GENERAL

AND

COMPARATIVE

Progesterone

ENDOCRINOLOGY

87, 300-311 (1992)

Metabolism in Vitro in the Decapod Crustacean, Penaeus monodon

N. J. YOUNG,* P. T. QUINLAN,~ AND L. J. GOAD*,’ *Department

of

Biochemistry, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, United Kingdom and; fvnilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford, MK44 ILQ, United Kingdom Accepted January 7, 1992

Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line detection of radioactive steroids were applied to identify metabolites of [4-‘4C]progesterone incubated in vitro with prawn ovary. There was extensive metabolism of progesterone by stage II (vitellogenic) ovary of Penaeus monodon. The most abundant metabolites were So-pregnane derivatives together with two minor metabolites, 20a-hydroxypregn-4-en-3-one and 1,4-pregnadiene-3,20-dione. In contrast, a much lower level of progesterone metabolism was observed in stage 0 (immature) ovary of this species. The hepatopancreas, gill, and abdominal muscle of P. monodon all metabolised [4-i4C]progesterone to varying degrees, generating materials similar to those produced by the ovary. A comparative study of progesterone metabolism in stage II ovary of Nephrops norvegicus indicated that one metabolite, 20a-hydroxypregn&en-3-one, was produced. o 1992 Academic PISS, 1~.

Both male and female decapods metabolise steroids. Tcholakian and Eik-Nes (1969, 1971) reported the metabolism of progesterone to 1 1-deoxycorticosterone, testosterone, androstenedione and 20~ hydroxypregn-4-en-3-one by the androgenie gland of the Blue crab, Callinectes sapidus. In a study of steroid metabolism by the testes, vas deferens, and androgenic gland of the shore crab, Carcinus rnaenas, the target tissues of androgenic hormone (testes and vas deferens) were found to be more active than the androgenic gland (Blanchet et al., 1972). These tissues contained a 17B-hydroxysteroid-oxidoreductase enzyme which catalyzed the conversion of Cis and C,, 17-ketosteroids to 17-hydroxysteroids (for example, androstenedione and estrone were converted to testosterone and 17B-estradiol, respectively). Also testes of the American lobster, Homarus americanus, metabolise proges’ To whom correspondence

terone and pregnenolone to 2Oc+hydroxyprogesterone and 20o-hydroxypregnenolone, respectively (Burns et al., 1984a). These observations were consistent with the identification of endogenous steroids in male decapods, for example testosterone in the serum and testes of H. americanus (Burns et al., 1984b), and have been taken to indicate that vertebrate-type steroids participate in male reproduction. Similar observations have been made in female decapods. Teshima and Kanazawa (1970) identified 17cy-hydroxyprogesterone, testosterone, 11-ketotestosterone, and 1 lphydroxyandrost-4-en-3,17-dione as metabolites of progesterone in ovarian incubations of the crab, Portunus trituberculatus. In another study, incubation of [4-‘4C]progesterone with ovaries of the same species yielded 17a-hydroxyprogesterone, testosterone, and deoxycorticosterone (Teshima and Kanazawa, 1971). In female crab, Carcinusmaenas, thegill,ovary, hepatopan cress, and muscle all metabolised [4-14C]

should be addressed. 300

0016~6480/92 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

STEROID

METABOLISM

and [6,7,7-2H,]progesterone in vitro. In all cases 20a-hydroxypregn-4-en-3-one was a major metabolite, with 1,4-pregnadien3,20-dione and Sol-pregnan-3,20-dione being produced by the gill. In addition, the ovary, gill, haemolymph , and hepatopancress incubated in viva with [4-14C] and [6,7,7-2H,]progesterone generated esters and polar conjugates of 20ol-hydroxypregn4-en-3-one (Hazel, 1986). 20a-Hydroxypregn-4-en-3-one, 17@estradiol, progesterone, androstenedione, and testosterone are all present in the ovary of Curcin~s maenas (Hazel, 1986). Recently, Swevers et al. (1991) investigated steroid metabolism in crustaceans. In Cancer pugurus, the vitellogenic ovary contained a 17@hydroxysteroid dehydrogenase (17@HSD) capable of converting androstenedione to testosterone. The hepatopancreas contained 201~ and 17P-HSD activity and also formed water-soluble steroid conjugates. Whole body homogenates of Mucrobruchium rosenbergii and naupilii of Artemiu sulinu produce apolar esters of pregnenolone (Swevers et uZ., 1991). Endogenous steroids have been detected in tissues of several species including progesterone, testosterone, and estrone in whole body extracts of Euphuusiu superbu (Nikitina et al., 1977), “estrogen-like” material in the ovary of the shrimp Purupenueusfissurus (Jeng et al., 1978), a number of steroids in the haemolymph of Astucus leptodactylus (Ollevier et al., 1984, 1986), and Sa-dihydrotestosterone, testosterone, pregnenolone, and 20a-hydroxypregn-4-en3-one in the ovary of N. norvegicus (Fairs et al., 1989). Moreover, vertebrate-type steroid titres in decapod ovaries are maximal during vitellogenesis in several species, including P. monodon (Couch et al., 1987; Van Beek and De Loof, 1988; Fairs et al., 1990). These observations, and the fact that exogenous steroids can stimulate vitellogenesis and spawning (Kulkarni et al., 1979; Nagabhushanam et al., 1980; Sarojini et al., 1986; Yano, 1985, 1987). may indi-

