GENERAL

AND

COMPARATIVE

Steroid

ENDOCRINOLOGY

371-385 (1976)

Biosynthesis by the Ovary of the Eur Anguilla anguilla L., at the Silver Stage’ EORENZO

Institute

28,

of Animal

COLOMBO Biology,

University

AND

PAOLA of Padua,

COLOMBO BELVEDERE Via Loredan

10, 35100

Padua.

Italy.

Accepted September 23, 1975 The ovary of the European eel, Anguilla anguilla, at the silver stage, was incubated either as an intact tissue preparation or as a homogenate with and without cofactors in the presence of [4J4C] pregnenolone and [4-W] progesterone. Intact tissue incubates displayed a more complex metabolite profile than reinforced homogenates, and deprivation of exogenous cofactors reduced the profile even further. Among the metabolites derived from pregnenolone, the following steroids were identified by their isopolarity and isomorphicity with standard compounds: 17a-hydroxypregnenolone; dehydroepiandrosterone; progesterone; 17a-hydroxyprogesterone; and androstenedione. The last three steroids plus testosterone, 1IjLhydroxyandrostenedione, and adrenosterone were identified using progesterone as a precursor. Metopirone inhibited the formation of 1 l-oxygenated androgens. Il-Deoxycorticosteroids were not found, indicating the absence of steroid 21-hydroxylase activity in the eel ovary. Integration of the product yield-time curves demonstrates that in vitro the activities of the enzymes 3p-, 17@-, and ll/Lhydroxysteroid dehydrogenases were less apparent than those of steroid 17a,20-C,,-desmolase, and 17a-, and to a lesser extent 11/lhydroxylase. Irrespective of the incubation conditions, pregnenolone produced more h5-3@hydroxy-than d4-3-ketosteroids, suggesting a predominance of the former biosynthetic pathway. Among the unidentified metabolites, water-soluble compounds were formed from both precursors in intact tissue incubates.

mong marine teleosts, a number of commercially important euryhaline species migrate for feeding into brackish or freshwater biotopes, but are unable to spawn unless they return to the sea. This seriously limits their cultivation in lakes, lagoons, or artificial ponds since population renewal depends totally upon the fry coming from the sea An extreme case is represented by the eel, Anguilla anguilla, which metamorphoses and differentiates sexually in Euroean inland and littoral waters but, supposedly, spawns in the vicinity of the Sargasso Sea (D’Ancona, 1960). At the time of their catadromous migration, eels acquire a characteristic silvery appearance, but their gonads are still far from being mature. In the ovary, oocytes are at the beginning of 1 A preliminary account of this work was presented at the Sixth International Symposium on Comparative Endocrinology, Banff, Canada, June 13-19, 1971.

vitellogenesis and their diameters are at the most 0.2 mm, i.e., one-fifth of grown condition (Fontaine et al., further follicular growth has been ob in wild specimens hindered from re the sea, whatever their age and size. other hand, in the few female eels captured in the sea, oocytes were slightly larger. Spermatogenesis was also more advanced in males (D’Ancona, 1960). Thus, it is conceivable that low salinity or other environmental factor (or factors) may activate a neuroendocrine mechanism inbibiti~ nadal maturation in the eel living in fresh or brackish water. A similar hypothesis has been recently proposed to explain why another euryhaline fish, Mugil capita, cannot spawn in fresh water but only in the sea. In this species, freshwater females are incapable of ovulation, and their ovaries contain a higher concentration of 1 l-ketotestoster-

371 Copyright C All rights

1976 by Academic Press. Inc. of reproductmn in any form reserved.

372

COLOMBO

AND

one2 (Eckstein and Eylath, 1969, 1970) and higher activity of the enzymes crucial to its biosynthesis, steroid 1 Ifi-hydroxylase (Eckstein and Eylath, 1969, 1970) and ll@hydroxysteroid dehydrogenase (1 lj?HSDH) (Blanc-Livni et al., 1969), than found in females caught in the sea. Since 11-ketotestosterone is a potent androgen in teleosts (Hishida and Kawamoto, 1970), Eckstein and Eylath (1970) have suggested that it may interfere, above a certain threshold, with the mechanism triggering ovulation in Mugil. On the other hand, Blanc-Livni et al. (1969) concluded, from their histochemical work on Mugil capita, that steroid hormone biosynthesis was probably deficient in the ovary of freshwater females. In the eel, the block in oogenesis is even more severe than that in the mullet, but it is not known whether ovarian steroidogenesis is concomitantly deficient or diverted towards the production of 11-oxygenated androgens. The present paper indicates that both alternatives may be realized in the ovary of Anguilla anguifla at the onset of its seaward migration. MATERIALS Reagents.

AND METHODS

Labeled steroids were purchased from

2 Trivial and systematic names of the steroids mentioned in the text: adrenosterone. androst-4-ene3,11,17-trione; androstenedione, androst-4-ene-3,17dione; cortisone, pregn-4-ene-17a. 21-diol-3,11,20trione; cortisol, pregn-4-ene-1 ljl, 17a,21-triol-3,20dione; dehydroepiandrosterone, androst-5-en-3Jl-ol17-one; 11-deoxycorticosterone, pregn-4-en-21-013,20-dione; ll-deoxycortisol, pregn-4-ene-17a ,21diol-3,20-dione; 21-deoxycortisol,, pregn-4-enellJl,17a-diol-3,20-dione; 2Oo-dihydroprogesterone, pregn-4-en-20P-ol-3-one; 1 lp-hydroxyandrostenedione, androst-4-en-ll@-01-3, 17-dione: 17a-hydroxypregn-5-ene-3P, 17a-diol 20pregnenolone, one; 21-hydroxypregnenolone, pregn-5-ene-3P, 21-diol-20-one; 1 lp-hydroxyprogesterone, pregn-4en-l Ip-01-3, 20-dione; 17a-hydroxyprogesterone, pregn-4-en-1701-01-3, 20-dione; 1 lp-hydroxytestosterone, androst-4-ene-1 l/3, 17p-diol-3-one; 1 I-ketotestosterone, androst-4-en-17j?-ol-3,11-dione; pregnenolone,pregn-5-en-3b-ol-20-one; progesterone, pregn-4-ene-3,20-dione; testosterone, androst-4-en17/l-ol-3-one.

