shaken vlgorous'ly for 20 mlns r IT5

of mechanlcal shaker to facfl ftate the transfer radfoactivity from the aqueous to the organfc phase' RadloactivitywasdetermlnedinaLKB-Wallac'RackBetaII liquÍd sclntfllatlon counterr Model No' I2L5'

2.L3 .2. L-Thyroxi ne radi oimmunoassay

P]agnathyroxineconcentrationsweredetermfnedusÍngthe Burke' Department of antisera which was donated to B'F'Good' by C'W' Endocrfnology,Radclfffelnfirmary,Oxford,England.Va]idationof (Burke and shakespêôFr 1976)' the assay has been described elsewhere

were added to DuplÍcate alfquots of plagna (10 to 50 ul) disposab.legìasscu]turetubes1o2xT5mm).Astockso.|utionoflmg

I ml NH4OH) was prepared' L-T4 in I ml EtOH/NH4OH (25 m] EtOH + 0.880 Standardsof0,9.32,rg.64,37.2g,62.!t93.2¡186.4nglm1Towere volumes of prepared by dilutlng the stock solution in appropriate T4Fs.Tol0u]ofstandardorp.lasnawasadded300ulofb.|ocker I 125I-T4 cPM per 300 u1) (see so.lutfon containlng (approximately r0,0oo (fÍnal dÍ'lutfon of l:3000) sectfon 2.!0.L.2.) and 200 ul of antf sera hrsr at 4oC. Free and bound and the mÍ>ture incubated for at least 12 contalning I mg each To were separated by the addftlon of I ml KPBS Dextran(PharmaciaChemicals)andCharcoa].Thetubeswereshaken then centrÍfuged at 800 x well and a'lloned to stand for 5 min and were I gfor20mln.Thesupernatant(boundfraction)wastransferedto I I (Ria and radloactivity determined Gamma l27l' i another set of nerv tubes ons cal cul ated u si ng sp'l f ne cu rve LKB-Wa'l I ac, F i nl and) and concentratÍ

Ì 116

ff ttf ng of the data as descrf bed f n Section 2.10.3.2.

Sensitfvity (2 x standard devlatfon of the blank) was 3I pg and fntraassay and fnterassay coefffclents of varfatfon were 6.7% and 9-L% respectÍvel y.

2.L3.3. Pltuitary hormones radf ofmmunoassays

2.I3.3,L. Iodlnatf on of pituitary hormones

pltultary hormones were fodfnated usfng Ch1oramfne-T method of

Greenwood et al (1963).

(i) Preparatfon of Bio-Gel columns for ge1 ffltratÍon of

iodlnated hormone PreParation

A. Preparation of columns 1. 10 m] disposabìe glass pfpettes were used wfth the top I inch of the plpette cut-off. 2. Columns were thoroughly washed Ín Bioch'lor, rinsed and drfed, 'l sf lconf sed w ith cotasl'1, and washed f n double di stll I ed water and drled agaln.

B. Preparatf on of Bio-Ge'l I. Approxfmate'ly 50 g Bio-Ge1 was placed f nto 600 m] beaker and and mfxed continued stirrf ng for 2 - 3 I 300 m] Ptss added wlth I hours. 117

2 The solutlon was alloued to settle and the excess buffer

removed from above the ge'ì. A new volume of Ptss was added to the gel and stírring recommenced for 15 minutes'

3 Step 2 was repeated 3 tÍmes to wash the ge'l solution'

C. Buil dfng col umns pipette I. A medium size gìass bead was introduced into a 10 mì which was pl aced i n a co] umn-hol df ng dev ice. A smal'l pf ece of silastic tublng (4.7 mm 0.D.,3.4 mm I.D.) was attached to the bottom of the plpette, and a screw clamp p.laced around the tube to control column flow. 2. Co]umn was rinsed with PBS. 3, Column was ff'lled with Bio-Ge'l by pourlng the slurry of Bio-Gel Ínto the column. 4. Alloned to settle (no movement of slurry apParent) to approximatelY B to 9 ml mark. 5. Released the c'lamp and allorved to settle further. was 6. Co'lumn was kept wet at all tfmes, and if too much BÍo-Gel in column¡ it was removed wfth a pipette to the recommended I

ml mark.

7. Column was washed wlth 20 m'l PBS' 8.1.5m]5%albumln(Sigma)inPBswasgentlyplpettedonto column-bedr below buffer, for better recovery of iodÍnated

protef n.

9. Column was washed with another 20 m'l PtsS and capped with paraffìm and stored at 4oC. Before use, co1umn was alloled

to warm to room temPerature 10. Co]umns were usua]1y prepared 24 hours prlor to use. tI8

( i i) Reagents

I. Purified hormone: of LH' FSH' TSH and Hfghly purffied hormone PreParatÍons prolactinwereused.Detailshavebeenclescribedseparately Ín the procedures under each hornrone' 2. Radioactfve iodlne: from Amershamr Australia' Sodium iodlde tl25rl was obtained was used' For each iociination 0'5 mCi in I2'5 ul 3. Chloramfne-T: AstocksolutionofCh.loramÍne-Twaspreparedbydissolvfng4 prior to each mg Chloramine-T in 5 ml PtsS' lmmediately

i odl nati on. (NarStOt): 4 Sodium metabisul Phlte dfssolved ln 5 ml PtsS prlor Sodlum metabisulphite (10 mg) was

to each f ocil nat'ion'

5 Transfer solutfon: ln 5 ml of 16% 50 mg of PotassÍunr fodlde was dÍssolved sucrose in PBSr prÍor to iodÍnation'

6. Ri nse sol ution: in 5 ml of B% sucrose 50 mg of Potassium fodide was dissolved in PBSr Prior to iodinatfon'

(iii) Iodlnation Procedures

I Theamountofproteintobeiodlnatedwasdisso.lvedin25ul of sPecffic Proteins PBS, in an EPPendorf tube' O.uantÍties 119

rFSH' 2'5 used were 2-5 ug of LH, 5 ug of oFSH or 2'5 ug of ug of Pr1 and 2 ug of TSH' 125t and nrlxed 2. 0.5 uCl sooium iodfde was added to the hornone well bY taPPfng on the tube' 3.Chloramfne-Tstocksolutlon .2ug/Sul)wasadded'mixedand left for I mfnute at room temperature' 4.Sodiummetabisuphite(50u1)wasadde

ThefractionswlthhlghestradloactlvÍtyÍnthehormone approxfmately peak were retalned and diluted in PtsS to gfve

20,000 CPM Per 100 ul. Flgure 2.4. Levelsofradloactivityinfractlonse]utedfromaBiogel l25lodin"' after column used to separate bound and free The iodinatlon of protefn hormone using chloramine-T' remalned on the unbound free iodfne was not elutedr and 6 and 7 column. With such an e'lutlon profiler fractlons were used ln radioimmunoassays' 200

180

¡-c 160 E ç r40 o F 1,20 x o 100 fc -¿ o BO : |o 60 o¡ 40

H 0 oN r 2 3 4 5 6 7 B 9 10 Fraction T2T

(oLH) 2.L3.3.2. 0vine Lutelnfzlng hormone radlolmmunoassay

( i) General method

400u.|ofplaslllaorstandardhormonewereplacedfn12xT5mm glassvia.lsusfngareagent.dlspensor(14ícronledicSystemsrHorshamr PA,U.S.A.),alongwith400ulofantjbociyclÍ.lutedinGe.lPBStogive approprÍatebindfriganddfsplacement.Iodinatedhornlone(I00u])was thenaddedwlthanotherdispenser,andtubesshakentoensuremfxing (22oC) f or 24 at roonr temperat ure of conter¡ts. Assay tubes were helci

hou rs.

from free hormone (ií) Separation of antlbody and hornrone

second antfbody Di'luted serum (I:1I with PBS) containfng (Antirabbftgammaglobullnsrafseclfnsheepagafnstrabbitgamma globulins)wasthenaddecltothetubes(100u])whichv¡eremixedwe.l] another 24 hours and then at 4oc for and he.ld at room temperature for afÍna]24hourperlod.Anti-rabbitganrmaglobulÍnsformeda the bound fractlon of the preclpÍtate wlth the antibody along with with 1.8 ml of FBS (at 40C) and hormone. The free hornrone was washed (2000 rpm for 20 nrÍn at 4oC)' The decanted after centrifugation pe.lletcontalnfngtheboundfractlonÍntheprecfpitatewascountedin the auto R.iagamma counter'

(iiÍ) Hormones

(LER-1056-C2) used for A highly purlfied fornr of 0vine LH L22

fodlnatlons was a glft from Dr. L.E. Relchert Jr.r Albany Medlcal

College, Albanyr New Yorkr L2208' U.S.A.. Standards of 0' 0.0196' 0.039,0.0781 0.1562,0.3!25,0.625, I.25t 2.5r 5t L0 ng/200 ul oLHr were prepared ln Ge'l PtsS, uslng ovlne LH (NIADDK-oLH-24) obtained from

NIAMDD, Bethesdar Marylandr U.S.A.

( iv) Antl sera

The antl-ovine LH serum nas obtafned from Dr. D.R. Lindsayr University of Western Austra'llar Nedlands' Austra'lfa. The antfserum exhibf ted 'lov cross-reactions Yrfth TSH (NIH-TSH-$9, 4.616) Prol actln

(NIH.P.S12, O.L7%) FSH (NIH.FSH.SI2T 0.926) and growth hormone (NIH.GH.SII, L.25Ð (0ldham et al, L979). The sensitÍvlty of the assay using thls antfsera tras 0.03 ng and the lnter and intra-assay coeffÍcfents of variatlon were 7.9% and 4.5% respectively.

(v) Calcu]atfon of resuìts

A. Non-speclffc bfnding comPonent

An estimate of the percentage of counts added which contribute to

the bound fractionr ln the absence of antibodyr tvas requfred so that a correction could be app'lied to the calcu'lation of actual counts bound. To assess this within each assayr 4 tubes contafnfng 200 ul of Gel PtsS L25I I00 u] ot LH and 400 ul of 0.7516 normal rabblt serum in Gel PBS,

were fncl uded. TB

B. Calcu'latlon of Í bound

The percentage of total counts bound to antibody' lnc]udlng correction factor for NSBñ was calcu'lated as fo]lows:

Tota'l counts bound to antlbodY

% bound x 100 Total counts added to the tube

Standard curves were constructed by p'lotting % bound against LH concentratfon. The% bound data was fitted ln a spllne curve p'lot of

go (% bound ln the absence of LH)

gx (% bound at each concentratlon of LH)

against LH concentrationr from which test samples were quantitated'

The counting and calcu'latlons vlere carrfed out 1n an LKB-ltla'llac automatfc mÍcrocomputercontro'lled two channel gamma counter dedlcated to RIA appl ications.

2.13.3.3. Rat Luteinizing honnone (rLH) radlofmmunoassay

Genera'l method, separation of antibody bound from free hormone

and calcu]atlon of the results are sfmllar to that used for oLH (Sectf on 2.L3.3.2.) except the volume of plasna used f n the assay ulas 200 ul. L24

Materla]sfortherLHradlolmmunoassaywereobtalnedfromthe Nationa]InstltuteofArthrltls,DiabetesandDlgestlveandKidney Dlseases(NIADDK)tUSA.Immuno-reagentswereelaboratedbyDrA.F. ParlowlDirector,PitultaryHormonesandAntlseraCentrerHarbour-UCLA Medlca.|CentrerTorrance,Ca]ifornÍa'UsAandaredescribedln Technlcal Report No' 143 of the Centre'

A. Hormone

HighlypurifiedratLutelnizinghormoneantigen(NIADDK-rLH-I-6) wasusedforlodlnatlons.Standardsof0,0.07,0.15l0.3lr0.62' trere prepared in Gel PtsS' using L.25, 2..501 5.00r 10'00 ng/200 ul FLHr rat LH (NIADDK-r'l-H-RP-2) '

B. Antiserum

Theanti-ratLHserumraisedlnrabbitswasNlADDK-antl-rLH-s-7. wlth TSH (NIADDK-rTSH-Ir The antiserum exhibfted lovr cross-reactions 5.2%)rpFolactln(NIADDK-rPRL-I,0.01%),FSH(NIADDK-rFSH-I'0.15%) (Technlca'l Report No' 143' and grorth hormone (NIADDK-rGH-L 0. oo4n asdetai]edabove).Thesensitlvityoftheassayuslngthisantisera was0.3ngandthefnterandlntra-assaycoefflcientsofvariatlon

were 7 .4 % and 5.0 % resPectivelY'

2.L3.3.4.0vineFo].llc.lestlmulatfnghormone(oFSH)radiolmmunoassay

Generalmethod,separationofantlbodyboundfromfreehormone andcalculatlonoftheresu]tsareslmllartothatusedforoLH L25

(sectfon 2.I3.3.2.) except the votume of plasna used in the assay l.,as 200 ul.

A. Hormone

Standard solutions of FSH were prepared from purified ovlne

(NIAI-4DD-oFSH-I5) FSH obtaf ned f rom NIAMDD, Bethesda, MDr U. S. A' ' standards of 0r o.78l-2t L.5625r 3.L25,6.25, L2.5,25' 50, 100 ng

FSH/200 ul were PrePared Ín Gel PBS. A highly PurÍfied form of Ovine

FSH (LER-1976-42) used for Íodination was a gift from Dr. L.E.

Relchert J r.

B. Antlserum

The antiserum was obtained from Dr. S.S. Lynchr Birmfngham and

Midland Hospltal for Women, Bfrmlnghamr which was raised fn rabbits agaÍnst hFSH. The cross reactivlty of the antfsera with oFSH was 100% whereas wfth rFSH it was 76%. ovine LH, TSHr GH, prolactin' rat LH' (McNeilly TSHr GH and prolactin shoved less than O.Uo crossreactivity

et al t 1976). The sensitfvity of the assay uslng this antfsera was 0.5 ng and lnter and intra assay coefficients of varfatfon were B'9%

and 6.0% respectivelY.

2.L3.3.5. Rat Fo]l icl e Stlmut atl ng hormone ( rFSH) radl of mmunoassay

Genera.|methodlseParationofantibodyboundfromfreehormone

and calcu]ation of the results are slmilar to that used for oLH (Sectl on 2.!0.3.2.) except the voJume of plasna used ln the assay was L26

200 u'l.

Materlals for the rFSH radioimmunoassay were obtained from the Nationa'l Instltute of Arthritlsr Diabetes and Digestlve and Kfdney A'F' Dlseases (NIADDK), USA. Immuno-reagents were elaborated by Dr

Par'low, Director, Pltuitary Hormones and Antisera Centre' HarbourUCLA Medical centrer Torrance, ca]ifornia, usA and are descrlbed ln Technical Report No. L44 of the Centre'

A. Hormone

HÍgh'ly purifled rat FSH antigen (NIADDK-rFSH-I-6) was used for L2'5' lodinatlons. Standards of O, 0.391 0'78' 1'56' 3 'I3 t 6'25' 25.00t 50.00 ng/200 u1 FLHr YreFe prepared in Gel PtsSr uslng rat FSH

(NIADDK-rFSH-RP-2).

B. Antiserum

The anti-rat FSH serum raised ln rabbits was

NIADDK-antl-rFSH-S-11. The antiserum exhÍblted low cross-reactions (NIADDK-rPRL-RP-2r 0'06%)', with TSH (NIADDK-rTSH-L I.67%)r prol actin (NIADDK-rGH-I-4' 0'02,6) LH (NIADDK-rLH-I-5 t 0.OL%) and growth hormone (Technlca'l Report No. L44, as detalled above)' The sensitlvity of the

assay using thls antfsera was 0.5 ng and the inter- and intra-assay coeff icf ents of varf atlon vJere 8.2 % and 5.6 % resPectlve]y' r27

2.L3.3.6. Rat Prolactf n (rPRL) radlolmmunoassay

hormone General methodr separation of antlbody bound from free used for oLH and calculatlon of the results are simllar to that was 50 ul' (Secti on 2.!3.3.2.), except the volume of pìasna used

from the Materfa.ls for the rpRL radloimmunoassay were obtalned and Kidney National Institute of Arthrftisr Diabetes and Digestive by Dr A'F' Dlseases (NIADDK)r usA. ImmUno-reagents were elaborated Harbour-ucLA Parlovlr Directorr Pftuitary Hormones and Antfsera centre' described in l,4edlcal centrer Torrance, ca]ifornlar usA and are Technical Report No. L20 of the Centre'

A. Hormone

Hfgh.lypurifiedratPRLantigen(NIADDK-rPRL-I-5)wasusedfor I'56r 3'L3' iodinations. Standards of 0r 0'098' 0'I95' 0'39' O'78' using rat 6.25, 12.50, 25.00 ng/50 u1 rPRL, urere Prepared in Gel PBS¡

PRL (NIADDK-rPRL-RP-3) .

B. Antl serum

The antl-rat PRL serum raised fn rabbits tvas than 0'oL % NIADDK-antl-rPRL-S-9. The antlserum exhibited less (NIADDK-rl-H-I-4) I FSH cross-reactlvlty with TSH (NIADDK-rTSH-I-3), LH (Technical (NIADDK-rFSH-I-3) and growth hormone (NIADDK-rGH-I-3) the assay Report No. !20, as detalled above). The sensftlvfty of using thfs antisera uJas 0.8 ng and the lnter- and lntra-assay L28

and 4'3 % respectlvely' coeff lcf ents of varf atlon YJere 8'5 %

2.L3.3.7.oVlneThyroldStlmu]atinghormone(oTSH)radloimmunoassay

Genera]method,separationofantlbodyboundfromfreehormone andcalcu]atlonoftheresu]tsareslmi.|artothatusedforoLH plasma used was 100 ul' (secti on 2.r3.3.2.), except the vo'lume of

obtafned from the Materlals for the radiolmmunoassay were and Dlgestive Diseases Nationa] Instltute of Arthritls, Metabo]ism (NIAMDD),USA.Immuno-reagentsweree]aboratedbyDrA.F.Par]ovll centrer HarbourucN-A Medical Directorr Pitultary Hormones and Antf sera are described in Technical Centrer Torrance, Ca] lfornlar USA and Report No. 139 of the centre'

A. Hormone

HlghlypurffledbovineTSHantlgen(NIADDK-bTSH-I-1)wasusedfor iodlnatlons.Standardsof0'0'39'0'78'I'5613'L2t6'25¡L2'50' were prepared ln Gel PBS, uslng 25.00t 50.00, roo.oo ng/100 ul TSH,

bovf ne TSH (NIADDK-bTSH-I-1)'

B. Antiserum

rabbfts vlas The antl-ovlne TSH serum ralsed ln exhfblted lovl cross-reactlons with NIADDK-antl-oTSH-1. The antlserum (oFSHr AFP-5679-C', O'03%)r pFolactln LH (NIAMDD-oLH-23 , 0.Lffi)' FSH (oGH, AFP-5285-c, 0.07r). (oPRL, o.O%l and grorth hormone ^FP-+22ÙC, L29

2.L4. HISTOLOGY

2.L4.I. Flxatives

Testlcular tlssue was preserved ln Boulns solutlon and Kidney

tlssue was Preserved ln Buffered Formalln ffxatfve'

Boui ns So] uti on

75 m'l saturated Pf crfc Acid oC) (1.5 gram percent in distilled water at 30

25 nl Forma'l i n 5 m] G'lacial Acetic Acld Tlssue was placed in Boulns solution for 24 hours and then

transferred to 70% Ethano'l for storage.

Buff ered Formal I n 8.0 I NaH,POO.2HZO2

13.0 g Na,HPOO

200 m] Formal ln

1800 ml Disti'lled Water Tlssue was pìaced lnto Buffered Fonnalin' and remafned ln the flxative during storage.

2.14.2. Ti ssue Processl ng

Blocks of tlssue were placed ln Tfssue-Tek cassettes and

dehydrated and cleared in an automatfc processor ln preparatlon for

embeddlng in paraff ln tvax as follows: 130

L. 707o Ethanol I hour 2. 80% rr I hour 3. 957ó rr I L/2 hour 4. 95% r rr I hour 5. Abs rr I 2 hours 6. Abs tr II 2 hours 7. 1:I Abs Ethanol/

Chl oroform I hour 8. Chloroform I 2 hours 9. Chloroform II 2 hours I0. Wax I 2 hours 11. l{ax II 2 hours

Tissue sectlons of 7 um thlck were cut with a microtome and stafned wlth haematoxylln and eosfn as follovrs:

I. Xyl o'l 2 mlnutes

2. Xyl o'l 2 mlnutes

3. Abso'lute a'lcohol I mlnute

4. 80% alcohol I minute

5. 30!B alcohol I mlnute 6. Runnlng taP water I mfnute

7. Mayers haemato>ryl I n 1.5 mfnutes

g. Runnlng taP water I mlnute o t!6 acld alcohol few droPs tll1 Plnk

10. Runnlng taP water I0 mlnutes

TI. Eosl n 7 seconds 13I

L2. 80ã alcohol rl nse

,.J. 13. Abso'lute al cohol 2 mfnutes

14. Absol ute a'lcoho'l 2 minutes

15. Xy'lene 2 mlnutes

16. Xyl ene 2 mlnutes

17. Mount ln DPX

2.L5. IMMUNOIIISTOCfrIEMISTRY

2.L5.L. Flxatives

Tissue to be examlned lmmunohlstochqnically was placed In %

Formaldehyde fn 0.1 M sodium phosphate buffer pH 7.0, contalnlng 15% picric acld as descrfbed by Stefaninf et al (1967) : ü,¡ù I

Plcrlc Acid/ Forma'ldehyde FÍxatlve

50 m1 AR Formaldehyde G0% solution)

500 m'l 0.2 ¡Ii sodlum phosphate bufferr pH 7.0 I50 m'l saturated aqueous solution of picric acld f l'ltered through Whatman No. I f i'lter paper

Made up to I I itre with distllled waterr and stored at 4 oc.

Sodlum Phosphate buffer Stock solutlon A : NaHrPO4.ZH2O 3I.202 g/1

Stock solutfon B : Na,HPOO 28.392 g/1

For pH 7.0r 39 ml of solutfon A and 61 ml of solutlon B were r

I L32

M NaOH or HCI' mlxed and adJusted to pH wfth I0

¡!

', Fixatlon: so that at least one Pieces of tissue were trlmmed placed lnto Picric Acld Formaldehyde dlmension was 5mm or lessr and oc)' was kept temperature (< 4 Tissue which had been maintalned at ice ln thls flxative for L6-24 hours'

Dehydratlonr Clearlng and Rehydratlon TlssuewasplacedinS0%EthanollYJlthchangesevery15 of the plcric acld was removed minutes untll the yellow colouratlon (4-5changes).Thetlssue|,asthendehydratedlc]earedandrehydrated

as follovls : 30 minutes l. 95% Ethanol ll LOOI6 il r d 2. r.b ll I 3. LOO7í r rr ll 4. Xylene I ll 5. XYlene II il 6. 1001ã Ethanol I tr Í 7 . LOOq, rr ll 8. loo% Í lrr g. 95Í rr 15 mlnutes ll 10. 809ó rr il 11. 50% rt tl L2. 25% tr ll 13. Distl'lled Water Í 14. PhosPhate Buffered Sallne I 0.1% sodlum azlde in Tlssue was stored in 30% sucl"osêr t 133

phosphate buffered sal lne'

PBS/Sucrose/ Azlde 8.5 g NaCl

1.07 g Na,HPOO 0.39 g NaHrPOO'2Hr0 300.00 I Sucrose 1.00 g Na Azlde water' Made up to t litre wlth distilled

1 ng ,] Ti ssue Processl i, 2.L5.2.

Cryostatsectlonsl{erecutat5umrdesslcatedundervacuumfor 4 oC for up to I week if necessary' 30 minutesr and stored sea'led at processed as fo'llows : r¡ Tissue was then '\t wlth normal sheep I 1' Pre-lncubated for 30'mlnutes serum (I0% in PBS) off sl ldes 2' Excess sheep serum dralned slides : 3. Antisera added to appropriate Rabbit-anti-oFSH (see 2'10'3'4') 50 u'l/sl ide at I:400 dilution Rabbit-anti-oLH (see 2'lO'3'2') 50 ul/s]lde at I:400 dllutlon

( ) Rabb i t-anti-oPRL NIAMDD-antl-oPRL-I 30 u]/sllde at 1:400 dllutlon (see Gramsch et a'l' 1983; Mouse-antl-h B-Endorphin glft to Dr J' Furness Per A' Hertz) I 50 ul/sl lde at I:400 dl'lutlon r 134

4. Incubated overnight at room temperature ln humfdltY boxes 5. Gently'washed !n Ptss wfth'stlrrlng 'for 15 mlnutes 6. F'luorescent conjugated second antibodles added to

approprfate sl ides : Sheep-antl Rabbit-FITC (trlellcome Diagnostlcs) 40 u\/ s'llde at I:80 df lutf on

Goat-antl Mouse-FITC (CaPPel ) 40 u'l/sl lde at 1:80 dilutlon I temperature in j 7. Incubated for I hour at room

¡ humfdftY boxes 8. Excess second antibodfes washed off with PtsS' 9. Gent'ly washed ln PBS wlth stirrlng for 15 mlnutes I0. Mounted in Buffered Glycero'l (pH 8.6)

Buffered G'lycerol (PH 8.6) Solution A - 0.5 M sodlum carbonate 53 g/1 NarOOt (PH I1.5) Solutfon B - 0.5 M sodfum blcarbonate 42 g/1 NaHCOt (PH 8.3) Solution C - GlYcerol

I one part solutlon B placed into a beaker and adJusted

i parts glycerol added to buffer I to pH 8.6 wlth so]utlon A. Two I so] uti on. I r t;

r 135

2.L6. STATISTICAL ANALYSIS

error of the mean (sEM)t All data ls presented as mean i standard unlessotherrrfsestated.Theprobabllltyofslgnlficantdifferences betweengroupswasca]culatedbyStudent|st-test.Treatmenteffects (ANOVA) or Analysls of were analysed by Analysls of Variance tissue graft survlval were tested Covariance (ANCOVA)' DÍfferences ln by Flnney (1948) and for slgnlflcance wtttr x2-tests as descrlbed Latscha (1953).

I rl 136

PRIVILEGED SITE CTIAPTER 3: THE RODENT TESTIS AS AN IMÌ'4IJNOLOGICALLY L37

3.1. INTRODUCTION

Prevlousstudlesontheimmuneprivflegedstatusoftherodent testishavehfghllghtedthe]ikelyimportanceofsomefactoror to factors in the interstitlal regfon of the testis. The experiments estab'lf sh a be reported in this chapter were undertaken to initial]y privi'leged routlne transplantation protocol, to conflrm the immune of the status of the rodent testisr and to then examfne components in interstitial reglon of the rodent testis that mfght be important creating the proposed immune privileged status of thls site'

3.2. CTIOICE OF TISSUE GRAFTS

The potential use of endocrine tlssue grafts ln an disorders or to immunologlcally privileged testls to treat hormonal continue stimulate endocrlne systems requfres that such grafts should to normal to secrete hormones after transplantatlon and to respond these studies endocrine stimuli. Two tissues were chosen for use in whichwou]da]]owvarlousendocrfneParameterstobemonltored. endocrlne Initially the pltuitary 91and, whfch is involved ln numerous hormones when specific actlvltfesr WâS chosen because it secretes some stimulfareprovided(eg.LHlFsH),andotherswhenspeclfic 'later the lnhibltors are removed (eg. Pro'lactin). In experlments' not only thyroid gland was chosen for lnvestlgation because ft bloodr which secretes hormonesr but also concentrates lodine from the can be readly monftored with the admlnfstratlon of radloactive

i odl ne. 138

3 .3. PITUITARY TRANSPLANTS

Threemethodsofpftultarypreparatlonweretested.Initfa]]y, lso]atedpltuitarlesu,eretreatedwlthco.|.lagenasedigestionto shovrn to retaln procure lsolated ce'lls which have been endocrinologfcalactivltyinvitro(Va.leetallLgT2l.Inasecond using trypsin digestion ln experiment, slmilar cells were obtained placeofcollagenasetoinvestlgatethepossibilitythatlnappropriate enzymetreatmentmightaffectcel]functionaftertransplantation.In tvas avoíded a'ltogetherrwith the a thlrd experimentr enzyme digestion use of quarters of whole pituitaries' transplantation from Donor pltuitarfes rtere obtained for castratedma]eratsuslngthemethoddetal]edin2.3.2.Theseanima.ls feedback on the pltultary should were used because the lack of sterold haverenderedthepltultarleshyperactÍvelntermsofgonadotrophin reclpients were secretion. In the ffrst two experimentsr hypophysectomisedatthetlmeoftransp]antation.Howeverrthisfn ftse]fmayaffectgraftsurviva]andthusrfnthethirdexperfmentr bothintactandhypophysectomisedrecipientswereused.Atthistfmer Se]awryandwhlttington(1984)publfshedareportsuggestingthat intratesticulargraftsofpancreaticls]etsweremoresuccessfu.lln cryptorchfdthanfnscrotaltestes,andthusthisthirdexperlment a]soinc]udedanlmalswithcryptorchidtestesasreclplents. 139

3.3.I. Experlmental Procedures

3.3.I.1. Anlmals

Male Porton rats were malntalned ln the departmental facilltfes pe'l'leted under a constant 12 hours I lght : 12 hours dark' A standard dlet (Mi1l lng Industrf es, Adelaide) and water were aval'lab'le ad

I i bitum.

3.3.I.2. Experiment 1. Co'll agenase-df spersed ce'll s

(200-250 grams) pltultarfes 1.¡ere removed from twenty male donors sacrlflced under ether anaestheslar and lndivldual cells fsolated uslng col'lagenase dlsperslon as described ln 2.4.2. Thfs dlgestlon yielded lO.5 X 106 ce]ls (determfned with a Neubauer haemocytometer) whlch were suspended fn 2 ml Du'lbeccots PBS. Twenty male reclpfents as Ã40-2gO grams), hypophysectomlsed one week before transplantatfon detalled ln 2.3.2.r l,{êFê randomly allocated lnto four grouPs of f ive anÍmals. One group was not transplanted and served as a control $Foupr 200 ul whl'le the other three groups were transplanted with 50r 100 and respectively of the cell suspensfon (250 O0o' 5oo 000, and I 000 000 cells) into one testls as descrlbed In 2.3.7.2. Al'l anfmals t{ere

welghed week'ly after transplantatlon and at three weeks after transplantatlonr surviving anima'ls were ræwelghed and venous b'lood cava as samples were obtained from both testes and the posterfor vena samp'les by described I n 2.3.5. Pro'lactln levels were measured ln these

radlof mmunoassay as descrf bed i n 2'L3'3'6' r40

3.3.I.3. Experlment 2. Trypsin-dfspersed cells

pltultarfes were removed from twenty male donors (230-300 grams) as in experfment I above. Indlvidual ceJls were lsolated usfng trypsfn

digestion as descrlbed I n 2.4.3. This digestlon ylel ded 20 X 106 cel'ls Ín 4 ml Dulbeccofs MEM. Twelve male recipfents (260-320 grams) were

hypophysectomlsed at the time of transplantatlon as detalled fn 2'3'2' (I A'11 animals were transplanted wlth 200 ul of the cell suspenslon

0OO OOO cel'ls) lnto one testls as descrlbed In 2.3.7.2. At thÍs tlme cells were also p'laced lnto culture as described ln 2.4.4'L' To assess their viabll ity at the time of transp'lantationr these ceìls were stlmulated with LHRII as described in 2.4.4.2. Three weeks after transplantatlonr survlvlng anlmals were rerlefghed and venous b'lood

samples were obtafned and Prolactfn levels monltored as for experfment

I above.

3.3.I.4. Experlment 3. Quarters of whole pltuftarfes

Ten pltultarles were obtained from adult male donors (270-300

grams) as described abover ônd the anterlor pltuitary cut into quarters. Thfrty adult male reciplents (2!0-330 grams) vlere randomly divided lnto sfx groups. Three of these grouPs were hypophysectomlsed at the tlme of transplantatfon as descrlbed itl 2.3.2.t and three were left lntact. t{ith each of these two treatmentsr one grouP received lntramuscular transplants of one pltultary quarter lnto the hlnd leg' one group received lntratesticular transplants of a pltuftary quarter lnto one testls ln the scrotum' and the thlrd grouP recelved transp'lants of a pltultary quarter lnto one testls made cryptorchfd as I4I

described I n 2.3.3. Transp'lants were performed as descrlbed ln 2.3.7.2. Three weeks after transp'lantationr survlvlng anlmals were rewelghed and blood sampìes obtafned as ln experiments I and 2 above'

Anima'ls were then castrated and further blood samples taken after another three weeks. Pro'lactlnr LH and FSH levels vlere measured using radloimmunoassays as described i n 2.L3.3.6., 2.L3.3.3. and 2'I3'3'5'

respectlvel y.

3 .3 .2. Resul ts

3.3.2.L. Experiment 1. Col l agenase-df spersed cel I s

There was considerable morta'lity amongst the anlmals ln this experiment. on]y one anlmal ln the control grouP of hypophysectomised' non-transplanted rats survlved the three week perlodr and this anlmal lost 20 grams in body wefght durlng this perfod of time. All other animals in thls group rapidly lost a similar amount of weight ln a considerably shorter tlme perlod and died in weeks I and 2 of the experlment. None of the animals receiving the lovlest transp'lantatlon the dosage of 250,000 cells survlved past the flrst week' Two out of five anlma'ls receiving the 500,000 ce1l transplants dled ln week 1' as did three of the flve animals recelvlng the 11000'000 cell transplants. The survivlng animals fn these two groups elther sustained their inftlal wefght or marginally galned or slightty lost welght over the three week perfod of study (Table 3'1')' No difference ln prolactin]evels could be detected in any of the samples obtalned

f rom these anf mals as seen f n Tab'le 3'1' r42

Tab'le 3.I.

transpl antatlon.

Change ln Pl asrna Prol actf n ( ng/ml ) Transpl ant Body Welght Grafted Non-grafted Peri Pheral (s) Testis Testis

500,000 cells +L2 L4 15 16 +I1 1I t: 10 -6 7 5 1,000,000 cells -2 10 T1 t1 +1 IO 6

- : no sample obtafned

Representative sections from the testfs of an animal receivlng 1,000,000 ce]ls are shown ln Figure 3.I. The graft slte fn the testis fs clearly marked by the small group of tubules that have pushed out through the entry ho'le made ln the tunica albuglnea (see Figure 3.1.a). Howeverr no grôfted ce]ls can be seen ln the region of transplantation, and tubules in this region and more generally in the testis (Figure 3.1.b) are typical of tubu'les seen fn untreated

hypophysectomised anlma'ls. There is no evidence for the actlons of any gonadotrophins ln these sections (from local or systemic sources)' suggestfng that grafts have not been technfcally successful' While there is no evldence of any lmmune reaction, there fs also no evidence of any graft tissue.

3 .3.2.2. Experiment 2. Trypsl n-d I spersed cel I s

Trypsin-dispersed cells fn culture dld respond to the addition of Flgure 3.1. Representatlve sectlons from the testis of a hypophysectomfsed rat three weeks after fntratestfcular transpl antatlon of 1r0001000 collagenase-isolated

pituitary ceìls. (a) The graft site fs clearly marked by a sma'lì group of tubules that have pushed out through the entry hole ln the tunfca albuglnea ( Þ). However, no graft cells are vfsfble Ín the reglon of transp'lantatfon. (b) Tubu'les are typlcal of those seen ln the testes of hypophysectomfsed anlmals and there is no evidence for the action of any graft-derlved gonadotrophins. X 80. 143

ì.È-' _s' -.r t 'lrtn L44

LHRI{ to the culture medf um as seen in Tabl e 3.2. suggestf ng that cel'ls were vfab'le at the tlme of transplantatfon. In thls experlment, elght of the twe'lve animals survived the'three weeks after transplantation, although all of these anÍma'ls lost weight fn this perlod (Table 3.3.).