IN

301

P. nowdon

cate that vertebrate-type steroids participate in the regulation of some aspects of ovarian maturation in invertebrates, The metabolism of [4-14C]progesterone by the tissues of P. monodon and N. nowegicus has been investigated to obtain further insight into the possible significance of steroid hormones in crustacean reproduction. MATERIALS

AND METHODS

Materials. Adult, female P. monodon, originating from farms in Sri Lanka, were kindly provided by Unilever Research. Adult, female N. norvegicus were abtained from the Marme Biological Station, Isle of Cumbrae, Scotland. Kieselgel60G for TLC plate preparation and precoated aluminium-backed silica plates (0.25 mm thickness) were obtained from E. Merck (Darmstadt). Berberine hydrochloride was supplied by Sigma Chemical Co., and benzoyl chloride by BDH Chemicals Ltd. [4-‘4C]Progesterone was obtained from NEN Ltd. and ah unlabelled steroids were supplied by Sigma Chemical Co. All solvents were of Analar grade and were redistilled prior to use. fa vitro incubations were carried out in a saline containing NaCl(540 m&f), KC1 (14 nut4), CaCl, (95 nuV), M&l, (11.3 m&Z), and Na, SO, (14.3 n&I). In vitro iticubations. Tissue (2-4 g) was lightly minced in saline (6-12 ml) and [4-r4C]progesterone (5 t&i) was added in a minimum volume of ethanol (typically 5 ~1). Mixtures were incubated for 5 hr at 2g” in the case of pp. monodon and 12” in the case of N. norvegicus, and terminated by freezing at - 20”. Lipid extraction and partition. Lipid was, extract& from the incubation mixture according to the method of Hara and ‘Radin (1978). The mixture was homogenized in hexane-propan5-ol (3:2, v/v, 70 ml), using a Polytron homogenizer (Kinematica GmbH, Lucerne) and filtered through glass wool. The residue was rehomogenized with a further 70 ml solvent and filtered, and the filtrates were combined. Solvent was evaporated in vacua to yield a crude lipid extract (ca. 70 mg). The extract was pa&ioned between hexane (50’ ml) and methanol-water (9:1, v/v, 50 ml), and each fraction was back-extracted with a further 50 ml solvent. The methanohc fractions were pooled’ and the solvent was evaporated in vacua to yield a lipid extract (ca. 30 mg). A fraction (ca. 1%) of the extract,was assayed gvr radioactivity. Typical recovery of radioactivity by thrs method was 65%. Analytica! TLC. Analytical TLC was performed on precoated aluminium-backed silica plates (0.25 mm thickness, 20 cm x 20 cm). A portion of the methanolic extract, (20,000+50,000 dpm) was spotted in ;a band (l-4 cm wide) 1 cm from the base of the plate along with a progesterone marker, and the plate was