BELVEDERE

New England Nuclear Corp. (Boston, Mass.) and repurified by bidimensional thin-layer chromatography (first system, benzene/acetone (82); second system, cyclohexane/ethyl acetate (1: 1)) shortly before use. [ 1 ,2-3H]Adrenosterone and [ 1,2-3H] 1 lp-hydroxyandrostenedione were prepared by sodium bismuthate oxidation of [1,2-3H]cortisone (53 Ciimmole) and [1,23H]cortisol (44 Wmmole) respectively (Idler et al., 1971). [1,2-3H]11-Ketotestosterone and [1,23-H] 1I/?-hydroxytestosterone were obtained by reduction with lithium aluminum tri-tert-butoxy hydride (LiAl(tBuO),H) of [1,2-3H]adrenosterone and [1,2 -3H] 1lb-hydroxyandrostenedione, respectively (Idler et al., 1971). These steroids were also purified as indicated above. Standard steroids were bought from Mann Research laboratories (New York) and Ikapharm (Israel), coenzymes from Sigma (St. Louis, MO.) and metopirone from CIBA (Switzerland). Noscreen medical X-ray safety films, type S, 18 x 24 cm, from 3M S.p.A. (Italy) were used for autoradiography. All solvents were freshly redistilled before use. Animals. Fourteen females of Anguilla anguilla in the silver phase were sacrificed for the experiments. Big eels (mean standard length of 85 cm and mean body weight of 973 g) were used for Expts A-F, and small eels (51.5 cm and 234 g) for Expts G-I (Table 1). Eels were captured in the lagoon of Comacchio, near Venice, during their seaward migration in November, and were kept in an outdoor tank with running water from the Venetian lagoon until sacrificed. Both ovaries were dissected out following pithing of fish. Incubations. Each incubation was performed with a 2-g sample of ovarian tissue derived from a pool obtained from two to five eels. A single pool of tissue was used for Expts A-C and another two pools for Expts D-F and H-I (Table 1). Ovaries, which are ribbonlike, were either cut into l-cm long pieces, preserving their cellular integrity, or homogenized as specified in Table 1. Homogenization was performed in half the volume of the final incubation medium by the aid of a motor-driven glass-Teflon homogenizer at low speed. Tissue remnants, unbroken cells, and nuclei were removed by centrifugation at about 7008 for 10 min. The ovarian preparations were transferred into 25-ml Erlenmeyer flasks, each containing 1 &i of [4J%Z]pregnenolone (52.8 mCi/mmole) or [4-‘C]progesterone (52.8 mCi/mmole) (concentration at the start of the incubation: 1.26 @4 for both precursors; tissue/ substrate ratio about 3.4 x 10s) (Table 1) previously dissolved in 200 ~1 of propylene glycol, plus 15 ml of medium. The latter was isosmotic with the plasma of the seawater eel and had the following composition: NaCl, 141.3 mM; KCl, 3.34 mM; Cacl,, 2.30 mM; MgCl,, 3.98 mM; MgSO,, 0.37 mM; Na,HPO,, 1.61 mM; KH,PO,, 0.36 mJ4; NaHCO,, 37.71 mM; glucose, 5.55 mM; phenol red, 2 mgil. As indicated in Table 1, some of the homogenates were supplemented with the following cofactors: NAD, 0.5 m&f; NADH,

OVARIAN

STEROIDOGENESIS

IN

TABLE SUMMARY

OF THE

Experiment A B c D E F G H I

0.05

m&f;

NADP,

INCUBATIONS

OF Anguilla

Preparation Intact tissue Homogenate Homogenate Intact tissue Homogenate Homogenate Intact tissue Intact tissue Intact tissue

0.05

n&f;

NADPH,

THE

373

EEL

1

OVARIAN

TISSUE WITH RAD~OA~XIVE PRECURSORS

Precursor

Cofactors

[ 4J4C]

Pregnenolone

+ -

[4-Y]