Once againr there was no c'lear evidence that concentrations of prolactin in the veins dralnÍng the testÍs containing the transplanted ceìls were any hlgher than in the venous blood from the ungrafted testl s or from Peri Pheral bl ood (Tabl e 3 .3. ) .

Table 3.2. The secretlon of LH by cultures of trypsin-fsolated rat pftuitary cells under basal and LHRH-stimulated condÍtions. Values are glven as Mean + SEM (n=4).

Treatment LH ( ng/m'ì )

Control 0.1I + 0.04

+ LHRH z.gs + 0.424

a: slgnlficant1y different from Control values (P < 0.05r students t-test).

Table 3.3. Changes in body weight over three weeks after tranpslantatfon of trypsln-dispersed pituitary cells lnto the testÍs of hypophysectomised male ratsr and plasna prolactln levels at three weeks after transplantation. Values are given as Mean + SEM (n=8)

Change in Pl asma Prol acti n ( ng/ml ) Body Wefght Grafted Non-grafted Peri pheral (g) Testfs Testis

_43+L2 11+6 L2+4 I1+5

The histological examinatlon did not locate any of the grafted r45

ce'lls¡ and as fn the testes of anlmals transplanted wlth col'lagenase-lsolated cells, there l{as no evldence for any

lmmunologica] reactf on. Testes vJere very slmllar ln appearance to those shcryn in Flgure 3.1.a and 3.1.b.

3.3.2.3. Experlment 3. Ouarters of who'le pituitaries

As ln earller experfments, considerable mortalfty of

hypophysectomfsed animals affected the outcome of this experiment' (Flgure I'Jhlle all normal anima'ls survlved and contlnued to galn wefght 3.2.1 durlng the perlod of studyr only 4 of the 15 hypophysectomlsed anfmals survlved (one intramuscular, two scrotal intratestlcular and 'lost one cryptorchld fntratestlcular graft reclplents). These anlmals welght durlng the three weeks of study (Flgure 3.2). There was no dlfference in body welght galn between the different groups of normal rats, and no dlfference ln body weight'loss of the hypophysectomlsed ratsr lrrespective of the locatfon of the tissue graft. Levels of LH (Figure 3.3.)r FSH (Figure 3.4.) and Pr] (Figure 3.5.) were slgnlflcantly reduced by hypophysectomy. Transp'lantatlon dfd not affect the]evels of any of these hormones ln efther normal or

hypophysectomised animals. Fo]lovling castrationr levels of LH and FSH rose slgniflcantly in normal anfmals. Prolactfn levels were sfgnlficantly reduced ln norma'l anfma'ls folloring castratfon'

14hole tlssue grafts are much more readlly ldentffied than grafts of lsolated cells, and pltuitary grafts were found in some of the graft reclpfents ln thfs experlment. No grafts 1¡ere recovered from intramuscular sltesr or from cryptorchid testesr although recovery Ffgure 3.2. Percentage change in body welght of normal (sol id bars)

and hypophysectomised (hatched bars) rats over the three week period after transplantatlon of one quarter of a rat

pltuitary into a leg muscle, a scrotaì testisr or a cryptorchfd testis. Numbers of anlmals survlvlng ln each group are lndicated fn brackets. 20

o ( 1 g) (1) 2 ) Ê 0 ( 5 ( ( 5 ) so 5 ) ct a -10

-?0

-30 Gryptorchid Muscle Scrotal HÞ oì Tr ansptant site Figure 3.3. Percentage change fn ptagna LH levels in normal (solld and

open bars) and hypophysectomlsed (hatched and

cross-hatched bars) rats, fol I orv ing transp'l antatf on of one quarter of a rat pltuitary fnto a 1eg muscle, a scrotal testlsr or a cryptorchld testis : (a) durfng the three

weeks between transp'lantation and castratlon (solld and hatched bars); and (b) over the three week perlod after castratfon (open and cross-hatched bars). 700

600

500 o o' 400 Ê o G o 300 òa 200

100

- 100 Muscle Scrotal C ry pt or chid H \¡à Transplant site Flgure 3.4. Percentage change Ín p'lagna FSH levels ln normal (solfd

and open bars) and hypophysectomfsed (hatched and cross-hatched bars) ratsr following transplantation of one quarter of a rat pituitany fnto a leg muscle, a scrotal testlsr or a cryptorchid testfs : (a) during the three weeks between transplantatlon and castratlon (solfd and hatched bars); and (b) over the three week perfod after castratlon (open and cross-hatched bars). < i;€æ--

600

500

400 o u) tr 300 G 'Ê o 200 òa

100

- 100 Muscle Scrotal Cryptorchid H 5 Transplant site co ,,tr ii

Figure3.5.PercentagechangelnpìagnaPro]actin]evelsinnorma.| (so]idbars)andhypophysectorrised(hatchedbars)rats overthe3weekperiodaftercastratlonandremovalof lntratestlcul ar pltuitary grafts'

I f - --æ ''' J

-10

o -20 g¡ c o E -30 o a -40

-50

-60

-70 Muscle Scrotal Cryptorchid HÞ \o Transpl ant site 150

from these testes was hampered by technlca] problems' Hotever' grafts were found in anlma'ls transplanted wlth pftuitary grafts lnto scrotal testesr partlcularly ln hypoPhysectomfsed anfmals. Flgure 3.6. shows sectlons from hypophysectomfsed reclpfentsr and as seen in Figure I 3.6.ar the pitultary tissue ls readi'ly identlfled. Whlle much of the central reglon of the graft apPears ]ìecrotlcr PerlPheral reglons are qulte healthy, and Ffgure 3.6.b shows a regfon of typlcal pitultary tlssue on the edge of a graft. Of partlcular lnterest fs the rlng of tubules around the graft (Flgure 3.6.a) whlch are shown in greater contafn any I detall ln Ffgure 3.6.c. These tubules do not appear to Serto1l cells or developf ng germ ce'llsr and there ls no f dentf fiab'le ; lumen ln most of thsn. Houeverr the centra'l reglon contafns numerous

sperm and both sperm heads and talls are clear'ly seen. Thls fs un'like'ly to be a flxatlon artlfact, slnce tubules ln the rest of the ü testfs (Flgures 3.6.a and 3.6.d) are tyPlcal of those found fn the r{& I testes of hypophysectomlsed anlmalsr and do not sho'¡ any of these

pecul f ar characterf stlcs.

3.3 .3. Di scussl on

The results of these experlments were dlsappointlng to say the 'least. That transplanted-hypophysectomlsed animals in Experfment I

were able to mafntaln body weight while non-grafted hypophysectomlsed anfmals'lost welght rúas encouraglng. However, the lack of any evldence for prolactln secretion by such grafts was dfscouraging. There ls evfdence that lnaPProprfate enzyme treatment during lsolatlon of cel'ls I importantìy ln ir can result ln a decrease ln hormone secretlon and more receptors to trophfc hormones(Va'le et a'l t L97\ Smlth and Va'ler 1980)'

T of hypophysectomlsed Figure 3.6. Histologlcal sections from the testes ratsthreeweeksafterintratesticulartransp.|antationof (a) tissue a quarter of a rat anterior pituitary' The graft is readlly fdentffied betow the tunica albuginear X 80' (b) and ls surrounded by seminiferous tubu'les' regions Healthy pituitary tissue fs seen in the perfpheral to the of the graft. X 320. (c) Tubules ln close proximity 'lumen' graft do not aPpear to contaÍn any germinal e]ementsorSerto]ice]]s,andyetspermarec]earlyseen. artÍfact X 320. This is unlikely to resu'lt from a fixation (d) are sÍnce tubules ln other reglons of the same testis typical of those found in the testes of hypophysectomlsed animalsanddonotshovlthesamecharacteristlcs.x320.

r 151

, !-

a '2

t-l I ¡lto I L52

Experlment 2 was deslgned to address thls problem. l{hlle co'llagenase treatment may have affected the vfabillty of tissue grafts fn experlment L there rlas no suggestlon from experiment 2 that trypsln digestlon tras more beneffcial in thfs regard. That such ceìls were able to respond to LHRH ln vltro was taken as evldence that these cells were not greatly damaged durfng fsolation. Howeverr there was sti'll no evldence for hormone secretlon by such cel'ls f n vivo' after transplantation. Experiment 3 was fnltfated to fnvestigate whether pieces of pltuftary would retain thefr endocrlne functÍon after transplantationr since such pfeces had been shcwn to secrete a wide range of hormones in vltror and to respond to stimulation by trophic hormones ln culture (see Hopkins and Farquhar, 1973). However¡ these grafts were a'lso apParently unable to secrete hormones after transplantatlon. Hypophysectomy of the reclplents fs not thought to have been contributfng to these problems in ôn! walr slnce the problern perslsted when normal anlmals were also used in Experiment 3. No general concluslons cou'ld be drawn from thfs thfrd experlment, about the usefulness of the testls as an lmmuno'loglcally prlvileged sfte over other recognlsed non-prfvielged sftesr such as muscle. In the absence of detectable hormone secretion, no ready means was avallab'le to assess graft survlval or function. It fs possÍble that not enough tlme was állowed for grafts to recover from fsolation and transplantatlonr and that thls had affected hormone secretlon by the grafts.

As dlscussed by Setchell (1978) the testicular caplllarfes aPpear to filter a fluld wlth a hfgh concentratlon of protefn. Testlcular lymph in a ram has more than two-thirds as much proteln as blood 153

plasna (Llndnerr 1963; Wa]lace and Lascelles, 1964) whlch ls much hlgher than lymph elsewhere in the body (except for the tiver)' It has aìso been found to be an lmportant route of exlt for conJugated sterolds ln the horse and plg testis (Setchell and Coxr L98\ Setchell et al, 1983). It ls quite possible therefore that graft àerived fn hormones may'leave the testfs ln testÍcular lymph rather than lymph fn venous blood. Because it is so dlfficu'lt to samp'le testlcular the lymphatfcs ln the spermatlc cord of small animals such as the rat (see Morris and Mclntoshr l97I)r such samples YÚere not avallable ln thfs studY.

The histology of the tissue grafts ls very lnterestlng. The presence of sperm ln the tubules closest to the graft |Yas unexPected glven the'lack of detectable hormone secretlon ln the b'lood' It

suggests that loca'l secretlon by graft tfssue may have been sufflclent to allow contlnued development of one or more llnes of germ cells' ln Horeverr that sertoll cells and other germ cells cannot be seen these tubules makes lt dlfficult to understand how such development of might have occurrred. An a'lternative explanation is that lnsertlon the graft caused a localfsed pressure on surroundlng tubu'les and may the have caused a blockage fn the tubule. This mfght account for genera'l 'lack of cells in the tubular epithel ium' However' vrhy sPerm This should survlve ln thls situatlon is dffficult to understand'

sftuatlon seems worthy of further lnvestlgatfon' Immunohistochemlstry

wou'ld have been useful in determfning the tikely role of hormone secretlon in this sltuatlon. Neverthelessr lt is lnteresting to note a the number of llterature reports whfch deal wlth tlssue grafts of of anterlor pftultary. In a number of prevlous reportsr the lmportance 154

addressed' neurosecretor stlmul I for the anterlor pltultary have been anterJor pltultary A number of wörkers have reported that transplanted 'capaclty lnto glands lose.thelr functlona'l after transplantatlon hypophysectomlsedanlma.lsrpresumablyduetoa]ackofsecretomotor (1936)' lnnervatlon. whlle Gardner and Hill (1935), Hi'll and Gardner for May (I935r 1937) and Greep (1936) reported normal functlon (L952) transplanted anterlor pltuitary tissuer Harrls and Jacobsen problems wlth re-examlned these works and found a number of technlcal approach of the methods employed and questloned the valldlty of the might be due these authors. They went on to Propose that graft fai'lure tothelossofcontactofthegraftwiththehypophyseal-portal neural contro'l of the vesselr which mf ght norma'lly be assoclated wlth anterfor pltultary. support for thls hypothesls was found when they the demonstrated that grafts of anterlor pltultary placed lnto cfnereum subarachnoid space under the medlan ernfnance of the tuber 'long perlods' while became rf chly vascu'larf sed and remalned vlab'le for Thls grafts placed lnto the pitultary capsule dld not survfve so wel'l' experfment would account for the lack of LH and FSH secretlon noted in 3 of the current studY. Prol actl n hovleverr should be readfly detectable in anterior pituitary grafts even wlth the above hypothesls. separatlon of the system rernoves the 91 and from the hypothal amo-portal prolactin-inhibltlng factors whfch usually control the secretfon of secretion of pro'lactln this hormone ln the lntact pitultary. Thusr the by Vleber et aJ shou.ld lncrease conslderably. Indeedr a recent report Prolactin (1983) has fn fact reported slgnfflcantly elevated levels of grafts under ln the venous blood of rat testes contalnlng pftuitary prolactln the capsule. These authors also found slgnlflcant levels of r55 fn the testls contalnlng the graftr and reported lncreased testosterone levels fn tfssue ln the vlclnlty of the graft over the levels ln the rest of the testls. Thfs ls'not surprlslng glven the gonadotrophfc effect of prolactln reported by Nlcolì and Bryant .lg72), Ìrho proposed that prolactin may be important fn the marunallan gonad ln providfng pools of precursors whfch can be mobÍ'lised by FSH in and/or LH for the production of sterofd hormones such as androgens the testfs. In experlment 3r the signlflcant reductlon of prolactin levels ln normal rats follorfng castratlon probably reflects the inverse relationshÍp reported to exlst between secretion of prolactin and gonadotrophlns (see Grandfson et a1t L977). The hfstologlca'l flndlngs from experlment 3r of sperm in tubules around the grafts' may be due to local effects on androgen productlon by graft-derlved prolactin as proPosed by l{eber et a'l (1983)' The dlscrepancy in blood hormone levels reported by weber et a'l

(1983) and those of the present studyr fs dlfflcu'lt to understand' It at I00 may be of consequence that Weber et al made thef r measursnents days after transplantationr in comparfson to the measurernents at 2L easfest days fn the present study. certainlyr the pltuftary ls not the tissue to work wfth. Not only is ft relatively dffficult to obtaln Ín the first fnstancer but lts care after fsolatlon requires Partlcular attentfon. Plagna concentratlons of prolactln have been shown to (MatthieJ change dramatfcally under varlous experlmental condltlons pro'lactln and swartsr IgTg)r âfìd there are problems wfth relylng on levels as an lndlcation of graft functlon after transplantatlon' 'levelsr stress can consfderably affect plasna prolactln as can anaesthesia (Seggle and Brownr 1975). These prob'lems would be of partlcular concern in lntact anfmals bearing pltuftary grafts slnce 156

the anlmalsl own pltultary may affect measurements of any graft derlved hormones. Such problems would have to be carefully addressed lf further studles l{ere to be performed wlth thfs tissue.

3.4. THYROID TRANSPLANTS

At this polnt I chose to lnvestigate the use of another tÍssue for transplantationi one that could be more easily obtalnedr and one ways whose functfon could be more easl'ly assessed ln a varlety of without necessarily termfnating the graft. The thyrold gland u'as an obvious candldate. Because lt concentrates lodlne from the blood' its functfon can be readlly and quantltatively assessed ln sltu by monltorlng the uptake of admfnistered radloactive iodfne' It secretes a number of hormones (eg. Thyroxiner TFI-Íodothyronlne)' the concentrations of which can be easily monltored in plôellâ¡ and it fs a the who'le tissue whlch f s readily accessfble surgfcally' One'lobe or g'land can be removed wfthout serf ous prob'lems for the donorr ârìd certafnly without causlng the levels of mortality reached with

hypophysectomY. Given the problems encountered wlth the pftultary transp'lants' the basfc aims of this proJect remalned to be fulfilled. A.routine transplantatlon protocol that was conslstant'ly effective had to be remalned deve'lopedr the lmmune prfvl'leged status of the rodent testls the to be conffrmed, and components of the lnterstitial reglon of rodent testis that might be important ln creatlng the proposed lmmune privl'leged status of this slte had stlll to be examfned.

In developlng a routine transplantatlon protoco'lr lt was decfded L57 to use a slngle entlre'lobe of thyrofd for transplantatlon. Entlre reclpfents were usedr and rather than measure hormone secretlon from the grafts (whfch would be comp'lfcated by the presence of the anfmalts own thyroid)' it was declded to use lodfne accumulatfon a,s a measure of graft functfon fn 3'ltu. Two experfments were performed ln the ff rst lnstance. Inltlally, the abllity to transplant thyrold lobes from one rat fnto the testÍs of another rat was checked' and secondly' the usefu'lness of the testis as a transplant slte vúas compared wÍth that of the kfdney capsule - a sfte used extensfvely by immuno'loglsts studying graft reJectlon (see Lafferty et al, 1975; Gose and Bach' 1984). A comparlson of graft survlval over time between these two sftes was made ln thfs second experiment wfth grafts belng termfnated at varlous times after transplantatfon.

3.4.I. Experfmental Procedure

3.4.1.I. Anlmals

Ma1e Porton rats were used in these experfments and maintained as described ln 3.3.I.I. These anlma'ls were not inbred.

3.4.L.2. Experlment 4. Survlval of thyrold allografts in the testls

Thyroid 'lobes were removed from three male donors under pentobarbitone anaesthesla as descrfbed in 2.3.I. and placed fnto sterile Dulbeccors PBS. Flve male recfplents (2I0 - 270 grams) were transp'lanted wlth one thyrold'lobe into one testls as descrlbed ln 2.3.7.2. Three weeks after transplantation' each animal was fnJected 158

procedures ,,lth I25Iodlne and grafts removed 24 hours later' These havebeendescribedlndetallln2.5.l.l.Transplantedtesteswere ln 2'L4' fo]lowlng processed for hfstological examinatlon as descrlbed both grafted. and control measurement of accumulated radloactlvity ln testes.

3.4.I.3.Experiment5.Acomparisonofgraftsurviva]fnthekidney and the testis

as for Thyrold lobes were reÍnoved from sf>teen male donors (340 470 grams) were experlment 4 above. Thfrty-two male recfpients - random]yallocatedtofourgrouPsofefghtanima]s.Eachanlma]was grouP half the animals transplanted with one thyrold lobe, and in each received transp'lants receÍved transplants into one testis and ha'lf I n 2'3'7'I' and 2'3 under the capsule of one kidney as described '7 '2' Atlr214randSWeeksaftertransplantationronegrouPofanlmals l25lodine grafts removed 24 hours was selected and lnJected wttft and in both grafted later as in experfment 4. Radfoactivlty was monltored 2'5'I'1' Grafted and nongrafted organs in each an{mal as detai'led in as described tlssues were then processed for hisologlcal examinatlon

1n 2.14.

3 .4.2. Resul ts

3.4.2.L.Experiment4.Surviva]ofthyroida]]ograftsinthetestls

accumulatlon Four of the flve anlmals grafted shored slgnlflcant ofiodlnefnthegraftedtestiscomparedtothecontro.|testlsas lndicatedbyaconcentratlondffferenceofmorethan4tlmes.This 159

measure of ltsfgnlflcancerf was adopted from the work of Lafferty et al

(1975) who correlated lodlne accumulation measurements wlth hf stologlcal eva'luatlon and found that a 4 fo]d or greater accumulatfon of lodlne ln the grafted organ over the control was lndlcatlve of a healthy graft. 0n hfstologlcal examlnatlonr fdentiflable thyrofd fol'llcìes contafnlng colloid were found ln the four anfmals wlth signiflcant fodlne accumulation. The fifth anlmal shcned an lodlne accumulation in the grafted testls of 3 times that of the contro'l testf sr and whl'le histologlcal studles suggested healthy tissue t.las present ln the graft, ft was not included as a successful graft under the above crlterla. Flgure 3.7. shovls thyrofd grafts ln the testis at the tlme of transplantation (Figure3.7.a) and at three weeks after transplantatlon (Figure 3.7.b). As llas seen wlth the pftuitary graftsr the centre of the thyrold graft has degenerated ln thf s tlmer although hea'lthy fo]l icles are found around the perlphery of the tlssue mass. It ls presumed that these are the folìlcles concentratfng the lodlne as dfscussed above. Flgure 3.7.c shows a graft recovered from an anfmal slx months after transplantatlon' and general regeneratlon of the central fol'l icles of the graft has occu rred.

3.4.2.2. Experlment 5. A comparf son of graft survlva] in the kidney and the testfs

As in experlment 4r graft survival uÚas assessed under the guldelfnes of Lafferty et al (1975). The percentage of grafts survfvlng ln the testis and kfdney at 1r2t4taîd I weeks after transplantatfonr as assessed by fodfne accumulatfon measursnentsr ls

shqln f n Ff gure 3.8. Intratestlcular grafts 1¡ere aPParent'ly unab'le to Ffgure 3.7.-Thyroid at'lografts ln the testes of adu'lt rats: (a) at the time of transplantatlon' (b) three weeks after transplantatfonr and (c) sfx months after transplantation. Whfle the central mass of the graft ls necrotlc at 3 weeks

after transp'l antatl on, heal thy fol I ic'l es surv lve on the edge of the graft. By six months, the who'le graft ls reconstituted. Dfsruptfon of semlnlferous tubules in (c) fs due to physlcal dÍsruptlon durlng tlssue Processfng. X

80. 09r Ffgure 3.8. Number of survivlng thyrold al'lografts in the testes

(sol f d cf rcles) and kldneys (so'lf d trÍangles) of adu'lt rats at It2t4, and I weeks after transplantation (n=4 anfmals per group). Survlval was measured by accumulation of radloactfve l25lodln", and a success recorded lf the grafted organ was found to contafn more than four tfmes .- the amount of radloactlvÍty measured ln the contralateral, ungrafted organ. Number of grafts surviving (out of 4' O Þ lu u) À

É UJ o o o- q) À dl Ér o t to UI t q¡ rñ a (o= o)

T9T L62

concentrate slgnfffcant quantltfes of lodlne at I week after transplantationr although all the testlcu'lar grafts 1r¿ere aPParently be noted concentratfng lodlne by week 2 of transplantatfon' It should that a'll fntratestlcular grafts t{ere accumulating lodfne qt I week after transplantatlonr but they did not meet the 4-fold crlteria this discussed earlier. There vJas aPparently some mortallty after concentrate tfme, with onl y 50'/ of lntratesticular grafts able to sfgnlflcant quantities of iodlne by weeks 4 and I of the experiment' In contrastr whl'l e 75ø. of thyrold grafts p'laced under the kldney capsule u,ere able to concentrate fodine at I week after after transplantatfonr thfs abf'lity rapldly declfned and by I weeks i n any transpl antati onr no i odi ne accumu'l atl on cou'l d be regi stered thyrold grafts placed under the kidney capsule. Figure 3'9'a shows a thyrold graft under the kidney capsule at I week after transplantationr and Flgure 3.9.b shows a similar graft at I weeks after transplantatfon. ReJection of the grafts is clearly demonstrated A close at I weeks with a massfve lymphocytfc lnfiltratÍon. the correlation tl,as seen between the presence of folllc'les and concentratlon of iodlne. Hlstology shored normal thyroid tissue fn all intratesticular a grafts at I and 2 weeks after transplantation. Ffgure 3'10'a shows region graft one week after transplantation. Necrosls of the central are seen has already occurred by thls time, a'lthough healthy folllcles in the perlphera'l regions of the graft' By weeks 4 and 8r grafts collold-contalnfng thyroid fol'licles were only found in those such a graft that had also accumulated lodlne, and Figure 3'10'b shovls the I week at I weeks. Note that there is not much difference between seen ln thls and I week grafts fn the testis, compared to the change Figure 3.9. Thyrold allografts transplanted under the kldney capsule fn aduìt male rats: (a) at I weekr ôrìd (b) I weeks after transplantatlon. Note the healthy thyrold fol'licles at I

week whlch have been destroyed by the massive lymphocytlc lnflltration by I weeks. Tissue grafts are lndfcated (T) to dlstfnguish them fro¡n normal kidney tlssue (K). X 80. 163

t .,\

t \rl ,.i'

ü û I [,1

of adult Figure 3.I0. Thyroid a'llografts transplanted into the testis I male rats: (a).at I weekr and (b) I weeks after transplantation. Necrosfs of the central region of the graft has al ready occurred by I weekr arìd no significant change ls seen from this time to I weeks after transplantatfon. Healthy foltlcles are seen on the edge I of the graft ln both cases' A crease ln the tissue graft section can be seen in the necrotic reglon of the ln (b). X 80.

I '164

l 'j,|

I I

I ,1 .l .l

\ \

ü ç rl

1." n- t; .. t .

I 165

tlme ln the kfdneY (Ffgure 3'9')'

3.4.3. Dlscusslon

that the thyrold The results of these experíments demonstrated vrasamostsultab.letlssueforusefntheproposedtransplantatlon studies.Itwaseasllyobtainedandtransplanted,itssurviva]was readflymonltoredfnvivo,andgraftswereeasilyidentlffedin hÍsto.Ioglcaìstudfes.Aroutlnetransp]antationprotoco]wasthus

av ai I abl e. Theresu.|tsofexperiment5conflrmedthepreviousreportsthat privilege' Likqriser the testis possesses some degree of immunological privileged (see chapter reports that the testis may not be completely ii present study with the loss of some grafts il I) are a'lso born out ln the ',1 ,] overaperiodofSweeks.Howeverrthatgraftswerecontinulngto after transplantation concentrate iodine in the testis sqne 2 months indicate that thÍs site whf'le those in the kidney were not is seen to doeshaveanfmmunologlcallyprlvi.legedstatus.Whythyroidgraftsin thetestisdldnota.l]accumu]atelodineatlweekafter transplantatlonfsdifficu]ttounderstand.Laffertyetal(1975) when placed reported that mouse thyrolds were rapid'ly vascularised underthekidneycapsulelandthiswouldnotonlybeessentla]for the graft for graft survivalr but also for radloactive lodfne to reach accumu]atlon.Thyroidgraftslnthetestfsmaynotbesufflciently transplantation to be I vascularlsed by the end of the flrst week after I I of the admlnfstered lodlne. The lymphatlc slnusofds i able to accumulate for these grafts rodent testls may provlde sufflcient nourlshment

k 166

durfng thls timer ôrìd may mafntafn grafts untll vascularlsatlon ls completed. Howeverr free lodfne should also be ab'le to enter this lymphatlc sPace and these grafts shoul d thus have had access to sufflcient lodlne. That the hfstology of atl intratesticular grafts r,las apparently normal at thls tlme lends support to the suggestion that grafts may not have seen sufficient iodlne at this time, and perhaps other factors Yrere lnvolved.

Comparlson of the histo'logy frorn the present experf ments wfth that reported by other authors is of lnterest. Dempster (1957) has pub'l lshed pfctures of thyroid autografts at 48 hours and 20 days after transfer to subcutaneous tissues and these are shown in Flgure 3'Il' In the present experÍments, necrosis of the maÍn mass of thyrold tlssue was found vrithln 1 week of transplant, and Dempster (1957) found this to have occurred wfthin 48 hours fn autografts as seen fn Flgure 3.11.a. Accordfng to Dempster, it is the remalnÍng perÍpheral foì'llcles that regenerate to reconstitute the tissue mass. It is fnteresting that he found thls regeneration to have occurred wlthin 20 days ln subcutaneous tÍssue (3.11.b). This was not found in the testis in the present study even at elght weeks after transplantationr but rras apparent by 6 months after transplantation (see Figure 3.7')' It is these findfngs that lend further support to the suggestion raised earl ler that tissue regeneratlon and probab'ly va'scularÍsation of the graft are somehow lmpaired fn the testfs. No explanation can rea'lly be offered at thÍs stage as to why thÍs might be so. Howeverr in a recent revlery on angiogeneslsr Flndlay (1986) has reported the angiostatic action of certain sterolds when copresented wlth heparÍn. As discussed ln chapteF Ir mast ce'lls are found in the testfsr and are known to secrete heparln, and lt may be that this allovs certaln steroids to Flgure 3.11. Thyroid autografts .transpl anted into subcutaneous tlssues: (a) at 48 hours and (b) 20 days after

transplantation. ltlhfle the mafn mass of the graft has necrosed by 48 hoursr there is vírtual reconstructfon of the lobe by 20 days. (a) X 95. (b) X 50. (from Dempsterr

1957 ) .

I I I

i 167

-,a1

t a a ?- ai r68

act as anglostatlc factors ln the testls and thus affect the also regeneratlon of fntratestlcular grafts. Such a mechanlsm mfght explaln the natural preventfon of vascu'lar lnfl'ltratlon of the semlnlferous tubu'les. These prop,ogals require further lnvestlgation' testis' They may a'lso be of consequence for lmmune reactfons fn the preparations' ln The presence of ìymphocytes seen in the hfstologlcal or around successfu'l and reJected tissue graftst requires mentfon' (1982) and such populations have a'lso been reported by Janney et al of Bobzlen et a'l (1983) and it seems that the mere Presence .lymphocytesaroundagraftcannotbeassumedtomeanthatgraft (1982) the survlval may be ln Jeopardy. Janney et al characterlsed lymphocytesfoundaroundxenograftsofpancreatlcls.letsof ( grafts Langerhansr and found that ln successful grafts where were malntalned normoglycaøn1a ln dfabetic mlce) the lymphocytes assfstlng largely suppressor lymphocytes and were likely involved in graft survfva]. In reJected grafts hovlever, the popu.lation were

predomf nantly T-cytotoxic lymphocytes'

A number of authors have demonstrated that allograft is lmmunogenlcfty can be reduced if the tlssue to be transplanted (see 1975; maintalned for a perfod in organ cu'lture Lafferty et alr a'l' 1983)' Gose and Bachr 1984; Leuker and sharpton, 1974; Lafferty et This ls thought to be due to a reduction fn the number of leukocytes carrfed ln the transplanted tissue durlng the perlod of culture (Laffertyeta'lr1983;Laffertyr1984).ltlhl'letheprocessiscertafnly beneficlalr there remalns a degree of graft morta]lty' Leuker and sharpton (1974) demonstrated signlflcant survlval of ovarlan allografts following organ culture, wlth sorne 50'|' of cultured antatl on' compared al 'lografts sti l'l functi onl ng 90 days after transpl 169 to tota'l loss of all uncultured a'llografts by 35 days after transplantatlon. As seen in Flgure 3.Lz.r the resu'lts obtalned fn these studf esr whlle .over a sl lghtly dffferent'tlme scale are slml'lar to those found fn experlment 5 of the present study (compare wfth Flgure 3.8.). It ls concefvable that the testls itself may provide some mechanism of rernovingr maskÍng or lnactivatlng graft-derived passenger leukocytes whfch allows the survfval of uncu'ltured a'llografts wfthin lts interstitial region. This would account for the simllarlty fn the results dlscussed abover and might also explain the apparently fncomp'lete immune prlvilege afforded grafts in this reglon' This proposal will be addressed in'later sections of this thesis'

ON 3.5. EXPERIMENTAL MANIPULATIONS OF THE TESTIS AND THEIR EFFECTS ALLOGRAFT SURVIVAL

In order to determine uhich components of the testis mÍght be important in protectlng allograft survlva'lr â fìuffiber of experimental technlques were used to a'lter varfous aspects of testlcular function' The effects on thyroid allograft survlval of cryptorchídlsmr lfgatlon of the efferent ducts, and an fncreased lymph flovl from the testis lnduced with human Chorionlc Gonadotrophlrr vlêFê tested' Graft survival ln testes in which Leydfg cells and/or macrophages had been specif lca'l1y destroyed was al so examl ned'

3.5.I. Efferent Duct Ligation

Ligation of the efferent ducts leadlng from the testis to the epldldymls results ln retentlon of the f'lufd which the testls secretes. The testls becomes dlstended and turgld, and ultlmately the I

Flgure 3.I2. Comparative survlva'l of ovarlan autografts (so'l f d

trlangles)r cultured allografts (solld dfamonds) and

non-cultured allografts (open dlamonds) for up to 90 days after transplantatlon (from Leuker and Sharpton' L974).

Note the slml'larlty f n the pattern of graft survival of cultured ovarian allograftsr to that seen with uncu'lturedr lntratestfcul ar thyrofd allografts presented in Figure 3.8. 100

BO G c ¡-o +. o 60 c -J ï- o Ë40 o l¡ u) I 20

o\¡ 0 70 90 10 30 50 Days after grafting 171

germlnal epithelfum of the semlnfferous tubu'les degenerates (Setchell, 1970). The blood-testis barrfer ls dlsrupted wlthin 2-3 days after ligatlonr but neforms wlthin 3 weeks (setchellr personal communlcation).

The most promÍnent changes fn the testlcular milleu fo'llowlng efferent duct llgatlon t+ere expected to arfse from changes ln factors contrlbuted from the semfniferous tubulesr and thus thelr importance Ín maintalnlng allograft survival could be assessed by this treatment

3.5.1.1. Experlmenta'l Procedure

The efferent ducts of both testes ln elght adult male rats (410 - 490 grams) were tfed as described ln 2.3.4. Thyroid lobes were

obtalned f rom donor ma]es as detaf led in 2.3.1. Four recf pl.ents were transplanted wfth one thyrofd lobe into one testis at the tlme the efferent ducts were ligatedr and the remalning four reclpients were transplanted with one thyrold lobe three weeks after the efferent ducts were tied. Three weeks after transplantatlon' each anfmal was l25Iodlne inJected wltfr as descrfbed I n 2.5.1.1. and the graft termlnated 24 hours later. Transplanted testes were Processed for hÍstology as descrlbed in 2.L4. fo1lowfng measurement of accumu'lated radfoactive lodfne ín both grafted and control testes.

3.5.L.2. Resul ts

Al'l anlma'ls showed some lodlne accumulatlon by the thyrold 172

grafts. In those anfma'ls transplanted at the tfme of efferent duct ìlgationr only three of the four shored slgnfflcant accumulatlon (ratfo > 4:r)r whlle a:ll anlmals grafted three weeks after duct grafts' ligatlon shqred sfgniflcant accumulatlon of lodlne by the Flgure3.13.ashowstheappearanceofthetestlsat3weeksafter efferent duct ligatlon. spermatogenesis is clear'ly dlsrupted ln this sectfon, and large sPaces are found beùreen the tubules and interstitfal tissue. Figure 3.13.b shows a thyrold graft ln the lfgated testfs at 3 weeks after transplantationr and healthy folllcles are c'l earl Y v f sÍ bl e.

3.5.I.3. Discussion

Destructlon of the germfnal epithel lum as lnduced by efferent the thyrold duct I igatlon does not appear to have been detrlmenta'l for allografts used ln thÍs study. The ffnding that the grafts survived and after when transplanted after distenslon and turgor had developed (three after the tubu'lar structure of the testls had collapsed weeks duct ligatfon)r lndlcates that the tubu'lar compartment of the testls is un]lkely to be fnvolved fn maintainlng the lmmune prlvlleged status of the testls.