302

YOUNG,

QUINLAN,

developed in chloroform-acetone (95:5, v/v). The RFs of a number of authentic steroids on this system are given in Table 1. Detection of radioactive metabolites. The distribution of radioactivity on the plates was determined by both autoradiography and plate scanning. Autoradiograms were obtained using Fuji X-ray film (RX) (18 X 24 cm). TLC plates were scanned for radioactivity using a RITA-3200 radio-TLC analyser (Raytest Ltd.) operating with a gas flow of 10% methane in argon. Identification of radioactive metabolites. Radioactive metabolites were identified on the basis of cochromatography with an authentic steroid on both normal-phase (preparative TLC, Kieselgel 6OG) and reversed-phase (RP-HPLC) chromatography systems. Authentic steroid (50-100 pg) was added to a portion (40,000-200,000 dpm) of the methanolic extract, and the mixture was applied to a preparative TLC plate in a 3- to 4-cm band. The plate was developed in chloroform-acetone (95:5, v/v). Markers were visualised by spraying with berberine hydrochloride (0.005% in ethanol) and viewing under uv light (254 nm). Lipids appeared as bright yellow/green areas against a darker background. The area of the plate corresponding to the authentic marker was scraped and the lipid was eluted from the silica in diethyl ether (4 ml). Solvent was evaporated under nitrogen and an aliquot (1%) was taken to measure the radioactivity. The extract from the TLC plate was then analysed by RP-HPLC with on-line detection of radioactivity. In cases when the authentic steroid was a 5a-pregnane the benzoate derivative was formed since this allowed detection of the steroid by monitoring at 215 nm. 5a-Pregnan-3,20dione was reduced with NaBH, prior to benzoate derivatization. Reduction of 5a-pregnan-3,20-dione. The material containing this steroid was dissolved in ethanol (1 ml), excess sodium borohydride was added, and the mixTABLE RFs OF AUTHENTIC

1

STEROIDSONANALYTICAL

Steroid Steroid fatty acyl esters 5@-Pregnan-3,20-dione So-Pregnan-3,20-dione Progesterone 20a-Hydroxy-So-pregnan-3-one Androstenedione E&one 1 ,CPregnadiene-3,20-dione 3@Hydroxy-So-pregnan-20-one 20a-Hydroxypregn-4-en-3-one Testosterone Su-Pregnan-3B,20adiol 17P-Estradiol

TLC RF 0.71 0.63 0.60 0.48 0.38 0.36 0.36 0.34 0.28 0.25 0.18 0.18 0.16

AND

GOAD

ture was left to stand (room temperature, 18 hr). Solvent was evaporated under nitrogen to leave the 5upregnan-3,20-diol. Formation of the benzoate derivatives of 5apregnanes. Benzoyl chloride reagent containing 1,2dichloroethane (10 ml), pyridine (1 ml), and benzoyl chloride (300 (~1)was freshly prepared prior to derivatization. This reagent (300 ul) was added to the steroid and the mixture was left to stand (room temperature, 30 min). The mixture was then diluted with 1,2dichloroethane (2 ml), washed with 0.1 M HCl(2 X 3 ml) followed by water (2 x 3 ml), and dried over anhydrous sodium sulphate. The solvent was decanted and evaporated under nitrogen. Derivatives were stored in a desiccator prior to analysis by RP-HPLC. RP-HPLC. RP-HPLC analyses were performed using a Kontron 414-T solvent pump with a Rheodyne injector, a Spheris ORB S3 ODS 1 column (Phase Sep.) and a Kontron Uvikon 740 LC detector. On-line detection of the radioactive metabolites was achieved using an Isoflo radioactivity monitor containing a flow cell packed with solid scintilator granules (NEN Ltd.). Underivatized steroids (A4-pregnenes) were analysed using an isocratic solvent system of methanol-water (3:1, v/v) at a flow rate of 0.5 ml/min and monitored at 254 mu. So-Pregnane benzoates were analysed using an isocratic solvent system of acetonitrile-water (95:5, v/v) at a flow rate of 1 ml/min and monitored at 215 nm.

RESULTS The in Vitro Metabolism of [4-‘4C]progesterone by Stage II (Vitellogenic) Ovaly of P. rnonodon

The lipid extract from this incubation contained 2.7 t-&i, representing a recovery of 54%. Of this, 2.3 t&i was recovered as a polar lipid fraction in the methanolic phase following partition of the extract. An autoradiogram of the analytical TLC plate used for analysis of this fraction is given in Fig. 1 and a radio-TLC scan of the same plate is shown in Fig. 2A. Eight distinct bands of radioactivity (not including the origin) were observed, one of which (band 2) appeared to cochromatograph with the progesterone marker. The RFs of the radioactive metabolites (Table 2) were compared with RFs of authentic steroids on the same TLC system (Table 1) and the majority of the radioactivity was associated with the pregnanes with

STEROID

METABOLISM

IN P.

