p. 585 to 589. Pergamon Press. Printed in ( NESIS IN OVARIAN TI~ A TELEOST, THE GUPPY ;4 RETICULATA G. D. LAMBERT AND M. G. E. POT 'or Comparative Endocrinology, State Un echt, Utrecht, The Netherlands (Received 8 April 1974) Almtract--1. Homogenates of ovaries of 3- and 12-month-old Iguppies we 6th pregueno- lone-7a-SH and progesterone-4-a4C, and with androstenedione.mdrostenedione- 1,2-all, re~, 2. From the double-labeled experiment, 17~-hydroxypre17 ~-hydroxypregnenolone, ~progesterone, dehydroepiandrosterone, androstenedione and testosterone vwere isolate ied by paper- and thin-layer chromatography, derivative formation and b2by recrystall astant specific activity. 3. In the ovary of the guppy the delta-5 pathway in ste~steroidogenesi .~s, as may be concluded fr6m the 8H/a4C ratios of the identified metabolitesmetabolites. 4. From the androstenedione-l,2-3H incubation, ovaries of 12-month- ppeared to be able to synthesize steroids with functional groups in the 11-1position (1 •one and 11 fi- hydroxytestosterone). 5. The androgen synthesis in ovaries of older guppies may Ibe consider le explanation of the sex conversion occasionally observed. INTRODUCTION Anoth~~ther problem in physiology of tlthe fish ovary tpacity for synthesizing ll-keto-11-ket and llfl- IN PREVIOUS~VlOUS studies (lambert et al., 1971), it was is its caI hydroxys~steroids. 11-Ketotestosterone has a strong shownL that the ovary of the viviparous teleost androgen,genie capacity (Idler et aL, 1961), and therefore Poeeiliaia reticulata contains aromatizing enzymes, might be considered as a male sex honhormone in fish. are able to catalyse the conversions of which , it has also been isolated ffrom ovarian stenedione* and testosterone to oestradiol. However androstenedio tissue (E3(Eylath & Eckstein, 1969); incubincubation experi- ver, direct evidence for complete steroid However ments pr,~roved ovaries to be capable of synthesizing ,'sis in the ovary of this teleost was not obtained. synthesis in the ovar 11-ketotestosterone (Eckstein, 1970; Eckstein & An incubationacubation experiment with the radioactive ~:,,lo*h 1 nd a4C-progesterone Eylath, 1970; Eckstein & Katz, 197]1971; Reinboth, precursors SH-preguenolone and hydroxytestosterone was considered necessary. Suchch a double-labeled 1972), and in some cases llfl-hydrox 1972). The experiment could also be used~ed to discriminate (Eckstein and Katz, 1971; Reinboth, could not be between a delta-4 and a delta-55 pathway in steroid synthesis of the mentioned steroids c demonstrated in ovaries of young guppguppies (Lambert synthesis. et aL, 1971). It is possible that in oldelolder animals the * The following trivial names aree used throughout this 11-hydroxylation is functioning. For tthis reason it paper: progesterone, pregu-4-ene-3,20-diov.3,20-dione;~ione preguenc~ has been attempted to demonstrate tlthe formation lone, 3fl-hydroxypregn-5-ene-20-oneone; 17a-hydroxypro- of such steroids in ovaries of virgin g~guppies which gesterone, 17 o~-hydroxypregn-4-ene-3,20-~me-3,20-dione;20-dione; 17~x- are at least 12 months old. hydroxypregnenolone, 3fl,17~x-dihydroxyprydroxypregu-5-ene-20-~ypreg one; dehydroepiandrosterone, 3fl-hydroxyhydroxyandrost-5-ene- 17-one; androstenedione, androst-4-e~trost-4-ene-3,17-dione;4-ene-3 MATERIALS AND METHOE[ETHODS testosterone, 17fl-hydroxyandrost-4-ene-3~t-4-ene-3-one;ae-3-one: adre,- Animals nosterone, androst-4-ene-3,11,17-trione; 11 fl-hydro- xyandrostenedione, 11 fl-hydroxyoxyandrost-4-ene-3,17- Virgin female guppies (Poecilia reticular,reticulata) were reared dione; ll-ketotestosterone, 17fl-hydroxyhydroxyandrost-4-ene- under laboratory conditions at 24°C and artificial light 3,11-dione; 11 fl-hydroxytestosteroneme, llfl,17fl-dihydro- during 12 consecutive hours per day. xyandrost-4-ene-3-one; eortisol, 11 fl,17~,21-trihydro- xypregu-4-ene-3,20-dione; cortisone, 17~,21-dihydro- Radioactive steroids xypregu-4-ene-3,11,20-trione; oestradiol,:radiol, 3,17fl-dihydro- xyoestra-l,3,5(10)-triene; oestrone, 3-hydroxyoestra- The following radioactive steroids, all pmrchased from 1,3,5(10)-triene-17-one. New England Nuclear Corp., Boston, Mas., U.S.A., 585 J. G. D. LAMBERTAND M. G. E. POT alayer chromato- Saponification of Lates was carried out : 17 Ci/mMol), with sodium hydroxi to Bush (1961). Ci/mMol), pro- ~1ol), oestradiol- Measurement of radZ strone-4-14C (sp. = (sp. act.: 55 Samples were essa'. aclear Chicago Mark I 55"1 mCi/mMol), scintillation counter tillator of PPO (4 g) and adreno- and POPOP (40 mg) 1.). Radioactive areas rtisol-4-14C and on TLC plates and I e located by means of on with sodium a Berthold thin layer tiogram scanner. 4-14C and ll- ared from 11 fl- Incubation procedure en°ster°ne-4-~4C The ovaries were decapitated guppies, ively by reduction with sodium borohydride, pooled, weighedw and t at 0°C in a medium (1 ml/1001/100 mg tissue) per litre aqua dest.: ts NaC1 (11(11.69 g), K( aCl~ (0.44g), MgCl~ (0.41 g), NaHCOa q 2Os (0.14g), glucose :hemicals used were of analytical grade; organic (0"10 g), nicotinamidn fumaric acid (0.01 g). s were redistilled once just before use. Cofactors AppropriateAppropri~ quantit i precursors and co- a the incubation mixture were obtained from factors (/ATP, NAE ADH and NADPH; ager, Mannheim, Germany. 