3 .5 .2. CrY Ptorch i d Í sm

l{hlle cryptorchidlsfn fs a natura'lly occurrlng abnormality in animals normally found wlth scrotal testesr lt fs easfly achieved experlmenta].lylnratsbyplaclngthetestisuplntheabdomlnal cavlty, and preventfng lts natural return to the scrotum' Figure 3.13. (a) Testis of an adult rat 3 weeks after I igation of the efferent ducts. Spermatogenesls fs clearly disruptedr ôrìd large exbravascular Ínterstitla'l spaces are seen between

the tubules. (b) A thyrofd a'llograft ln the same testis shown ln (a). Healthy thyroÍd folìic'les can be seen fn the graftr which does not appear to have been affected by 'llgatfon of the testlcular efferent ducts. X 80. 173

t.,lt8,.: I t'

tt'ít'i ìt t74

on the testls The most wldely recognfsed effect of cryptorchldlsm are rapidly is the lnductlon of semfnlferous tubule damage. Germ cells deptetedfnthecryptorchldtestls,althôughthesertollce]]shave The sertoll ce]ls been found on]y marginally altered morphologicalìy' ln the testis and some spermatogonla are the on'ly cells remalning Sertol I ce]'l after l ong term cryptorchidi sm (clegg, 19611 1963) . (within a few days of function ls affected by thls treatmentr since rates (Hagenas et the procedure) ABP secretlon ls reduced to very ]ow a1lL977).Thesemlnlferoustubu]eshoweverarenottheonly in Leydf g ce]'l testlcular components to be affected. Marked changes (see 1983)' Leydig cell morpho'logy and functlon a'lso occur sharpe, organelles involved volume lncreases with hypertrophy of the cellu'lar leve'ls are in steroldogenesls (Kerr et a'|, 1979). Serum testosterone cells together often lon, to normalr desplte these changes ln Leydig (1983) concluded that with lncreased clrculatlng leve'ls of LH. Sharpe and probably the changes ln Leydlg cel'ls were loca'lly lnducedr cells' Farrer involved changes in the factors secreted by the serto'll eta.|(1985)reportedimpairedtestosteronebiosynthesisfnthe lnvolved ln cryptorchld rat testls, and ldentlfled a number of enzymes cryptorchld testlsr testosterone synthesls which were fnhibÍted ln the blrth whlch but the animals studied were made cryptorchid soon after in the testls that may result ln dlfferent effects to those observed Kerr et al (1979) have also ls made cryptorchld ln the adu'lt anlma'|. fndlcatedthatdamagedsemlnlferoustubulesmaymetabo]fse and thls mlght testosterone to other metabolltes such as oestradlol' Leydlg cell account for the apparent dlscrepancy between increased p'lagna leve]s' production of testosterone and normal or decreased the 'level of the changes in the clearance rate of testosterone at t75

testlsareun]lke.|ytooccurslnceb]oodflowchangeshavenotbeen consequence of cryptorchidlsm demonstrated fn eutherlan mammals as a (Í,laltesandSetche]l,1964¡Setchel]eta.|11966;Waltesetall tg68).

Cryptorchidismisanothermeansbywhicha]terationsintubular of the disruption is functlon can be achieved, although the aetfology quitedifferenttothatachfevedwithefferentduct.l.lgatlon.

3.5.2.1. Experímenta'l Procedure

Tenadu]tmalerats(300-350grams)rmaintainedasdescrlbedin the method described 3.3.r.r. were rendered bilateral'ly cryptorchld by 1n2,.3.3.tryroto]obeswereobtalnedfromdonormalesasdetal.ledfn 2.3.!.Fivereciplentsweretransplantedwithonethyrold]obeinto placed Ín the abdomen' and the one testis at the tlme the testis was with one thyrold'lobe lnto remalnlng ffve recipients Y{ere transplanted thetestlsthreeweeksaftertheyweremadecryptorchld.Threeweeks wittt t25lodfne as after transplantationr each animal was lnjected describedin2.5.l.l.andthegrafttermlnated24hours]ater. Transplantedtesteswereprocessedforhistologyasdescrlbedin2.L4. fo.llo*ingmeasurementofaccumu]atedradfoactivelodlnefnboth grafted and control testes'

3.5.2.2. Results

A]]anlmalsshcnedslgnificantaccumulationofiodinelnthe graftedtesteswhencomparedtotheungraftedtestls,andlnall L76

c€tsêsr the ratlo between both testes tras greater than 4:I. There was no apparent dlfference between grafts transplanted at the tlme of placlng the testfs fnto the abdomenr and those grafted three weeks after the testls was placed fn the abdomen. Collofd-contafnfng thyroid follicles yrere found fn al'l grafts. Hlstologlcallyr the grafts and testes were very simllar to those shcnn Ín Figure 3.13. after efferent duct I igation.

3.5.2.3. Di scussi on

These results suggest that the lmmune prfvlleged status of the cryptorchfd testfs ls as effective as that of the scrota'l testis. Graft survlval does not aPPear to be affected by lncreased temperaturer or by the destruction of spermatogenesls seen fn experlmentally-lnduced cryptorchÍd testes. Thls fs fn agreement wlth the flndlngs of Selawry and Whlttfngton (1984) who found the cryptorchld testls to be a suftable slte for pancreatlc lslet al'lografts ( see a'l so 3.3. ) .

3.5.3. Human Chorlonfc Gonadotriphin

Lymph flow frorn the rat testls is lower than ln other mammals (Rat - 0.5 ul/glh Setche'll and Sharper 198I; Ram - L ullglh Lindner, 1963; Boar - 10 ul/g/h Setchet'l et al, 1983). This might possibly al'low a1'lograft antlgens to be presented to the host lmmune system ln

a manner that would fnduce lmmune tolerance rather than reJectlon.

Human Chorlonfc Gonadotrophfn (hCG) has been shown to cause an accumulatlon of interstltla'l flufd in the testis beglnning 8 to 12 L77

hoursafteraslnglesubcutaneouslnJectlon(SharpeL977alL979l the rlses ln vascular Permeabillty 1n I9BO)r câüsed by appreciab]e Sharper ln lymph flow (Setchell and testlsr and accompanled by rlses I hours after inJectlon of hCG' I98I). These changesr whlle apparent peakaround24hourspost-lnJectlon.Subsequentstudieshaveshown thattheeffectonvascularPermeabilitylsmaxlmalatabout30hours afterinJectlonofhCGandnormalva]uesarefoundagafnby4Shours post.lnJection(Sowerbuttsetal'1986).Ifaprotocolcou]dbe deve]opedtomaintainanincreasedlymphf]owoveranumberofweeksr theimportanceoftherateoflymphf.lovlforallograftsurviva]cou]d be assessed.

lYmPh flovr wlth hCG 3.5.3.1. Maintenance of increased

HcG]eadstoadownregulationofltsreceptorsontheLeydig cel'ls(Sharper1976¡HsuehetalrIgT6)andareducedandrogenlc responsetosecondandsubsequentfnJectlonslfgivenduringthe4S hoursfol]ovlfnganlnftlalhccinJectlon(Hsuehetal,L9l7\.Itwas to lnvestigate the effect of repeated therefore necessaryl initlal.ly, inJectionsofhCGontesticu]arlymphfloYllinterstftia]fluidvo.lume andvascularpermeabllltyinadultma]eratsrlnordertodetermlneif anincreaselnlymphf]owcou]dbemalntalnedoveragreaterperiodof time.

3.5.3.1.I. Experfmental Procedure

Adu]tmalerats(2so-570grams)weremalntalnedasdescribedln four groups of ten rats recelved 3.3.I.I. In the flrst experimentr 178

dally subcutaneous lnJectlons of 50 f.u. hCG as described ln 2.6. for elther lt 2t 3 or 4 days. Ten uninJected rats served as controls wlth 'l measúrements of vascul ar Permeabf f tyr lymph f'low and f nterstf tl al . flufd volume being nade 20-24 hours after the last hCG ÍnJectlon (see 2.L0., Z.LL., 2.!2.). In a second experiment, fOUr grOUps Of f f ve animals received efther one dose of 50 l.u. hCG or 2 or3 siml'lar doses with either 48 or 72 hours between lnJectlons; measurements of albumfn c'learance were nade 24 hours after the last hCG iniectfon (see 2.I2.).

3.5.3 .L.2. Resul ts

3.5.3.L.2.L. Effect of dally doses of hCG

r25I-hSA The resu'lts show a marked response of spaces and 125t-f,SA ha'lf-tlme of clearance of 20 hours after the flrst hCG lnJectionr lndfcatlng that there were fncreases ln vascular SIC.-EOTR permeabilfty and'lymph flotv (Tab'le 3.4.). l{hfle the sPaces tend to lncrease over the treatment perlod, these changes were not signfficant. Albumln clearance rates dfd not remaln elevated after the first day of treatment and ln fact feìl signiflcantly below control values by day 3 of treatmentr returnfng to contro'l values by day 4. In contrast to the orlgfnal method of Setchell and Sharpe (198I)r the values used to calculate lymph flotl were obtained from two dlfferent groups of anlmals. The values presented are thus calculated on mean values only and standard errors cannot be determlned. Hovever they suggest that lymph flqr was also elevated only on day I of treatment

whereas vascular permeabilfty peaked at 24 hours and then sloltrly volume or tspacer cr-EDTA and albumfn, ha'lf tlme of Tab.le 3.4. Measured changes f n one-hour df stributfon _of after clearance from the testls and the calcu'lated limph flow and vascu'lar Permeabl'llty albumfn group). repeated dally inJections of hCG (50 i.u.) (Means + SEM, 5 anlma'ls Per

Hal f-time of Lymph Vasèul ar Days Testi s Dlstribution Volumes 'low A'l bumí n cl earance f permeab'l I ity of wef ghts Cr-EDTA Albumfn (¡r'llg) (min) ( ¡r1lg/mi n) ( p1lmln/g) hcG (g) (pllg) treatment

+ 2.95 208 + 23 0.66 0.47 + 0.07 0 1.30 + 0.04 198.95 + L2.L9 34.90 * * * + 8.61 65+13 2.53 1.4r I 0.30 1 1.30 + 0.03 237.50 + 25.54 82.98 * * 4.15 I87+ 6 0. 89 o.g5 + 0.14 2 1.35 + 0.04 239.r4 + 22.56 58.53 ! l( * * + 540 + 3I 0.51 0.92 + 0.ll 3 1. 16 + 0.05 334.49 + 31.65 58.70 3.42 * + 2.06 r87 + 32 I.07 0.66 t 0,04 4 I .13 + 0.05 288.63 + 1I.11 49.52 14.59 62.86 0.32 L. S. D. n. s. lì. s.

(agalnst used to detect sfgnfficant treatment Analysls of varlance and covariance testls welghts)-was llsted the Least slgnfflcanl Difference va'lue (L.s'D'r 5% level) ls H dlfferences, and where approprlate the control value are { (n.s. lndicates no slgnfficant dlfference). values differlng signlflcant'ly from \o (*). lndlcated treatment grouPs. consequently group values Lymph Flow are ca]cu'lated from the results of two dlfferent for and of sfgnlffcanõe cannot be detêrmlned' means have been used ln ca]culations and standard errors levels I80

returned tovards normal.

3.5.3.L.2.2. Effect of doses of hCG eVery 2 or 3 days

Elevated c'learance rates of albumfn from the testfs after the

second and third doses of hCG were seen if the frequency of the 48 hours or 72 hours (Tab'le ÍnJections ',Jas reduced from 24 hours to

3.5. ) .

3.5.3.I.3. Discussion

The present resu'lts on the effect of one lnJectlon of hCG on

a'lbumfn space and albumln clearance conffrm the observatfons prevlously reported by Setchell and Sharpe (I98I) and Sowerbutts et al found an lncrease in lnterstltlal fluld ü 0986). Prevfous studfes also I volume at 20 hours after a sfngle hCG inJection (Setchell and Sharper IgBl). tt,hi'le the results obtalned fn the present study show a similar trend (SlC"-fOtA spacer Tab'le 3.4.)r îorìê were found to sfgnlflcantly SlC.-'fOtR differ from control values, but the control values for the space are higher in the present study than those found by Setchell and sharpe (1981)r ôrìd thls may contrfbute to the lack of significance. No explanation can be offered for thls dffference.

Following repeated admÍnfstratfon of hCG as used in the first

experlmentr there Ytas a sustalned elevatlon in albumln space and vascular permeabillty, although both tended to return towards control, and at no tfme were the effects as great as those followlng the first fnjectlon. By comparlng these resu'lts with the effects of a single

Þ r81

ìl

Tab'le 3.5. Half-tlme of clearance of albumln from the testls 24 hours after one dose or the Jast of a serles of repeated doses of hCG with lntervals of 48 or 72 hours between doses (Means + SEM).

Days of treatment Al bumln cl earance wlth hCG (Tt/Z r mln)

i., + 0 208 ! 23 +x. t 65+13 * Iand3 6s! r ì( il land4 I07+ 2

, * ï I'3 and 5 88 + 1 * I'4 and 7 86+ 3

+ from Table 3.4. * All va]ues are slgnificantly (P < 0.00I) less than the control value, the values ln llnes 4 and 5 are nigner than I 1ne 2 (P < O.OOI and P < O.0I respectively).

I t82

fnJectlon of hcG (sovlerbutts et alr 1986) lt appears that second and subsequent lnJectlons uúere t{lthout effect on these parameters' søerbutts et al (1986) reported an a]bUmln'space of 53 + 3'I7 at 48 hours after a slngle lnJectlon of hcG which fs not sfgnfflcantly dlfferent from the value fn the present study of 58.53 + 4.75 at 48 (see hours after the flrst and 24 hours after the second hCG dose Tab'le 3.4.). The fallure of lnterstitlal flufd vo]ume to lncrease slgnlffcantly over the perlod of treatment as reflected by the 5IC.-EOTR spâc€r desplte slgnlflcant changes fn vascular permeabllitV' by suggests that changes in vascular Permeabillty were accompanied be so concommltant changes ln lymph flor. llthy a'lbumfn clearance should dramatÍcally reduced by day 3 of treatment ls difflcu'lt to understand' and presumably Vascular PermeabÍ'lfty fs still lncreased at thÍs tlmer other factors may also be lnvolved ln regulatlng lymph flow and albumln c'learance f rom the testis. {d't ,î (seCond If fnJections are admfnlstered every 48 hours or 72 hours to experlment) rather than every 24 hoursr the response by the testls

each dose of hCG, in terms of albumin clearance ( ref'lecting lymph flovl in part) ls simflar to that in anlmals recefvlng a single dose' dal]y This means that the loss of response found ln anlmals recefvlng after the lnJections of hCG can be fu'lly recovered withfn 48-72 hours prfmary hcc inJectlon. setchell and Sharpe (198I) proposed that the

effect of hCG fn the testis is an indÍrect effect on vascular by the permeabil ity with changes due to certafn substances secreted proteln testls ln response to hcG. steroids, prostaglandins and of I synthesfs have not been found to have any influence over the effect {r testls hCG on hormone uptake or f nterstltia'l flufd vo]ume fn the rat

r r83

(VelJola and RaJanlemlr 1985; Sowerbutts et alr 1986). However these

authors have recently demonstrated the inhibitory effect of an antagonlst to 5jhydroxytryptam'lne on albumln sPace lncreases afte¡: hCG treatment ln the rat testls whlch may fmPlfcate mast ce'lls ln the actfons of hcG on vascular permeabÍllty in the testis. If secondary substances are responsible for the lncreased vascular permeabillty

after hcG adminlstration, the repeated hcG doses may have affected production and/or release of these substances. Setchell and Rommerts

(1985) have found that the hCG-induced fncrease in permeabillty of testlcu'lar blood vessels ls completely abollshed by pretreatment wlth ethane dlmethane sulphonate (EDS)r â colìpound whfch ellmfnates the Leydig cells from the testls (Molenaar et al, 1985). However, an

effect of EDS on other cell types has not been excluded. The c'lose assocfatfon of Leydlg cells with testfcular macrophages (Nfernl et al' 1986) and mast cells (see Nlstal et a'I, !984¡ Maseki et alr 1981) may tl a'lso be lmportant for the actlons of hCGr and would also be affected

by treatment wlth EDS.

A reductlon ln testicular bindlng of hCG within 24 hours of treatment has prevfously been demonstrated (Hsueh et a'l , L976' L977 ¡ Sharper 1976) and thfs is re'lated in part to loss of LH (hCG) receptors. There Ís also a large decrease ln the capaclty of the testis to secrete testosterone when stfmulated agaln at this time (Sharper I977b) and the two events may be re'lated. The loss of LH receptors persfsts for long perlods and is not maxfmal until 2 days after treatment (Hseuh et alr L977). Whfle the vascular response may I present study shol It be medlated via hCG receptors, the results from the that a second hCG dose at the tfme of maxlmaì receptor loss produces

r r84

the anticf pated increase f n vascu'lar permeabl'lf ty. Thf s suggests that

the normal numbers of hCG receptors on the Leydlg ce'l'l are not

.neêessarf ly fequf red for the :vasoactlve.actlons,.of hCG Jn the testf s,. It ls fnterestlng to note that Bergh et a'l (1986) and Wfdmark et al

(1986) have recently presented evldence whfch impl lcates leucocytes fn

the hCG induced changes fn testlcular vascuìar permeabÍlity.

3.5.3.2. Effect of lncreased lymph flow on a'l'lograft surviva'l

Havlng establ lshed that lnJectlons of hCG every second day

maÍntained an increased lymph f'low from the testls (as reflected by albumin cìearance rates), the effect of this treatment on thyrofd

a'll ograft surv ival I n the testf s was i nvestf gated. ir 3.5.3 .2.I. Experlmental Procedure

Twenty adu'lt male rats (300 - 420 grams) were mafntaÍned as described Ín 3.3.1.I. Thyrold lobes for transplantatfon were obtafned

from adult maìe donors as descrfbed ln 2.3.L. Recf plents were random'ly allocated to four groups of ffve anfmals. Two groups recefved one thyroÍd lobe transplanted lnto one testfsr while the other two groups did not recelve transp'lants. One of transplanted groups and one of the non-transpl anted groups recelved no further treatment. The other

transplanted group received subcutaneous lnJectfons of 50 i.u. hCG every second day as descrfbed In 2.7.t as dfd the other untransplanted

group. HCG fnJectfons were contfnued for three weeks after transplantatlon. Three weeks after transplantation and 24 hours after

the last hCG lnJection, all transplanted anlmals vúere lnJected wfth 185 l25todfne as descrfbed ln 2.5.I.I. Twenty four hours laterr testes

¡¡ere removedr and lodfne accumulatlon by the lntratestfcular grafts was monltored. Grafts were then processed for hfstologfçal examfnatlon as described ln 2.L4. At the time of graft removal¡ clearance of radÍoactlve iodlnated albumfn from the testls rvas monltored ln both groups of the non-transpìanted animals as described 1n 2.L2.

3.5.3 .2.2. Resul ts

The half-tfme of clearance of albumin from the testis ln hCG

fnjected animals u,as 56f6 of that in the unfnJected control anlmals (IO8 + 7.35 mfn : L92 + 19.6 mfnr P < O.0I - students t-test) All transplanted anfmals showed signiffcant lodlne accumulation in the grafted testls when compared to the control testis (ratfo > 4:I). There rlas no slgnlflcant difference between graft survlval as

measured by the lodfne ratlo fn the transplanted-control group and the

transplanted-h0G f nJected grouP Q.43 ! 0.32 z 4.67 t 0.52) Histo'logy shwed co]'lofd-contaÍnf ng fol'llcles ln al'l graftsr frrespective of treatment. Grafts fn untreated anlmals were no dlfferent to those shovrn previousìy fn Figure 3.L4. As seen in Figure

3.14.ar hCG treatment did not alter the morphologlcaì appearance of

the testis, whfch vJas very slmilar to that seen ln control anfmals. Likewfse the grafts were not great'ly different to those of control

anlmalsr and Flgure 3.14.b shows a graft after 3 weeks of hCG

i nJ ect f ons. Flgure 3.14. (a) Section of the testls from an adu'lt rat injected

subcutaneously with hCG every second day for 3 weeks. The appearance of the testis fs no dffferent to control

testes. (b) A thyrofd allograft in the same testis shown fn (a). Healthy thyrofd follfcles can be seen ln the

graftr whlch does not appear to have been affected by hCG

treatment. X 80. 186

{t r. 187

3.5.3.2.3. Dlscussf on

By meagurf ng the half-tlme cllea¡.ance of albumln f rom the testls and the.volume of fnte¡rstltlal fluld (one hour Cr-EDTA spâce)r ô quantitatlve measure of lymph fìovr from the testis can be made (see 2.L2.). However, half-tlme clearance of albumln alone fs lndfcatlve of the state of lymph f'lorv (see Tabl e 3.4.). The maJorlty of albumin ÍnJected lnto the testis'leaves via the'lymphatfcs (Setchell and Zuppr unpub'lished observatlons) and an fncreased clearance of albumln from the testisr r{fth a constant or increased fnterstitial flufd volumer must reflect an lncrease fn lymph flow. In the anfmals fniected with hCG for 4 consecutfve days there was no slgnfffcant change ln interstitlal fluld volumer although the va'lues do appear to have

fncreased somewhat over controJs. Whl'le such measurqnents were not made in anlmals lnJected wfth hCG every second day for two weeks, lt was assumed that fnterstitla'l ftuld volume dfd not fall durlng thls time. Thus, the increased clearance of albumln from the testls fn these anlmals ls likely to ref'lect an lncreased lymph flow, and provfdes conffrmation of the effectlveness of treatment. It was also

assumed that the transplanted anfmals recelvfng the same hCG treatment

would also have shovln this response had they been tested (iodfne admfnistered to monitor graft survlval prevented this). Sfnce the surviva'l of grafts fn the hCG lnJected animals tras no dlfferent to that in the unlnJected animalsr lt appears that an fncreased lymph flov ls not detrfmental to graft survfvaì. It ls therefore'llkely that the slovrer rate of lymph flow fn the rodent testls comPared to that

found ln other mammals and other reglons of the bodyr is not of importance fn provldlng the lmmunologica'lly prfvfleged envlronment ln r88

the rodent testfs.

3.6. IMPORTANCE OF LEYDIG CELLS AND/OR MACROFHAGES FOR ALLOGRAFT SURVIVAL IN THË TESTIS

Leydigcellsspontaneous]yadhereto.lymphocytesand non-specifically suppress prol lferation of mitogen-stfmulated and a'llo-antlgen stlmulated lymphocytes ln vftro (Born and wekerler 1982)'

Androgens and other sterold products of these ce'lls have been shown to

be fmmunosuppressfve (Clemens et al t L979; Roubinlan et al' L9771' consequentlyr the Leydlg ce]'l has been proposed as a I ikely contrlbutor to local lmmunosuppressfon ln the testfs (Head et a'l' 1983c; Head and Bi'l1f ngham, 1985). The drug ethane-dimethyl-su1 phonate (EDS) has been used extenslvely fn recent years to speclffcally destroy Leydig cells fn rodents (Morrfs et a'lr 1986¡ orLeary et alr 1986; Molenaar et al' 1985). },lhi'le serfous and sometfmes fatal slde effects occur fn other specfes (eg. plgs dle after EDS adminlstratlonr Dr. C.J.G. Wenslngr Dept. Veterinary Anatomyr State University Utrechtr The Netherlands' personal communlcatlon)r no other effects have been noted in the rat' It was of lnterest to examfne al'lograft survival in anlmals ln which Leydf g cel'ls had been destroyed by EDS'

Hoyreverr a number of other ce]'ls are found ln the fnterstitlal tlssue of the testls, and of Partlcualar lnterest are the macroPhages (see Nlemf et al' 1986). Whlle these cells do have phagocytfc actlvities, they cannot be assumed to be the same as other rovlng

macrophages of the fmmune system. They are resldent fn the testls' and as dlscussed fn the lntroductory revlew (chapter 1) are found ln close 189 assocfatlon wfth Leydlg cel'ls. They have even been proposed as Leydfg cetl precursors fn the rat (Clegg and MacMfllan, 1965a). The posslb'le role of these màcropha.ges ln providlng the lmmune Prlvllege In the rat testls has not been prevlously examlned. Thelr common contaminatlon of l-eydfg celì cultures mlght contrÍbute to the reported

lmmunosuppressive ef fects of Leyd.lg ce'l'ls f n v f tror dl scussed above. Hovatta et al (1986) have reported the effects of sfl ica-lnduced

damage to macrophages ln the testes of rats. Alllson et al (1966) and gehler et a'l (1978) have prevlously demonstrated the cytotoxfc effect of fntraperftoneal inJections of sflica on macroPhage actlvityr wfth assoclated loss of;natural kilter actlvfty. Horäver, Hovatta et al (1986) reported that testlcular macrophages vtere. apparently destroyed by fntratestfcular fnJections of sllicar although the evÍdence was not concluslve. It was felt warranted no*"u"., to include an lnvestlgatfon

of the effects of f ntratestlcular inJectlons of si'lica o,n allograft ' su rv iv al ,,

3.6.I. Experfmenta'l Procedure

Twenty eight adult male rats (350 -'480 grams) were malntalned as described ln 3.3.I.I. Thyrold lobes for transplantatlon u,ere obtafned from adult male donors as described,,in 2.3.L. In an lnitial trfal, two groups of four anlmals,were transplanted with one thyrold lobe into one testisr wlth one group recelving intraperftoneal lnjectlons of EDS

as described I n 2.8.r ônd the other grouP recelvlng lnJectlons of vehlcle only (DMSO/water). trn the second experfmentr recfpients were randomly a'l'located to one of four treatment grouPs wfth five anlmals ln each groupr and agafn transplanted wfth one thyrold'lobe fnto one I90

testls. One group remalned untreated as the control $Foupr one grouP was lnJected wlth EDS as descrlbed ln 2.8.r orìê grouP was f nJected wlth sltlca lnto the testls to be graftedr as descrlbed ln2.g.r ôrd the final group recefved both fntraperltonea'l EDS and fntratestlcular

Sf I f ca. Sl'llca f nJected anima'ls u,ere lnJected f nto the ungrafted testls with a sfmilar vo]ume of saline vehicle to monltor the effect of this technlque on the testÍs. All anfma'ls were transplanted 3 days after treatmentr âtìd grafts termÍnated 3 weeks later. Animaìs were l25lodine lnJected witn 24 hours prÍor to graft removal' as descrfbed In 2.5.I.1. After examining iodlne accumu'latfon in both testes of all anlma'lsr testes were placed in Bouins ffxative and processed for

hl stol ogf ca'l examl natlon.

3.6.2. Resu'lts

Graft survfval was assessed by the 4:1 ratÍo rule of Lafferty et a'l (1975) discussed in 3.4.2.2. Results are presented in Table 3.6.' grouping results of both experfments. One contro'l anlmal in the inltfa'l trlal dld not survlve the experfmentr and one of the anfmals in the second experfment receiving both EDS and intratesticular Si'lica was reJected at the tfme of graft removal because the ungraftedr sallne injected testis had regressed consfderablyr and could not be

used as a contro'|.

When graft survlval ln all EDS treated anfmals fs compared to

that in all non-EDS treated anfmals, graft survfval was slgniflcantly 'lica reduced by EDS (P < O.OI). Such comparisons of survlval ln al'l Sf treated versus non-Si'l lca treated shovls no slgnlficant dffference. 19r

Table 3.6. Efiect of EDS and/or Sl'llca on thyroid allograft survlval. Graft Survival Treatment Success Fall

Control 7 I a EDS 3 6 Sfl fca 3 2 a S¡ ica + EDS 0 4

Combf ned non-EDS contro'ls IO 3 b Combf ned EDS treatments 3 10

Comblned non-Silica controls 10 7 Combfned SlIica treatments 3 6

a - slgnlflcantly dlfferent from Control (P < 0'05) b - sfgniffcantly dlfferent from combined non-EDS controls (P < 0.0r) (Signfficance tested In 2x2 contingency tables wfth X2 ana'lysis as descrlbed by Finney (1948) and Latscha (1952) ) 192

Three weeks after EDS admlnfstratlonr tubules aPPear relatlvely grossly hea.lthy, Figure 3.15.a. The fnterstftlal regfon ls underpopulatedandthep¡edomlnantcel'ltvlê.lsthetestlculan.'' macrophage (Flgure 3.15.b). Tlssue grafts'are slgnlflcantly infiltratedbyl'ymphocytesasseenlnFigure3.Is.candnohealthy fo]llcles are obvlous. At greater magnificatlon however' the odd grafted testlsr while follicle remnant ls seen (Figure 3.15.d). In the graft, there does the lmmune reactlon fs concentrated around the aPpeartobesomedamagetotubu]essurroundlngthegraft¡andthe fnterstitfal tlssue ln the vicinlty of the graft also contafns a sfgnlflcantnumberof.lymphocytes(Flgure3.15.c).Thlsfsnotthe (Figure case ln other regfons of the testis 3'15'b)' Sflica ínjections into the testis apPear to have dlsastrous effects at the sfte of fnJectlon' Figure 3'16'a shows an tubules at lmmersion-flxedr salfne lnJected testisr and healthy dlfferent siages of spermatogenesls are clearl! sêêrìr fn assoclation the with relatively normal fnterstltfal tlssue. Flgure 3'16'b shows slte of a sflica inJectlon lnto the testis. spermatogenesls has been tubules' and completely abollshed fn what remafns of the semlnlferous of the lnterstitlal tlssue appears to be lnfiltrated by large numbers survival horeverr lymphocytes. This treatment has not affected graft amongst and as seen ln Flgure 3.16.d the graft ls easity ldentffled

thls mess of damaged tfssuer wlth healthy collold-contalnlng the slte of fol]ic'les. It appears that the damage ls loca]ised to qulte unaffected by inJectlon, because the rest of the testls aPPears delinfatlon the treatment. As seen ln Flgure 3.I6.cr there is a c'lear

of the damaged regf on and norma'l testls tf ssue' combination of The combined use of EDS and sillca has produced a Figure 3.15. Sections from the testis of an adult rat 3 weeks after treatment with EDSr and transplantatlon of an intratesticular thyroid a'llograft. (a) seminiferous tubules are relative'ly healthyr and so¡ne contain sPerm. x (b) 80. The fnterstitial regÍon is gross'ly underpopulated

and the predominant cell type ls the testicu'lar

macrophage. Some regenerating Leydig ce'l'ls are apparent' x320.(c)Thethyroida.l]ografthasbeendestroyedby inf iltrating 'lymphocytesr and no hea'lthy foll ic]es can be seen.Thisreactiona]soaPPearstohavespreadÍntothe surrounding interstitial tissue. x 80. (d) At greater magnifÍcatfon, some follfcle remnants are obvfous (F). X 320. te3

-. I V a. a nl f \rì \ ' :¡i'- t-. , '';' *;, þ{ rr'i tI', ' lv ¡ t. 'l I ? I

d. -Æ

.'-D :; Figure 3.16. Sectfons from the testes of an adu'lt rat 3 weeks after an lntratesticular lnJection of SilÍca' and the

transplantatlon of an f ntratesticular thyrold allograft. (a) Control sectlon from a sallne fnJected testis shoving heatthy tubu'les and interstitial tissue. (b) Site of injection of Si'lica partÍcles Ínto the testfs.

SpermatogenesÍs has been complete'ly abolished in what

remains of the serniniferous tubules, and the interstitlal tissue fs heavily fnfiltrated with lymphocytes. (c) The

reaction to Sil ica is very speclf f cr and clear'ly delineated. Tubu'les Ín other regions of the testfs do not appear affected by the treatment. (d) Likewiser the thyroid allograft aPPears unaffectedr despite being placed in the region damaged by Si'lica. Healthy thyroid folticles are seen fn the peripheral regfons of the

graft. X80 {

I ¡ iu i

(

I 4,: t. fli I,. tù.

I I

Þ6t r95

The the effects seen when these treatments are used lndlvldua'lly' evldent ln locallsatlon of the sl:!lca-fnduced damage ls agaln clearly ii celJs ln th9 lnterstltfSl Ff gure 3.Ì7.a and 3.17..c.'The'1ack of Leydlg EDS treatment reglon and the atypfca'l appearance of thls tlsbue after lsagalnseenfnFlgure3.lT.b.ThegraftisseenlnFlgure3.LT.c, seen when EDS was used and while the severfty of the lmmune response a]one(seeFlgure3.15.)lsnotobvfousinthissectionrnohealthy thyrold fo.|]lc]es can be seen. Horvever as rl|as noted ear]ler, some and a rfng of fol'llcle remnants can be seen at higher magnificatlonr foìllcular epithellal cells is clearly seen amongst the lymphocytic/flbrobl astlc mass ln Flgure 3'17'd'

3 .6.3. Dl scussf on

d Theseresu]tssuggestthattheLeydfgce.l]sarelmportantfor l thyroldallograftsurvlva]intherattestfs.Whetherthesece]]s maf ntaln perform speclflc lmmunosuPPressive functions' or secretlons remalns fmmunosuppressfon by the actlons of thelr steroid tobee]ucldated.Importantlnteractionshavebeendemonstrated successful destructlon between Leydig cells and macrophagesr and the usefu'l experlment' Horeverr of macrophages by sfllca would have been a That graft the actions of sllfca treatment aPpear very localÍsed' taken to mean survival was not affected by this treatment cannot be provisfon of an that macrophages are not lmpllcated ln the Indeed' ln the immunologfca]ìy prfvlteged environment in the testls' qufte slllca-treated testes most of the lnterstltla'l reglon appeared I unaffected and the macrophages (and Leydlg cells) ln these {r normal when slllca was areas may have been responsfble for graft survlval'

ï Figure 3.I7 . Sections from the testis of an adult rat 3 weeks after transplantation of an intratesticular thyroid allograft, an intratestlcular fnjection of Sflica, and treatment with EDS. (a) As noted previous'lyr the region of

Sil ica-lnduced damage Ís very ìocal lsed. X 80. (b) The

ll lack of Leydig cells ln the interstitlal regionr and the $ ,il atypfcal appearance of this tissue after EDS treatment is clear'ly evfdentr although there aPpears to be a more genera'lfsed presence of lymphocytes fn the interstitial

region than seen when EDS, or Sil ica is used alone. X

'l 320. ( c ) The thy roi d a1 ograft has been rej ected, and infl'ltrated by lymphocytes. X 80. (d) As noted with EDS treatment a'lone ( see Figure 3.15. ) ' some f ol'l icl e remnants can be seen in graft zone (F). X320.