monodon

303

dPm

0 dr

A

SF

2ocm

P

FIG. 1. Autoradiogram of the TLC plate following separation of the lipid extract from the in vitro incubation of stage II ovary of P. monodon with [4-‘4C]progesterone. The plate was developed in chloroform-acetone (95:5, v/v). The RF of authentic progesterone is shown as P and the bands indicating radioactive metabolites are numbered (l-g). For identities of bands see Table 2.

little, if any, associated with androgens and estrogens. The mobilities of the authentic steroid and radioactive metabolite were then compared on RP-HPLC. Representative RP-HPLC data are given for band 1. Band 1 had an RF (0.59) consistent with that of 5c+pregnan-3,20-dione (0.60). Authentic 5a-pregnan-3,20-dione (100 kg) was added to a portion (30,000 dpm) of the polar lipid extract and the mixture was subjected to preparative TLC. The eluted Sa-pregnan-3,20-dione contained 10,470 dpm and a part (4000 dpm) of this material was reduced, benzoylated, and analysed by RPHPLC. The results of this analysis are shown in Fig. 3. Three peaks were observed in the uv trace, these were due to the C-3 and C-20 epimers of 5ol-pregnan-3,20diol dibenzoate which resulted from the reduction of the corresponding dione at the C-3 and C-20 positions. Similarly, three peaks which cochromatographed with the

FIG. 2. Radio-TLC-scans of 20-cm TLC plates following separation of the lipid extracts from in vitro incubations of [4-*4C]progesterone with (A) stage IX and (B) stage 0 ovary of P. monodon. Plates were developed in chloroform-acetone (95:5, v/v). The RF of authentic progesterone is indicated by P..Qr, origin; SF, solvent front. For identities of peaks see Tab& 2 and 3.

authentic compound were detected by the radioactivity monitor. This indicated that the radioactive metabolite which was in band 1 was 5a-[4-‘4C]pregnan-3 ,2+dione. The RF of band 2 was identical with that of progesterone (0.48). Authentic progesterone (100 p,g) was added to a portion (40,000 dpm) of the polar lipid extract and the mixture was subjected to preparative TLC. The eluted progesterone contained 5520 dpm. A part (3000 dpm) of this material was analysed by RP-HPLC and the single peak detected by the radioactivity monitor cochromatographed with the added progesterone. This indicated that band 2 was unmetabolised starting material, [4-14C]progesterone. The RF of band 3 (0.38) corresponded with that of a reduced product of band I, namely 20a-hydroxy-5o-pregnan-3-one (0.38). Authentic 20a-hydroxy-5ceFpregnan 3-one (100 eg) was added to a portion (30,000 dpm) of the polar lipid extract and the mixture was subjected to preparative

304

YOUNG,

QUINLAN, TABLE

AND GOAD 2

IDENTITIESANDRELATIVEPROPORTIONSOFMETABOLITESPRODUCEDONINCUBATION [4-'4c]P~o~~s~ERoNE in vitro WITH STAGE IIOVARY

RF

Band

Identity

0.59 0.48 0.38 0.34 0.28 0.25 0.18 0.15

TLC. 3-one dpm) form HPLC.

Sa-Pregnan-3,20-dione Progesterone 20a-Hydroxy-Su-pregnan-3-one 1,4-Pregnadiene-3,20-dione 3j3-Hydroxy-Sa-pregnan-20-one 20a-HydroxypregM-en-3-one 5a-Pregnan-3P,20a-do1 Unidentified

The eluted 20ol-hydroxy-5a-pregnancontained 8490 dpm. A part (3000 of this material was derivatized to the benzoate and analysed by RPOne major peak was detected by

3 0

I 5

10

15 Time (mid

FIG. 3. HPLC analysis of the benzoate derivatives of (A) band 1 produced on in vitro incubation of [4-‘4C]progesterone with stage II ovary of P. monodon (detected by the radioactivity monitor), and (B) authentic 5a-pregnan-3,20-dione (detected by the uv monitor).