Progesterone + -

0.5 mM

and

niacinamide, 10.0 a. One incubate received metopirone, 25 $f. Incubations were carried out in a Dubnoff metabolic shaker at 15-16” under an atmosphere of 95% O*-5% CO,. Aliquots of 1.5 ml were withdrawn from the incubation media or the homogeates after 7.5, 15, 30. 60, 120, 240, and 360 min and were replaced by an equal volume of fresh medium WitbOUt pFecuFsoF. In the incubates with intact tissue, the latter was separated from the medium at the end of the incubation (360 min). Metabolism was terminated by mixing thoroughly with 3 vol of ethanol, and samples were stored at -20” until processed. Extraction. Before extraction, 7 yg each of the following steroids were added as carriers to the incubation aliquots, the homogenates and the media remaining after the removal of the last aliquot, and to the incubated tissues: progesterone, l’la-hydroxyprogesterone, ll@~ydFoxyprogesterone, 2Oa-and 2Ojdihydroprogesterone. 1 I-deoxycorticosteFone. 1 ldeoxycortisol, 21-deoxycortisol, androstenedione, Il~hydroxyandrostenedione, adrenosterone, testosterone, BljI-hydroxytestosterone, and Il-ketotestosterone. When [4-V]pregnenolone was used as a precursor, the following carriers were also added: pregnenolone, 17a-hydroxypregnenolone, 21hydroxypregnenoione, and dehydroepiandrosterone. To monitor recovery and to facilitate identification, the following tritiated steroids (104-l@ dpm, to keep [3H]/[14C] ratios within 1 and 20) were added to samples: [ 1,2-3H]progesterone, [ 1,2-3H] l’la-hydroxyprogesterone, [ 1,2-3H]androstenedione, [ 1,2-3H] 1l/Jhydroxyandrostenedione [1,2-3H]adrenosterone, [1,2VIltestosterone, [ 1.2-3H] 1ljl-hydroxytestosterone, and [ 1 ,2-3H] 11-ketotestosterone. Samples with [4-l*C] pregnenolone received aIso [7-3H]pregnenolone. [7-3H] 17a-hydropregnenolone, and [7-3H]dehyroepiandrosterone. Extractions were conducted as previously described (Colombo et al., 1972). 77z’hin-&er clzroomatography (TLC). Extracts were

Metopirone (25 &&I) +

spotted in a small volume of benzene on TLC plastic sheets (20 x 20 cm) with a 250Fm thick layer of silica gel FZS4(Merck). The deposition of fat-laden extracts was carried out by alternating spotting with developments in cyclohexaneibenzene, 1:l (direction A). After spotting was terminated, nonpolar fats were fully displaced from the chromatographic origin by 2-10 developments in the above solvent system. Excessive amounts of fats were removed from the plate by turning it 90” to the left and pouring methanol with a Pasteur pipet above the fat contour, taking care to exclude the area near the origin. Methanol was left to percolate from the plate through its lower edge which had been previously cut with 0.5cm deep indentations. After the methanol washing, the edge was leveled again and the plate was left to dry. Steroids were first separated with two to three developments in benzene/acetone, 8:2 (direction B, at 90” with respect to direction A), and then with two to three developments in cyciohexane/ethylacetate, I:1 (direction A). This procedure minimized contact of steroids with large and tailed fat spots. Autoradiography (exposure time from 6 hr to 20 days) and carrier visualization was done as previously described (Colombo er al.. 1972). [14C]Labeled carriers were removed from plates by cutting the spots with scissors and hanging them on a bent needle inserted under the tip of a 25 ml buret. Elution was performed with 10-15 ml of acetone oF methanol. Identijication of metabolites. Radiochemical identity and homogeneity of [F4CJmetabolites were inferred from their isopolarity and isomorphicity with aothentic [3H@ompounds diluted with carriers. Isopolarity was demonstrated .by the constancy of the [3H]/[‘*C]ratio after acetylation, NaHB, reduction, or CrO, oxidation of the metabolite (Colombo et al., 1972). Coincidence between the transformed carrier and [l*C]F~~o~~t~vit~ after chromatography was also checked by autoradi~~raphy. Isomorphicity was established when, after recrystallization from acetone-water or pyridine -

374

COLOMBO

AND

water, the [3Hl/[W] ratios of three consecutive crystal crops and of the last mother liquor were within r5% of the mean. This procedure was applied to a pooled sample when the metabolite was formed from the same precursor in different incubates. However, before pooling, the chemical nature of the compound was always verified by formation of a single derivative and autoradiography. The residual precursor was identified only by its mobility in the first bidimensional chromatogram. Calculation of yields. Percentages of unused precursors and yields of identified metabolites were calculated on the basis of precursor initial radioactivity, and were corrected for procedural losses through the recovery of the corresponding [Wltracers. Incubation aliquots were corrected also for dilution. Liquid scintillation counting. Radioactivity was counted by means of a Packard Model 2425 Tri-Carb Liquid Scintillation Spectrometer System. In the absence of significant quenching, the counting efficiencies for 14Cand 3H were 60.9 and 43.3%, respectively. The 14C spillover in the 3H channel was 9.9%, and that of 3H in the 14C channel was 0.02%.

BELVEDERE

RESULTS The percentages of initial precursor radioactivity extracted with ethanol and ethyl acetate from the different fractions of the incubates of Expts A-F is shown in Table 2. In the intact tissue incubates (Expts A and D), a major fraction of the label was taken up by the tissue. The time course of this incorporation can be deduced from Fig. 1. Up to 30 min of incubation, tissue uptake rapidly increased, but decreased steadily thereafter. Thus, tissue retention was greater for precursors than for their distal metabolites. This difference was more marked with pregnenolone than with progesterone. Formation of water-soluble compounds (highly oxygenated or conjugated steroids) was tested in the final samples of all incu-

TABLE PERCENTAGES INCUBATION END

OF INITIAL AND FROM OF THE

PRECURSOR RADIOACTIVITY EITHER THE MEDIUM AND

INCUBATION OF EXPERIMENTS A-F, ETHYL ACETATEQ AND [%]/[‘*C]

I Aliquots taken during incubation Experiment A B C D E F

Extractant* EtOH EtAc EtOH EtAc EtOH EtAc EtOH EtAc EtOH EtAc EtOH EtAc

%[T] (10.6p 10.6 (43.0) 43.0

(44.8) 44.8 (12.4) 12.4 (39.2) 39.2 (42.1) 42.1

2

RECOVERED THE TISSUE, AFTER RATIO

FROM ALL THE ALIQUOTS TAKEN OR THE HOMOGENATE REMAINING EXTRACTION WITH OF THE EXTRACTS

II Medium or homogenate at the end of incubation %[“C] 36.6 33.8 46.7 47.2 48.2 48.9 25.8 24.9 55.0 52.9 54.3 52.9