T 196

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O Ia

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al.a

.t ttl (t '4 rl '.ff ,r a\ b oa ' :i.. t .;.j. I : ' ¡0: l r 'r Ì I r97

thyrold used ln conJunctlon wlth EDS, the reduced survfval of allografts appeared to be due to the actions of EDS rather than of on sf llca. More iJetälled lnvestlgatf ons of the actlons 'Slllca can be macrophages are obviously requlred before their lnvolvement not possfble dfscountedr and unfortunately further lnvestfgatfons were in this studY. status of The effects of EDS treatment on the lmmune prfvlleged of the rodent testls requf re further examinatf on' l'lhÍle the reJectlon allografts after such treatment may relate to the effects of EDS on (1987) on macrophage Leydlg cellsr the recent flndings of Kerr et al As seen numbers ln the testls after EDS treatment requlres dlscussion' ln Flgure 3.18.r macrophage numbers fn the testls are slgnlflcantly are also increased 3 days after EDS treatment. Most Leydig cells completely destroyed by thls stage. blhi'le thls lncrease in macrophages rapldity mlght be due to prolfferatlon of testlcular macrophagesr the of the fncrease ln numbers suggests that they may be circulating the testls to macrophages of extratesticular origln that infi'ltrate supported by phagocytose the degeneratfng Leydlg cel'ls. Thls ldea fs theapparentdec]ineinmacrophagenumbersbyT-l0daysafterEDS of admfnlstration. Indeed, Kerr et al (I9S5) report the destructlon Leydig cells with EDS to el lcft an immune response involving of lymphocytes macrophage-lymphocyte lnteractlons, with 1 arge numbers fnflltratlng the fnterstitial tlssue 7 days after EDS treatment' The destructlonofLeydfgce]lsbyEDsmayaffectthefunctfonof testicularmacroPhagesrandcauseoral]ovlaccumu]atlonof extra-testicular marcrophages ln the lnterstltial regfon' Thls mlght thena]lownormalprocesslngofgraftantigentothehostlmmune systemascfrculatlngmacrophagesareProcessedlnlymphnodesl Figure 3.18. Numbers of Leydig cells and Macrophages in the testes of adu]t rats up to 70 days after EDS admfnistration (from Kerr et al ' f987 ). 30

25 Lcydlg cell¡ / llacr

20 (o o F x (,)15 J J l¡¡ o \ t-' 10 r tt'"- I 5

ro @ o 7 14 21 28 42 70 Control 1 3 Days after EDS r99

generat{ng productlon of sensltized cytotoxlc lymphocytes, and ultimate graft reJectlon. This ls a very excitlng hypothesls' and the lmp.ortanceof:Leydlg.cel]s.andmacrophagesforthefmmunologlcally prlvi'leged status of the rat testls warrants further fnvestlgatlon' 200

CHAPTER 4: THE OVINE TESTIS AS AN IMIVIIJNOLOGICALLY PRIVILEGED SITE 20r

4.I. INTRODUCTION

Al'l prevlous r,iork publ ished on graft survival in the testis has (see chapter used only rodents and reìated species as the recipients abil of the l). To my know'ledge there is no description of the íty ighted Ín the testis in other mammals to support al'lografts. As high] introductory review to this thesis, the anatomy of the interstitial regionoftherodenttestisisverydifferenttothatofother animals for various mammals. To use tissue grafts in the testis of studies of endocrine function and possibly growth manipulation would Likewise' the require them to have immuno]oglca]1y prfvileged testes' the human medícal imp'lications of much of this research would require testis to be an immuno'logically privileged site' Theanatomyoftheinterstitialregionintheovinetestisis mammals' with much mo.re representative of that found in the larger usefu'l to sheep readily available as a resoulcêr it was obviously was also examine the immune status of the ovine testis. Likewise' it mÍght be of interest to examine testicular lymph for any hormones that secreted by tissue grafts in the testisr given that there was no indication in the earlier experiments that such hormones were leaving earlier the testis in venous blood. such studies were hampered in the lymphatics studies because of the inability to access the testicular authors have in the rat, due to their small size. However, a number of ram described the cannulation of the testicular lymphatics in the (Lindner, Lg63, L967t 1969; Cowie et a'l , Lg64; Morris and Mclntosh' (Hall and I97I; Ball and setchel'1, 1983). Morris and co-workers Morris' i4orris, 1962, 1963r 1965; Ha]1 et al, L967; Lascelles and extensively t96I; Pederson and Morrisr 1970) have also used the sheep 202

specles would be in lmmuno'logica'l studfes, and ft was felt that this 'lymph cannot most sultable for the studies requfred. whíle testicular be assumed to be exactly the same as the ftuld fn the lnterstftlal reglon of the testisr it should provide an indication of changes occurring in the interstitium that might be important during a'llograft after surviva'l or rejection. This would be useful in following events initiatlng various experimental manipulations of the interstitial the environment. Horvever before one could pursue the involvement of lymphatics in transporting graft-derived hormones out of the testis' elements in and the importance of lymphoid and interstitÍal fluÍd to providing immune protectection in the testisr it was necessary

demonstrate allograft surviva'l in the ovine testis'

Thischapterdiscussesthesurviva]ofautograftedand allografted thyroid tissue, and allografted pituftary tissue in the ovine testis.

4.2. EXPERIMENTAL PROCEDURE

4.2.I. Animal s

Adult Merlno rams were obtained from the Institute flock and ad libitum and housed indoors in individual Pens. water was avaÍlable (Milling animals were fed lucerne hay and sheep pellets Industriesr the Adelaide) once per day. All experlments were performed durlng season but months of Aprl] and May tovrards the end of the breeding

when animals were still sexually active' 203

4.2.2. ThYroid TransPl ants

'thyroldectolnised 7n 2'3'L't , Twelve rams were totally as descrlbed be used as graft and the recovered thyroid lobes were p'laced on ice to materia].Transplantationalwaysoccurredwithintenminutesafter and thyroid removal. six animals received intratesticular autografts fnto one six received intratesticular a1'lografts of one thyroid lobe testÍs, at the tlme of thyroidectomy as described in 2'3'8' The animal' contra'lateralr ungrafted testis served as a contro'l Ín each Threeweeksaftertranp.lantationanimalsu,eregivenaTRH chal'lenge as descríbed ln 2.5 '2' Fourweeksaftertransplantationra]tanima.|sreceivedan (see and were intravenous bolus of I moi lodine-I3l 2.5.1.2.) Austral ia) castrated 48 hours 'later under ha'lothane (F'luothaner ICI the anaesthesia. Thyroid hormones are largely transported in circulation Ín associatlon with serum bindlng proteins such as albumin (Robbins et al11978) and Galil et al (198I) have shown that over 95% Because of albumin injected into the ram testis is cleared in lymph. of thisr testicular lymph was thought possibly to be an important hormones avenue for the transport to the general circulation of and lymph samp'les produced by the grafts in the testis. To al'low blood tobecol]ectedlpolyvinylchloride(PVC)orpolyethylene(PE) (2.3.5'2') and a catheters were inserted into a testlcular veln of both testicular lymphatic vessel (2.3.6.) in the spermatÍc cord vÍa testes prior to castratfon. Jugular venous blood was also sampled plagna and lymph samples an f ndwel-ling catheter ( see 2.3.5.2.). Blood the distribution were stored at -zOoC until assayed. After castration Tissue of I-I3I was assessed in each testis as described in 2'5'L'2' 204

from the transplant site of the grafted testfs was then processed for histo'logical examination as described ln 2.I4' Paraffin sections Were stained wlth haemotoxyl ln and eosln and examlned 'by,i ight'mlcroscoçiy. Blood samples were taken at weekly intervals fro¡n thsse twelve anfma'ls and four other contro'l rams of the same age and from the same to flock, for hormone analysis. sampling was begun six weeks prior transplantatfonr and continued through the experiment until four weeks after castration.

4.2.3. PituitarY TransPlants

Inasecondexperimentranotherfourramsreceived intratestfcular pituÍtary al'lografts. Four hea'lthy Merino males castrated soon after bfrth (wethers) were selected as pftuÍtary 'lack donors. In these anlma'ls, the of steroíd feedback on the pituitary should have rendered the pituitaries hyperactive in terms of gonadotrophin secretion. The protocol adopted in this experiment was initia'lly to prepare the testis of the recipient anfmal in the same

way as used for thyroid transplants (see 2.3.8.). Donor pituitaries and were obtained just prior to transplantation as described in 2'3'2' p'laced on lce until transplanted as described in 2.3.8.' and no more than ten minutes elapsed between the removal of the donorfs head and the transplantaion of the fsolated pituitary into the recfpient testi s. To ensure that the pituitaries were functfoning normally prior to transplantation, donor wethers were challenged with an intravenous bolus of Lutefnislng Hormone - Releaslng Hormone (LHRFI) three weeks prfor to surgery as descrfbed In 2.6. At the same time, the rams that 205

LHRH as were to be used as graft reclpients were infused with LHRI-I which' if described i n 2.6.2.L. to check that a concentratfon of 'local infused into the testlcular artery to provide sufficient by viab'le concentrations of LHRH to stimulate gonadotrophin secretion peripheral transplants ( see 2.6.2.2.), would not be sufficient in the circulation to stimulate the animalrs own pituitary into secreting

gonadotroPh i n s.

To Four weeks after transplantation al'l animals were castrated' an expanded examine graft function before the testis was removedr surface of the catheter was Ínserted into the testicular artery on the (see 2'6'2'3') for testis and LHRH was infused through this catheter viable one hour to stÍmulate gonadotrophin secretion by the grafts' pituitary grafts shou'ld secrete protein hormones under such a stlmu'lus, and these would probably leave the testis in testicular .lymphandvenousb]ood.CatheterswerethereforeplacedÍntoa and 2'3'6' testlcular vein and lymphatic as descrÍbed i n 2.3.5.2 continued for two Samplfng was begun 20 mín prior to LHRH infusion and hours afterwards at 10 minute intervals' After castratlon, tÍssue from the graft site and a comparable pieceoftissuefromthecontro.ltestiswereplacedinPicric as Acid/Formaldehyde fixatíve and processed for immunohistochemistry

described i n 2.L5-

Bloodsamplesweretakenweeklyfromthesefouranima]srand the same age throughout the experiment from four other control rams of and from the same flock, to monitor gonadotrophÍn levels' 206

4.2.4. Immunohi stochemi strY

cryostat sections of graft tisgue and normal Pltultary were prepared and processed for immunohistochemica] loca'l isation of (LH)' Foll ic'le-stimulating hormone (FSH), Lutelnising hormone Prolactin (Prl) and p-Endorphin (End) as descrÍbed in 2'L5'2' Sections were mounted in buffered glycerol and examÍned by fluorescent

microscopy.

4.2.5. Hormone AssaYs

Plagna and lymph concentrations of LHr FSH, and TSH vlere measured in using doub'le-antíbody radioimmunoassay procedures as described 2.L3.3.2, 2.L3.3.4 and 2.13.3.7. Concentrations of these hormones are (NIADDK-oLH-24)' expressed re]ative to the follorving standards : LH radfoimmunoassay FSH (NIADDK-oFSH-I5), TSH (NIADDK-bTSH-I-I). A di rect

was used to measure serum TO as detailed in 2'I3'2'

4.3. RESULTS

4.3.I. ThYroid TransPlants

4.3.1.1. TRH chal'lenge

To The TRH chal'lenge 3 weeks after transplantation stimulated secretlon fn intact anlmáls as seen in Figure 4.L., but dld not have any effect in the thyroldectomised thyroid-transplanted animals' whether the grafts Yúere auto- or a'llografts' Flgure 4.1. Plasrna TO levels fn Control (solld and open dfamonds), Thyrofdectomlsed-thyrofd autografted (open squares) and Thyroldectomlsed-thyrof d al lografted (sol id squares) Merlno Fâ¡tsr fo'llotring intravenous lnJection of either 2ml physlologlcal sal ine (one group of Contro'l anf mals on'ly,

open dfamonds) or 200 ug TRH fn 2ml physlologlcel saline

(second group of Contro'l anlmalsr sol f d diamondsr and a'll transplanted anlmals, solld and open squares). Values plotted are means (n=6) I standard error of the mean. t40

TRH r20 + ^ -E 100 o) \/c BO ç Þ o 60 Êo o -o. 40

N 0 {o - r20 0 1,20 240 360 480 600 Time (min) 208

4.3 .L.2. Iod I ne I ocal f satl on

Typlcal photographs obtained with computerlsed gamma photography are shown in Ffgure 4.2. While five of the six autografted testes (Figure shovred specíf ic iodf ne loca'l f sation Ín the region of the graft 4.2.ùr fìofìê of the six allografted testes showed any such localisation (Figure 4.2.br P < 0.025 by X2 test). A cross-sectional scan of tissue counts through the testis shows a signÍficant concentration of counts at the autograft sÍte when compared to background (Figure 4.2.ù while the allograft counts cou'ld not be 'leve'ls df stlngulshed f rom background (Ff gure 4.2'd) '

4.3.I.3. Hi stoì ogY

Figure 4.3 shovrs rePresentative sections of the grafts at four weeks after transplantatfon. l,lhi'le groups of c'learly defined' heaìthy thyroid fo1l lc'les containing colloid can be seen in the autograft (Figure 4.3.a) no such tissue was found in the a1'lograftr although follfcle remnants could be seen (Figure 4.3.b).

4.3.L.4. Plasrna and lymph hormone concentratlons obtained at castrati on

No significant leve'ls of thyroxine could be detected in the

venous blood or lymph obtained from any of the testes at the tlme of surgery (see Figure 4.4.). Figure 4.2. Representatíve computerised gamma photographs of iodine accumulation fn testes of thyroidectomised Merino rams contalnlng thyrofd autografts (a) or thyroid allografts 131lodin". (b), 48 hours after administration of I mOi The outer heavy lfne shows the outlfne of the testls and the smaller open circle the sÍte of the suture in the tunfca. In the cross-sectfona'l scans of counts for both grafts (crd)r ordlnate values indicate units of field wfdth, and abcÍssa values fndicate ratlo of counts relatfve to background. Abcissa va'lues Ín (c) are not dÍrectly

comparab'le to those in (d)r where background values

have not been subtracted. i {

I I I'

I 0 0

ht 0t

ir 4\ i1 t! t1

6,02 Figure 4.3. Representative histologÍcal preparations of intratestfcular thyroid autografts (a) and thyroid al'lografts (b) four weeks after transp'lantatf on. X 320.

H ,î

Ì I

! 21Q

¡ $ r, .il',

,] ilt l,'l I ,t

u t I

;

Ì Flgure 4.4. Plasrna levels of TO fn perlpheral blood (solld bars) and

testlcular venous blood from transplanted (open bars) and control (hatched bars) testesr three weeks after thyrold transplantatlon. Values pìotted are lndlvfdual levels for Autografted (numbers 1-4) and Aìlografted (numbers 5-8)

I rams.

ti 7

A -E o) vc o ,= x o l¡ .ç l- t

0 7 I I 2 3 4 5 6

N Ram number H H Autografts All ogr af ts 2L2

4.3.1.5. Plasrna hormone concentratlons in weekly samples

Levels of plasna TO and TSH mgasured fn weekly blood samples from all animals are shown Ín Figure 4.5. Control anlma'ls showed a reasonably stable plasna TO leve'l whi'le assocf ated TSH levels v"ere basal (Figure 4.5.a). There was no difference in the hormone profiles of autografted (Flgure 4.5.b) or allografted (Flgure 4.5.c) anlma'ls and in al'l transplanted animals, TO leve'ls fe'll to basel ine values within two weeks of thyroidectomy. TSH levels rose as a consequêrìCêr peaking 4-6 weeks after thyroldectomy.

4.3.2. PituitarY TransPl ants

4.3.2.I. LHRH chal lenge

Figure 4.6. shc¡ws the response of donor wethers and recipient

rams to the pre-transplant LHRH challenge. A]l wethers showed a (sÍng1e significant LH secretion in resPonse to the injected LHRH dose of 5 ng/kg bw). The one-hour infusion of LHRH (tota'l dose of 2 ng/kg) into the'latera'l saphenous veln of reciPient rams did not stimulate LH

secretion sign lf icantl Y.

4.3.2.2. Plagna and lymph hormone concentrations

'The.infusion of LHRH into the testfcular artery failed to fnduce

any secretion of LH or FSH in any of the anlmals' Unstlmulatedlevels to in venous blood and lymph from grafted testes were no different those from samples from the control testes or Peripheral blood' Fi gu re 4.5 . Plagna levels (nglml) of TO (solld squares) and TSH (open squares) fn weekly blood samples collected from (a) control, (b) thyroldectomfsed-thyrold autografted and (c) thyroldectomf sed-thyrold allografted Merf no rams. Samples were not taken ln weeks 9 or Il. In treated anlmals, T lndlcates the"tfme of thyrof dectomy and transp'lantatlonr and C lndicates the tlme of castratlon. Values pìotted are

means (n=6) * standard error of the mean. 2L3 100

o ^ 100 @ E T C o, BO V Y vÊ c o 60 t-+. o b 40 +t e o C) 20 Ê o o 0

100 T C 80 V V

0 0?4 6810t?t4 Time (weeks) Figure 4.6. Leve'ls of LH (ng/ml) in Jugular plasna of castrated

(l¡lethersr sol f d df amonds) and entÍ re (Ramsr open dlamonds)

male Merfno sheep follorvlng intravenous admfnfstration of

LHRH. l{ethers recelved a slngle boìus injection of 5ng

LHRFI/ kg bw lnto the Jugu'lar veinr and rams r€cefved an intravenous fnfuslon of 2ng LHRH/mfn for I hourr fnto the lateral saphenous vefn (see text for detafls). Time of fnJectfon or begÍnning of infuslon is lndicated. values plotted are means (n=4) J standard error of the mean. 20 LHRH

16 +

,A E gt T2 vÊ tJ o E B o o l- o.

N HÞ 0 B0 100 r"o -20 0 20 40 60 Time (min) 2L5

4.3.2.3. Immunohl stochemi strY

F.luorescent.conjugatedantlbodiesfai]edtorevea]any contro'l slgniffcant regfons of survlving tissue in any of the grafts' 4'7" and pituitary tissue hovlever was well stafned as seen in Figure ned f or the given showed cl ear'ly def i ned cel'l s specif ical I y stai some stainfng' this was hormone tested. white the graft reglons showed to be largely non-speclfic and any speciflcally stained cel'ls appeared

invasive PhagocYtes.

4.3.2.4. Plagna hormone concentrations in weekly samples

in pituitary grafts had no impact on circulatlng LH or FSH leve'ls

any of the anlmals.

4.4. DISCUSSToN

reJection of The results of these experfments clear'ly demonstrate direct contrast endocrine allografts by the ovine testÍs. This is in (Dib-Kuri et a'l t L975; to similar experiments performed ln rodents appears that the Head et a.l, rgg3a; whltmore and Gittes, rgTg). rt to that of rodents immune competance of the ovine testis ls superior the mamma'llan and it is not correct to make the generalizatlon that testis is an immunological'ly prlvileged site'

as judged by iodine Because thyroid autografts were not reJected, transplantation accumu'lation and histologyr the technfques adopted for wou]dapPeartobesatlsfactoryalthoughitwassurprislngthatthe Ffgure 4.7. Immunohf stochemlstry of representatlve sectlons of control Testls (arb), control Pituitary (c'd) and fntratestlcular

pf tuf tary A'llograft (e'f) treated wlth f'luorescent- conJugated anti-ovÍne FSH; and control Pltuitary (grh) and intratestlcular pitultary Allograft (f'J) treated wfth fluorescent-conjugated antl-human End. Each fluorescent image (arcrergrl) is accompanfed by lts phase-contrast

image (b,drf,hrJ). X 180. gLZ 2L7

were thyrold hormone proflles of autografted and allografted anfmals not dlfferent. Howeverr a number of explanations may account for thfs' have been too The tlme between transpl antatlon .and graft renrgval may short to allow sufficlent recovery of graft tlssue f9r To .secretlon' It is more:llke'ly that the survlvlng pockets of thyrofd tlssue ln the autografts may have secreted TOr but the small amount of tissue meant that leve'ls produced were below the sensitlvity of the assay. 2t8

TESTIS GENERAL DISCUSSION CTIAPTER 5. THE IMMUNOLOGICALLY PRIVILEGED - 2I9

5.1. DISCUSSToN

The processes fnvolved fn inftiating and effecting thq reJectlon response arç.stll'l not completely understood. l{hile efflcient grafts lymphatiC drafnage ls necessary for prompt reJection of some (Barker and Bilinghamr fgdgl, there is now considerable evidence that the critlcal events in the rejection response occur ln the graft itse'ff (Pederson and MorrÍs, L9703 Ascher et alr 1981). A number of ce]1 types have been shovn to participate in allograft reJectlon (Hayry et al, 1984) lncluding T-ce'lls, Natural Kilter cells and

Macrophages. However the authors of such reports have also lndicated that reJectfon ultimately depends on the development of a genera'lised inflammatoF}Fesponserinitiatedandregulatedbyspecific antigen-related lymphocytes. There are therefore numerous processes occurrlng locally in the graft that mÍght be subiect to interference from the envfronment of the graft and previous workers have rodent demonstrated that the immunologically privileged status of the testls is probably due to some factor or factors within the testls Itself. Testicular-derived factors that may contrfbute to such Ínterference and account for this privlleged status have recently been (1985)' revfewed by Head et a'l (1983a, 1983c) and Head and Bitlingham Suggestlons inc'lude the Leydig cel'ls because of theÍ r abil ity to 'lymphocytes adhere spontaneously to and to suppress non-speciffc prol iferation of mitogen-stimulated and alloantÍgen-stlmulated lymphocytes ln vltro (Born and l{eker'ler 1982); the germ cel'l populatfon because of their abillty to impair T-cel1 responses to

'lymphocytes, and 'lymphocyte responses to mitogens (Shearer and products Hurtenbachr L98\ Hurtenbach and Shearer, I}S!); and varfous 220

t{hlch can be shown Such as androgens, progesterone and Prostaglandlns tobefmmunosuppresslveinvitro(Clemenseta.l,L979;Roubinianet a]r1977).Experimentsalterlngendogenoussteroid.levelslor ln affecting various cells of thg lmmqne system have been employed attemptstodetermfnethemechanlsmsunder.lylngtestlcularimmune L982)' privi'lege (Stites and Siiteri, 1983; Hurtenbach and shearerr

TheexperimentsreportedÍnchapter3haveconflrmedthatthe prfvilege. The rodent testis does possess some degree of immune tubules are resu'lts presented demonstrate that the seminlferous of unlikely to be invo'lved in protecting allograftsr since impairment duct 'ligation their normal functions by cryptorchf dlsm and efferent The destruction of germ has not adversely affected graft survival. graft survivalr cells by experlmenta'l cryptorchfdism has not affected immune and they are thus unllkely to be involved in conferring the various protection on allografts in the lnterstitium. Likevllser two treatments suggest that effects on sertol I ce] 1 s caused by these an immunologically they are unl ikely to be the prime instlgators of their privileged environment ln the interstitial tissuêr although site preclse role in contributíng to the privl'lege afforded thls (1985) concluded that cannot be altogether discounted. whitmore et al privi'leged sertoli cells were responsible for the immunologlcally only status of the testisr after studies in rats with serto'll-cel] results fn testes created by in-utero irradiatlon. This treatment cells sterlle testes, but does not adversely affect the lnterstitlal of these (Means and Huckins, Lgl4'.)r âfìd therefore the conc'lusions probably authors about the importance of the sertoll ce]l is 'lymph flow f rom incorrectly founded. In the present study' the s'lovler 22L the rat testis has also been found to be of llttle importance fn protectlng a'lìografts in the testis from fmmunologicaì rejection' f s that The.conclusion drawn from these lnltf a'l lnvestigatlons the rat the factors responslb'le for the lmmune prlvftege afforded testis are probably located withln the lnterstitia'l reglon of the products testls. As indicated, the Leydig ce]ls and their androgenfc great'ly affected are possib'ly impl icated. Graft survfval in rats was bythedestructionofLeydlgcel]swlthEDswhichmayimplicatethe from Leydig ce]ls and/or thelr products in protecting allografts accumulation of immune reJection in the testis' Howeverr the after this macrophages and lymphocytes in the lnterstitial tissue may react treatment (Kerr et a'1, 1985) suggests that the immune system as a consequence of to EDS treatmentr ând that grafts are rejected ce1l this treatment, rather than as a resu'lt of the loss of Leydig to occur functfon. Apart from the Leydig cellsr other cel] types known ln the testicu]ar lnterstftium may aìso be involvedr and the mast region must ce'llsr macrophages, and mesenchymal cel'ls found in thls chapter 3 a'lso be considered. PrelimÍnary experiments described ln uslng attempted to examine the involvement of testicular-macrophages by Hovatta si'l ica to destroy these resident macrophages as described et a'l (1986). Howeverr thls treatment was found to be very'loca'lised (1986) (who in ln its action - a finding not reported by Hovatta et al factmakenomentlonofthehistologyofthetestfsaftersuch aìlograft treatment). The importance of testicu'lar macrophages for survlval ln the testls remaf ns to be determined'

ovine testis The resu'lts presented in chapter 4 fndicate that the testis can no is not an immunologlcally prlvi'leged site. The mammalian 222

longer be generally referred to as lmmunologlcal'ly prlvlleged since one exception has now been demonstrated, and from an anatomlcal perspectlver the Privileged testls may in fact be a rodent-speclflc ovlne and rodent phenomenol. Jhe dlscrepancy.ln lmmune :tqtus of the testfs ls unllkely to lnvolve sterolds as the so]e factor ln and immunosuppression. First'lyr the types of steroids present, thelr relatfve concentrations, would have to be appreclab]y different between sheep and rats to account for the dlscrepancles in a1ìograft survlval reported fn the prevfous two chapters. Howeverr such data as fs availab]e suggests that the steroid profile in these two species fs not greatly dlfferent (see Setchell' 1978). SecondlYr the importance of sterof d invo'lvement rernains questlonab]e because of the unPhyslologlcallyrr¡igh|lconcentrationsneededln-vitrotoproduce 1982) It is lmmunosuppresslon (Wyle and Kentr L9773 Holdstock et al ' ' lnterestfng that Head and Bl'll lngham (1985) have reported the reduced pretreated survlva'l of f ntratestlcul ar parathyrold a'l'lografts in rats wlth a sing'le inJectlon of oestrogen. These animals had low clrculatlng levels of testosterone although Íntratestfcular and testlcular venous leve'ls of testosterone do not apPear to have been 'l the examined. The Leydig ce]l is the f ke]y source of oestrogen in (1974) adult rat testfs (Payne and Valladares, I98O) and Mulder et al have demonstrated oestradfol receptors on Leydfg ce1ls. Lou¡ Periphera'l levels of testosterone after oestrogen treatment have been shown by other authors to be due to dfrect inhlbitlon of Leydlg ce]l function (Kalla et al, 1980; Tsai-Morris et alr I9S5). Head and Blllingham (1985) proposed that local]y produced hormones might be the key to survfva'l testlcular lmmune prlvilege. They suggested that the reduced of lntratestlcular parathyrofd allografts ln rats inJected 1-2 days 223

'level prfor to graftlng wlth oestradiol cyplonate was due to a reduced of fmmunosuppressfon in the testls due to the ìower lntratestlcular levels of androgen. However, exogenous oestrogen treatment allso de Jong et al' suppresses pltuftar:y LH secretlon (Gay and Devert L97Ii and body I975)r ârìd might therefore affect other cells ln the testis cul ar I eve'l s of genera'l 1y. Kal I a et al ( I98O) have al so reported testf testosterone to be reduced after oestradÍol treatment' but found pregnenolone and in particular progesterone levels to increase than signlflcant'ly. Pregneno'lone ls more lmmunosuppressive ln vitro suggests testosterone (wy1e and Kentr L977¡ PavÍa et a'l, L979) which that the results of Head and Bil'lingham (19S5) mfght not be simply due to a lack of steroid inhibition of the immune response. rn 1969r on macrophage Vernon-Roberts revlewed the effects of sterold hormones actlvfty.Oestrogenshaveastrongstimu]atingeffectonthe in the macrophage system general'ly, and oestrogen injectlon results sp1 een and 1 iver and appearance of I arge numbers of macrophages i n the (Helmy and Nicolr the mobilizatfon of these cel'ls Ínto the cÍrculation a sing'le 1g5I; Nicol and Helmy, 1951; Nicol et al, Lg52l. Following vernon-Roberts subcutaneous inJectlon of 17p-estradiol in the ffiousêr in the (1969) observed two peaks of lncreased numbers of lymphocytes perftoneal cavity on the first and sixth days after inJection' Both by an peaks of increased lymphocytes were followed 24 hours later Íncrease in the number of cel'ls intermediate in appearance between 'lymphocyteandmacrophagerand4Shourslaterbyanincreaseinthe (1985) would number of mature macrophages. Head and Bi'llingham therefore have made thelr grafts at a time of increased lmmune actlvity. Leydlg cel'l function rÚas also affected at thls tlme and testosterone leve'ls were slgnlflcantly reducedr which may have 224

affected other ceìl types ln the testfs and reduced the

lmmunosuppression normally found ln this sfte. These reports lndlcate : that the effects of an oestrogen inJection are not confined to the LeydÍg cell, and ft ls therefore un¡¡lse to assume that the reduced survlval of parathyrold allografts fn the testis after oestrogen treatment was only due to an impafrment of Leydig cell function. other factors besides steroids are lfkely invo'lved ln fmmune suppressfon in the testis.

There rsnain therforer two fundamenta'l areas for examination and (if comparison between the two specles used fn the present experlments not in other speclesr as Yrell). FirstlYr We need to be ab'le to compare the interstitial envfronments of the ovine and rodent testisr examining the various physlological aspects of their functlon' sterold and other factors r{ productfonr and types and leve'ls of steroids lit I produced; and secondly to examine the various immunocompetant cel'ls in the interstitfal regfon of the testisr and to understand their functions, and relationshfps with other cetl types of thÍs reglon'

ù 'ffi;..i- r .::.'.nT+*- --*- 225

CIIAPTER 6: MATERIALS AND METHODS

,ll rl,T

lr 226

In addftfon to the materlals and methods outlfned in chapter 2' the follovlng materla'ls ánd methods were used ln the studies to be presented fn thfs section.

6.I. REAGENTS

Testosterone, Pregneno'lone, hCG, and Coomassfe Brllliant Blue

C-25O were obtafned from Sfgma Chemfcal Co.r St. Loufs, Mfssouri'USA' 3H-Testosterone, 22Nu-sodlum l4c-Mannfto], RadÍo'l abel I ed Ch] orider 3H-Wut"., und 125I-Sodium lodide were obtained from Amershamr

Austral i a.

6.2. HEATING OF TESTES

oC Local ised heating of the testes at 43 for 30 minutesr was

achfeved by anaesthetising the animals with sodium pentobarbitone (Nembuta'lr Abbotts; 60 mg/kg bw) and placlng them on two sheets of oC. perspex ìying across a water bath that was maÍntafned at 43 Th' sheets of perspex vúere slightly separated so as to allow the animals just scrota to hang down into the water whfch was kept at a level Anfma'ls be1ow the perspex to ensure complete warmf ng of the scrotum' were left in this posÍtlon for 30 minutes. They were then returned to thei r cages after recoverY.

6.3. PREGNENOLONE t I I Pregnenolone was dissolved fn sesame ofl, and administered to I hypophysectomised animals wlth dafly subcutaneous lnJections for

! 227

fourteen days. Animals were hypophysectomfsed as described fn 2'3'2' and pregnenolone lnJections were commenced on the day of hypophysectomY.

6.4. TESTOSTERONE IMPLANTS

Lengthsofmedical.gradesllastictubing(4.7mm0.D.r3.4mm by I.D., Dow Corningr Michlganr usA) were prepared Prlor to fllling closing one end wfth silastic adhesfve (Silicone Type A adhesive' Dow corning, Mlchiganr usA). A gnal'l funnel was used to pack testosterone (Sigma) Ínto the tublng, and when we]1 packedr the imp'lant was sea'led with another plug of silicone adhesive. Imp'lants were stored fn a oC dessicator at 4 untll used. Anima]s were anaesthetised wfth sodium pentobarbitoner and a srna'l'l incision made ln the skin over the abdomen. Using a blunt-ended prober a subcutaneous pocket was created of sufflclent size to contain the required implant. After feeding the lmplant into the pocket' the opening was sutured closed.

6.5. CELL DISPERSION TECÈINIOUE

A fluid can be obtained from the testls after dlspersÍon of testicular cells and centrifugatfon. The testicular capsule },as possible' carefully removed wlth as much of the testicular artery as (Setchellr The The rete testls is also removed with the capsu'le 1978)' resultfng tubular mass was carefulìy transferred to a 5 ml dfsposable syringe to whfch was attatched a 21G needle. The tlssue ln the syringe

was expelled through the needle lnto two Eppendorf tubes, and 228

immedlately centrffuged at 8000 g for 5 mfnutes. The resultlng oC supernatant was col'lected and stored at -20 until assayed.

6.6. HORMONE ASSAYS

6.6. I. Testosterone radi oimmunoassay

Plagna testosterone concentrations were determined usfng antisera raised in a ewe against a testosterone-3-carboxymethyloxlme-BSA conjugate (Dr. R.I. Coxr Hormone Assay Development Groupr csIRo Divislon of Anfmal Productlon, B'lacktown, N.S.|{.). The specifÍclty of this antfsera Ís shovln in Tab'le 6.1. The details of the procedures followed rrere as described by Dr0cchio (198I). Brieflyr duplicate aliquots of p'lagna (from 5 ul to 100 ul dependlng on testosterone (L2 concentration) were added to disPosable glass culture tubes x 75 adding I m] mm) and the volume adjusted to I00 u1 wlth gel PBS. After of toluene : hexane (2:1) mfxburer the plas,ma was vigorously extracted 'l for I minute on a vortex. The plasna was then f rozen in a iquid nitrogen-ethano'l bath and the solvent extract was decanted in to another set of cu'lture tubes. The solvent mix was then dried under a oC). stream of nitrogen gas (40 Standards (0 to 1600 Pgr testosterone the in I00 ul gel Ptss) were also extracted and drled the same way as (n)- 3H) samp'les. These standards were mixed wfth (¡ 2, 6, Z ptss, testosterone (approx. 20 x r03 dpm fn 200 ul gel Specific Activlty 93 Cilm mol) and antisera (fn 200 ul gel PBS; 1:20'000 final dilution). Equfvalent amounts of radiolabe'lled testosterone and antisera were a'lso added to drfed extracts of samples. Assay tubes oC were incubated at 4 for at least L2 h. Free and protein-bound 229

Tabte 6.1. Specificlty of data for testosterone antlsera

Steroid % cross reactionx AB ** Testosterone 100.00 I00.00 5a-D i hyd rotestosterone 3 1.00 35.00 4c¡-androstene-3 F' 17Ê-d i o1 3 0.00 4c-androstene-17Þr I9-d i ol -3-one 3.5 Androstenedf one 1.3 2.0 Epitestosterone ( 17cr-hyd roxy-4- androstene-3 -one) 0. r1 Etl ochol ano'lone ( 5 p-and rosta ne-3 c-ol -17-one ) 0. r0 Androsterone ( 5c-and rosta ne-3 Ê-ol -17-one ) o.02 Dehyd roe p f an d roste rone ( 5- and rostene- ol -17-one ) <0.0r 0estradÍ ol -17Ê 0.10 0.07 0estrone <0.003 Oestri ol <0.003 Progesterone <0.004 Corti sol 0.003

tf Calculated from the amount of sterofd required to supPress maxfmum 2 bfndfng of'H-testosterone by 50%. *tr Values for testosterone. arbitrarily set at 100%. Cross reactivfty determfned by (A) Dr. M. Wong, Hormone Assay development Groupr cslRo, Divlsion of Anfmal Production, (B) Dr. M.J. DfOcchio, Department of Animal Sciencesr Waite AgrÍcul!!lul Research tnstltute. This table ¡las adapted from Îuî.J. DrOcchio (I98I). 230

sterolds were separated by the addltlon of 200 u'l of PBS contalnlng

Dextran and Charcoal rc2.5 mg Dextran, 625 mg Charcoal ln I00 ml of oC PBS). The tubes were allowed to stand at 4 for 15 min and were then centrifuged at 800 x g for 20 min. The supernatant (bound fraction) was transferred to scintÍ1'lation vla'ls and radf oactivity was determfned uslng the scintillatfon system descrÍbed in Sectlon 2.r3 .1.4.

Sensftivity (2 x standard deviation of the buffer blank) was 5 Pg and fntraassay and interassay coefflcients of varfatlon (standard

devfatlon/mean x LOO%) were 6.0% and 8.9%t which were calculated from ten repllcates of a sample run ln the same assay and from three samples of lowr medium and high concentratfon, run fn dup'llcate' ln each assay respectfvelY.