OF OF

P.monodon Relative abundance (%I 34.9 13.8 28.3 1.1 12.7 1.2 2.2 5.6

the radioactivity monitor and this cochromatographed with the authentic steroid benzoate. These data indicated that band 3 was 200-[4-‘4C]hydroxy-5a-pregnan-3-one. The RF of band 4 (0.34) was consistent with that of 1,4-pregnadiene-3,20-dione (0.34). Authentic 1,4-pregnadiene-3,20dione (50 pg) was added to a portion (200,000 dpm) of the polar lipid extract and the mixture was subjected to preparative TLC. The eluted steroid contained 2090 dpm. On RP-HPLC analysis of this fraction one peak was detected by the radioactivity monitor and this cochromatographed with the authentic steroid. These data indicated that band 4 was 1,4-[4-14C]pregnadiene3,20-dione. Androstenedione and estrone have RFs which are similar to those of 20o-hydroxy.5a-pregnan-3-one and 1,4-pregnadiene3,20-dione (Table 1) and thus, may also have been present as radioactive metabolites in bands 3 and 4. However, on benzoylation of the 20a-hydroxy-Sa-pregnan3-one fraction (band 3) androstenedione, if present, could not form a benzoate derivative while any estrone would give a monobenzoate, and these compounds would separate from 20a-hydroxy-5apregnan-3-one benzoate on subsequent RPHPLC. Similarly, androstenedione and estrone do not cochromatograph with 1,4pregnadiene-3,20-dione on RP-HPLC . As

STEROID

METABOLISM

radioactivity cochromatographed only with 20a-hydroxy-.Sx-pregnan-3-one benzoate and 1,4-pregnadiene-3,20-dione on analysis of bands 3 and 4, respectively, it can be concluded that little or no metabolism of progesterone to estrone and androstenedione had occurred. The RF of band 5 (0.28) was consistent with that of another reduced product of 5~ pregnan-3,20-dione, namely, 3@hydroxy5ol-pregnan-20-one (0.28). Authentic 3phydroxy-5a-pregnan-20-one (100 p,g) was added to a portion of the polar lipid extract (40,000 dpm) and the mixture was subjected to preparative TLC. The eluted 3@hydroxy-5a-pregnan-20-one contained 5 110 dpm. A part of this material (3000 dpm) was derivatized and analysed by RP-HPLC. The one peak of activity detected by the radioactivity monitor cochromatographed with the authentic steroid, which indicated that band 5 was 3p-[4-i4C]hydroxy-Sapregnan-3-one. Band 6 had an RF (0.25) consistent with that of a reduction product of progesterone, namely 20a-hydroxypregn-4-en-3-one (0.25). Authentic 20a-hydroxypregn-4-en3-one (50 pg) was added to a portion (200,000 dpm) of the polar lipid extract and subjected to preparative TLC. The eluted steroid contained 2390 dpm. On analysis of the fraction by HPLC a radioactive peak was detected by the radioactivity monitor and this cochromatographed with the authentic steroid. This indicated that band 6 contained 20a-[4-i4C]hydroxypregn-4-en-3one. Band 7 had a RF (0.18) which was consistent with that of Sol-pregnan-3l3,20ol-diol and testosterone. However, investigation showed that the RP-HPLC system employed for steroid benzoate analysis easily resolved the dibenzoate of Sa-pregnan3@,20o-diol from the monobenzoate of testomsterone. Thus, authentic Sol-pregnan3/$2Oo-diol (50 pg) was added to a portion (200,000 dpm) of the polar lipid extract and

IN P. monodon

305

the mixture was subjected to preparative, TLC. The eluted 5ol-pregnan-3P,20a-diol contained 4310 dpm. The fraction was derivatized to form the dibenzoate and applied to the RP-HPLC system. One peak was detected by the radioactivity monitor and this cochromatographed with the authentic Sol-pregnane dibenzoate. This indicated that band 7 was 5ol-[4-‘4C]pregnan3p,20cu-diol and that there was negligible radiolabelled testosterone. The RF of the minor metabolite (0.15) forming band 8 did not correspond to any of the RFs of authentic steroids. From the mobility of this compound on TLC, however, it appears that it may be a polyhydroxylated steroid but it was not investigated further. The identities of the metabolites of [4-i4C]progesterone and their re1ativ.e amounts (the radioactivity recovered with the authentic steroid expressed as a percentage of the total radioactivity applied to the preparative TLC plate) are summarized in Table 2. The in Vitro Metabolism of [4-‘4CJProgesterone by Stage 0 (Immature) Ovary of P. monodon The lipid extract from this incubation contained 2.9 l&i, representing a recovery of radioactivity of 57.1%. The radio-TLC scan produced after chromatography of an aliquot of the extract by analytical TLC is shown in Fig. 2B. The distribution of radioactivity was confirmed by autoradiography of the plate. Four bands of radioactivity (not including the origin) were observed and their identities were determined by comparison of their RFs with those of the metabolites identified after incubation of the same steroid with stage II ovary. Identifications made in this way were confirmed by comparing the RT of the metabolite with that of the authentic steroid on RP-HPLC. The identities and relative amounts of the. metabolites are given in Table 3.