[3H]/[‘4C] 0.63 0.69 2.11 2.07 1.77 1.75 0.88 0.93 1.30 1.33 1.48 1.53

ETHANOL

DURING AT THE

AND

III Tissue at the end of incubation %[W] 39.8 36.1 58.8 44.8 -

[3H]/[‘4C] 0.34 0.37 0.15 0.20 -

I + II + III 87.1 80.6 89.8 90.3 93.0 93.7 97.1 82.2 94.2 92.1 96.4 95.0

a Samples were extracted 2-4 times with 3-10 vol of ethanol (extracting hydrophilic and hydrophobic [“C] metabolites) and, following evaporation of most the ethanol, the aqueous residues were extracted 4 times with 4 vol of ethyl acetate (extracting hydrophobic [‘“Cl metabolites only). All recoveries were corrected for procedural losses. * EtOH is ethanol; EtAc is ethyl acetate. c Incubation aliquots were extracted with ethylacetate alone, and it is assumed that with ethanol extraction recoveries would have been at least equal to those obtained with ethyl acetate.

OVARIAN

STEROIDOGENESIS

IN

THE

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375

F~cl. Recovery-time curves of ethyl acetate-extractable radioactivity and of nonutilized precursor contained in the medium during incubation of intact ovarian tissue of Anguilh with [4JT]pregnenolone (Expt A) and j4T]progesterone (Expt D).

bates by comparing the [ 3H]/[ ‘“cl ratio of the aqueous ethanolic extract with that of the ethyl acetate extract of the aqueous residue left after evaporation of most of the ethanol. Since the tritiated compounds added as tracers were relatively water insoluble, an increment in the ratio means that some [‘“cl labeled compounds were hydrophilic. As reported in Table 2, the increment was either small or nonexistent for the homogenates, whereas the [“H]/[‘4C] ratio increased consistently in both the medium and the tissue of the intact tissue incubates. The chemical nature of the water-solubie compounds remains to be determined. Pregnenolone was metabolized more rapidly than progesterone in the homogenates (Expts B and C versus E and F), as illustrated by their disappearance curves (Figs. 4-6). Exogenous cofactors were essential for precursor utilization, which was virtually complete in their presence (Figs. 4

FIG. 2. Yield-time

and 5) and negligible in their 6). In the intact tissue inc cursors disappeared exponenti medium, pregnenolone faster terone, as a consequence of both tissue retention and metabolism (Fig. 1). At the end, the precursors were almost en metabolized (Table 3), apparently wi support of endogenous cofactors. Homogenization reduced the tota tabolite profile displayed by the intact sue, and deprivation of exogenous cofactors diminished it even further. Comparison of Figs. 7-10 for pregnenolone and Figs. II- 14 for progesterone makes this point evident. As regards the carrier steroids investigated, pregnenolone was co verted only to 17a-bydroxypreg~e~o~o~e by the homogenate without cofactors (Fig. 10). In the supplemented homogenate (Fi 9), and in the intact tissue incubate (Figs. 4 and 8), dehydroepiandrosterone, progester-

curves of the identified metabolites released in the incubation medium in Expt A.

376

COLOMBO

FIG. 3. Yield-time

AND

BELVEDERE

curves of the identified metabolites released in the incubation medium in Expt D.

FIG. 4. Recovery-time curve of nonutilized precursor and yield-time curves of identified metabolites contained in the reinforced homogenate of ovarian tissue of Anguilla during incubation with [PC]pregnenolone (Expt B).

Tim.

,..A”,

5. Recovery-time curve of nonutilized precursor and yield-time curves of identified metabolites contained in the reinforced homogenate of ovarian tissue of Artguilla during incubation with [4‘Tlprogesterone (Expt E). FIG.

one, 17a-hydroxyprogesterone, and androstenedione also were formed. Similarly, progesterone was converted only to 17a-hydroxyprogesterone by the homogenate

without cofactors (Fig. 14), while the reinforced homogenate (Fig. 13) and the intact tissue incubates of Expts D, G, and H (Figs. 11, 12, and 15) also synthesized androstenedione, 1 lfi-hydroxyandrostenedione, adrenosterone and testosterone (Table 4). The yield-time curves of the identified metabolites were also affected by the incubation conditions. The difference between unsupplemented and reinforced homogenates (Figs. 4 and 5 versus Fig. 6) was clearcut, as expected. Differences exist also between the yield time curves of the reinforced homogenates (Figs. 4 and 5) and the intact tissue incubates (Figs. 2 and 3), although a quantitative comparison is not possible since only the steroids released in the medium were measured in the latter system. This difficul-

OVARIAN

STEROIDOGENESIS

IN

THE

377

EEL

FIG. 6. Recovery-time curve of nonutilized precursor and yield-time curve of the main metabolite contained in the unsupplemented homogenates of ovarian tissue ofArzgui//a during incubakn w&h ~~-14C~p~e~e~~~o~~ (&@ @) and [4-Ylprogesterone (Expt F).