Plagna testosterone concentratlons were determined wlthout

chromatography after solvent extraction. Hor¡ever the only steroids whfch have been shovln to cross react with the antisera used are 5 a-dlhydrotestosterone and androstenediol (Table 6.I) (DtOcchfor 1981). (the Since the'levels of these androgens in ramsr boars and rats specfes of interest ln the present studies) were very low re'lative to testosteroner they do not contrfbute signfflcantly to the testosterone values (Falvo and Nalbandov, L974¡ Tremblay et al' 1970; Schanbacher'

1976).

6.7. PROTEIN ASSAYS

Protein measurements were made using an adaptlon of the 23L

Protefn-Dye blnding Mlcroprotefn Assay of Bradford (1976).

6.7.L. Proteln Reagent

Coomassle Bril'l iant B'lue G-250 (I00 mg) was dissolved in 50 ml

95% ethano'l. I00 ml of 85% (w/v) phosphorfc acid was addedr and the solution dÍluted to a ff na] volume of I litre'

6.7 .2. Protel n AssaY

Protein solutlon containfng up to I mg/ml protefn fn a volume of 20 u1 was dfsPensed lnto I ml cuvettes. I m'l of the Protein reagent (6.7.I.) was added to each cuvette. After mfxing by inversionr

absorbance at 595 nm was measured after 2 mfnutes and before 15 minutes, agalnst a reagent blank prepared frorn 20 ul of the appropriate buffer and I ml of the protefn reagent. Measurqnents were

made uslng a Perkin-E'lmer Lambda 5 UV/Visua'l Spectrophotometer'

Standards of 0, 0.2r 0.4r 0.61 0.8 and I.0 mg/m1 Proteln were prePared uslng bovlne serum albumln. By plotting the welght of protein against the corresponding absorbârìCêr a standard curve tras generated that could be used to determlne the protein ln unknown samples' Such a curve is shown fn Figure 6.1. Comp'lex stabllity was rePorted by Bradford to be satfsfactory for up to one hour after mlxing with sample. All samples in this study were measured by t5 mlnutes after addftlon of Protefn reagentr ârìd as seen in Figure 6'2't absorbance

vras quite stable durÍng this perlod of tfme' Figure 6.1. Standard curve for the measurement of protein using the mÍcroprotein method of Bradford (L976). Absorbance (595 nm) OÞlUUl \ulg)!æ(o O

! t o ru * o 9. 3 o o a o À o 3 a atrf o IF -l o o) = ^ì3 t0 3 v- co

zEz Figure 6.2. Absorbance spectrum at 595 nm for dye-protein comp'lex' indicating complex stabllity for more than 30 minutes. Absorbance (595 nm) ¡ I o IJ l\'

CÐo

I { -t -l ll : 1,,, ll:l¡i -¿ o | ¡r :!l!r! I rA

, l'! I

v=.=

.¡ o iil:ii,¡l=i,t':t rrr|*,t ttt I t! il ¡ : r I rr-iilrir I 19

?--+-1-.+ ff+T-l: -::-f-i-t

o i. r . r-,-:.

Et¿ 234

6.8. MEASUREMENT OF SODIUM AND POTASSIUM LEVELS

Sodlum and Potasslum,levels ln blolpglcal flulds were kindly measured by the Dept of Bfochemistry and Chemlcal Patho'logyr Fl lnders lr4edical centre. Measurements were made wlth an lon-se]ectlve e'lectrode using an automated Beckman Astra-8 analyser'

6.9. ADMINISTRATION OF RADIOISOTOPES IN PHYSIOLOGICAL STUDIES

In a number of studfesr one or more radlolsotopes were admfnistered to anfmals to monitor changes ln concentratfon of extracellu'lar flulds. The flve radio-labelled tracers used were 125l-No.tu'l 3H-Wut". (ToH), 3H-Testosterone, I4g-Mannlto'I, Rat Plàsrttâr und 22Nu-Sodfum Chlorfde. Equfllbration times al'lowed for markers to partltion between vascular and extracel'lular compartments were based on descrlptlons by Chandrasekhar et al (1986) for Testosterone; and covle et al (1964)r Setchell et a'l (1969)r èfìd setchell and Sharpe 22Nuc1 L2íI-NRS, l4c-Mannlto]' (1981) for TOHr ' und

6.9.1 3 H-l¡rtater

Trftiated water (100 uci/ml) was admlnistered by intraperftoneal inJectfon of I mt. Equllibration for at least 3 hours was ensured' lng with some anfmals al'loyred overnf ght equil ibratlonr prlor to sampl of any flufds. 235

6.9.2. 3 H-Testosterone

Labelled testosterone. was admlnlstered by the radiolabe'l'led sterofd lnfuslon technique of Horton and Talt (1966)' A Teflon cannula was placed fnto the Jugular veinr under sodium pentobarbitone 3H-Testosterone anaesthesia. A priming dose of 0.5 uçi of 1121617 (specfffc activfty 370 uQí/ug) in 0.25 m] of 8% ethanol saline was lnjected lnto the Jugular. Ha]f an hour laterr a constant infuslon of 3H-Testosterone was started (0.5 uCi/ml 1n 8% ethano'l sal fne) wlth a i usA) Harvard i nf usf on pump (Harvard Apparatus co. , l4ode'l I100r Mil l s' fftted with a 2 ml infusfon syrlnger at the rate of 18 u'l/mln into the same Jugular vein. This infusion v,as malntalned for two hours initlallyr to achieve equillbration (see Chandrasekhar et alr 1986)' and contfnued durfng any extended sampl lng procedures after equil ibration.

6.g.3. r4c-Mannito'l

Because mannitol is rapidly cleared from blood by the kfdneysr animals fnjected with thfs marker had their ureters severed under

sodÍum pentobarbitone anaesthesÍsa¡ prior to adminfstration of the mannlto]. This ensures recirculatÍon via perltoneal f'luld. Followfng l4O-Mannltol this procedurer I uÇl in 250 ul physfologfcal sallne was fnJected intravenously. Two hours equl'libratlon was allowed prÍor to sampìing of anY fluids. 236

L25 6.9.4. I-Norma'l Rat Pl asma

Normal rat pìasrna tras obtained from donor a.nimals, and lodlnated using the method employed for radÍoimmunoassay proteln hormone I25I-NRP fodlnation as descrÍbed 1n 2.13.3.I. In thf s ÍìitrìrìêFr *u' prepared at a fÍna'l concentration of approximately 140 uCi/mì.

Incorporation was monf tored as described by Thorrel'l and l-arson

(1978)¡ with some 88!ã of 'labe'l'led protef n recovered after TCA precl pftatlon.

Iodinated-NRP (5 uCi) was adminlstered by lntraperitonea'l injectfon. Equiìibratlon tlme fo. 125I-Atbumfn is at least 6 hours (see Coyie et alr L964i Setchel'l and Sharper 1981)r although diffusÍon time from plasna for I0O% of plaslna albumin fs 16.6 hours (Landis and

Pappenheimerr 1963i see also 6.9.5. betow). Thus animals were inJected wtttr 125-NRP in the evenlng of the day before sampling was requlred, to allow overnight (14-16 hours) equilibrationr Prior to samp'ling of any fluids.

6.9.5. 22Nu-sodfum chlorfde

Landis and Pappenhefmer (1963) have published data summarÍsing the dlsappearance from the circulatory system of various 'l ipid-insoluble substances whlch df stribute primarfly in extracellul ar ftuid. Such substances 'leave the vascu'lar system at rates which vary inversely wfth molecular sfze. Disappearance from plasna is

accompanled by simultaneous appearance of the test moìecules Ín tlssue spâcesr again at rates whfch vary inversely vJith molecular size.

Sodium chìorlde equfllbrates extremely rapldly with a half time 237

(compare clearance of less than 2 mlnutes from rabblt arterlal p'lasna (1975) with albumfnr as dlscussed ln 6.9.4.). Godhino and setchelt 24Nucl reported a half-tlme clearance for extractlon from ram (1985' testicular caplllarles of I mlnute. In addltlonr Bustamante see 22HuC1 a'lso Bustamante and Setchell, 19SI) found a 601z extraction of on a sfngìe pass of p'lagna through the rat testis. These two reports 22Nuc1 suggest a rapid equl'llbratlon of between blood and interstitial fluid in the testls. In the present studiesr labelled sodium ch]oride for (2 uCi) was lnjected fntravenous'ly and equfllbration ¡¡as allowed one hour before any fluids were sampled'

6.10. STATISTICAL ANALYSIS

(sEM)' Atl data is presented as mean ! standard error of the mean unless otherllse stated. The probability of sfgnlficant differences

between groups was calcutated by studentls t-test and treatment (ANoVA) of effects were analysed by Analysfs of variance or Analysls Covarlance (ANCOVA). 3¿ U3IdYHC NOIÐIU -¡llulsulrNl 3l-tl No itocNll't Y - olnÏ ]ÚI$lsulINI

8EZ 239

7.L. INTRODUCTION

Wlth the discrepancy in the lmmune status of the fnterstltlal reglon of the sheep and rat testfs determined in the earlier experfmentsr it was of lnterest to examine lnterstltla'l fluids for factors that might be of consequence for allograft survlva'l' In thfs

FegaFdr sterold hormone'levels were of particu'lar lnterestr since a number of sterofds have been found to be fmmunosuppresslve ln vitro (see Kitzmlller and Rocklin, 1980; Stltes and slfterfr 1983) ' To of examine sterofd levels ln lnterstitfa'l fluld requfres the samP]ing such f'luld. The most wldely used technlque is that of Drfp Collection (Pande et al, 1966)r which tl,as also detal'led fn the fntroductory review, and is seen in Figure 7.I. While the Drlp system works wel] (fn terms of ftuld recovery) on a rodent testisr lts appllcatlon ln other specfes ls I lmlted. This ls Probably due to the Presence of the large lymphatfc sfnusolds ln the rodent testls and the lack of the connectlve tissue that fs found in the larger mammals such as sheep and pfgs. Howeverr as dfscussed ln chapter L thls technique has a least of number of other Possfble physioìoglcal short-comingsr not the whlch ls the necessity to remove the testis from its btood supply' Thls rrfaulttt fs shared by almost a'll technlques Prevlously used' It is of concern that the testosterone leve'ls rePorted for lnterstitla'l f'lufd collected by thls technique are much hlgher than those found ln testicular venous blood or testicular lymph (see Llndnerr 1963r 1969; tt,allace and Lascelles, L964¡ Hagenas et alr 1978; Sharper L979, Sharpe

and cooper, 1983; Sharpe et alr 1983; Turner et al' 1984). The c]ose assocfatlon of Leydlg cells wlth blood caplllarfes (see Mclntosh' yet 1969; and Chapter I) must be of some physlotogfcaì lmportance and tt i:i I

Figure 7.I. Drfp-collectlon method emp'loyed by numerous fnvestlgators to obtaln interstitfal f'lufd from the testes of rats. Some investlgators perform steps A and Br and then al'low gravitatlona'l coì'lectlon at 4oC for up to 17 hours or more. Others proceed on to step Cr and use centrlfugatlon

at 54 x G at OoC for 15 minutes to obtaln the ftuld exudate. Not to scale (adapted from Turner et al, 1984). 24Q

T¡

"Drip" Gollection

E f f erenl Spermolic Ducls Cord & \ \

A B c 4 Cenlrif uge xG 54 TIF "X" lncision

j i i

I I t,

T, I

I 24L

the Drfp collectlon system appears to assume that ftufd formatlon and

hormone secretlon will contlnue to be physlologlcally normal for up to 17 hours at 4 oC without a blood supply.

For these Fêâsorìsr lt was decf ded to explore an a'lternatfve

method of lnterstitia'l fl uld co]'lection.

7.2. fHE. PUSH-PULL CANNULA

7.2.I. Introduction

For some !€âFSr neurophysfologists were faced with the necessity to collect extracellular flufd contfnuously from the brafn - a problem not dlsslmllar to that for testlcular physiologfsts attempting to

,lÀ ùl 1961r Gaddum the use of a t,l, collect interstitla'l fluld. In J.H. Proposed rrPush-Pull Cannularrr which consfsfted of two concentrfc tubes. It was

based on a system developed by Fox and Hllton (1958) for the perfuslon of subcutaneous tissues between two parallel needles 1.5 - 2 cn apart.

Accordfng to Gaddum (196I)r the push-pull cannu'la had four dfstlnct

advantages ( for neurophysio'logy) : tt(I) It detects the substances liberated under fafrly normal conditions. Q, It gfves an lndlcatlon of the turnoverr rather than the storesr and can be used to study the effects of factors such as nervous stlmulation. (3) It should be possible to 'local lse the site of 1 fberation of substances ln such tfssues as the t by Ì centra'l nervous system more Precfsely than

; other means. (4) The blood-brafn barrfer presumably does not i ntervene. rl

Ì 242

push-pull cannulae have now been developed ln varlous fonns, and

have been wfde'ly used ln numerous appì ications. Phi'l1lpu (1984) has revlewed these applications, and summarises that rr... probably the

mafn advantage of working 1llith push-pu1ì cannulae lies in the versatflity of the techniquer ... The use of the push-Pull cannula for detectfon of endogenous neurotransmftters fn the perfusate (has)

opened a neu perspective fn studylng the dynamlcs of the release of neuroransmitters in distinct brain areas.rl

The sfmi'larity of the origfnal problem in both discfplines' and the solutlon provided by the push-pu1l cannula for one of these disclplfnes, made thfs technlque worthy of closer exa¡nÍnatlon' The abillty to measure endogenous substances in the perfusate obtaÍned by a push-pull cannula tvas of obvious slgnificance.

'.L iif t,¿

7 .2.2. Constructl on

As already mentionedr the push-pull cannula orfglnal'ly descrfbed

by Gaddum (1961) conslsts of two concentrfc tubes open at one end only

I (Figure 7.2.). The fluld fs drlven through the fnner tube. The approprlate adJustment of the dfstance between the tips of the outer and inner tubes generates a siphonr which is important for the further transport of the flufd and for a constant rate of outflovÚ as wel]. This technfque has been extensively descrlbed (Szerb, L967¡ Dudar and szerbr L970i Myersr L972¡ Yaksh and Yamamurar L974). Szerb (1967)' (L974) a'l t Dudar and szerb (1970), Yaksh and Yamamura and Honchar et I I (1979) examined the experimental conditions necessary i have thoroughly for a constant rate of flow, and for the recovery of substances

! FTgure 7.2. Section of Gaddumts push-pull cannula. Arrorys fndicate directlon of flowr and the hatched area is the area between the perfusate and tlssue. The outer tube fs a No. 20 or No. 18 needler the fnner tube a No. 28 need'le (from Phil I lpu' I984).

tl I

\ a'

€þz 244 diffusing from the tfssue fnto the push-puìl cannu'la. 0f most lmportance ls the relatlonship of the two components of the cannula. Since the'lorer tfp of the inner cannula protrudesr an artificfal cavlty is created around the tip (Figure 7.2.). The outflotr contafns substances that were present in thls cavity. To reach a constant flow rater two pumps are needed : one to push the fluid into the cannu'la and the other to puìl the Perfusate from the outer tube into the collectlng tube. Recovery from the Perfusate of substances Present in the tlssue is goodr provided that the tip of the lnner tube protrudes to some extent (Philllpu, 1984). Indeed, the reproduclbility of the results depends to a great degree on the position of the lnner tube (Szerbr L967¡ Dudar and Szerb, !970', Yaksh and Yamamura' 1974) ' Hoyleverr the greater the dfstance between the tlPs of the inner and outer cannulae and the hlgher the rate of perfusfon' the more dlfflcu'lt lt ls to obtain a constant rate of outflow (Yaksh and

Yamamurar 1974).

Two maJor problems were Percelved with the designs used by (see neurophyslo'loglsts. FlrstlYr the dead space fn the outer cannula Flgure 7.2.) mlght affect reproducibflity between samples. Secondly' the commerciaJly avaflab]e cannu'lae (klndly provided by Dr Richard

Dyerr ARC Institute of Physlologyr Babrahamr cambridge, u.K. and availab'le from Cl ark E'lectromedical Instrumentsr Pangbourner Readf ngr U.K.) were deslgned to a'l'lovr the inner cannula to be unscrewed and

removed from the outer cannula. While this ls important for the lntroductlon of such cannulae into brainr lt also creates the potentlal for different distances to arfse between the tips of the lnner and outer cannula between dffferent samplingsr unless the

components are screwed together to the same posftion each tlme' In the 245

studies to be presented ln thls thesisr push-pull cannu'lae were constructed by adaptlng stainless steel needles. I9G needles were used for the outer cannula and 25 G needles for the inner cannu'la' In both côsêsr 'luer-lock flttlngs were removed, and the sharp, polnted ends yúere removed and squared (see Figure 7.3.). The first problem described was overcome by introducing the inner cannula through the side of the outer cannu'la. In this Walr suction on the outer cannula would draw perfusate up from the rrcavityn ¿'rectly out to the samPllng point. The second problem related to separatlon of the two component cannulae. This separation was not required for the present studÍes' and thus the permanent fixing of the two cannulae (solderlng of the fnner cannu'la to the outer cannula at the point of entry) overcame thÍs dlfffculty and ensured constant results for each cannula constructed. The fnner cannula vtas allg¡ved to protrude past the outer cannula for I.0 mm. Thfs a'llowed a Perfusfon surface area of 2'8 mm3 (1967) around the cannuta tfp, calculated by the method of Szerb as seen in Figure 7.4.

7.2.3. Apparatus

The apparatus used to samp'le fluid from testes by the push-pu'I1 cannula fs shown ln Flgure 7.5. For each cannular the lnfusate was

pumped lnto the fnner cannula (see Ffgure 7.3.) with a Minfpuls II peristaltlc pump (Gllsonr France) wlth a s'low head. connectlon between (I.D. run' the pump and the cannula t{as made t{ith potyvinyl tubÍng 0.8 (I'D' o.D. I.2 mm). The outer cannula was connected by Teflon catheter I.0mmro.D.I.6mm)toa2m.lglasssyringeonaHarvardlnfuslon Both pump (Modeì 1100, Harvardr Minnfs.r USA) for perfusate withdrawl' Figure 7.3. A diagrammatic representation of the push-pu1l cannula in a testlsr demonstrating the prfnciples of mixfng and diffusion across the perfusate fnterface and resultant dilution of collected fluid. 246

The Push-Pull Cannula

Testis

t a , t I t

I I t a , t I , I ,

t \t a I a I a

a t Ffgure 7.4. Method of calcuìation of perfusion surface area of a push-pul'l cannula as described by Szerb (1967). The

dfagram shqrs the tlp of the cannu'lar and the measurements

made for calculatloni arrows indlcate dlrectlon of flowr and the broken I lne shqs the cross sectlon of assumed lnterfaclal area. 247

it I L-e-

2 2 a +(h+hr) Surface Area = E 2¡ (b + 0.025)21 + [ un 2 - (b + 0.025)r (b + 0.025) + (hr)21

where : a = Insfde radlus of outer needle = 0'45 mm b = outslde radlus of lnner needle = 0'25 mm h = protruslon of lnner needle = 1 mm ht = [ (b+0.025)h]/ta-(b+0.025)l = 1'571

2 Surface Area of constructed cannul ae = 2'788 ßìlll . Figure 7.5. Arrangement of apparatus used to samp'le fnterstÍtial fluÍd from rat testes wÍth a push-pull cannula.

W = V'later bath used to maf ntain stage (S) temperature. L = Cold-tfght source.

M = Mfcro-manlpulators used to posÍtion push-pull cannul ae. $= lfater-Jacketed perspex stage used to hold testes durlng cannulatfon and samPling. IP= Infuslon pumps used to infuse perfusate through cannulae. One was a hlgh-revolutfon pump for loadfng and flushing of the cannulae, and perfusion and sampl ing t f nes; and one was a slow-revo'lution pump for constant low-level infusion durfng sampl ing.

SP= Syrfnge pumps used to wfthdraw flufd from the cannul ae. 244

T' 249

pumps yrere adJusted such that fnfusfon and wlthdrawal equilfbrated at approxlmately l0 ullmln. The cannulae were attatched to micromanipulatorsr usfng p'lasticine (see Figure 7.6.a). A perspex (Figure stage was constructed to ho]d the testes for cannulatlon 7.6.a)r âîd warm water tllas reclrcu'lated from a heated water bath through the stager ¡lith water temperature adjusted to maintain testis (aPProxfmately temperature on the stage at normal scrotal temperature oC; 33 Kormano, 1967c).

7.2.4. Use of the Push-pu1l cannula

Rats were anaesthetlsed wfth sodlum pentobarbitone and each testis exposed through an lnclsion ln the scrotum. Anlma'ls were then posltfoned wlth thelr testes placed lnto the two grooves in the. - (Figure ppe-wâFmedr persPex stage, with theÍr tail under the stage 7.6.b). A small entry ho'le was made ln the tunlca of the testis to be cannu'latêdr usfng a 25 G needler and avoiding all obvious blood vessels. By carefully inserting a pair of Pointed forcepsr this hole was kept patent whlle the cannula was inserted using the mlcromanfpulator. The forcePs Ì'Jere withdrawnr so that the tunica enclosed the outer cannula. The cannula was lnserted to a depth of

approximately 5 mmr and Figure 7.6.b shqs a cannu'la in posltion ln a rat testis (see also Figure 7.3.).

7.3. INVESTIGATIONS IN THE RAT

As mentloned in the introductlon to this chapter, the prlmary afm of these studfes t{as to examlne steroid leve'ls ln fnterstftlal fluid' Figure 7.6. (a) Close-up view of mlcro-manipu'lators wlth cannulae held in posftlon wlth p'lastf cf ne ( Þ). (b) Cannulae ln posltlon ln the testes of a rat which have been withdrawn from the scrotum and placed fnto the grooves fn the perspex stage. ¿50,

E ,"1 -

ìrr_- 25r

and testosterone yJas chosen as the sterofd to be measured fn inltfal studfes. To monltor physfologfcal aspects of the technlque, measufements of protelnr Sodfum and potassium were also made.

7 .3.L. Experlmental Procedure

7.3.I.I. App'l icatf on ln the testís

As out'lined in the dlscussions on the theory behind the push-pull cannular the assumptfon in use is that components Ín the fluid fn which the cannu'la fs placed wfll cross the interface with the infusate around the extended tip of the inner cannula, and appear ln the sampìed perfusate. This has been found to occur both Ín vftro and in vfvo ln braln (see PhlltlPu, 1984). It was lmportant to examlne the performance of thÍs theory ln the testis. It was aìso Ímportant that perfuslon would reach throughout the fnterstltium, and not slmply f'lush out a partlcular regfon. ThÍs was checked by infusing a solutfon of Pontamine Sky Blue dye ( 0.5% ln lsotonfc mannlto'l) through a push-pu1l cannula posftfoned ln a testis. To monltor the extent of tlssue damage caused by cannulation of the testisr a testis tras processed for histologÍca'l examlnation after a push-pul'l cannula had been in place for one hour.

7 .3.L.2. Measurqnent of Dl I uti on

It was apparent from the I iteraturer and the lnitlal studles usfng the push-pul1 cannu'la ln the testfs that a certafn amount of dilution occurs wlth thÍs process. It would obviously be important to 252

flufd are to monltor thls effectively if actual levels in the orlginal be assessed. I physlologlcal A common technlque for measurfng ftuld dllutlon in âñd allow studiesr ls to admlnlster a radloactlve tracer to an anlma'lr of interest' ft to equilibrate between blood and the fluld compartment the levels of Assuming that tissue barriers do.not affect equilibration' fluld' The radfoactivity should be the same in blood and collected samples of blood and d.ifference Ín levels of radioactlvity measured in during diluted f'luÍd will provÍde a measure of the dilutlon occurring of di'lut'ion of collection. This technÍque was used to measure the amount InitÍally' interst.itÍal fluÍd collected with the push-pull cannula. ín push-pu11 trÍtiated water (3H-Water) was used to monitor dilutÍon turnover studies' samples since thís tracer is widely used ln metabolic ín 6'9'l' Administration and equilibration tfme were as discussed l4c-Munnitol, Folloring these studies, it was decided to a'lso use 125I-labelled 3H-Testosterone, und norma'l rat plasma to monltor 22Na-Sodium dilution. rn latter experiments, Ch1orlde was also used' to compounds These tracers were chosen because of their similarity beingusedinthepush-pulltechnique(see7.3.1.3.)orthefr (see 7'3')' sfmi'larity to compounds of interest in lnterstitial fluid section Adminlstratlon and equllfbratlon times were as discussed ln by gamma (LKB 6.9. Levels of radfoactivfty where monitored in samples (LKB counting as 1271 Rlagamma) or scintlllatlon I2I5 Rackbeta II) injected wlth approprlate to the tracer used. ltlhile some animals were rrcocktailn inJectlons of two only one tracerr other anlmals recelved tracer tracers. In these situatlons, samples were counted for each as lndlvldualìyr and corrected for cross-channel contamination separately on all determlned from standards of each tracer counted 253

channel s.

7 .3.1.3. Cholce of Infusates

'leve'ls To monftor steroids, proteins, and sodfum and potasslum in the co]lected perfusatesr ft was necessary to use a medium that would not influence these results. At the same tlme lt was lmportant to use an fnfusate that would not be physiological'ly distressing to the testis. Two flufds were chosen for study. Isotonfc MannÍtol was chosen because it'lacks a'll of the compounds of fnterest. castrate Rat Plasma

was also chosen for infusion, as a substance that mfght be more like that ffltered by testlcu]ar blood vessels to produce fnterstitlal flufd.

7 .3.L.4. Protocol used

Fo1lowing admfnlstratlon and equflibratlon of tracer(s)' anfmals were re-anaesthetlsed as needed, and a push-pull cannula placed into each testls as requfred. Push-pul1 sampling was routine'ly perfonned lnto for one hourr tfmed for each cannula from the tfme of insertfon f rom the the testis. After samp'l lng, interstltial fluld was col'lected holdlng syringe and collectfng cannula by backflushing down the line after remova'l of the push-pu1l cannula from the testis' Testlcu'lar venous and peripheral vena cava blood samples were obtafned as

described f n sectl on 2.3.5.I. A'll samples were lmmedf ately measurements of centrlfuged. A1 fquots of supernatant were samp]ed for oC radioactlvity, and the rernainlng volumes stored at -20 until

assayed. 254

Levels of Testosterone were measured ln a'll samples col'lected' as descrfbed ln 6.6.I. In those sampìes obtalned wfth mannitol infusate, proteln:levels were measured fn interstltlal fluld and testlcular venous plasna as descrlbed in 6.7. These samples were also monltored for sodium and potassfum levels as detalled in 6'8'

7 .3.2. Resul ts

7.3.2.!. Appl ication ln the testis

Infusion of dye through the push-pul'l cannula resulted in a generallsed colouring of the testls as seen ln Flgure 7.7. While there pofnt was a greater concentratlon of dye in the region closest to the of fnfuslonr there trlas evidence of more wldespread dlstribution' The collected perfusate 1¡as more dilute than that Ínitlally infused into the cannula (Ffgure7.7.). These results suggested that transfer of versa' f'luld t+as occurlng fron the cannu'la into the testls' and vice The hlstological examfnatlon dld not reveal any significant appears to damage to the testls. As seen in Figure 7.8. the cannula separate the tubu'les a'long natural sheerplârìêsr wlth most structures left lntact. Any damage is llmlted to the lmmedfate vicfnlty of the cannula and nefghbouring tubules aPpear quite norma'l'

7 .3 .2.2. Measurement of Di I uti on

Table 7.I. shcrus the amounts of varfous radioactive markers appearlng fn the push-pul1 perfusater collected wlth either isotonfc mannito'l or castrate rat plasna lnfusate. The results fndicate that i

Figure 7.7. Demonstration of the mixfng and dfffusfon occurrlng between perfusate and interstltfa'l fluid in the testis with the use of the push-pu'll cannula. The vÍal in the upper left of the plcture contalns the Pontamine Sky B'lue/Mannftol infusate, and the vlal ln the uPPer rfght contalns the sample col'lected from the push-pull cannula. The tover 'left of the photogragh shc¡ws a control testis. The lorer rfght of the photograph shows a testfs after one hours sampllng of fluid wlth the push-pull cannula usÍng Ë \È blue t the PSB/Mannitol infusate. Note the dlstÍnct co]ouring over a large area of the cannulated testfs, and the dilution of the dye colour in the sampled f'luid

compared that infused. This suggests that the perfusate fs mixfng with lnterstitlal f'luÍd throughout the testis, and that dlffuslon across the interstltla'l fluid/perfusate lnterface does occur. 255

-1 r[I

! Figure 7.8. Sections from the testis of an adult rat after Ínsertion of a push-pull cannu'la. (a) shows the polnt of entry of the cannu'la, and the separatÍon of semlniferous tubules

after cannulation (X 30). Some tissue debrÍs fs seen Ín

the cannula ho1e. Hoyreverr tissue damage is very localised

and as seen in (b), tubules seem to have parted along natural sheer-planes (X 80). The cannula tvas coated fn

indfan ink prfor to insertlon, and particles of ink can be seen in thÍs regfon. The re'lationship of tubules and ür.ü I fnterstitlal cells in regions surrounding the polnt of

cannul ati on appears quite norma'l . As seen f n (c) r tubu'l es separated from their neighbouring cells by the push-pulì cannula appear fntact, and have mafntafned thefr structure (X 320). (d) shows partlcles of indÍan ink whÍch have been carrfed away from the sfte of cannnu'latlon (X 320). These

particles are seen between two tubules Ín close apositfon,

which fs evldence for the abil fty of substances to move readfly through the interstltium of the rat testfs.

! 992 + _ _-.!t= +-ia"*-

Table 7.1. Amounts of varlous radloactlve markers appearlng fn the effluent from a push-puìl cannula lntroduced lnto the testls of an anaesthetlsed rat. Values are expressed as a percentage of levels Ín testicu'lar venous blood sampled at the same tÍme, and are presented as means wÍth the standard errors of the ßêèrìsr and the number of observations.

Infusate used Isotonlc Castrate rat mann ltol serum

3H-water 17.l + 3.Uo (8) 3.92 + 0.61% (5) r4c-mannftol r.76 ! 0.3870 (4) l.o2 t 0.3% (3) 3H-testosterone 6. l9 ! 2.07l (4) 2.75 ! L.sr% (3)

125r-""t serum protein 0.82 + 0.I4% (4) 0.20 + 0.IIÍ (4)

22¡r"ct 5.16 + L,2L% ( r0)

N ('r\¡ 258

the amount of radloactfvlty enterlng the fluld passlng through the cannula depends on both the markerr and the type of infusate. Vfhlle

T0Þl passed rapldly lnto mannltôl lnfusater lt df.d' not'transfer so' readlly lnto castrate rat serum. This relationshlp held true for all other markers (P < 0.05).

7.3.2.3. Analysis of Interstitial F'luid

Havfng determlned levels of testosterone, protefnr sodlum and potasslum ln perfusates and blood samples, corrections had to be made fn the push-pull coJlectlons for the dllutlon created wlth the technfque. The resu'lts discussed f n 7.3.2.2. indicated that lt was necessary to correct for the dflution fn a collectfon uslng a marker approprlate to the substance of fnterest. When the concentratlon of albumfn fn fnterstltial flufd ulas ca'lcu'lated usfng the dilutfon of to lodlnated rat Plasma Protefn Ínto fsotonic mannltolr values slmflar b'lood p'lasma were obtaf ned (Tab'le 7.2.). When the concentratlon of testosterone fn lnterstltlal f'lufd was calculated using the dflution of 3H-testosterone lnto either mannltol or castrate rat plasnar the va'lues obtalned were lovrer than testf cular venous blood values 'levels measured under the respectlve treatments. WhÍle the calculated than from castrate rat Plasma infusates were sfgnlffcantly higher those obtalned wfth mannitol infusates, the venous blood leve'ls were also signif fcant'ly higher in these anf mals (Tabl e 7.2. ). To ensure that these results were not biased by an unequal distrlbution of 3H-testosterone markerr 3H-testosterone 'levels were measured fn blood and drip-collected fnterstftlal fluid followfng lnfusfon of the marker as described ln section 6.9. No sfgnlficant dlfference (P < 0.01' n=4) was found in the levels of marker ln any of the samples collected. Sodium levels in fnterstitial flufd urere corrected for dilution of 22NuCl into mannítol lnfusate. WhÍ1e no dfrect marker was avallable for measurement of potassium dllution, ft was assumed for ca'lculations that sodium and potassÍum ions would diffuse at simflar ratesr and Tab'le 7.2. -fhe ca1culated concentratlons of protefnr testosteroner sodlum and potasslum ln testlcular fnterstltlal fluld, wfth measured values for testlcular venous p'lasma. values are presented as means wlth the standard errors of the ffiêôrìsr and the number of observations.

Interstltlal f'luld, Testf cu]ar venous col I ected usl ng blood plasma Isotonlc Castrate rat Isotonfc Castrate rat mannltol serum mannitol serum

(4) (5) Proteln (mg/m1 ) 75.3 + L2.9 58.4 ! 2.8

+ (9) Testosterone (ng/ml) 1I.6 + 4.2 (g) 233 + 60 (8) 23.4 + 5.2 (7) 591 68

(e) Sodlum (mmol ) r4I.7 + 18.2 (9) 137.8 1 r.9 (9) Potasslum (mmo'l) L2.2 ! 2.5 (9) 4.4 + 0.1

('lN ro 260

corrected for thus potassium leve'ls ln lnterstitfa'l f'luld have been 22Nucl. fluld dllutlon agalnrt The resu'lts suggest that fnterstltlal potasslum contafns slmflar amounts of sod'lum but,proportfonatly more (Tab1e than b'lood Pl agna 7 .2.\ '

7 .3 .3. Dl scusslon

Theseresultssuggestthatthelevelsoftestosteroneln interstitla'l f'luid are no hlgher than those of testlcular venous blood - a ffndfng that ls ln dlrect contrast to those reported by workers calculated uslng the drip collectlon method (Tabl e 7.3.). The values the lovl from the mannito'l lnfusates may be falsely loÏJ because of lmportant blndlng capaclty of this f'luid. The dilutlon factor is a'll in these studles ln determfnfng the final concentratlons of substances in push-pull perfusatesr and ca]culatlon of absolute concentratlons wfth push-pull collection may need refinement. The validation of the dÍlutlon 3H-testosterone correctlon and the reasons for using to determine dilutfon correctÍon for testosterone levels have already been dlscussed' However' the disparlty in fina] levels determinedr in comparfson to reports by other authors uslng other techníques (see Table 7.3.), requÍres careful consideration. It cannot be entirely attrfbuted to error of dilution correction, although some error must no doubt be aì'lowed. It is of fnterest

that when castrate rat serum was infused Ínstead of mannito'I, testosterone levels in the collected f'luid íncreased sfgnificantìy. This may mean that protein is important for testosterone transportr añd that protein should be

added to the perfuslon medfum. Indeed, Ewing et al (1979) have suggested an important role for protein in testosterone transport. However, the ratlo of testosterone ln lnterstltlal fluid / testfcular venous blood was comParable to that obtained wlth mannitol infusate, whlch suggests that this ls not a problem. That a'll values were signlficantly lncreased when castrate rat serun Table 7.3. Testosterone levels ln testfcu'lar lnterstltla'l ftuld and testlcular venous blood of male rats, wfth the dffferent technlques used to obtafn lnterstltlal fIuld. Values are presented as means wlth the standard errors of the rlêârìsr and the number of observatf ons.