306

YOUNG, TABLE

QUINLAN,

AND GOAD

dpm

3

IDENTITIES.~NDREI.ATI~I~PROP~RTI~NS 0F METABOLITESPRODUCED ON~NCUBATIONOF [4-'4c]PROGESTERONE in vitro WITH STAGE 0 OVARY OF P. monodon

Band 1 2 3 4

Identity

Hepatopancreas

Relative abundance m

5a-Pregnan-3,20-dione Progesterone 3P-Hydroxy-Sn-pregnan-20-one 5a-Pregnan-3P,20a-diol

0.7 95.4 2.3 1.7

Time-Course Study of the Metabolism of [4-14C]Progesterone by Stage 0 Ovary of P. monodon

Incubations were performed at 18” for 30 min, 1 hr, 2 hr, 4 hr, and 8 hr and the recoveries of radioactivity from these incubations were 4.3 l&i (86%), 4.0 PCi (80%), 4.2 t&i (85%), 4.5 t&i (90%), and 4.7 t&i (94%), respectively. Aliquots (ca. 1%) of these extracts were examined on analytical TLC and the distribution of activity was assessed by TLC plate-scanning and confirmed by autoradiography. The identities of the radioactive metabolites tiere determined as in the previous incubations. A progressive increase in the amount of 3phydroxy-5a-pregnan-20-one was seen as the incubations proceeded. However, in the 8-hr incubation an additional metabolite was detected in small amount and identified as 5a-pregnan-3l3,20a-diol. The in Vitro Metabolism of [4-14C]Progesterone by the Gill, Hepatopancreas, and Abdominal Muscle of P. monodon

The lipid extracts from the gill, hepatopancreas, and muscle contained 3.7, 3.0, and 3.2 $i, respectively, representing recoveries of radioactivity of between 60 and 75%. Radio-TLC scans produced after chromatography of portions of these ex-

1

&Q0

SF

Or

1 20cm

i

4. Radio-TLC-scan of the 20-cm TLC plate following separation of the lipid extracts from in vitro incubations of [4-‘4C]progesterone with hepatopancress, gill and muscle of P. monodon. The RF of authentic progesterone is indicated by P. Or, origin; SF, solvent front. For identities of peaks see Table 4. FIG.

tracts by analytical TLC are shown in Fig. 4. A comparative radio-TLC scan for ovary from the animal at the same developmental stage is shown in Fig. 2B. Autoradiograms of the plates confirmed this distribution of radioactivity. The RFs of the bands for each tissue were compared with those of radioactive metabolites identified after incubation of [4-14C]progesterone with stage II ovary of P. monodon, and their identities were confirmed by RP-HPLC as before. The identities of the radioactive metabolites and relative amounts for each tissue are given in Table 4. An additional metabolite, noted in the case of the hepatopancress (peak 1, Fig. 4), had an RF (0.70) consistent with that of a low-polarity steroid ester. One of the prominent metabolites produced by the hepatopancreas could not be identified with the range of authentic steroids available.

STEROID

IDENTITIES [%14G]P~o~~s~~~o~~

METABOLISM

IN P. monodon

307

TABLE 4 AND RELATIVE PROPORTIONS OF METABOLITES PRODUCED in vitro WITH HEPATOPANCREAS, GILL, AND ABDOMINAL

ON INCUBATION OF MUSCLE OF P. monodon Relative

Tissue

Band

Identity

abundance (%I

Hepatopancreas

1 2 3 4 5 6

Fatty acyl steroid ester So-Pregnan-3,20-dione Progesterone Unknown 3&Hydroxy-5-a-pregnan-20-one So-Pregnan-3P,20a-diol

6.1 9.2 33.6 13.1 4.7 11.6

Gilt

1 2 3 4

5a-Pregnan-3,204one Progesterone 3P-Hydroxy-5o-pregnan-3-one 5-ol-Pregnan-3/3,20a-diol