ty is aggravated by the fact that the tissue/ medium coefficients of partition of the metabolites at the end of the incubation ranged from 0.5 to 8.5 dependkrg approximately on their polarity. Polar androgens (testosterone, 11/J-hydroxyandrostenedione, and adrenosterone) were retained the least by the tissue (Table 3). vertheless, it is fairly safe to assume t yields were mostly higher in the reinforced homogenates as, in some cases, yields of metabolites were more than 10 times higher than those obtained by i tissue preparations. The increase of ydroxypregnenolone and, especially, ydroepiandrosterone and androstenedione from pregnenolone (Fig. 2 . 4), and of 17a-hydroxyproges, to a certain extent, androstenterone (Fig. 3 versus rder of magnitude. On conversions, in the reinhomogenates, of pregnenolone to ield, uncorrected for of progesterone to testosterone (0. Is%), BlJ&hydroxyandrostenedione (0.04%) and adrenosterone (0.02%) were not increased significantly (cf. Table ld values of progesterone in Expts A of B1 jL=hydroxyandrostenedione and sterone in Expts D and E were not sufficiently high to lot yield-time curves. Integration of the curves of Figs. 2-6 demonstrates that precursors were transformed predominantly into their 17a-hy-

droxylated derivatives in all ex~er~rn~~ts” Although ~7a-hydroxypregne~o~o~e was always formed and metabohzed faster than 17a-hydroxyprogesterone, it was converted to deby~oepia~drosterone (Figs. 2 a 4) to a lesser extent than ~7~-bydrox~ gesterone to a~droste~edione (Figs. 3 and 5). This may be due to differences in substrate affinities of the enzyme system. The temporal distribution of product yield- time curves and tial emergence of the curves latent period (Figs. 2 and 5) are with the following biosynthet pregnenolone * 17~2 -+ dehydroe~ia~dr nenolone -+ progesterone -+ ;7 progesterone + androstenedione terone. The ~r~dorn~~a~ce of synthetic pathway is suggeste olone produced more d”-3@-

androstenedione,

smce

COLOMBO

AND

BELVEDERE

of 1 lj-hydroxyandrostenedione, substance A, was also diminished (Fig. 16). Comparison of Figs. 1.5 and 16 demonstrates that metopirone altered the metabolite profile significantly, both depressing and favoring ~ the accumulation of various unknown cornpounds. It also affected yields of identified products, especially androstenedione (Table 3). In our experiments, no radioactivity was associated with any of the remaining carriers, in particular with 1lj&hydroxytestosterone and 11-ketotestosterone. On the other hand, an increasing number of unidentified compounds was observed with increasing incubation time, especially in the intact ,tissue incubates. The range of their polarities was wider than that of the investigated carriers (Figs. 7-9, and 11-13). DISCUSSION

The present study demonstrates that the ovary of Anguilla anguilla at the silver stage is able to perform a well-diversified set of steroid conversions known to be catalyzed by the following enzymes: 3pHSDH, d 5-ketosteroid isomerase, 17$‘HSDH, 1 lb-HSDH, steroid 17a-hydroxylase, steroid 1lb-hydroxylase and 17a, 20Cz,-desmolase. From a qualitative standpoint, this biosynthetic system is interesting for two reasons. First, it includes steroid 1 l@-hydroxylase and 1 lp-HSDH activities, which seem to have an ovarian localization only among teleost fish (Eckstein and Eylath, 1969; Eylath and Eckstein, 1969a,b, 1970; Blanc-Livni et al., 1969; Eckstein, 1970; Eckstein and Katz, 1971), whereas the remaining five enzymes have been reported also in the ovary of other j vertebrates (Ozon, 1972). Secondly, it is devoid of the enzyme steroid 21-hydroxylase required for the formation of 1ldeoxycorticosteroids, namely 1 l-deoxycorticosterone and/or 1 I-deoxycortisol, which have been shown to be important products of ovarian steroidogenesis in other teleost species (Colombo et al.,

OVARIAN

STEROIDOGENESIS

IN

THE

EEL

FIGS. 7-8. Autoradiographs of the TLC profile of all the metabolites released in the medium (Fig. 7) or retained by the tissue (Fig. 8) after 360 min of incubation of intact ovarian tissue of Ana~illn with [4-‘“C]pregneno~one (Expt A). FIGS. 9- 10. Autoradiographs of the TLC profile of all the metabolites formed from [C’“C]pregnenolone after 360 mitt of incubation with the reinforced (Fig. 9, Expt B) and the unsupplemented (Fig. 10, Expt C) homogenate of ovarian tissue ofilngulla. Symbols: 0 = chromatographic origin; 1 = pregnenolone; 2 = 17~-bydroxypreg~eno~one; 3 = dehydroepiandrosterone; 4 = progesterone; 5 = 17a-hydroxyprogesterone; 6 = androstenedione; 7 = 11/3hydroxyandrostenedione; 8 = adrenosterone; 9 = testosterone; IO = 11/3hydroxytestosterone; Ii = ilketotestosterone; 12 = 11-deoxycorticosterone. Broken circles indicate the position of catrier steroids associated with contaminating 14C label or without 14Clabel.