Testosterone leve'ls (nglml)

Method I.F. Testls Vefn Reference

Mfcropuncture 150 + 27 (l7) Comhaire and Vermeu'lenr 1976 Drlp / Centrlfuge r37 + 25 ( 10) Hagenas et a'l ' L978 73 + s (26) 28 + 5,3 (10) Turner et a'ì, 1984, 1985

Drlp / Gravlty 315 + 29 (4) Sharpe et al, 1983 590 + 20 (8) 99+20 (8)

Push-pu'll cannu'la 1r.6 + 4.2 (9) 23.4 + 5.2 (7)

N or H 262

vras infused, tvas at first surprisfng. Howeverr the high levels of gonadotrophlns ln castrate p'lasfna may be lmportant. LH leve'ls were measured ln the castrate plasna fnfusate of these experlments and were found to bé slgnfflcant (> 8 ng./ml). It would appear that the lnfuslon of castrate rat plasna dlrectly fnto the testls provfded a signiflcant gonadotrophin stlmulus resu'ltlng in an fncreased secretfon of testosterone. That the ratlo between lnterstitial fluld and venous b'lood remalned simllar to that ln the unstlmulated testfs cannulatlons was indeed lnterestlng, and suggests that Leydlg cel'ls may ln fact secrete proportionately more testosterone dfrectly lnto blood vesse'ls than fnto lnterstltial fluid. The results also suggest that the much higher testosterone concentratlons f n testlcul ar interstltial fluld compared wlth testlcular venous blood reported prevfously (Sharpe et alr 1983; Turner et a'|, L984, f985) may be artefacts due to the unphysfologfcal technfques used for coìlectlng the lnterstftfal fl uid.

Prevlous measurements of tota'l protein ln drfp collected fluld

have been varfable. Pande et al (1966) found'levels ln rat f'luld to be almost twlce those of serum, and leve]s ln human flufd to be almost half those of serum (Pande et alt L967). The slmllarlty in protefn levels measured fn lnterstitial fluid and venous b'lood fn the present study conflrms a prevfous report by Sharpe (1979). Thfs fs possibly of slgnlficance ln determlning the physfologlcal usefulness of the

technlque. Yaksh and Yamamura (1974) reported Proteln leve'ls fn push-pul'l perfusates ln braln to be a useful lndfcator of tlssue

damage wfth increased levels of proteln reflectfng sone degree of

tissue damage. The present resultsr ln conJunctlon wlth the hÍsto'loglcal studlesr would suggest that tlssue damage ln the testfs 263

fs negllgfble with the push-pull technfque. The potassium concentratfons reported ln drip collected lnte¡:stltlal fluld (Sharper L979i. Pandel 1966) are probably falsely high. Potasslum levels ln testlcular lymph are comparable to blood

pì asna I eve'lsr although tubuìar I eve'ls are sf gnlf icantìy greater than blood p'lasna leve'ls (Setchell and Waltesr 1975). However, the levels found fn lnterstitfal fluld fn the present studfes are sti'l'l more than twlce those of blood plasrna. Thfs ls most surprlslngr and is so far

unexplaf ned. I'lhlle lt mlght be f ndf catfve of cel'l or tubublar damage, as already dfscussed the hlstologfcal examfnatlons have not revealed any extensfve damage that mfght contrfbute to such levels.

7.4. Concl usfons

The results presented lndfcate that testosterone leve'ls ln

interstftlal fluld are unlikely normaìly to exceed testlcular blood levels. Assumlng that fnterstltlal fluld ls the fìuld from whfch testlcular 'lymph orlglnatesr thfs theory would account for the reports by Lf ndner (1963r L967, 1969)r l{allace and Lascelles (L964) r ârd

Setchell et al (1967), of testosterone levels fn testlcular 1ymph, belng some 70 % of testlcular venous b'lood levels. Buh'l et a'l (1982) and Marsha'l'l et al (1984) have reported malntenance of spermatogenesls

fn hypophysectomised rats and stalk-sectioned monkeys glven exogenous testosterone, trhen lntratestfcular levels of testosterone were I0 - 20

% of levels prevlously reported as normal. In 1979r Cunnfngham and Huckins reported the perslstence of complete spermatogenesls ln lntact rats treated wlth testosterone proplonate fn whlch fntratestfcular levels of testosterone were2O% of normal. Rea et al (1986) have 264 recently reported quantltatlve malntenance of spermatogenesls ln hypogonadotrophfc adu'lt rats treated with testosterone lmplants, where intratestfcular levels of testosterone were 15% of normall' The results from the present study suggest howeverr that the Prevlously rePorted rrnormalrt levels may have been overestlmated by up to 10 tlmesr and that the above studies (Cunningham, L979¡ Rea et a'lr 1986) may in fact 'levels be measurlng more norma] of Íntratestf cular testosterone. It is important to consider hotf this dlscrepancy might arise. As mentlonedr the present study suggests that Leydig cel'ls secrete testosterone more selective]y fnto blood vessels than fnto interstitial fluíd/testicular lymph, and thls hypothesis ls presented in Ffgure 7.9. In thls modelr lnterstftlal fluid levels of testosterone are more a consequence of levels of testosterone ln blood, and changes ln blood flow and vascular Permeability are thought to have dramatic effects on testosterone levels fn lnterstitlal f'lufd. ThÍs being the caser the drip collectlon system is faced wfth an lmmediate problem' Isolation of the testls from the b'lood supply would prevent thfs route of secretlon. Physlo'loglca]ly, the Leydf g cel'ls ln close proxlmity to blood vessels (and Mclntoshr 1969 reported the maiorlty of Leydfg cells to be in such a relationshipr see also Figure 1.2.1 might lndeed be affected by the lack of blood florv in these vessels, under these condltions. Assuming that they can continue to secrete testosterone norma]ly (a questlonable assumption fndeed)' the secreted

product is un'l ikely to f f nd f ts way into a b'lood vesse1r ârìd would rrinterstitf The more than likely accumu'late f n the a'lrr fluid' concentratfon of such secretion fnto lnterstltfal fluid would elevate the concentratlons measured ln drip co]lected fluid to an abnormal level. ft may be that values obtained from drfp collected fluid Figure 7.9. Dfagrammatlc representations.of the proposed mechanfsms of testosterone secretfon in the rat testfs. (a) The exfsting hypothesis proposes that the LeydÍg cells prÍmarÍly secrete androgen Ínto the extravascular fnterstftfal flufd (arrons). This creates a high concentratfon of androgen fn thÍs flufd which supplies

the semlniferous tubules wlth the high levels needed for spermatogenesÍs. B'lood levels of testosterone arise frqn the diffusfon of testosterone from the intertitlal

fluid Ínto the blood caplllarfes. Some mechanfsm must also exist to restrlct the amount of testosterone secreted ln testÍcular 'lymphr since the levels of testosterone measured fn this fluid are no greater than venous blood 'levels (see text for dfscussion). (b) The new hypothesis proposed from the work of this thesfs. Leydfg cel'ls preferentiaìly secrete

testosterone lnto blood vessels (arrows). Some Leydfg cells wiìl secrete dfrectly fnto lnterstftial fluidr and some may supp'ly semlnÍferous tubu'les di rect'ly. Howeverr interstitiaì fluid levels of testosterone are

comparable to blood levels and arise more as a consequence of blood flow rater ôrìd capiìlary permeability. These factors appear to be carefulìy regulated by the seminlferous tubulesr and cel'ls fn the Ínterstitiaì tlssue. 265

<-( B loo d T

ey dig cel

mph << Ly T

Seminiferous tubule

rl \È ,i

<< Blood T

eydig cel

Lymph <{ T

Seminiferous tubule

r 266

rrstoresrr rather than normal represent intratestlcular of testosteroner lnterstltial flufd leve'ls. Figure 7.I0. shorvs sectlons from a testls to after drfp collectfon was malntafned for 18 hours. It ls lmportant a certafn amount note that whfle the morphology ls reasonably normal, This may of shrinkage of the tubules has occurred durlng col'lection' affect intercellular communlcation and df sturb normal physio'logica] processes in the interstítia'l region'

of All of these points ralse serious doubts as to the usefulness It is drip collectfon in physiological studies on interstitial fluid' also lmportant to note that the push-pu1l system descrfbed here perfusate probably requires reffnsnent. The process of dllutlon of the absolute 'leve'ìs of makes f t unsuitab'le for some studies. The determined' and testosterone calculated depend on the dilution factor degree of dlfference d are probably subject to some error. However, the iil t in values obtaíned by push-pu1l collectlon compared to drip-coìlectlon cannot be accounted for simply by error of dilution correction' Further studles are required (see chapter 8)r but thfs work has the demonstrated that a re-evaluation of our understanding of steroids environment of the endocrine testis may be needed' Leve'ls of in interstltial fluidr and the mechanisms of secretion found in the endocrine testÍs also requlre further investigatlon'

t T

r Figure 7.10. Sections from the testes of an adult rat. (arb) Control sections from a testfs flxed by vascu'lar perfusion at the tfme of castration (a - X 80, b - X 320). (crd) Sections from a testfs in whfch the blood was flushed from the vasculature wfth saline at the time of castrationr and the tissue ffxed by vascular perfusion after 18 hours of oC Drip-collection of fnterstÍtial fluid at 4 (c - X 80, d - X 320). Note the conslderable shrfnkage of tubu'les after drfp-collectÍon (c'd) when compared to controls ü i.È I (arb). The cel'ls in the lnterstftial region appear to lose a considerable amount of contact with the

semlniferous tubules under such condltÍons, whfch may

affect cel'l-ce'll communicatf on and paracrf ne control.

I 267 268

CHAPTER 8: TESTOSTERONE LEVELS IN INTERSTITIAL FLUID - EFFECTS OF

VARIOUS EXPERIMENTAL TREATMENTS

d ir; I

! 269

8.I. INTRODUCTION

This chapter reports on a series of experfments undertaken ffrsgy to test the performance of the push-pu'l.l cãrìrìulãr and secondl! to examfne the hypothes'ls of testosterone secretfon ProPosed fn the last chapter. Hypophysectomy with pregnenolone suPPlementatfonr treatment with hCG, treatment with EDS, heating of the testesr efferent duct lfgatlonr and Ínsertion of testosterone implants' were used to lnduce various changes ln the testÍs, the interstitlal fluid compartment, or in testosterone product'ion/secretion. The push-pul1 cannula tras used to obtaln interstitlal fluid from testes after these treatmentsr and compared urith drlp collectfon in some instances. Testosterone levels were measured fn the collected f'lufd and fn testfcular and Peripheral venous blood.

8.2. EXPERIMENTAL PROCEDURE

8.2.L. Effect of anaesthesla and push-pu]l cannulation on testlcul ar venous concentratlons of testosterone.

Levels of testosterone ln testicular venous blood obtained from testes after push-pu'll co'llectfon of lnterstltial fluid were lourer in the lnf tia'l exPerlments than mlght norma'lly be expected (see 7.3.2.3.). To investlgate thls furtherr slx grouPs of four animals (330-500 g) were used to examine the effects of pentobarbÍtone anaesthesla and push-pul'l cannulation on testicular venous blood levels of testosterone. One group of anfma'ls vlas processed for each one of six treatments. This lnvo'lved sampl f ng testf cular venous b'lood elther lmmediate'ly the anlmal was anaesthetlsed, one hour after 270

anaesthesla was fnduced with testes kept fn the scrotum, one hour after anaesthesla was lnduced wlth testes exposed on the pre-warmed perspex stage detafled ln 7.2.4.r after one hour of samplfng with the push-pull cannula¡ after two hours of exposure on the perspex stager or after one hours exposure on the stage and one hours sampl fng wlth the push-pul ì cannul a.

8.2.2. Hypophysectomy wÍth pregnenolone suppl ementation

The fmportance of pltultary lnvolvqnent fn testosterone productlon has already been dlscussed ln chapter 1. The gonadotrophin LH fs necessary for a number of steps fn androgen productfonr but u'ltfmately acts on a sPecf f lc step f n the transformation of cholesterol to pregnenolone. It does not however, lncrease converslon of pregnenolone to testosterone (Ha'lt, 1966). SupplementatÍon of

hypophysectomised anlmals wlth pregnenolone (Img/100 g bwr dafly for 14 days) maintalns low Perlpheral plasna testosterone levels and normal rete testls testosterone levels (Harrfs and Bartker L975; Anthony et al r 1984).

Four groups of four anlmals (300-435 g) were hypophysectomlsed as

descrÍbed ln 2.3.2. Pregenenolone ln 0.1 ml of Sesame Oflr Yúas administered to three grouPs at 0.5, I.0, and 2.0 mg/100 g bw/ day for 14 days as described fn 6.3.(see also Stefnberger et alt L975; Anthony et a'|, 1984). The fourth grouP was fnJected wfth the vehfcle on'ly. Injections u,ere begun on the day of hypophysectomy. InterstltÍa'l flufd

yras co'llected f rom both testes wfth the push-pull cannu'la 24 hours after the last inJectlonr as descrlbed ln 7.2.4.t and testlcu'lar 27L

venous and perlpheral blood coJlected as descrlbed in 7.3.1.4.

8.2.3. Human Chorlonic GonadotrophÍn

A slngle lnJectfon of hcc Ínto a male rat has been shown to produce a strfking fncrease in the permeabllity of the testlcu'lar blood vessels to albumin, in the amount of extracelìular f'lufd in the testi s, and f n testlcu'l ar I ymph f'lorv ( see 3.5.3. r ôrìd Setche'll and Sharpe, 198I). In additfon, testosterone concentratfons ln testlcular venous blood and periphera'l b'lood are increased at 2t 16 and 72 hours after such an fnJectfon (Hodgson and de Kretserr L982rL984; Hodgson et alr 1983; Risbrfdger et al' 1981; Soryerbutts et a'lr 1986). It ls likely horvever, that these responses are under different mechanfgns of control (see 3.5.3.1.3. )

Twelve grouPs of four animals (300-450 g) were fnJected wlth 50

i. u. of hCG subcutaneously. Interstitlal fl uf d was col'lected f rom a group of anfmals (from both testes) at 0r 2t 4t 8t L2t L8, 24, 30, 36, 48,72t and 96 hours after inJectfon¡ uslng the push-pull cannula as

described f î 7.2.4., and venous blood collected as descrfbed ln

7 .3.L 4.

8.2.4. Ethane Dímethane'sulphonate (EDS)

As a'lready descrÍbed in 3.6., EDS PrimarÍly lnJures Leydlg cel1s.

Rommerts et al (1985) have reported that EDS speciflcally inhÍbits the LH-regu'lated functfonal propertfes of the mature Leydlg cell, possibly vfa alkylation of protefns. Molenaar et al (1985) have rePorted the 272

complete degeneration of Leydfg ceì'ls by 72 hours after EDS lnJection' with no signfflcant testosterone productlon detectable at thfs time.

Two groups of four rats (280-360 g) were usedr wlth one grouP given intraperltoneal lnJections of EDS (75 mg/kg bw) dfssolved lñ

DMSO/water (I:3) as described ln 2.8., and the other grouP injected wfth DMSo/water only. Three days later¡ Ínterstitial fluid and venous

'l b'l ood were col ected as descri bed I n 7 .2.4. and 7 .3 . I.4.

8.2.5. Heat treatment

Heating the testls primarily causes damage fn the seminfferous tubulesr although not a'll cells there are damaged to the same extent (Co'llins and Lacyr 1969). The interstitlal tlssue apPears been heated or made cryptorchid. ni hypertrophled after the testis has l'ù I IndÍrect tests of androgen productlon suggest a moderate fall wfth

sometimes a translent increase I0 to 15 days after heatfng (see Setchellr 1978). Kerr et al (1979) found cytoìogfca'l changes fn Leydig cells of the cryptorchfd testfs that r+ere suggestfve of fncreased sterof d secreti on. Howeverr perl phera'l 'l evel s were normal or I owered in these animals, suggestlng a biosynthetic block fn testosterone production, on conversion of testosterone by damaged tubules to other metabolites. The concentratlon of testosterone in the sera of rats oC whose testes had been locally heated to 43 for 30 mlnutes dfd not

show any significant changes fn a 7 week perlod follovfng heatfng (Main et al ' 1976¡ Setchellr 1978).

i Eight groups of four anfmal s Q70-425 g) were subjected to r 273

oC locallsed heatlng of the testes at 43 for 30 mfnutesr as descrfbed 1n 6.2. Interstitial fluÍd was collected from a group of anfmals (from both testes) at 0, 7, L4, 2L, 28, 35, 49t and 63 days after heatlngr uslng the push-pul'l cannula as descÉf bed 1n 7.2.4.r ôrìd venous blood collected as descr{bed fn 7.3.I.4.

8.2.6. Efferent Duct Ligation

Lfgation of the efferent ducts of the rat testls causes disruptlon of spermatogenesis with maximum atrophy of the serniniferous eplthelium occurrÍng at 21 days after efferent duct ligation (Smlth' L962¡ Mafn et alr L978¡ Maln and Setchel'l' 1980; see also 3.5.1.). coltins et al (1978) and Co]llns and Tsang (1979) have reported the 'light mlcroscoPfc appearance of the Leydfg cells to be normal 2l days ,.} after efferent duct llgatlonr and their abflity to Produce androgens u (1978) FSH 'leve'ls I Ín vltro to be unf mpaired. Maf n et al have reported to lncrease from 5 to2L days after duct ligation. Rlsbrfdger et a1 (1981b) found lncreased levels of serum LH two to four weeks after efferent duct ligatfonr along wfth an increased sfze of Leydig ce]ls and an enhanced testosterone response to gonadotrophin stfmulation in vitro despfte a marked 'loss in LH-hCG receptors. Serum testosterone 'levels were not affected two weeks after either unilateral or bilateral ligatfonr remaining unaltered at four weeks ln the unilaterally ligated anima'ls but sfgnlficantly decreased Ín the bilaterally ligated anlmals. The results suggest that changes in Leydlg ce1'l function after efferent duct I igation are due to loca'l

T changes ln the testisr probably lnvolvlng the Paracrine control system I (Risbridger et al, I981b).

3 274

In four groups of four anlmal s (280-425 g) the efferent ducts of both testes were tigated (bilateral ligation) as descrfbed fn 2.3.4. Interstitia'l fluld was col'lected from a grouP of anlmals at 0, 7r L4t and 2I dayê after lfgatlonr usfng the push-pU'lì cannula as descrfbed in 7.2.4.t and venous blood co]lected as described in 7.3.I.4.

8.2.7. Testosterone Impl ants

Testosterone-fflled sflastic Ímp'lants have been used in a number of studies to investfgate the role of thfs hormone in the maintenance and restoratlon of spermatogenesis under a number of experfmental conditions (Ahmad et al t L973; Buh'l et a'l , L982). Depending on the length of these lmplantsr serum testosterone levels can be suppressed

.'t (Huang and Nieschlag' 1986). Sun et fi s'l ight'ly, or elevated 6 to 30 fold \r¡ al (1986) have also lnvestlgated the effects of such lmplants on plasma testosterorìer LHr and FSH levels, and on testosterone levels Ín Ínterstitial f'luid col'lected wfth the drfp co'llection method. Huang and NÍeschlag (1986) monitored changes over an 8 week perfod' and the majoríty of changes fn testosterone and gonadotrophin levels seen after I weeks apPear to have occurred within the lst week of

impl antatí on.

'length Four testosterone-filled silastic fmplants per of 2t 4, 8,

L2, and 16 centfmetres were PrePared as descrfbed in 6.4. One group of four X 2 cm implants were not fflled, and were used as controls. SÍx I groups of four anÍmals (400-580 g) were lmp'lanted with the capsules as ir descrfbed ln 6.4. One week later, interstitial ftuíd was collected

I 27s

from one testls ln each anfmalr using the push-pul'l cannula as

descrlbed I n 7.2.4. Follorvlng collection, testicular venous blood was collected as descrlbed fn 7.3.I.4. The testis vtas then forced through

a 21G needle to,dlsPerse cel'ls as detalled lrì 6.5.r the resultant suspenslon lrunedlately centrlfugedr and the suPernatant collected' trn each animal, interstltia'l fluid was collected from the remaining testls uslng the drip collectfon technique described in I'2'5' (see a'lso Flgure 7.I.). Perf pheral venous blood was coìlected as described in 7.3.1.4.

8.3. RESULTS

8.3.I. Effect of anaesthesfa and push-pu1ì cannu'lation on testlcular venous concentrations of testosterone.

The concentratfon of testosterone was signÍficantly reduced under anaesthesiar as seen in Figure 8.I. Exposure of the testis for one or two hoursr sampling of interstitial fluid wfth the push-Pull cannula, or a combination of these two treatments dfd not have any greater

effect on the I evel s of testosterone, than those al ready caused by anaesthesla. Slnce all these other treatments also involved 'lowered anaesthesfa, ft is unl ikely that they have fn themselves i i testosterone I evel s.

g.3.2. Hypophysectomy w ith pregnenol one supp'l ementati on t ï The increasing doses of pregnenolone supplementation to

hypophysectomised anima'ls resulted fn an fncreased testosterone production. As seen ln Figure 8.2.r provlsfon of 2 mg t Figure 8.I. Testlcular venous btood leve'ls of testosterone fn adult rats : (IMM) Ímmediately after induction of anaesthesia with pentobarbÍtoner (ANA) after one hours anaesthesia' (EXP) after exposure of the testis of an anaesthetÍsed animal for one hourr e/P) after one hours sampling of interstitfal flufd from the testis of an anaesthetfsed animal with a push-pull cannula, (2EXP) after exposure of

testes for 2 hours¡ (P/P + EXP) after one hours samp'l ing of IF fluid with a push-pu'll cannula and one hours exposure of the testf s. Alì treatments Ínvo'lved anaesthesiar which sígnificantly (P < 0.00I, t-test)

'l owered venous testosterone I evel s. Howeverr no treatment

reduced testosterone 'levels more than was Índuced by

anaesthesia alone' and levels measured after some

treatments re¡nained significantly greater (P < 0.05) than

those measured under anaesthesia alone (x). Values are

mean t standard error of the ffiêârìr n=4. 200 *

= E 150 u) \/c o c o 100 L o +t o o +t 3T' 50 o þ

0 IMM ANA EXP P/P 2EXP P/P+E N\¡ Treatment o\ Figure 8.2. Testosterone levels fn peripheral blood plasna (open bars)r testicular venous b'lood p'lasna (hatched bars) and testicular lnterstltial fluld (solid bars) of

hypophysectomised adult rats treated with 0' 0.5, I.0r or

2.0 mg Pregnenolone/1009 bw/day for 14 days after

hypophysectomy. Values are mean:t standard error of the

mêônr n=4. *: va]ues sf gnif lcantly different (P < 0.05)

from untreated animal s. 50

45 A 40 -E * u) 35 \/c o 30 Ê o 25 L o 20 +¿o o +t 15 .* o o þ 10 * * 5 * .r n.d n.d :* 0 0 0.5 1.0 2.0 0 N \¡ Pregnenolone (mg / lOOg/ daY) 2t8

pregenenolone/IOO g bw/day was sufffclent to produce levels of testosterone fn testlcular venous blood comParable to those leveJs fn anaesthetlsed control anfma'ls (see Ffgure 8.1.). fn all groups ln whÍch testosterone levels were detectable¡ lnterstltial fluld levels were 4-6 times lower than testlcular venous blood levels, but greater than peripheral venous'levels. As seen ln Figure 8.3.r the 2 mg dose of pregnenolone was able to mafntafn testfs sÍze' but accessory organs (semfnal vesfcles shown) have regressed to the same extent as those Ín vehfcle treatedr hypophysectomised-control animal s

8.3.3. Human Chorfonfc GonadotrophÍn

Follovlfng a sfngle lnJectfon of hCG, 'levels of testosterone peaked fn perfpheral venous blood at 2, l/t and 72 hours (Figure 8.4.a). Whfle there fs a suggestion of a peak a'lso occurring at24 hoursr this ln most likely caused by'lovr values at 18 hours glvfng a false nloyrn, and spllttfng the peak normally found around 16 hours (see Sowerbutts et al' 1986). trlhlte both the testis vein and lnterstitia'l f'luid samples show changes (Ffgure 8.4.b)r these do not parallel the peripheraì testosterone levelsr partÍcu1arly at 72 hours. Levels of testosterone ln testlcu'lar venous blood peaked at 2 and I hours after hCG. Againr low values at 18 hours apPear to comp'licate interpretation of the results. Interstitfal f'lufd levels of testosterone shoved sfmflar trends to those observed in testlcular venous blood. However these levels are agaln consistently lower than testicular venous blood levels. The adminfstration of hCG in fact appears to increase the discrepancy between the interstftfa'l fluid levels and the venous blood levels. When the ratfo of testosterone ln I

Figure 8.3. Testes (T)r Semlnal Vesicles (S) and Adrenaìs (A) from control (C)' hypophysectonlsed (H)r and hypophysectomfsed - pregnenolone supptemented (P, 2ng/L009 bw/day) adult male rats. Adrenal sfze is aPparently unaffected by hypophysectomy or pregnenolone supplementatlon. Testls and seminal veslcle size are slgnificantly reduced by

hypophysectomy. Pregnenolone supplementatlon of

hypophysectomlsed anlma'ls mafntains testls slzer but

seminal vesicles regress to the same extent seen ln untreated hypophysectomised anfmals. d H c

S V

6L¿ Figure 8.4. Testosterone levels up to 96 hours after a singìe subcutaneous fnJection of 50 i.u. hCG. : (a) in perÍphera'l bìood plasnar and (b) in testicular venous b'lood (hatched bars) and interstitial fluid (open bars) Perfpheral blood p'lasma levels (so'l id bars) are also represented in (b) for dlrect comparlson of concentrations in the different f'lulds. Values presented are means ! standard error of the

rìêôrìr n=4. x : va]ues sf gnif f cantly different (P < 0.05) from untreated anfmals. The ratio of mean testosterone levels in interstitial fluid compared to those in testicular venous blood is shoYrn fn (c)r ârìd reflects the relatlve changes in concentration of testosterone fn these two fìuid compartments. 280

2 E o' Ê 0 o o?4A12182430364A7296 c o 1500 L o *¿ o f350 o +t L o o t- 10

900

750

¿50

300 t I L

o24A1218243036497296

0 I

o o.70 +t G t 0 .60

a 0 .50 t-a o-4 l!¡ a 0 .30

0 .20

0.1

024 at218 24 30 36 48 72 96 Time after hcc (h) 28r lnterstftial fluld / testls vein 1s plotted agafnst tlme (Flgure 8.4.c), the ratf o decl lnes for the f irst 4 hours follorvf ng hCG admlnfstratfonr fndlcatlng a greater lncrease ln.venous blood levels compared to lnterstitia'l fluld levels. From 4 to 24 hours post hCG' the value of the ratlo increases agafn (onlttlng the dec'llne at 18 hours) r whi'le testosterone I evel s i n venous b'lood and i nterstitlal f'luid peak at I hours and then fal'l (Ffgure 8.4.b). This suggests that the changes ln interstftia'l leve'ls are not as great as those ln venous b'lood. The increase fn interstÍtia'l flufd testosterone from 4 to efght hours fs proportionately greater than the increase in venous blood'levels at this tfme. The ratfo value contlnues to increase between I and 24 hours when testosterone levels are falling fn both venous blood and fnterstitial flufd, and suggests that the dec'llne ln venous testosterone 'levels ls proportionate'ly greater than that seen in lnterstitfat flufd. Fron 24 hours, the ratfo dec'lfnes to 36 hoursr and slowly begfns to rise to 96 hours post hCG.

8.3.4. Ethane Dfmethane Sulphonate

Three days after admlnfstration of EDS, testosterone levels were slgnificantly reduced and were almost undectable ln ÍnterstÍtfal fluÍdr testicular venous blood and peripheral venous blood (Table 8.I.). That the levels fn these three fluids are not signlffcant'ly different suggests that thÍs basal 'leveì of testosterone f s not of testfcul ar orfgf n. Table 8.1. Levels of testosterone ln posterior vena cava and testÍcular venous blood plasmar and testlcular interstitial fluld in adu'lt rats 3 days after treatment with DMS0/water (Control) or Ethane Dlmethane Sulphonate (EDS, 7íng/kg bw fn DMSO./water). Values are means + SEM, n=4.

Testosterone (ng/m1 )

Testfcul ar venous Interstltl a'l Posterf or blood plasma f'lufd Vena Cava

CONTROL 37.52 + 6.4I 22.63 ! 4.24 2.05 + 0.34

EDS 0.329 + 0.05 0.32 + 1.00 0.32 t 0.06

N No 283

8.3.5. Heat treatment

Perfpheral levets of testosterone were not greatìy affected by heatlng of the testes (Ffgure 8.5.)'. Up tq 28 days after heatlngr perlpheral testosterone levels tvere no dlfferent to control levels.

(P < From 28 days to 63 days there u,as a sl ightr but slgnlficant 0.05), increase fn peripheral testosteroner although the leve'l measured at 49 days after heating was not signiffcantly different from contro'|. Testicular venous'levels were lncreased by 14 days post heatlngr and remained elevated until 35 days after heating. The levels measured ln the testis vefn at 49 and 63 days were sfgnlficant'ly lower than control levels. Interstitfal fluid levels of testosterone are possibly the most lnteresting data from thÍs experiment. By 7 days after heatfng, lnterstÍtfal levels of testosterone are slgnfflcant'ly higher than control leve'ls, and continue to increase to 35 days after heatÍng. At this timer they are not signifcantly different from testicul ar venous 'leve'lsr and thÍs relationshlp is sti'll found at 49 days post heatlng. By 63 daysr interstltfal leve'ls of testosterone are agaln lower than testls vefn leve'ls, but are stil'l signfficantly

h i gher than contro'l I evel s.

8.3.6. Efferent Duct LÍgation

Results obtained after bilateral efferent duct Iigation are shc'¡n ln Flgure 8.6. Perlpheral leve'ls of testosterone were signlffcantly reduced at 7 and 14 days after treatmentr returnfng to control leve'ls 'levels by 2l days after duct lfgatlon. Testis vefn of testosterone were a'lso sfgnlficantly reduced by 7 days after treatmentr returning Figure 8.5. Levels of testosterone ln perÍpheraì blood plasna (open bars), testlcular venous b'lood plastna (hatched bars)r ôfìd testlcular lnterstltfal flufd (so'lld bars) of adult rats up to 63 days after heatfng of both testes at 43oC for 30 mlnutes. values presented are means * standard error of the mean, n=4. * : va]ues slgnlflcantly dlfferent (P < 0.05) from untreated anima]s. 100

^I E 80 g' 70 ¡* vc o 60 * * tr * .r o 50 l¡ o +t 40 3n o .* +t 30 *i* o * o * Þ 20 :r .r r* 10 * * :r

0 0 7 L4 2t 28 35 49 63 N 5o Days after heating ,il lr

Figure 8.6. Levels of testosterone in perlpheral blood plasma (open I bars)r testfcular venous blood plasna (hatched bars)r ârìd i, testicular interstltial fluid (solld bars) of adult rats at 0,7,L4, and 21 days after bilateral llgatlon of testlcular efferent ducts. Values presented are means ! standard error of the ffiêôlì¡ n=4. * : values slgnlflcantly dlfferent (P < 0.05) from untreated animals.

I -da * --={=

50 t

,A 40 -E o) vÊ o 30 c o t- o P 20 o o * +t o * o * l- 10

* *

0 7 t4 2t 0 N (rl@ D ay s af ter EDL 26

to control 'levels by 14 days after EDL. Howeverr these ìevels contlnued to rfse, and were slgnlflcant'ly increased by 2l days after at: treatment. Interstltlal ftuid levelsr ulêFê sfgnfffcantly lower than testls vefn levels at all tlmes. Howeverr the profile of testosterone levels fn interstÍtlal f'lufd was much more like that of perlpheral levels, than of testis vein concentrations. Interstitfal fluid levels of testosterone were signlficant'ly reduced by 7 and 14 days after duct lfgatlonr returnlng to control 'levels by 2I days.

8.3.7. Testosterone Imp'lants

Testosterone-fflled sl'lastic fmpl ants raf sed testosterone I evels in perlpherat venous blood slgnlflcantly wlthfn 7 days (Table 8.2.1t and there was also a sfgnfficant (P < 0.01) lfnear correlatfon between

I sfze of lmplant and perfpheral levels of testosterone. Testlcular $,{E I venous leve'ls of testosterone were sf gnf f lcantly lncreased ln anlma'ls

wlth the 12cm and 16cm lmp'lants. Whlle perlpheral venous levels were signifÍcantly (P < 0.05) 'less than testlcular venous levels fn control animals, they were slmllar to testlcular venous levels ln animals

Ímpl anted w fth 2cm, 4cm, 8cm, and 12cm fmpl antsr and were slgniflcantly (P < 0.05) greater than testfcular venous levels fn animals wÍth l6cm Ímplants. ThÍs suggests that ln the control anÍmals, the testes are the maJor source of testosterone, and that androgen productfon by the testes was sfgniflcantly reduced fn the presence of testosterone lmplants. That peripheral levels of testosterone are greater than testlcu'lar venous levels ln anlmals wlth l6cm fmplants I suggests that testfcular productfon of androgen fn these anlmals fs I less than that released from the lmplants' and may in fact have ceased

r ,g -.-æ

Table g.2. Leve'ls of testosterone ln flulds col'lected from adu'lt male rats impìanted with varlous sfzes of testosterone-filled sl'lastic capsules for seven days. In each anfmalr samp'les of testlcular lnterstftla'l fluld were co]'lected from one testis with a push-pu'll cannula (I.F.) and from the contralateral testls by drlp co1'lectlon (Drfp). Posterlor vena cava (PVC) and testlcular venous (TV) blood plasma were also sampled, as tras the supernatant col'lected after dlspersfon of the testlr rurpìed wfth the push-puil cannula (Sup). Values presented are Means + SEMr n=4 anima'ls per group. Anaiysls of varlance war used to detect treatment differences' and where approprfate the [east Slgnlilcant Difference va]ue (LSD, 5f) is lfsted (n.s. lndlcates no slgniflcant dlfference).