2/zMol of each) wer~ ~-5 ml incubation vials, to which tthe homoge ted. Incubations were ~tography carried otout at 25°C losphere or in a 95% O2 and 55% CO~ ga continuous shaking. •layer chromatography (TLC) was carried out on After 1 hr,hi addition; 12 ~Mol) of cofactors :ed plates (10x20cm) with silica gel, F254 were adde~added and after r the enzyme reactions , A.G.) or cellulose MN 300, F 254 (Antec, A.G.) were terminatedterrr by of dichloromethane :ated tanks using the following systems: system 1 : (10 ml). omethane-methanol (97 : 3); system 2: benzene- ,' (8:2); system 3: benzene-methanol (9: 1); Extraction and fractionation system 4: benzene--cyclohexane (1 : 1); system 5: benzene--ethylacetate (7 : 3); system 6: hexane- Labeled and unlabeled carriers were added to the ethylacetateetate (1 • 1); system 7: cyclohexane-ethylacetate incubation mixture before extraction with dichloro- (1 : 3); system 8: ethylacetate-cyclohexane-ethanol (45 : methane (3 × 10 ml). The combined dichloromethanedic 45 : 10);~; system 9: toluene-acetone (7:3); system 10: extracts were evaporated in vacuo and the residue petroleumam ether (40--60°C)-tert. butanol (1 : 1); system dissolved iin aqueous methanol (70%; 8 mll and stored at 11 : petroleumtroleum ether (40--60°C)-tert. butanol (3 : 1); - 18°C ovovernight to precipitate triglyceridq'cerides. Following system 12: toluene saturated with propylene glycol, centrifugal~tion the supernatant (the steroid fraction) was Only cellulose plates were developed in system 12. evaporatec~orated to dryness, and then fractionatedfractio into a The spot3tted cellulose plates were dipped up to the origin phenoliclic aand a neutral fraction (see LambertLambel et al., 1971). in a mixtureixture of methanol-propylene glycol (5 : 1), and allowed to dry for 10 rain before development. After RESULTS chromatography, the 3-keto-A4-steroidsroids were located by u.v. absorption, whereas the other steroids or derivates I. Incubation with pregnenolone-7~-at~e-7~-SH and Pro- were detected by spraying with primuline-imuline according to gesterone-4-14C as precursors Wright (1971). (190 mg) of eight, Paper chromatography (PC) wasvas carried out on The homogenate of the ovaries (190 J Machery and Nagel M.N. 263ff. paper (Diiren, 4-month-old virgin guppies was incubaincubated with 3H- Germany) in the system petroleumam ether (40-60°C) - pregnenolone (4-99/zCi; 10.1/zg) and 1414-C-progeste- methanol-water (5 : 4 : I). Steroidsas were detected by rone (1.15/zCi; 6"3/zg) in the presence of cofactors absorption of u.v. light. in an atmosphere of 95700 O2 and 5% C(CO~. After the Gas-liquid chromatography (GLC)~) was applied in the incubation the following steroids (100 /zg of each) quantification of the steroids durin g the recrystallization were added as carriers: pregnenolone, progesterone,r procedure, by using a Hewlett-Packardlckard 402 gas chro- 17c~-hydroxypregnenolone, 17o~-hydrydroxyprogeste- matograph with a flame ionizationa detector and a 4 ft rone, dehydroepiandrosterone, androstenedione, 3% SE 30 column. testosterone, oestradiol and oestrone.estrone. Following the extraction and removal of triglglycerides the Microchemical reactions steroid extract was fractionated into a phenolic Oxidation was brought about withth chromium trioxide fraction and a neutral fraction. according to Eckstein (1970). Methylation was carried out withth dimethyl sulphate Phenolic fraction as described by Brown et al. (1957). Formylation was performed by¢ dissolving the dry Following TLC in system 3 the oeoestradiol and steroid in formic acid 98% (0'5 ml).p. After 2 hr at room oestrone fractions were methylated. After TLC in temperature the formylation is completed. system 8 it appeared that the SH as wewell as the 14C genesis in ovarian tissue of Poecilia reticn :ed carriers of identical to andros1 d dehydroepiandro- low to permit sterone formate res oreover, a constant specific activity wa~ er repeated crystal- lizations (Table 1). Androstenedione. em 1 of the andro- stenedione fraction R least three radio- 1 fraction was active areas, one of, ~onds with authentic only the areas androstenedione. ' enedione area was ~rs were eluted eluted and the acti' 1 appeared to resist 7a-hydroxypro- oxidation and forn rally, the substance aydroepiandro- showed a constant ivity after repeated :; (3) androstenedione; (4) pregnenolone; crystallizadlizations (T~ le purified andro- ogesterone. TLC in system 2 of the first stenedion,stenedione was ma labeled; the 8H/14C n affected a separation in a combined 17~- ratio is 33. ~ypregnenolone and testosterone fraction and Testost~tosterone. Fo: ! the fraction con- 7~-hydroxyprogesterone fraction• After TLC taining ththe carrier l delded a compound em 1, it was possible to separate 17cx-hydro- with the same mo thentic testosterone menolone from testosterone.