1973), amphibians (Colombo and Belveere, 1974), reptiles (Colombo et al., 1974; Colombo and Yaron, a) and mammals (Lucis et al., 1972; Colombo and Pesavento, 1973). uantitative considerations are more difcult to make owing to the limitations inherent in an in a/itro technique. Since the

irreversible metabolism sf an ished labeled precursor for a few closed system precludes the att both chemical equilibria and ste radioactivity measurements ma ly transient situations. Moreover, the lack of blood flow in the incubated tissue to remove secretory products favors their

2.75

7.88

16.14

3.58

13.40

Dehydroepiandrosteroned

Progesterone

17a-OH-Progesterone

Androstenedione VIII/l and VII/l

bVIIII2r and VII/l VIII/l and VII/l xl2 and V/l -

(Experiments

Incubations with [4-14C] Pregnenolone

17a-OH-Pregnenolone

PROGESTERONE

RECRYSTALLIZATION

A-C)

12.93

16.31

6.72

2.50

[*H]/[W]

Acetylation

[4-W]

AND

BD TLC”

AND

FORMATION

Initial

PREGNENOLONE

BY DERIVATIVE

[3H]/[‘4C]

[4-W]

Metabolite

IDENTIFICATION INCUBATION

[3H]/[14C] WITH

II6 and xl3 -

VU2 and VII/l VIII/2 and VII/l

BD TLC

3.65

7.09

2.37

[3H]/[‘4C]

CrO, Oxidation

Derivative formation

AFTER

4

TO CONSTANT

TABLE

VII/l and II/2 VW4 and II/2 Ix/l and II/2

-

-

BD TLC

12.97

3.50

15.73

[3H]/[‘4C]

FROM

1st: 2.41 2nd: 2.40 3rd: 2.49 1st: 6.81 2nd: 6.64 3rd: 6.52 1st: 16.20 2nd: 16.41 3rd: 16.20 1st: 3.78 2nd: 3.76 3rd: 3.75 lst:13.51 2nd:13.44 3rd: 13.57

[3H]/[14C]

Crystals

13.64

3.76

16.45

6.94

2.38

[3H]/[14C]

3rd Mother liquor

Recrystallization

OF THE METABOLITES FORMED TISSUE OF Anguilla anguillu

R ATIO

NaHB, Reduction

OVARIAN

Fs E

8 z $ g

3.91

2.45

2.30

8.58

41.55

Androstenedione

Testosteroned

1 lp-OH-Androstenedione

Andrenosterone

Adrenosterone

-

VIII/I and VII/l X/3 and III/l IV/6 and X/5 -

-

(Experiments

D-H)

2.19

I .98

3.56 xl5 and VU2 IV/6 and x/5 IV/5 and Xl5 -

VI/2 and VIII2 _-

6.21

2.10

2.03

I.33

IV/5 and XL5 -

-

VIII2 and II/2 Ix/l and III2 -

5.46

3.5@ 3.67

1.34

1st: 1.43 2nd: 1.41 3rd: 1.40 1st: 3.78 2nd: 3.79 3rd: 3.68 1st: 2.01 2nd: 1.98 3rd: 2.00 1st: 2.18 2nd: 2.04 3rd: 2.10 1st: 6.26 2nd: 6.26 3rd: 6.19 lst:49.36 2ndz47.67 3rd:48.16

--

47.81

6.22

2.21

2.03

3.76

1.42

n BD TLC = bidimensional thin-layer chromatography. * Chromatographic solvent systems: I = cyclohexaneiethyl ether (1: 1): II = cyclohexaneiethyl acetate (6:4); III = cyclohexaneiethyl acetate (1: 1); IV = cyclohexaneiethanol (8:2); V = benzene/ethyl ether (1: 1); Vl = benzene/ethyl acetate (6:4): VII = chloroform/ethyl ether (8:2); VIII = chloroform/acetone (9:l); IX = chloroform/acetone (85: 15); X = chloroform/ethanol (99: I). c Number of developments per solvent system. d Recrystallized as acetate derivative. c [WI/[ *%]ratios of the two major compounds obtained after reduction of andros&enedione. f Adrenosterone formed in Expt II.

1.40

[4-“C] --.__

17a-OH-Progesterone

Incubations with progesterone

is E

? E 0 6 8 F t2 z ?

z

E

0

382

COLOMBO

AND

BELVEDERE

OVARIAN

STEROIDOGENESIS

metabolic recycling along with an artificial diffusion and incorporation into the oocytes. The latter phenomenon was probably the determinant of the tissue/medium coefficients of partition of the identified steroids (Table 3). These biases were further amplified in the homogenates, where pools are dispersed without intra- and extracellular distinctions. Under our experimental conditions, the activities of the enzymes 3/j’-HSDHisomerase, 17jGand 11/S-HSDH were less apparent than those of steroid 17cr,20-C2,desmoiase, 1761- and, to a lesser extent, 1IjJ-hydroxylases. In fact, the 3j-HSDHisomerase was unable to compete successfully with the 17ahydroxylating system to metabolize pregnenolone both in the absence and in the presence of exogenous cofactors (Figs. 2 and 4; Tab. 3). When competition became more stringent, as in the unsupplemented homogenate, no A 4-3-ketosteroid was produced concomitantly with 17a-hydroxypregnenolone. However, since significant amounts of 17a-hydroxyprogesterone and androstenedione were found in the intact tissue incubated with pregnenolone (Expt A), the level of 3j3HSDH-isomerase activity was a minor but not negligible one. 17,8-HSDH failed to increase testosterone accumulation in the reinforced homogenate of Expt E above the level found in the intact tissue incubate of Expt D, despite an enhancement of androstenedione synthesis and the availability of excess cofactors.