(ng/ml Impl ant Length Testosterone )

(cm) I. F. Drfp TV PVC sup LSD

Contro] g.g4 + 0.95 10I.36 + 7.78 L2,63 ! 2.05 1.0I + 0.19 20.50 + L.23 8. 16 + 4.89 2 10.38 + 2.47 L4.44 + 0.75 8.09 + t.I4 6.75 + 1.14 19.03 2.69 + L,42 2.83 4 7.94 + L.25 L4.42 + 0.70 9.99 + 0.59 8.12 + 0.98 I4.7r I 12.18 + 5.02 16.84 + 3.39 15.94 + 2.94 18.35 t 1.98 L5.28 + 0.89 7.69 + 8.23 L2 L3.69 + 3.I9 16.93 + l.l4 L9.77 ! 2.94 27.66 + 5.70 16.31 0.58 + 3.83 16 t6.2L + 0.72 22.98 + 3.20 24.72 + 0.81 30.03 + I.zL 18.41 0.92 n. s. LSD n. s. 9.I2 4.77 6.27

N {o 288

altogether. Drlp collected fnterstltfal fluld levels of testosterone were slgnfflcantly reduced in anfmals wfth fmp'lants' and there t{as no sfgnlflcant dlfference ln the leve'ls of testosterone ln drip-co'l'lected flufd betueen any fmplanted anfmals, regard'less of lmplant slze. Drlp collected testosterone levels remaÍned greater than perlpheral and testicular Venous blood levels wfth the use of 2cm and 4cm fmplants;

were the same as peripheral and testicular venous levels with 8cm and l2cm implantsr and were lower than blood levels wfth the use of l6cm fmplants. In contrast, push-puìl collected fluld levels of testosterone urere slgnfficantty greater than perlpheral venous blood 'levelsr but not slgnfflcantly dlfferent from testfcular venous blood levels 1n contro'l anfmals. Furthermorer push-pull f'luld levels were not signiffcantly dlfferent from elther perfpheral or testicu'lar venous blood levels wlth the use of 2cmt 4q¡r 8cm or 12cm imp'lantsr

and were sf gnlf fcant'ly 'less than perlpheral and testicuìar venous blood 'levels ln anima'ls wlth 16cm fmp'lants. Desplte an apparent fncrease ln push-pu'|1 fluid leve'ls of testosterone wlth increaslng implant sfze (Tab'le 8.2.), 'levels of testosterone fn push-pull flufd were not signiflcantly dlfferent between any of the treatment grouPs or the control group. Use of l2cm and l6cm impl ants caused a slgnlflcant increase ln testosterone'levels fn testfcular venous b'loodr but push-pul'l fluid levels were not affected. The levels of testosterone ln drlp-co'llected flufd from control anfmals v',ere slgnificant'ly (P < O.OOI) greater than tevels in push-pu'll col'lected flufd. Howeverr ln fmptanted anfma'ls the testosterone concentratlons fn these two flulds were not sfgniffcantly different. The concentratlon of testosterone from control anfmals, fn testlcular flufd collected as supernatant from dfspersed cellsr was slgnlficantly 289 greater than that ln perlphera'l venous plasna, but not slgnificantly dlfferent from testicular venous Plasna levels. There YJas no signiflcant dlfference ln supernatant levels of testosterone between control anlmals and any of the lmplanted anfmals. Supernatant leveìs of testosterone were signifÍcantly greater (P < 0.05) than push-pull collected levels fn controlr 2cmt and 4cm imp'lanted anfmals, but were not signiffcantly different in anfma'ls with Scmr 12cm and 16cm implants. Drfp-collected fluid levels of testosterone were slgnÍflcantly (P < 0.OOl) greater than supernatant levels fn control anÍmalsr but not ín lmplanted animals.

8.4. DISCUSSIoN

The results of these experfments have all provlded support for the theory proPosed fn the prevfous chapterr that Leydíg cells are llkely to secrete as much testosterone lnto testicular venous blood as they do fnto fnterstftlal flufd. The general effect of anaesthesía on lowerlng of blood testosterone levels fs unfortunate. Setchell and Gall'l (1983) have also reported on the problem faced with pentobarbitone anaesthesia and the measurernent of testosterone production and testlcular b'lood flow¡ due to effects on gonadotrophfn secretion. Ether has been reported to cause a rapÍd decline ln testosterone production ln rats (Bardfn and Petersenr L967¡ Farlss et al, 1969). Setchel'l et al (1965) have reported light pentobarbitone anaesthesla to lovrer testosterone secretfon ln rams in the days fol'lolrrÍng anaesthesfar although no dÍfference was noted at the tfme of inductlon. The effects of other anaesthetfcs are worthy of closer investigatlonr although it woul d obvious'ly be usefu'l if a method coul d 290

be developed for sampllng ln the unanaesthetised anlmal. Prevfous reports of pregneno'lone supplementatfon after hypophysectomyr have found fntratestlcular levels of testosterone to be maintained with the hÍgher doses of pregnenolone despite low peripheral testosterone levels (Harris and Bartker L975i Anthony et al, L984; Turner et a'|, 1985). The maximum dose of pregneno'lone used by these authors rlas equiva'lent to I mg/100g bw/day. At thfs dose¡ testis vein levels of testosterone rúere lower than control values and the results of the present study agree wlth the prevlous studies Ín this regard. However' when the pregneno'lone dose was lncreased to 2 mg/Ioog bw/day in the present studyr testls vefn levels of testosterone ÌJere not slgnfficantly different from normal controls. Turner et a'l (1985) collected testfcular lnterstÍtla'l f'luid from pregnenolone-lnjected hypophysectomlsed anlmals by drf p collection' and found interstitfal levels to be slgnlffcantly greater than testlcular venous blood leve'ls, a'lthough leve'ls in both these flulds were less than in norma'l control anfmals. In contrast to the findfngs of Turner et al (1985)r the testosterone levels in push-pul'l fnterstftfal fluid samples obtafned in the present study were signif icant'ly 'lorer than those in testicular venous samples. Holever' all vatues were less than those obtalned from normal controlsr whfch is in agreement wfth the report of Turner et al (1985). This agafn suggests that in drip coìlected fluld, metabolfsm may contfnue after isolation of the organr ônd the resultant metabolites (eg. testosterone) accumulate in the collected flufd. As raised earlfer' these elevated levels may also reflect the rrstoresrr of testosteroner rather than actual leve'ls ln the lnterstitium. Testls vein levels of testosterone are apparentty normal wfth 2mg Pregnenolone/I009 bw/day 29r despite a 'loryer than normal concentration of testosterone fn fnterstitlal ftuid which may reflect a reduced blood flow. The effect of gonadotrophins on testfcular blood fìotr fs controversfal (see Daehlin et alr 1985). However, Bindon and WaÍtes (1968) have reported testlcular blood flow to be decreased after hypophysectomy in mice, and Setchell et al (1969) observed reductlons in testlcular blood flotY ln rats after hypophysectomy. Setchell and Galfl (1983) have provlded some evfdence for the involvement of semfnlferous tubu'les in regulatfng blood flowr and whfle pregneno'lone admfnlstratfon to hypophysectomised animals can malntafn tubular Æ!P concentrations and qualitatlve'ly malntaÍn spermatogenesls (Anthony et al, 1984)r other tubular factors may not be mafntalned by such treatment. Any gonadotrophf n-dependant Processes f nvolved in regulatfon of fnterstitlal hormone concentratfons Ís I ikely to be affected fn hypophysectomised anlmalsr and may not be compensated for by the provfsion of pregnenolone. The fmportance of blood flow for hormone secretion fs also apparent fn the results obtaÍned after hCG admfnlstratfon. $lhile both lnterstitlal fluld and testfs vefn levels of testosterone shqed

changes after hCG (see Figure 8.4.), they dld not parallel the changes ln periphera'l 'levels of testosterone. 0f partÍcular note f n thf s regand are the values at72 hours after hCGr when perfpheral leve'ls peaked for the third tlme, without concommltant peaks in interstftial or testicu'lar venous blood. Thfs suggests that other effects such as a change ln blood floul or testosterone clearance rate contrfbute to the peak found fn peripheral blood samples at this time. The ratio of fnterstftia'l fluld testosterone / testicular venous levels provfdes further evfdence of the effect of vascular parameters on interstftial 292 f'lufd testosterone levels. As was noted from Ffgure 8.4.r the peak fn testis vefn testosterone at I hours after hCG administration tvas followed by increasfng levels of testosterone fn interstltlal fluld as ref'lected by the increasing value of the ratÍo up to 24 hours after hcc. Thf s ratio data Ís represented in Flgure 8.7., a'long with recent'ly publ ished data of Sorverbutts et al (19S6) shouring changes f n vascular perîneabitity after hCG (as determined by measurement of I hour albumfn spaces). Note that the perfod of increasfng vascular permeability whÍch fs assoclated wlth the second maJor peak fn perlpheral blood 'levels of testosterone, also cofncldes wfth the perfod of increaslng testosterone levels in lnterstftÍa'l flufd reflected în the fncreasfng ratlo values. The data obtafned from thÍs experiment suggest that a number of factors may be involved in the responses noted after an inJectfon of hCGr and the followfng hypothesfs is provfded as an atternpt to lncorporate known events with the observed responses. The fnÍtla'l peak in testicular venous blood probably reflects the LH-]Íke stfmulatlon of Leydfg ce]ls by hCG, resultfng in secretion of testosterone prfmarfly lnto the testicu'lar vascular bed. From 4 to 24 hours, fncreasfng vascuìar Permeabllity combÍned with continued secretion by Leydfg cellsr results fn fncreased levels of testosterone in fnterstÍtial fluld. The possÍb'le dynamfcs of this event have been previously addressedr and are likely to involve 'leukocytes and other lnflammatory resPonses (see 3.5.3.1.3.). Durfng thfs time, a second peak fn peripheral levels occurs. Increasing levels of fnterstltial ftuid during thfs tfme probably increase receptor occuPancy by hCG. According to Sharpe (I9BO), testÍcular bound hCG increases llnearly for 2 to I hours after inJectf on, p'lateaus f rom I -16 hours and then dec'lines towards 40 Figure 8.7. The ratio of the mean leve'l of testosterone fn testlcular venous blood compared to that ln testicu'lar interstitlal flufd (open trlangìesr represented from Ffgure 8.4.)r ôîd the l-hour albumfn space ln the testes (solid trlangles, from Soryerbutts et a'l' 1986) ln adult male rats at varlous tlmes after a sfngle subcutaneous fnJectlon of 50 l.u.

hCG. Va'lues for albumfn spaces are means ! standard error

of the Íìêâtìr n=4. 150 L

B Ag,

a- v 1oo 'lt = 6 a o (t { o ¡ Ct t o 4 a tr o a- + 50 I' E o Jl= ¿ -

T 2 4 10 20 40 100 N \o Time after hCG (h) t¡, 294

hours. t{hlte thÍs mfght mafntaln Leydfg cel'l secretion, testicular venous levels fn fact are not malntafned durfng this tlme. Blood f'lomr

has been.'reported to be f ncreased at this tlme.whfch would reduce levels of testosterone measured per unit volume. Setchel'l and Sharpe (1981) found testÍcu'lar blood flow to be unaffected at 12 hours after

hCG, but sÍgnfficantly lncreased at 16 and 20 hours after hCG, to about twice control levels. Damber et al (I98I) have also reported sÍgniflcant increases fn testicular blood f'low 20-24 hours after hCG. However ft is more like'ly that Leydig cell secretion fs sfgnÍficantly reduced at thfs tÍme. As shown in 3.5.3.I.2.L., lymph flow is increased 24 hours after an lnjection of hCG' and the peak fn periphera'l venous testosterone levels at 16 - 20 hours may be due to lymphatÍc clearance of interstltial ftuid contalnlng unusualìy high levels of testosterone at thls tfme. After thfs inftial 24 hours, down regulatfon of LH receptors on the Leydfg cells has been reported (Hseuh et al, L976; Sharpe' 1976) whfch may prevent further stimulation of the Leydfg cells. Other factors such as testicular LHRH-Iike actfvity are also tikely to lmpaÍr Leydig cell functÍon at this tlme (see Sharpe and Fraserr 1980). Subsequent changes after thÍs timer are probably due to varÍations in testfcular b'lood flow and testosterone clearance ratesr especfatly for the peak Ín perfpheral

testosterone at 72 hours after hCG as a'l ready dÍscussed The admlnlstration of EDS reduced testosterone to almost undetectable levels 3 days after adminfstratfon, whlch supports previous reports (MorrÍs et al' 1986; Mo'lenaar et al, 1985; OfLeary et al, 1986). Interstltfal levels of EDS measured Ín f'luid collected by push-pull cannulae rúere not sfgnifícantly different to venous levels 'levels (testfcular and peripheral), whfch suggests that these low are 295 of extra-testlcu'lar orlgln (probably adrenal)r sfnce Leydfg cell destructfon fs complete by 3 days after EDS admfnlstratlon. The use of the push-pu1l cannula to obtaln lnterstitlal fìuld from testes after locallsed heating, has again revealed an lnterestlng response not previously reported. The tack of conslstent change in peripheral 'leve'ls of testosterone has been establ ished by other authors (see Maln et al, !978¡ MaÍn and Setchel'l' 1980; Galll and Setchell, 1987). The testlcular venous levels of testosterone were inltfally unaffected by heating, and then rose from 14 to 35 days after heatfng. Gall'l and Setchell (1987) reported simfìar findfngsr and correlated thls rise wlth a decrease fn testls wefght. AccordÍng to these authorsr testis wefght after heating beglns to fall wlthin 2 daysr is lowest at 2L daysr and partfa'lly recovers by 56 days. Thfs weight reduction fs due to dls¡uption of spermatogenesfs. The fnterstltlat levels of testosterone determfned ln the Present study have lncreased sfgniflcantly as testis wefght decl fnesr and at 35 days after heat treatment are not sfgnf ffcant'ly dlfferent from testicu'lar venous levels. Setchell and Galil (1983) reported changes fn blood flov durfng thls time periodr and suggested that blood flov mlght be regulated by tubular mass. Thusr as sPermatogenesfs ls disrupted and tubular mass decreases, testlcular blood flow would also decrease'

These authors ralsed the probìem of accounting for an inabÍllty of Leydig cells to comPensate for reduced blood flow by only partiaìly increaslng testicular venous concentratlons. The results of the present study suggest that greater levels of hormone in fact accumulate in interstltfa'l fluid. It is of interest ln this regardr that the perfpheral levels of testosterone l{ere also margfna'l'llr but signiflcantly increased at these times of greater fnterstitial levels 296

(see Figure 8.5.). Thfs may be due to lymphatlc clearance of interstÍtial ftuid testosterone. Bergh (1985) has recently descrfbed the tikely paracrfne reguìatÍon of Leydig cells by senfnlferous tubulesr a'lbelt at specffic stages of the spermatogenic cycle. It is possib'le that such effects mlght occur more generally in damaged testesr particularly durÍng reinitiation of spermatogenesis and repopulation within the tubules. The hístologfcal presentatfons in Figures 8.8.arb. show the changes fn rat testes at certain times after oC a 30 minute exposure to 43 heat. The gradual dlsruptfon of spermatogenesis over tfme fs clearly vfsible, and by 7 days most dÍfferentiatlng cel'ls have dlsapPeared. Late Pachytene prfmary spermatocytesr round and e'longated spermatfds and the spermatozoa are absent. Whfte a few earlier stages of prímary spermatocytes perslst up to 7 and 14 daysr they are absent by 2L days. 0f lnterest fs the dramatlc change with tubules regeneratlng between 28 and 35 days' and by 42 days after heatlng normal spermatocytes and developing spermatids are seen fn the maJorÍty of tubules. By 56 daysr the testis is hlstologically normal. That the change between 28 and 35 days should also occur at the time of maxfmal lnterstÍtial fluld levels of testosterone may be due to paracrfne stfmulation of Leydig cel1s by the tubules. By 49 days after heatingr fnterstitial levels u,ere stl'lì the same as testlcular venous levels' but both were signfflcantly loner than at 35 daysr and venous levels were sfgnÍficantly lower than control levels. Thfs suggests that blood flow may be increased during this stager and ln fact Setchelì and Galil (1983) have reported this to be the case. By 56 days, these authors found b'lood flow to have returned a'lmost to normal rates. In the present studyr fnterstitial levels of testosterone are again reduced below testicular venous I

i i I

l Flgure 8.8.a Histologfcal sections from adult rat testes at varlous tlmes after heatlng at 43oC for 30 minutes. (a) Control, (b) 7 daysr (c) 14 days, and (d) 2L days follctting heat treatment. X L25. (from Galil ' L9823 see also Ffgure g.g. b). @

L6Z Figure 8.8.b Histological sections from adult rat testes at various tlmes after heatÍng at 43oC for 30 minutes. (e) 28 daysr (f)35daysr$)42daysr(h)56daysfolloringheat treatment. X 125. (from Gal il ' L982). @

-'.-.a ' '

a6z 299 levels by 63 daysr although they are still sfgnlffcantly greater than control levels, and the testls veln concentratfons are stlll signlficantly tower than control levels. By thls tfmer spermatogenesfs Ís again norma'lr aìthough testis weight is stf tl reduced. Galil (l'982) reports that some tubules failed to regenerate by 42 days after heatlng, and contalned only Sertoli cells' sPermatogonfa and spermatocytes. Thfs may account for the contfnued dffferences fn testosterone levels at 63 daysr compared to control levels. Llgatfon of the efferent ducts of the testis causes flufd secreted by the testis to be retafned lnside the semÍnfferous tubules and rete testlsr resulting fn enlargement and eventual degeneratlon. Setchell et al 1977) and Mafn et al (1978) have reported that serum testosterone levels are not consistent'ly affected by EDL. Rlsbrfdger et a'l (I98I) found serum testosterone levels to be siml'lar to control levels at 2 weeks after EDL, but sfgnificantly reduced by 4 weeks. Mafn and Setchelt (1980) found no dlfference ln testicular venous levels of testosterone between bilaterally lfgated animals and control animals at 21 days after ligatlon, but dld find sfgniflcant differences between unilateralìy llgated and control anlma'ls at thls time. These results suggest that great fluctuatlons fn secretion of testosterone occur after bilatera'l ligatlon and comparÍsons of data from dffferent authors need to be made at simlla¡'time points. The effects of bilateral ligatfon on gonadotrophfn responses probably

makes comparfson of unf'lateral and bilateral data of questionable value. The resu'lts of the present study show minor varfatlons ln perfpheral levels of testosterone whlch were sllghtly reduced at 7 and 14 days after duct lfgatlon, but not sfgnlflcantly different from control levels by 2l days. This fs not dÍssfmilar to previous reports' 300

0f fnterest ls the slgnfflcant reductlon fn testfcular venous levels at 7 days after I igatlonr whích return to normal by 14 days ' and are then sÍgnfficant'ly lncreased by 2L days. Because fnterstltial fluld levels do not shol a sfmfliìF Fêspons€r it is unlikely that the venous leve'ls are directly attrÍbutable to changes in Leydig cell secretion. It is more likely that a'lterations in blood flw are of consequence fn thls regard. Indeedr wang et al (1983) concluded that durÍng aspermatogenesis followlng irradfatfon (as with heat and efferent duct ligatfon) the capacity of the testfs to secrete testosterone is severely limlted by decreased testlcular b'lood flowr not by the abitity of the Leydig cells to release testosterone fnto thefr fmmedlate envfronment. Wang et al (1985) have also reported b'lood flow to play a role fn changes of testosterone secretlon seen after unf'lateral llgation. Efferent duct ligatfon and heat seem to fnduce very sfmllar changes ln the testfs, and lt ls not surprfsfng that responses sfmflar to those found after EDL were also noted in heat treated anlmals in the present study. Heat-treated animals dÍd not shor the reductlon fn testlcular venous levels of testosterone at 7 days after treatmentr whfch may relate to a difference fn the aetiology of disruption ln spermatogenesÍs. HouJever fn other respects' the changes fn testosterone 'levels aPPear very slmilarr further highl ightlng the 1 lkely importance of tubular-fnterstltial

i nteracti ons.

Much of the data on venous levels of testosterone affected by the treatments used fn thfs studyr supports existfng literature. New informatfon or fdeas have been generated largely from the data collected wfth the pushLpull cannu'la. Thfs data has consistently 30r

supported the hypothesfs ralsed earìÍer regardfng partftlonlng of testosterone levels between venous blood and lnterstftlal fluld.

Fûrther support comes from the ffnal experiment with the use of' testosterone lmplants. This experfment was deslgned to compare the drlp-collectfon system and the push-pull system dfrectly. The initial dfscussion on the drÍp-collectlon method rafsed the I ikely problern of continuing metabollsm after isolation of the organ. Deprived of blood flory, the various products of the testis would have to end up fn the fnterstitfal fluid - a factor most I Íkely to contribute to

artlflcfally high leve'ls of substances measured f n such f'luid. A number of authors have dernonstrated that testlcu'lar productfon of testosterone fs inhibited fn normal anfmals wfth exogenous

admf nlstration of supranormal 'leve'ls of thÍs hormone by lnJection or

subcutaneous fmplant (see Cunningham and HuckÍns, L979¡ Rea et al¡ 1986). If contfnued testfcular productlon of testosterone fs ü {Ë J contrÍbutlng to hlgh levels fn drfp-collected fluld from normal animalsr levels ln drlp-collected flufd from testosterone-supp'lemented anlmals should be dramatlcalìy reduced since lntratesticu'lar

metabo'llsm wl'l'l be suppressed. The resu'lts of the present study suggest that thÍs is indeed what happens. As soon as exogenous testosterone was provfded to anlmals, drip col'lected f'lufd leve'ls fell substantialty. In a recent abstractr Sun et al (1986) reported the use of testosterone implants for 50 days, and found the same result' namely that drlp collected testosterone levels were signiffcantìy reduced in impìanted anima'ls. In planning this experiment¡ it was envisaged that testosterone levels in drip coìlected fluid and push-pull collected f'lufd would be very sfmilar fn fmplanted anfmals;

particularly when the implants were large enough to suppress 302

lntratestlcular productlon/secretlon of testosterone. Agaf n' as seen in Table 8.2., the levels measured in flufds collected by these technfques were not slgniflcantly dlfferent in lmplanted animals. Thls is lnterpreted as support for the theory behÍnd the use of the push-pu1l cannula, and provldes further evfdence that the high levels of testosterone found in drfp-collected fluid from unimplanted animals are artffactual due to probìems with the technique. It is interestfng that the testosterone levels in supernatant fluid from cell-dfspersed testes are not slgnlficantly dffferent from the drip co'lìected fluld levels Ín fmplanted anfmals. This supernatant f'lufd contafns both fnterstftial fluld and tubular flufd in rough'ly equal proportions, although interstitlal fluld probably comprises a

greater proportlon f n aspermatogenfc testes (Ga'll1 and Setchel'1,

I9B7). Comhafre and Vermeulen (1976) and Davfes et al (1979) reported sÍmllar concentratlons of testosterone fn tubular and interstltfal d ',ù I fluid and the results in the present study provide further evidence for thfs. In 1978r Setchel'l et al reported a speciffc' saturable carrler-mechanismr probably lnvolvlng facllitated dfffuslonr to be ., fnvolved ln the transport of testosterone lnto semfniferous tubule fluld and rete testfs fluid. ThÍs study was based on comParisons of f'luid / plasna ratios of labelled testosteroner Sc-dÍhydrotestosterone 'l and Sc¡-androstan-3cr17p-df ol and the lpid df strf butlon coeff fcients of these steroids (see also Setchell, 1980). FluÍd / plasma ratios of labelled testosterone were reduced wfth increased levels of cold testosterone in plasmar and the authors concluded that entry into tubular f'lufd was by a saturable carrÍer mechanfsm. Since entry was T I a'lways down a concentratfon gradlentr the process vlas thought to I lnvo'lve facil itated dfffusfon. Although these studles did not monitor

! 303

fnterstitial fluid, the authors consfdered thls mechanfsm to be

probabl y 'located i n the semf nl f erous tubull es. Androgen b i ndi ng protei n ci dld not appear to be fnvolved. The results of the present study suggest that tubular and fnterstitfal fluÍd levels of testosterone may not be greatly dffferentr and the rrgradÍentrt fn fact aPPears to occur

between the vascu'lar and interstitial compartments (especÍally wlth the large exogenous admfnistratfon of testosterone from 16cm implants). It appears that a saturable carrfer mechanls¡n does exist' but that it may ln fact ìfe ln the capillary endothelfum. Thfs fs suggested because fnterstitial fluid and cel'l dfspersed f'luÍd leve'ls plateau in lmplanted anlmalsr tvhfle testicular venous and perÍpheral blood levels continue to rfse. It is also possible that two carrÍer systems operater with one transportlng testosterone fnto lnterstitlal fluld, and the other lnvolved ln transport of testosterone lnts the tubu'les. Testosterone admfnistratfon by fmplants or injectlon to rd I normal anlmals reduces gonadotrophin secretion (Buhl et alr 1982; Rea et al' 1986)r iìrìd if one or other of these carrfer mechanf sms t'las regulated by gonadotrophinsr ft mlght exp'lafn some of the differences

noted between the present study and the earlier studies of Setche'll et al (1978). More lnvestfgations are obvlously requfred, but this fs certafnly a fasclnatlng ldea. That testosterone levels ln supernatant flufd from control anfmals are hfgher than testicular venous blood levels ln the present study, may be due to cell damage durfng

df spersion. The contribution of testosterone f rom damaged ce'l'ls would not be such a problem in implanted animals when testicular honnone

productÍon tvas supPressed by the imp'lants.

r 304

CHAPTER 9: INTERSTITIAL FLUID IN RAMS AND BOARS - USE OF THE

PUSH-PULL CANNULA

! 305

9.I. INTRODUCTION

The fnvestigations fn the rat with the push-pull cannula suggested that the technique was more than adequate in provfding access to fluid in the interstitia'l regÍon under physfologfcal condftfons. Gfven that drfp col'lectfon does not work effectively wfth other species (J. Closset, Unfverstly of Liege, Belglum - personal communÍcatlon) it was of lnterest to extend the use of the push-pul1 cannu'la to other specfes. Because of the Ínterest in the lmmune status of the ram testisr this species tYas an obvious chofcer and two animals were avaflable for push-pul1 cannulation. At this tlme' two boars were also avai'lable for study and were included because of the comp'letely different interstÍtlal anatomy of the boar testis (see chapter 1). In additfon to push-pull samples of fnterstitial fluld, blood sampìes were taken f rom a peripheral vef n and testfcu'lar velns' and testicu'lar

lymph samples vJere also coì'lected. In the boarsr testicular venous blood was col'lected from an fnternal spermatfc vefn fn the spermatic cord.

9.2. MATERIALS AND METHODS

Most materÍaìs and methods have been previously descrfbed in chapters 2 or 6. The apparatus requfred for push-pull cannu'lation had to be modffied somewhat to allow for the different sfzes of the anfmals. A large persPex stage was constructed to hold the testes durlng cannu'lation. The stage and mfcromanfpulators were attatched to a large metal frame that was mounted on the operatfng table across the lt¡' rear of the anfmal. This frame was positioned so that the testes cou'ld 306

be p'laced on the stage but breathlng movements of the anlma'ls could not move the stage or micro'manipu'lators. Thf s was important because the breathlng movements of the anfmals during anaesthesfa contlnualìy moved the testes in relatÍon to the cannulae ff the testes rested on the anfmal.

9.2.L. Rams

The night before surgellr each ram was given an lntravenous l2sl-human injectÍon of I00 uCf TOH and 90 uCl serum aìbumfn. Rams were kept overnfght wlthout food or water¡ Ín metabolign crates Ín order to collect any radfoactive ¡vaste. Two hours prior to surgery, 22HuCì another lntravenous fnJectlon of 100 uCi was admlnistered. Rams were anaesthetised wfth sodlum pentobarbitone as described ln 2.3.L. Testes were lso'lated through a scrotal incfsfon' and catheters inserted fnto lymphatlcs fn the spermatlc cord as descrfbed in 2.3.6. Testlcular veins rrere exposed by resecting the head of the epidÍdymis.

Using a 26G needle, an entry hole was made fnto a strafght veln, and a polyethylefìe cannula (0.2 mm I.0.r0.5 mm 0.0.) contalning heparlnfsed saline, was fnserted for a dlstance of 3 - 5 cm. Once a'll catheters were inserted and checked for sampling' push-pull cannulatlon was

performed f n much the same way descrfbed f n 7.3.L.4. for the rat. Greater use of pofnted fonceps to aid entry of the cannula vlas necessary due to the thicker capsule of the testis. Push-pul'l sampling was performed sfmultaneously on both testes in each anlmal for one hour. Blood and lymph samples were collected every 15 mfnutes. Í{hen collectfons were concludedr pieces of testls where cut from the site of lnsertlon of the push-pulì cannula, placed fn Bouins fixative, and 307

processed for hf stologlca'l examlnation. Animal s ïJere then euthenased.

9.2.2. Boars

In the experfments w ith the boars, lt was necessary to anaesthetlse the animals prior to adminfstratlon of fsotopes. The neuroleptic Stresnal (Azaperoner Janssen Pharmaceutica, Beerser Belgluw 2n1/20 kg) was glven by fntramuscular fnJectlon to subdue the anima'l. An ear was then shaved, and warm water used to dilate the vefns. Sodfum pentobarbitone (Nembutal, Abbot, 60mg/ml) was Ínjected lnto a vefn to lnduce anaesthesfa. An ear vein was then surglcally exposed and cannulated wfth a polyvinyl catheter (0.8 mm I.D.,I'2 mm O.D.) usfng a method sfml'lar to that descrlbed for cannulation of the 125l-hu..n lateral saphenf c vein I n 2.3.I. Ioo uçl of serum a'lbumf n 22NaCl and 100 uçl were fnJected through the ear veln cannula, and two hours equillbratlon a'llowed. Testes were then exposed and cannu'lae Ínserted fnto lymphatics and velns ln the spermatic cords and fnto f nterna'l spermatf c velns as described for sheep in 2.3.5. Push-pul1 cannulatlon and blood and lymph samp'ling was performed as descrfbed ln g.2.L Micromanlpulators were unable to be used to position cannu'lae in boars. The tunfca rras so thick and tough that each time the cannulae uere pushed through the entry hole, the plastlcine holdÍng the cannulae onto the mlcroanipulators parted from the mlcromanfpulators. So once an entry hole was made, cannulae were carefully posltioned by hand. Figure 9.1. shows a boar wfth push-pull cannulae ln posltlon. Tlssue samples from the site of lnsertfon of the push-pull cannula were placed ln Bouins fixatlve and processed for histologlcal examlnation. Animals were then euthenased. Figure 9.1. Photograph shovling push-pu'|1 cannulae (arrow) positioned in the testes of an adult boar. 809 309

9.3. RESULTS

Due to the 'lfmlted number of anlmals avaflabler testosterone levels were the only measurements made ln thfs study. The values obtafned for each anfmal are shown in Table 9.t. Values Presented for

push-pul'l samp'les have been corrected for df lution as monitored by 22¡tuO1. Testosterone infusÍons have not been performed in these speciesr and thus fina'l dilutlons can not be accurately determfned.

However values corrected for the ratio of testosterone to sodfum

dilutfons as determfned fn ratsr are Presented for comparison. In both rams and boarsr testosterone levels in fnterstftla'l fluid were slmilar to testlcular venous blood levels. Lymphatlc levels of testosterone were also comparable to testicular venous blood levels fn both specfesr but in the boar, were hfgher than internal spermatfc venous level s. Histology of the ram and boar testis after push-pull cannulation fs presented in Ffgures 9.2. and 9.3. These two species have greater

amounts of fnterstitial connective tfssue than the rat, and tfssue

damage at the point of fnsertlon is more obvfous ln these two species (compare wlth rat histology in Figure 7.8.). Hoyever the damage appears very 'local ised and does not seem to be sufficlent to affect

hormone'levels f n the collected fluÍd.

9.4. DTSCUSSIoN

The findings fn these experfments conffrm Prevlous reports of testosterone levels fn testicular flulds in rams and boars. Lindner (1963) investigated the partltion of androgen between ìymph and venous Tab'le 9.I. Levels of testosterone (ng/ml) ln perlpheral and testicu'lar venous b'loodr testicuìar lymph and testlcular lnterstltlal fluld samples from adult rams and boars. Indfvldual values, and mean values + SEM for each speclesr are presented.

Samp'le Ram No.I. Ram No.2. Ram Means Boar No.l. Boar No.2. Boar Means

Bl ood Perl pheral 2.0 2.2L 2.ll + 0 .lI L.57 2.59 2.08 t 0.51 Testls Vefn 30.1 26.54 28.32 + I .77 35 .06 47 .77 4L.42 I 6.35 Spermatic Vefn 2L.4L 22.60 22.0L + 0.59

Testlcul ar Lymph 31.8 23.88 27.84 I 3.96 26.62 44.71 35.67 + 9.04 * Interstitial F'luld 23.83 17.43 20.63 t 3.19 15.60 16.05 15.83 + 0.22 Corrected f. F.+ 28.60 20.92 24.76 + 3 .84 L8.72 L9.26 18.99 + 0.26

* : values corrected for dflutlon durlng col'lectlon as measured by dflutfon of radloactlve sodlum ch]orlde (see main text for details). * : corrected for the ratlo of testosterone to sodlum ch'lorlde dl'lutlons as determfned ln rats (see maln text for detail s).

(¡ o Figure 9.2. Histological sectlons of the testls of a ram after push pull cannulatfon: (a) thls sectlon shows the norma'l structure of the ram testlsr whlch does not aPpear to have

been affected by perfusfon samPllng with the push-pull cannula; (b) section from slte of lnsertlon of push-pull cannula. illost tubules appear lntactr and damage from cannu'latlon ls very local lsed. X 80' Ir€ Flgure 9.3. Hlstologlcal sectlons of the testls of a boar after push pull cannu'latlon: (a) thls sectlon shows the normal structure of the boar testisr whfch does not appear to have been affected by perfusion sampl fng wlth the push-pull cannu'la; (b) section from site of insertion of push-puìl cannula. Most tubules appear lntactr and damage from cannulation is very locallsed. X 80' 312

'f- i t¡!

.'¡

H ,I

r 313

blood fn the testes of rams and found testosterone levels ln ìymph to (ratio be lorer than the levels fn plagna of spermatic vefn blood 0.61). Setchell et.al (1983) studled levels of sterolds In boar testfs lymph and venous blood, and found levels of testosterone in lymPh to

be scrne twofold hlgher tnan levels in spermatfc velns' Thfs ffnding was seen in one pig fn the present study, but not in the second anfmal. It fs of interest that levels in the lnternal spermatlc velns of both plgs were sfgnlficantly lwer than levels ln testicular venous b'lood taken from a vefn on the surface of the testls' Free and Jaffe (1975) have reported the transfer of testosterone from spermatfc

venous btood lnto testlcular arterÍa'l blood ln the ratr and Jacks and Setchell (1973) have confirmed thÍs fn the ram. Internal spermatlc vefn cannulae are lnserted well up the cordr and would likely sample b'lood from a number of vefnsr after transfer had occured' The results of the present study suggest that venous-arterfal transfer may also ürü I occur ln the boar. Untf'l recently, it was assumed that such transfer occurred by passlve dlffusfon (Setcheì'|, 1978), although Free et al (1976) dfd suggest that blood mfght pass between non-metabo'l ic channels between artery and pìexus. Anatomical evldence has been found for dlrect arterio-venous connections between testlcular artery and pampiniform p'lexus or branches of the epididymal arterles and the

pamplniform plexus, in the sPermatic cord of the ram' bull and boar (Wensf ng and DiJkstra, 1981; l¡lensf ng et al ' 1981; Noorduizen-Stassen'

! rg84).

L The small number of animals avallable for study limfts the general fnterpretations of the data. Likewlse, lt fs necessary to

T I cal ibrate labelled-testosterone transfer into lnterstltlal fluld ln I these specÍes before further studfes are undertaken' Høleverr the r 314

and suggest that levels of testosterone in I results are encouraglng, the interstltial fìuid ln rams and boars are also comParable to the levels ln testtcular venous blood. The results a'lso suggest that the push-pull cannula ls llkety to be of use ln studying the interstltlal fluld of a number of specles, and ls not llmlted to use ln rodents'

¿,

315

AND RAMS CI-IAPTER 10 : IMMUNOCOMPETENT CELLS IN THE TESTES OF RATS 316

IO.I. INTRODUCTION

prlvileged The factors most 1 lkely to contrlbute to the lmmune status of the rodent testls were revlened in.the lntroduction to this thesfs (chapter t), and most interest aPPears to focus on endogenous (RoubinÍan 19773 immunosuppressive agents such as sterofds et a1t l{hitmore et alr 1985; Head and Billingham, 1985). The nork in this - thesis has demonstrated that the ovine testls¡ in contrast to the rodent testfs, ls not an immuno'logically prfvileged sfte' since both (rat animals have effective lymphatfc drafnage from the testis - Tilneyr L97L., McCu]lough, 1975¡ ram - Llndnerl 1963; MorrÍs and in Mclntoshr 197I) this suggests that some lmPortant difference exlsts the lnterstltia'l regfon of the testis fn these two sPecies' Results wlth the push-pull cannula have not found any signiffcant dlfference in testosterone levels between these speciesr and while the data is not completely conclusfver no other reports are known to detall large dffferences ln the sterold profiles of adu'lt male rats and rafns' (chapter Elimination of Leydfg cells ln the rat testÍs wlth EDS 3) effectively removed the lmmuno'logfcal protectlon from the testls' and grafts were rapldly reJected. Howeverr the ram testis also contains Leydig ce]lsr and this suggests that some other factor must be impllcated fn thls result. of Macrophages have been identiffed ln the lnterstitial tlssue the rat testfs (chrlstensen and Gillum, 1969; Clark' 1975i Nfemi et alr1986)ritfìdhavebeenshqntocontainclassllmaJor (Fc) histocompatablllty antfgen (Nl€mí et al, 1986) and lmmunog'lobulln receptors on thelr surface (Mlller et alr 1983). The presence and composition of lymphocyte populations have not been systematlcally 317 investfgated, although sorne rePorts make reference to their Presence in the testfcular interstltfum (Ritchfe et a'|, 1984; Nierni et a'|, 1986). Macrophages act fn genera'l by removlng and digestfng excess antigen, and their role ln antlgen Presentation for the fmmune response has been discussed fn chapter I. However¡ ff antfgen Ínteracts indÍscriminaetly with'lymphocytes in a location unfavourable for cell co-operation, tolerance fs fnduced lnstead of immunity

(Fawcett' 1986)

The study to be reported in this section was undertaken to

examine the presence of macrophages and the various classes of

lymphocytes in the. ram and the rat testÍs, fn order to investigate possible differences ln the Ímmune-ce'l'lular compostitfon of the interstitfal tissue comPartments of these two specfes whÍch mlght account for the different resPonse to tlssue al'lografts.

I0.2. EXPERIMENTAL PROCEDURES

The experiments to be described were initiated durfng a vfsft to the Department of Anatomy, Instftute of BfomediciÎêr UÎiversity of Turku in Flnland. Much of the work was performed by Dr Passi Po1lanen and Professor Nisni after my return to Austral ia. I am most grateful for their efforts on my beha'lfr and for thef r permissÍon to Ínc'lude the work in thfs thesis.

10.2.I. Anlmal s

sprague-Dawley rats and Finnish Land-race sheep were used in these studies. 318

L0.2.2. CytochemistrY

Macrophages were fdentfffed usfng hlstochqnfca'l demonstratlon of non-specfflc acld-phosphatase actlvfty (Cohn and Ílelner, 1963).

I0.2.2.L. Preparatf on of Tlssue

Rat testes and pfeces of testes from slaughtered rams were frozen fn I fquid nftrogen and 5 um sections were cut in a cryostat. Sections oC) were drfed at room temperature and fixed in co1d acetone (-20 for 15 mfnutes.

L0.2.2.2. Acf d-phosphatase detectl on

Acid phosphatase actfvfty was demonstrated with the lead sa'lt method of Gomori (1941)r ârìd the azo-dye coup'llng method of Barka and Anderson (L962).

I0.2.2.2.I. Gomori method

L0.2.2.2.L.L. Incubatlon medfum (pH 5.0)

0.5 M veronal acetate buffer, PH 5.0 I0 ml.

Sodi um B-gl yceroPhosPhate 32 mg.

Lead Nitrate 20 mg.

Lead nitrate must be completely dissolved fn the 319

buffer before addlng the sodfum B-glycerophosphate'

L0.2.2.2.L. 2- Procedure

oC t. Sections were p'laced fn incubating medlum at 3l for 30 minutes to 2 hours. 2. Sections were washed well in dfstilled water.

3. I% ammonium sulphÍde (freshly prePared) was added to sections for 2 minutes. 4. Sectlons rúere washed again in distflled water. 5. CounterstafnÍng was performed Ylth 7" Methyl green

(ch]oroform extracted) for 5 mf nutes. 6. Sectlons were washed fn tap water. 7. Dehydratlon in a'lcohol and clearlng of sectlons in xylene was carried out prÍor to mountlng in DPX'

L0.2.2.2.2. Azo-dye coupl ing method

I0.2.2.2.2.L. Incubation Medi um

oC) Stock Solutfons (stored at 4

Solution A : Pararosanilin

Pararosanlì ln HCI 2gn 2NHCI 50 ml Heat gentl y to bol I i ng poi nt, cool and f i'lter. 320

Soìutlon B : Sodlum Nltrlte

NaNO, 400 mg Di sti]]ed water I0 ml

Make fresh everY four daYs.

Solution C : Veronal Acetate Stock

Sodlum acetate (CH.C00Na.3H20) I.95 gm Barbitone sodÍum J 2.95 gn Distilled water I00 ml

Solution D : Substrate

Napthol ASBI phosPhate (Sfgma) 50 mg DÍmethyl Formamfde 5 ml

Med f um

I. Solution A 0.4 m'l Solution B 0.4 ml Walt one minute

2. Add So'lution C 2.5 m'l Solution D 0.5 mì DistÍlled Water 6.0 ml AdJust pH to 4.7 uslng I N NaOH and fllter'

L0.2.2.2.2.2. Procedure

oC l. Sectfons were placed in Íncubatlng medÍum at 37 for 10 - 60 mfnutes. 2. Sections were then washed well in dfstilled water. 3. Nuc'lel were counterstaf ned Ylth % methyl green for 5 minutes. 4. Sectlons washed brfefly fn tap water. 5. Secti ons were then dehydrated f n a]coho] r cl eared Í n 32t

xylene and mounted Ín DPX.

L0.2.3. ImmunocYtochenf strY

Speclfic monoclonal antfbodles 1¡ere used to examfne lymphocyte 'loca'l popul ations and the I satf on of maJor hf stocompatabi'l f ty compl ex antigens in testes from rats and rams.

10.2.3.I. Preparatlon of tissue

sma'll pf eces of f resh tissue were f lxed by immersÍon in phosphate-buffered 416 p-formaldehyde overnight. Tfssues were kept in an arabicum-sucrose medium until sectioned in a cryostat.

I0.2.3.2. Detectf on of Cell type-specif lc antigens

Indirect fmmunoperoxidase stafning was used to visualise specifíc antigens. Antibodfes used are detafled in Table 10'1'

Procedu re

I. Frozen sections were rehydrated by incubating s'lides ÍnPBSfor3-5mÍnutes. 2. Non-specffic binding sftes were blocked by incubation of sections Ín 5% normal rabbit serum' 3. Prfmary antibody was appl ied to sectionsr which were then fncubated for 60 mfnutes at room temperature' control sections rúere Íncubated wfth norma'l mouse 322

Tabl.e Io.l. The ànt{bodfes used to ldentlfy varlous subtyPls of lymphocytesandtheMHCllantfgen-posìtfvecel.lsinthe rat and the ¡:am testls.

Antigen SpecÍficfty Reference

* 1983 MRC 0x 17 rat lFlC II antlgen Mayrhofer et al, t( Da]lman et al 1984 MRC 0x 19 rat thymocytes ' a sT-l' all ram T-cells Miyasaka et alr 1985a T T-80' ram T-he'l Per/ i nducer 1983 1 ymphocytes Miyasaka et al, + ST-8 ram cytotoxlc lYmPho- cytes Miyasaka et al' 1985a I E-53' ram B cel]s MÍyasaka et al' 1985a r sB-I' ram c'lass II l,tlO antlgen Miyasaka et alr I985b

*: Kindly provfded by Dr. A.F. t',illiams, sir william Dunn Schoo'l of Pathoiogy, University of Oxford' U.K' +: KÍndty irovided by Di. M. Miyasakar School of Medicine, Hamamatsu UnfversitY' Japan. 3B

serum. 4. Sectlons were washed 3 tlmes wlth PBS. 5. Peroxldase-conJugated rabblt antl-mousè

lmmunog'lobul ln (DakoPattsr Copenhô$erìr Denmark)

dfluted 1:50 Ín PBS contalning I% bovine serum a'lbumfnr was fncubated wfth sectfons for 30 mfnutes

at room temperature. 6. SectÍons were again washed 3 times with PBS' 7. Sections were fncubated in 3-3-DÍamlnobenzÍdÍne so]utÍon (see Farr and Nakane' I98I) for 5 mlnutes' 8. After another wash with PBS' sl ides were rinsed fn dÍstilled water. 9. Copper suìphate solution u{as applied to sections and incubated for 5 minutes. 10. Sectfons were again washed wlth PBS and rfnsed fn distilled water.

11. Mayers haemalum t{as used to counterstain sections which were then dehydrated ln alcohol and mounted'

10.2.4. Semithln sections

Isotated testes of adult rats and rams were perfused through the testicular artery, initially with salfne (rats 30 seconds, rams 2 minutes)r ârìd then wÍth sif gluteraldehyde + 3% p-formaldehyde f n 0'2 M Cacodylate buffer, pH 7.2 (rats 15 mlnutesr rams 60 mlnutes)' Pieces of testis of approximately I mm3 were cut and immersed in the same fixatfve overnlght. These were then washed in bufferr postflxed ln 2 % osmlum tetra-oxide, dehydrated Ín alcohol and embedded ln Epon. 324

semithin sectfons were cut at I umr stalned wlth Totuidfne Blue' and examlned by 1 fght mfcroscoPY.

L0.2.5. Paraffln sections

(see pieces of rat and ram testis were flxed 1n 4% p'formaldehyde L0.2.3.I.) or Bouins fixative (see 2.L4.I.). Tissue was then then embedded ln paraffln as dscrlbed in 2.I4.2.r sectloned at 7 um, dehydrated, cleared and stained with toluidlne blue to reveal metachromatic mast cells.

10.3. RESULTS

i n Hi stol oglca'l sectl ons after cytochemf cal staf nf ng are shovln Flgure 10.t.a. Tolufdlne bluæstalned sernfthin sectlons revealed cel'ls with the morphologfcal characterfstlcs of macrophages in rat testes' as seen ln Ffgure lO.I.a.1. These ce]ls were relatively large and round in shape, and contalned round nuc'lef bearing small lndentatlons' The cytoplasm contafned numerous densely stafned granules and vacuoles. The cells were often assoclated wfth Leydfg cel1s. No such cells were found in the toluÍdine b'lue-stalned sernlthin sections of t{ere ram testis. occasfonallyr elongated cells with irregu'lar nucl€i¡ seen in seen f n the rat testls (Ffgure I0.1. a.2.). Sfmflar ce'l'ls were the ram testis, with light coloured nuclei and a thÍn cytop'lasn

contaf nf ng numerous granules. In sections treated to revea'l acid phosphatase actfvityr numerous (Ffgure large, round cells stained positive fn the rat testls IO.t.a.3.). These ce]ls were similar to those observed in the semithin Figure 10.1.a Histologfcal sectlons from the testes of rats and rams after cytochemical stafnfng for lmmunocompetant cel'ls. (I) Toluidine blue staÍnfng ln a semithin pìastic sectfon of a rat testis. Note the round macrophage (arrovr) in the border of the lymph sÍnusoid and the fnterstitial cel'l cluster, and its close assocÍation with the adJacent Leydlg cell. X 1300. (2)ToluidÍneb]uestalnfnglnasemfthinplastic section of a rat testis. Note the elongated granuìe-filled macrophage (arrow). X 1500.

(3) Acid PhosPhatase activftY in a para-formaìdehyde-flxed frozen section of rat testfs. Note the elongated positive ce1'l (arrow)' X

150.

Acfd activitY in a 10, PhosPhatase para-formaldehyde-fixed frozen sectlon of ram testis. Only elongated posltive cells are present in the ram testis (arrovl). X I50. 325

. .¡ çrF

* ¡ t

t a. - F . i:.,.-;

,l:

.t", , .'. .} ",. -'# èr,. '"'þ"'* 'i!i; 'i

ì 326

sectlons and described above. As in the case of the semithln sectfons in the ram, no such cell were found fn the sections of ram testis stalned for acld phosphatase actfvlty. Indeed, only a few phosphatase posltive cells were seen fn.the ram.testis, and these were elongated in shape and contalned gnall granuìes (Figure 10.I.a.4.). Simllar cells were also seen fn the rat testis (Figure 10.1.a.3.) In the ratr the round acfd-Phosphatase cel'ls were scattered throughout the fnterstltium of the testis. The e'longated cells seen fn both ram and rat testes, appeared only in the Ínterstitial tlssue in 'l both specf es, al though occasi onal stai ned cel s were f ound I n c'l ose associatlon with the tamina ProPrfa of the sqniniferous tubules' Stalned cells were never observed wlthin the tubules in either sPecies.

Macrophages of the rat testÍs comprised some 25 % of all fnterstitial ce]ls, and formed the maJorlty of phosphatase-positlve cells. In both rat and tamr the e'longated ce'l'ls comPrf sed some L-2 % of the total lnterstitial cel'l popu'lation.

Figure lo.I.b. sho'rs histo'logica] sections after indf rect

immunoperoxidase staining. T-lymphocytes (ram : ST-l+. rat : MRC 0x 19+) were only occasfona'lly observed ln testes of both the rat and the

ram (Flgures IO.I.b.5.r 10.1.b.6.). They were usually found in close assocfation wfth blood vesse'ls, and were never seen fn the tubules' Very few T-helPer/inducer (T8O+) or cytotoxfc (ST-8+)lymphocytes were seen ln the ram testis. B-'lymphocytes (E-53+) were not found ln the

ram testls. No study was made of these cells in rat testfs. MaJor hÍstocompatabÍl ity antfgen in rat testis (MRC 0x 17) was found on endothel ial cells and macrophages (Flgure lO.1.b'7')' The l*lC Ffgure 10.I.b Histologfcal sectlons from the testes of rats and rams after lndfrect immunoperoxidase stainfng for

immunocomPetant cel I s- (5)ST-IpositiveT-.|ymphocytesfnanacetone-fixed frozen section of rat testfsr courìterstained wlth Mayerts haemalum. X 500. (6)ST-IposltiveT-lymphocytesinanacetone-ffxed frozen sectlon of ram testisr couñtorstaÍned with

Mayerf s haqna'lum. X 200.

(7) MRC 0x-17 posftlve class II MIC antlgen-posftive cells fn an acetone-fixed frozen section of rat testis, counterstained with Mayerrs haemalum' X

150.

(8) SB-1 positive cells fn an acetone-fixed frozen sectÍon of ram testis. X I50. ,. , 328

antigen ln the ram testfs (SB-I) was locallsed on the endothellal cells and occaslonal lnterstitlal cells (Flgure I0.I.b.8.). No antfgen was found f n the.seminfferous epithe'lfum of the ratr nor ln the gérmlna'l ePf thel lum of the ram. Ín No mast cells were identified on the basÍs of metachromasla' toluídlne blue-stalned paraffin sectfons of rat or ram testis' Considerabìe numbers of mast cells have been demonstrated in the rat testÍcular capsule in the vfcinlty of the testicular artery by other investlgators (see sowerbutts et alr 1986) and other more specific methods of identiffcation may be requlred'

Tab'le L0.2. summarlses the Presence of various lmmune cel'ls found ln the course of the Present studY.

10.4. DISCUSSIoN

The present flndings demonstrate dlfferences ln the magnftude and compositlon of the testfcular macrophage popu'lation between the ram and the rat. Previous reports have ldentified macrophages Ín the rat testfs by varlous means (chrfstensenr L975¡ Bergh' 1985; Niemi et a'|, 1986)r âfìd found that they comprlsed some 25% of all lnterstftial ce11s. The flgure obtafned in the present study usfng cytochemícal demonstration of acfd phophatase activfty, has confirmed these earlier reports. Howeverr ln contrast to these ear'ller Fepo¡tsr two morphologlcally different types of cells whÍch exhibit acÍd

phosphatase activity have been found ln the rat testfs . In addition to the large, Foufìdr phosphatase posltive macrophagesr a sma'll

proportlon 1.1-2 %) of the interstitial cells were s¡nall and elongated, found but exhÍblted acfd Phosphatase activlty. Simflar cells were also in the ram testfs, although no large round phosphatase posltfve

macrophages were seen ln ram testis. These srnallr elongated ce'lls 329

Table !0.2. The lmmune cells demonstrated ln the rat and the ram testf s. NI i,:

Ram Testls Ceìl Type Rat Testls

Macrophage + I arge and round srnal'l and elongated + +

Lymphocytes T cells +- + B cells ND Mast cells I

ND = not determlned

l I ¡ i

t, I t,

L t' I

I 330

resemble the tlssue histlocytes described by Stieve (1930) and may represent the stem ce]ls of testicu'lar macrophages and Leydlg cells (Clegg and MacMillan, I965a, b), Whlle Paff et al 1947) reported mast cells to a'lso exhÍbÍt phosphatase activftyr no cells contafnfng metachromatic granules were observed in the sectlons of the present

study. The observation that macrophages present in the rat testis are not found in the ram testis demonstrates a signfficant dlfference fn the composÍtion of fmmunocompetent cells fn the testes of these two species. It fs of great interest to speculate on the role of I testicular macrophages fn providfng the fmmunologfcally prfvileged envÍronment of the rat testis.Oehler et al Q977) have reported the rat spleen to contain a sub-populatlon of macrophages whÍch suppressed lymphocyte pro'llferatlon ln mixed tymphocyte cultures. Sfmllarlyr ,I macroPhages to il Fernbach et a'l (1976) reported perftoneal-exudate rI I fnhiblt proliferation ln mixed lymphocyte cultures ln mlce' Macrophage productlon of prostag'landin E, has also been shovn to lnhibit lymphoprolfferatfon (Bray, I98O). Nelson (1976) has revfewed non-specffic lmmunoregulation by macrophages and thelr Products' and cites a number of cases where the inhÍbitory ro]e of macrophages has

been demonstrated. Hersey and MacLennan (1973) reported Protectlon of certain tumour celts from lymphocyte toxicity by macrophages' ltlhere target cells are kÍlIed solely or primarily by cytolytlc effector T cells (as ln the case of many tissue allografts) the presence of excess macrophages and/or activated macrophages can lnhÍbit cyto'lysis

(Klessl f ng et a'|, L974¡ Vasudevan et al t L974) '

I In I982r Born and wekerle reported that Leydlg cells i non-specf f ically suppressed mÍtogen- or allogeneic ce]l-induced r 33r

lymphoproliferatfon. They suggested that the Leydfg cells, whlch have also been shorrn to adhere non-specfffcally to lymphocytes and

leukaemlc cells (Rlvenson'et al, L9743 Born and Wekerle, 1982)r ¡{ould provide an fmmunologica'l'ly weak zone ln the testicular lnterstitlum - a regfon fn whfch leukaernfc cet'ls often ffrst reappear after

chernotherapy (Plnkel, L97l). Holever, these authors characterised their Leydfg cell preparations with criterfa part'ly attributable to

macrophages (Molenaar et alr 1984)r ôrìd found L0'20 % non-Leydfg cells. Testfcular macrophages are often found ln c'lose associatfon wÍth Leydfg cells and Míller et al (1983) reported portions of Leydig ce1'l cytoplasm to be endocytosed by macrophages. The lmmunosuppressive activlty of spleen derf ved macrophages in norma'l (Oehter et al' 1977', and leukaemic (Glaser et al t L975) rats tempts one to suggest that the

fmmunosuppressfve activlty reported for the above Leydig cell

preaparatlons may have been caused by contaminatfng macrophages. dr¡t uf

The lnterstltfal macrophages of the rat testls have been shoYn to

express c'lass-Il MHC antigen. The Presence of SB-1 antlgen fn the ram

fs well correlated wlth the Presence of l'tlC-II antigen (Dr Miyasakar personal communfcation to Dr F. Pol'lanen) and cells bearing the SB-1 antigen are occasslonally seen ln the ram testls. The data obtalned fn the present studyr suggests that the e'longated cells expressing the acid phosphatase actlvity fn ram testfs are possib'ly macrophages' and mlght be capab'le of antfgen exPressfon to T-lnducer'lymphocytes' It is unllke'ly howeverr that they would act ln the manner ProPosed for the large, round macrophages of the rat. The absence of l¡tlO antigen ln the T I germfnal epfthellum of the two species would afd in protecting the I immunologlcal'ly forelgn germ cells from immune surveillance' sfnce r 332

lnltlation of an lmmune reactlon would be fmpafred. Pollanen and Nlsnf (1987) have recently reported that the human germlnal eplthellum ls devold of I'tlC-trI antfgen. T-lymphocytes have been found in the testis of both the ram and the rat, although they are few ln number. Since these are the lymphocytes of consequence ln allograft reJectlon, it is unllke'ly that

difference ln lmmune status of the testfs fn the ram and rat fs due to dffferences ln lymphocyte populations. Indeed, Head et al (1983c) have reported long-established lntratesticular alìografts in rats to be promptly reJected follovring specfficr acute immunisation of the host' which would support thls vfew.

The macrophage appears to be the one cel'l which shqs maJor dlfferences in the fnterstitfat tissue of the rat and ram' and evidence has been presented which suggests that it may welì be { l involved ln 'localfsed fmmunosuPPressÍon. The physfologfcal and immunological lmportance of Leydig ce'|1-macrophage fnteraction rqnaîns to be determined, and further studies fn this area are certalnly warranted.

r Ì

;

333

CHAPTER 11 : CONCLUSIONS 334

The studles reported f n thf s thesf s were f nf tf ated to ræexamf ne the fmmunologically prlviìeged status of the testls. Thelr Pursuft has fnvolved not only a study of the lmmúnològy of the endôcrfne testls, but a reassessment of aspects of the physfology of this reglonr particularly wfth regard to androgen secretlon'

The testls of the rat has been conflrmed as an lmmuno'loglcally prfvileged sltee ârìd thyrold and pituitary allografts have been found to survlve for the three weeks of most studlesr and longer (> 6 months fn one instance). It does aPPear that vascularlsation of tlssue grafts in the testis fs not as rapld as seen fn other reglons of the bodyr and thls may be of lmportance ln the fmmune prfvilege afforded thls sÍte. It may also explafn the apparent lack of hormone secretion by tfssue grafts examfned 3 weeks after transplantation¡ when other

f ndÍcators of graft functlon shwed tissue vlabll ity' The dlsruptlon of sPermatogenesis by a number of experlmenta'l treatments dld not alter graft survival. That thyrofd aìlografts survfved ln abdomfnatly-Placed testes suggests that the lower temperature of the testis ls not an important component of its lrrnune privi'leged envfronment. During the present studyr Head and Blllingham (1985) a'lso reported the survfval of parathyroid allografts in abdomlnally-placed testes. These authors Provlded further evidence that temperature fs not critlcalr when they demonstrated prornPt reJectlon of parathyrold allografts placed ln subcutaneous pockets in the ear - a sfte that ls also of lower ambient temperature' Whfle efferent duct 'l fgatfon created sfgnlffcant damage to the sqnfnlferous tubular eplthelium, allograft survival was not affected. Sertoli cells are not morphologlcally a'ltered wlth such treatmentr although there ls 335

(see evidence that the secretory functions of these cells are affected chapter 3). While the fnvo'tvement of tubular elements could not be totalìy discounted, the results did suggest that factors responsible for lmmunosuPpression were.probably located outsfde the blood-testis barriêrr withln the lnterstitial tÍssue. Evidence that cellu'lar associations/fnteractions in the interstitial reglon were probably important came from the findings that hCGrinduced lncreases ln vascular permeabilfty and lymph flon had no effect on al'lograft su rv Íval .

A number of authors have cited the Leydig cells as possibly being responsible for fmmune regulation in the testfs. Their role in sterofd secretion and thefr apparent abitity to adhere to lymphocytes are two attributes that would be useful in thfs regard. In the studÍes of thfs thesfsr EDS was used speclffca'lìy to destroy the Leydlg ce]ls. Thls led to reJectlon of thyrold allografts in the rat testlsr conffrming the involvement of these cells ln maintalnfng an lmmune protection in thls reglon. Hovlever the effects of Leydig cell destructlon on the functions of other cell types fn the testis were not assessedr and macrophages in partfcular have been shown to be closely assoclated wfth Leydfg celìs fn the testis, and mlght welt be altered by such treatments. It ls dlsappofnting that the use of Íntratesticular

'l w so i nJ ecti ons of sf lca specf f f cal I y to destroy the macrophages as unsuccessfu'|. Thls technlque appears to be of llmlted applfcation fn the testls since fts effects are very localfsedr and other methods of

macrophage destruction are needed.

A study of graft survlval ln the testis of the ram indlcated that

the fmmune status of the rat testis fs not necessarfly a general 336

marmalian characterfstlc. Ílhfte thyrofd autografts survlved ln the ram testisr and concentrated lodlne 3 weeks after transplantatlonr thyrofd and pltuitary allografts were reJected wfthfn this tfme. These results led to an examfnatlon of the fnterstitlal region of the testis ln rats and rams to determine what differences exfstedr if anyr that might exptafn the superfor lmmune status of the ram testis.

The lnvestlgatfons of fnterstitial fluld requfred inltfal studles on the physiology of the endocrfne testis. Avallable technlques for samplfng interstltial ftuld were found to be unsatisfactory, and somewhat unphysf ologf ca]r ôrìd the Push-puìl cannu'la used by neurophysiologfsts was adapted to investlgate the lnterstltlal f'luid of the testis in ratsr with a s¡nall trfal lnvolvlng rams and boars' A number of experÍments were performed with the cannular whfch resulted fn the development of a new hypothesis on testosterone secretion ln the testls. The studies suggested that Leydlg cells secrete testosterone prlmarl'ly fnto b'loodr and that interstftial fluid levels arfse more as a consequence of blood levels. The first part of thls proposal ls in itself, not entlre'ly nevl. In the early studies by Fawcett et al (1973), these authors stated that fn the animals with lnterstltial organisatlon I lke that of the ratr the movement of testosterone from the Leydlg ce'lls could be envisaged as a release of androgens into btood caplltarfes and Ínto the protefn-rlch extracellular flufds that move from the blood vascular system fnto the lymphatlc slnusolds whfch surround the sernlnfferous tubules. They suggested that the lymphatic route of testosterone transport is the primary source of androgens for the tubules ln these animals. In anfmals wlth Ínterstitial arrangements lfke the ram and man' blood 337

capillarÍes are fn much closer contact with the tubules and may supply testosterone directly to the tubules. In these specfes, lymphatlcs would be more fnvolved in the return of extracellular flufds to the general circulatfon than fn dfstrlbutfon of testosterone. The large mass of Leydfg cells fn the boar was presumed to be able to supply androgen dÍrectly to the tubules wlthout the need for an indlrect transport system such as the tymphatics or blood vascu'lar system (see also Connell and Connell, L977). Thus the potentfal fmportance of blood transport vras recognÍsed in the early anatomical studies, However it appears that the physfologfcal importance of vascu'lar transport of testosterone was'lost in the reports of high testosterone levels fn drip co'llected interstftial ftuÍd (Hagenas et al t 19783 Sharpe et al, 1983; Turner et aI 1984). These reports aPpear to have

been suffÍclent cause to neglect the potentlal problem of accountfng for hlgh lnterstitfat levels of testosterone which can not be measured in testlcuìar lymph or venous blood. The studles in the rat have a'lso led to the hypothesis that signfffcant leve'ls of testosterone are required for spermatogenesfs, and that these levels are provlded for ln interstitfal fluld (Stevens and Steinbergerr 1983). Howeverr others report evidence to the contraryr suggesting that interstftial levels of L045'¿ of frnormalrr can quantitatlve]y maintain spermatogenesis (Cunnfngham and Hucklns, L979¡ Rea et alr 1986). Comhalre and

Vermeulen (1976) make reference to studies reportlng testosterone concentrations ln interstitfal tissue which exceeded the leve'ls they found ln rrcell-freerr fnterstltÍal fluid collected by mlcropuncture. They concluded that Leydfg ce'lls must contain consfderable pools of testosterone whích would contribute to levels measured in tfssue. It is more than I ikely that these pools also contribute to the high 338

leve'ls of testosterone measured fn drlp collected ftuid. Thls study has hopefully provided sufffcfent fncentlve to ensure a reevaluatlon of the contributlon made by the vascular network fn the testfs for the dlstrfbution of testosterone. The second part of the hypothesfs developed ln this thesls proposes that fnterstitial fluld levels of testosterone arise more as a consequence of blood levels. The studies wÍth the push-pu'll cannula demonstrated that fnterstitial fluld levels of testosterone are no greater than testicular venous blood levels' and may in fact be

somewhat less; and are also more comparable to those levels found ln testicular lymph. Results from studles uslng such treatments as heat'

hCG fnJectionsr ônd testosterone fmplants to alter testlcular function, have also suggested that blood flow and capillary permeabiìity may be fmportant factors in determfnlng lnterstitia'l fluld 'levels of testosterone. These factors are also regulated by ce1ls ln the sernfnlferous tubules and change fn relatlon to various stages of spermatogenesfs (Setchell and Galll, 1983¡ Bergh and Damber' 1984; Bergh 1985). The present studies, wlth support from the

I iterature, suggest that testosterone requi rernents for spermatogenesf s are not met by excessfvely high levels of the hormone fn interstftfal fluid as previous'ly assumed (see above). The semfnlferous tubu'les undoubtably requfre greater 'levels of testosterone than are achÍeved e'lsewhere fn the body, and levels of testosterone ln testlcular fnterstltial f'lufd are certaÍnly greater than the extracellular fluld levels anywhere else ln the body. However, ft seerns that these levels

are much lower than prevlously thought. Requirernents of testosterone for continued spermatogenesfs seem to be controlled by lntrlcate regulatfon of b'lood flow and capfllary permeability' and close 339

paracrine communlcatfon between Leydlg cells and the serninfferous tubul es. The level of testosterone ln the fnterstitlal fluid of rams and boars was found to be slmilar to that fn the ratr and no evfdence could be found in the literature to suggest that the steroid proffle of rams and rats, or the leve'ls of steroids invo'lved would dlffer sufffciently to account for the dlfferent immune status of the testls in these two specles. Further studies are obvfously requlred before the 'lack of sterofd lnvolve¡nent can be generalìy concluded. Howeverr from the avallable lnformatfon¡ the present studÍes do suggest that the importance of the Leydig celt for the immune prlvflge of the rat testfs ls not necessarlly because of its steroid secretlon. The interest ln immunosuppressfon by steroid hormones has large'ly arlsen as a hypothesls to account for survlval of the fetus ln the womb - lt ls after all an allograft. Howeverr the subject fs some,what controverslal. Accordlng to the in vitro studfes of Pavfa et al (1979)r progesterone and oestradiol were effectlve inhÍbitors of cel'l-mediated lympholysis in mÍce at concentratlons of 5 -I0 ug/m'|. Cortisol was also effective at these concentratlons, but testosterone was not at all fmmunosuppressfve. Part of the controversey of the role of sex sterofds ln fmmunosuppression during Pregnancy revo'lves around physio'loglcal versus pharmacologlcal concentrations. For example, the levels of progesterone required for fmmunosuppression fn mice cells fn the study by Pavla et al (1979) are unphysiologfcalty hlgh. Even in

the pregnant mouse when progesterone levels should be at thelr greatestr cfrculating levels are only 6 - 70 ng/ml (Parkening et al' 1978). It is certain that reproductive sterolds do have cytotoxfc effects on lymphocytes at dose ranges of 2 - 50 uglml (Kltzmfller and 340

Rock'lin, 1980). HoYever, lt ls also apparent that sterolds alone cannot account for the fmmunosuppressfve effect of pregnancy serum

(Schlff et al, 1975). If thfs ls a Problem ln Pregnancy-related fmmunosuppresslon, then it fs an even greater problem for immunosuppression in the testfs. The requfred leveìs of these hormones are un'likely to be found in the interstftial tissue. CertaÍnlyr the measurements made wlth the push-pull cannula need to be extended to cover other steroids. Howeverr if the testosterone results are fn any way related to the levels of other sterolds, the push-pull data would suggest that there are going to be even lower'leve'ls than prevlously antlcipated. In summaryr the data obtalned from the present studyr ln conJunctÍon wfth numerous lfterature reportsr côsts severe doubt on physÍologlcal role of sterofds as modulators of host responsfveness to tissue allografts fn the testls. Furthermorer steroid lmmunosuppression tends to prevent lymphocyte prol fferationr ônd as discussed by Kltzmlller and Rock'lin (1980)r these effects fn vlvo wou'ld not fnhibit the effector part of the immune response. Inhlbitlon of this arm of the response Ís important for graft survfva'l ln the testlsr sfnce Head and Blltfngham (1983) have demonstrated reJection of vlable intratestfcular allografts followfng extra-testicular sensitfsatlon of the host against the graft donor - an efferent response. Thereforer other mechanisms are required to exP1afn the success of tissue allografts fn the testls, and for that matter the success of the fetus ln the womb of a Presensltised host; and these mechanlsms should fnhÍblt an effectorr not a Prolfferatlve, resPonse-

The reports of macrophages ln the lnterstltlal reglon of the testis, and thef r close involvement with Leydig ce'l'ls prompted an 34r

lnvestigatlon of the fmmunocompetant cel'ls of the rat and ram testls. This investfgation reveated that rams do not posses the large, round macrophages found fn the rat testis. A number of llterature reports have provlded evfdence for an lmmunosuppresslve role of tissue-speciffc macrophages. As discussed fn chapter I' initlatlon of an effector resPonse by the immune system is dependent on macrophages processing of antigen presented ln the afferent immune resPonse.

Macrophages in the testis may Process antlgen locally wfthout subsequent presentatÍon to effector cells, and thereby significantly reduce the strength of the afferent response. fndeed, under such cfrcumstances tolerance may be fnduced' rather than reJectÍon.

Howeverr the effectiveness of extratesticular sensitlsatlon in initiating graft reJectÍon suggests that a state of tolerance may not necessarlly exlst. Cytotoxfc T-ce'll medfated cytolysis of target tissues can also be prevented by the presence of large numbers of macrophages, as dfscussed ln chapter 11. ThÍs cytotoxlc T-cell response ls also a comPonent of the efferent response of the immune system - the response that needs to be control'led accordlng to Kltzmlt'ler and Rocklin (1980). It ls the concìusion of thls thesfs' that the macrophages of the rat testisr probably Ín conjunctfon wfth the Leydig ce'lls, are of key lmportance fn provfding an Ímmunologlcally prlvileged envlronment ln the rat testls. The

mechanisms fnvolved fn thfs fmmunoregulatfon and the significance of Leydig ce]l involvement have yet to be determined. Horever the intlmate assoclatlon of Leydlg cells with macrophages' may affect

macrophage functlon and explafn why allografts were rejected after Leydig cell destruction with EDS. That the ram does not appear to contafn testÍcular macrophages of the type found predomlnantly in the 342 rat testf sr and reJects tf ssue allografts prmpts further lnvestlgatlons fn other specles. Horyever, lt may mean that the potentlal use of tlssue allografts fn agr,fculturally lmportant specles to stlmulate grorth and modlfy aspects of endocrlne functfon ls llmfted. The recent report of macrophages fn the human testls whfch are slmfJar to those fn the rat testis (Pollanen and Nlemlr 1987) means that the use of the human testls as an fmmunologfcaì'ly prfvfleged sftee F€trâlns an exclting prospect.

343

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