IN

THE:

EEL

Testosterone was almost undetecta when pregnenolone was used as a precursor. Similarly, the ~~~-~SD~ activity produced only a small accumulation of adrenosterone in spite of variability in the yield of 1lp-hydroxyandrostenedione. Although these indications are not conclusive and steroidogenes~s from cholesterol and acetate remains to be explored, it is conceivable that the dominance of the A 5” 3@-hydroxysteroid pathway over the d 4-3ketosteroid route, the presence of a steroid 1 lj?-hydroxylase acting on androstenedione and the deficiency of 17JLHSDH activity may lower the production of androstenedione and testosterone. Since these steroids ermediates in estrogen biosynatter also may be redu ve been shown to sti hepatic synthesis of calcium-bindi pholipoproteins necessary for yol tion in teleost fish (Bailey, 1957; Ch Ho and Vanstone, 1961; Rack and Fraser, 197l;Placketal.. 1971).Inthisgroup,therefore, estrogens are believed to be involved in the process of vitellogenesis under the control of the pituitary gland (Dodd, 1972), Whether oocyte growth in the eel is blocked at the previtellogenic pituitary-independent stage by a gonadotropin ins~f~~ien~y ar inadequate estrogen secretion or both will be answered by further investigation. S estrogens have been tentatively identi the ovary of Anguilla nnguilla (GoI 1963,

where

they

(Breuer and Bzsn,

can

b

1965).

FIGS. II- 12. Autoradiographs of the TLC profile of all the metabolites released in the medium (Fig. I I, Expt G) or retained by the tissue (Fig. 12, Expt D) after 360 min of incubation of intact ovarian tissue of Angrcilla with [4-WJprogesterone. The profile of the incubation medium of Expt D was comparable to that of Expt G but 1I-oxygenated androgens were less evident. FIGS. i3- 14. Autoradiographs of the TLC profile of all the metabolites formed from (4-Vlprogesterone after 360 min of incubation with the reinforced (Fig. 13, Expt E) and the unsupplemented (Fig. 14 Expt F) homogenate of ovarian tissue of Anguiilu. FIGS. 15-16, Autoradiographs of the TLC profile of all the metabohtes released in the medium after 360 min of incubation of intact ovarian tissue of Anguiiiu with [CWlprogesterone in the absence (Fig. 15. Expt H) and in the presence of metopirone (Fig. 16, Expt I). Symbols as in Figs. 7- 10. Spots marked by crosses were made with a radioactive solution colored with Sudan III in order to correlate the autoradiographs with the thin-layer plates. A = CrD,-nonoxidizable contaminant of 1lb-hydroxyandrostenedione

384

COLOMBO

AND

plete ovarian maturation and ovulation were induced in this fish after injection of carp pituitary extracts (Fontaine et al., 1964; Lopez and Martelly-Bagot, 1971). and Ozon, 1965). Moreover, complete ovarian maturation and ovulation were induced in this fish after injection of carp pituitary extracts (Fontaine et al., 1964; Lopez and Martelly-Bagot, 1971). The absence of steroid 21-hydroxylase activity and the consequent lack of 1 ldeoxycorticosteroid biosynthesis in the ovary of Anguilla are not surprising. In teleosts, 21-hydroxylated derivatives of progesterone have been implicated in mediating gonadotropin induction of oocyte meiotic maturation and ovulation (Sundararaj and Goswami, 1971; Goswami and Sundararaj, 1971; Hirose, 1972; Colombo et al., 1973) and these stages are far ahead of the maturational condition of the ovary of migrating silver eels. Crucial to this argument is to establish whether or not a steroid 21-hydroxylase is acquired following gonadotropin treatment. In other fish, synthesis of ll-dexoycorticosterone and 11-deoxycortisol was found to be much greater at the end of than during vitellogenesis (Colombo and Belvedere, in prepration). Our experiments confirm the reports by Eckstein and his co-workers on the ability of the teleost ovary to form 11-oxygenated androgens, though, in our case, the basic androgen was androstenedione and not testosterone. At present, the significance of these steroids in the eel is uncertain. We have found yields that are considerably lower than those reported in Mugil cupito living in fresh and sea water (Eckstein and Eylath, 1970) and in Tilapia aurea before and after spawning (Eckstein and Katz, 1971). Thus, it is doubtful whether they may have as much physiological relevance in Anguilla as in the other species. On the contrary, taking into account that sex diferentiation occurs rather late in the life cycle of the eel, usually after silvering

BELVEDERE

(D’Ancona, 1960), and that the male gonad possesses strong steroid 1 lJ-hydroxylase and llJ3-HSDH activities (Colombo et al., 1972), these enzymes in the ovary might simply represent testicular remnants. ACKNOWLEDGMENTS Work aided by Grant 72.01033.04 115.4918 from the National Research Council (C.N.R.) of Italy through the sponsorship of the Institute of Marine Biology of Venice. We are grateful to Prof. Bruno Battaglia for encouraging this research, to Prof. Armando Sabbadin for the use of the facilities of the Hydrobiological Station of Chioggia, and to Prof. Howard A. Bern for reading the manuscript. We are indebted to Mr. Umberto Arezzini for photographic assistance.

REFERENCES Bailey, R. E. (1957). The effect of estradiol on serum calcium,, phosphorus and protein of goldfish. J. Exp. Zoo/. 136, 455-469. Blanc-Livni, N., Abraham, M.. Leray, C., and Yashouv, A. (1969). Histochemical localization of hydroxysteroid dehydrogenases in the ovary of an euryhaline teleost fish, Mugil cupito. Gen. Camp. Endocrinol. 13, 493. Breuer, H., and Ozon, R. (1965). Metabolisme des hormones steroides androgenes et oestrogenes chez les vertebres inferieurs. Arch. Anat. Microsc. Morph. Exp. 54, 17-33. Chung-Wai Ho, F., and Vanstone, W. E. (1961). Effect of estradiol monobenzoate on some serum constituents of maturing sock-eye salmon (Qncorhynchus nerka). J. Fish. Res. Bd. Canad. 18, 854-864. Colombo, L. (1965). Identihcazione di estrone e 17pestradiolo negli ovari e nei testicoli di Anguilla anguilla L. Boll. Zool. 32, 1163-1173. Colombo, L., and Belvedere, P. (1974). Biosynthesis of 1I-deoxycorticosterone by the ovary of Triturus alpestris alpestris (Laur.). Gen. Comp. Endocrinol. 22, 342. Colombo, L., Bern, H. A., and Pieprzyk, J. (1971). Steroid transformations by the corpuscles of Stannius and the body kidney of Salmo gairdnerii (Teleostei). Gen. Comp. Endocrinol. 16, 74-84. Colombo, L., Bern, H. A., Pieprzyk, J., and Johnson, D. W. (1972). Corticosteroidogenesis in vitro by the head kidney of Tilupiu mossambica (Cichlidae , Teleostei). Endocrinology 91, 450-462. Colombo, L., Bern, H. A., Pieprzyk, J., and Johnson, D. W. (1973). Biosynthesis of 1 l-deoxycorticosteroids by teleost ovaries and discussion of their possible role in oocyte maturation and ovulation. Gen. Comp. Endocrinol. 21, 168-178. Colombo, L., and Pesavento, S. (1973). Biosynthesis in vitro of 1 I-deoxycorticosteroids by ovarian and

OVARIAN

STEROIDOGENESIS

IS

THE

EEL

placental tissues of different vertebrates. Gen. Endocrinoi. 21, 214. Colombo, L., and Yaron, Z. (a). Steroid 21-hydroxylase activity in the ovary of the snake Storeria dekayi during pregnancy. Submitted to Gen. Camp. Endo-

enate. steroid precursors and metabolites, and gonadal and adrenocortical steroids. J. EXp. Zuoi 178, 461-478. Hirose, K. (1972). Biological study on ovulation in vi:rr; of fish. IV. Induction of in vitro ovulation in crinol. Oryzias lafipes oocyte using steroids. Bu!i. Jap. Colombo, L., Yaron, Z., Daniels, E., and Belvedere, P. Sot. Sci. Fish. 38, 457-461. (1974). Biosynthesis of 1 i-deoxycorticosterone by Hishida, T.. and Kawamoto, N. (1970). Androgenic and the ovary of the yucca night lizard, Xantusia male-inducing effects of 11-ketotestosterone on a vigih. Gea. Comp. Endocrinol. 24, 331-337. teleost, the medaka(Qiy:ins latipes). J. Exp. &cl. D’ Ancona, IJ. (1960). The life-cycle of the atlantic eel. 173, 2799284. Symp. Zoo/. Sot. Londoiz 1, 61-75. Idler, D. R., Horne, D. A., and Sangalang, G. E. Dodd. I. M. (1972). The endocrine regulation of (1971). Identification and quantification of the magametogenesis and gonad maturation in fishes. jor androgens in testicular and peripheral plasma of Gen. Comp. Endocrinol. Suppl. 3, 675-687. Atlantic salmon (Salmo sub) during sexual maEckstein, B. (1970). Metabolic pathways of steroid turation. Gen. Cnmp. Endocrinol. 16, 257-267. biosynthesis in ovarian tissue of a teleost, Tilupia Lopez, E., and Marteily-Bagot, E. (1971). L‘os aureu. Gen. Comp. Endocrinol. 14, 303-312. cellulaire d’un poisson teleosteen, Anguiila nnguilla Eckstein, I?.. and Eylath, LJ. (1969). Metabolism of 1 lL.: III. Etude histologique et histophysique ati ketotestosterone in ovaries of a teleost, Mugil cours de la maturation provoquee par injections capita, from the sea and from fresh water. Gen. d’extrait hypophysaire de carpe. 2. Zel&rsrh. Comp.

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Eckstein, B., and Eylath, U. (1970). The occurrence and biosynthesis in vitro of 1I-ketotestosterone in ovarian tissue of the mullet, Mugi/ capita, derived from two biotopes. Gen. Comp. Endocrinol. 14, 396-403. Eckstein, B., and Katz, Y. (1971). Steroidogenesis in post- and prespawned ovaries of a cichlid fish, Tilupiu aurea. Comp. Biochem. Physiol. 38 (2A), 329-338. Eylath, IJ.. and Eckstein, B. (1969a). Isolation of llketotestosterone and dehydroepiandrosterone from ovaries of the common mullet, Mugii capita. Gen.

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Eylath, II., and Eckstein, B. (1969b). Steroid biosynthesis in vitro by ovarian tissue of mullets (Mugil sp.) from sea and freshwater. Gen. Comp. Endocrinol.

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Fontaine, M., Bertrand, E., Lopez, E., and Callamand, 0. (1964). Sur la maturation des organes genitaux de 1’Anguille femelle (Anguilla anguilla L.) et P&mission spontanee des oeufs en aquarium. C. R. Hebd.

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Goswami, S. W., and Sundararaj, B. I. (1971). In vitro maturation and ovulation of oocytes of the catfish, Heferapneustes fossilis (Bloch): effects of mammalian hypophyseal hormones, catfish pituitary homog-

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Lucis, 0. .I., Lucis, R.. and Kerenyi, N. A. (1972). Deprivation of environmental light and biosyxthesis of steroids in the adrenals and gonads. Gen. Ozon, R. (1972).

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570-573. Young, D. 6.. and Hall, P. F. (1971). Steroid bydroxylation in bovine adrenocortical mitochondria. Competition between side-chain cleavage of cholesterol and 1l,&hydroxylation. B&hem. LO, 1496 1.502.