1.3 60.3 29.5 8.9

5ol-Pregnan-3,20-dione Progesterone 3&Hydroxy-So-pregnan-20-one 5a-Pregnan-3I3,20ol-diol

1.2 92.7 I.5 4.5

Muscle

The in Vitro Metabolism of [4-14C]Progesterone by Stage II Ovary of N. norvegicus

The lipid extract from this incubation contained 3.2 pCi, representing a recovery of radioactivity of 64%. On radio-TLC scanning of the analytical TLC plate two bands of activity were detected and autoradiography of the plate confirmed this distribution. ,Band 1 had an RF (0.49) which corresponded to that of authentic progesterone (0.49) and band 2 had an RF (0.27) which corresponded to that of 2Ocr-hydroxypregn4-en-3-one (0.26). An aliquot of the extract (500,000 dpm) was taken and authentic progesterone (200 pg) and 20o-hydroxypregn4-en-3-one (100 p,g) were added. This was chromatographed on preparative TLC, the areas of silica corresponding to the two markers were scraped, and the radioactivity associated with each fraction was determined. It was found that 97.4 and 2.6% of the recovered activity cochromatographed with progesterone and 20a-hydroxypregn4-tin-3-one, respectively. The fractions were analysed by RP-HPLC as shown in Fig. 5. ‘The two radioactive compounds co-

chromatographed with the marker steroids indicating that band 1 was unchanged [4-‘4C]progesterone and band 2 was 20~ [4-‘4C]hydroxypregn-4-en-3-one. DISCUSSION All the tissues of P. mono&n studied metabolised [4-r4C]progesterone but to varying extents. Stage II ovary of P. m?onodon displayed a relatively high rate of metabolism, converting 86.2% of the added progesterone in 5 hr, with the most abundant metabolites being Sa-pregnane derivatives. The formation of similar steroids from progesterone has been demonstrated in the ovary and pyloric cecae of the starfish, Asterias rubens (Fleming, 1976; Schoenmakers and Voogt, 1980; Voogt et al., 1986), and in the ovary of the hagfish, Myxine glutinasa (Kime and Hew, 1980) The present study is the first to report the production of such metabolites in crustaceans. Two minor metabolites were identified as A”-pregnenes, specifically ~OCYhydroxypregn-4-en-3-one and I ,4-pregnadiene-3,2@dione, The former steroid has also been identified as the only product of

308

YOUNG,

QUINLAN,

GOAD

A

A *

10

AND

15

1 20

tlhqlpdhk

I 25 Time(min)

, 10

IJ 15

I 20

, 25 TimeCmin)

FIG. 5. HPLC analysis of (1) band 1 and (2) band 2 detected after in vitro incubation of [4-14C]progesterone with stage II ovary of N. norvegicus showing (A) radioactive metabolite (detected by the radioactivity monitor) and (B) authentic steroid (detected by the uv monitor).

ovarian progesterone metabolism in the shore crab, Curcinus maenas, and the latter as a product of progesterone metabolism by the gill of the same species (Hazel, 1986). From the structures of the radioactive metabolites identified in this study, a pathway for the metabolism of progesterone by stage II ovary of P. monodon can be proposed (Fig. 6). The major steps involve (i) reduction of the A4-bond in progesterone to form 5a-pregnan-3,20-dione, (ii) reduction of the 3,20-0x0 groups of the latter steroid to form either 3P-hydroxy-Sol-pregnan-20one or 20a-hydroxy-5a-pregnan-3-one, and (iii) further reduction of the latter two steroids to form 5a-pregnan-3l3,20ol-diol. These steps would require So-reductase, 3-oxo-steroid reductase, and 20-oxo-steroid reductase enzymes. The minor steps in-

volve (i) the introduction of a double bond at the Al-position in progesterone to form 1,4-pregnadiene-3,20-dione, and (ii) reduction of progesterone at C-20 to form 20~ hydroxypregn-4-en-3-one. These steps would require desaturase and 20-0x0steroid reductase enzymes, respectively. Further reduction of 2Oo-hydroxypregn-C en-3-one to form 20ol-hydroxy-5a-pregnan3-one, catalysed by a So-reductase enzyme, is also a possibility. The pattern of progesterone metabolism in stage 0 ovary differed from that observed in stage II ovary and may be of some significance. In stage 0 ovary only 4.7% of the added progesterone was converted during the incubation compared with 86.2% in the more mature tissue. In the case of the immature tissue only the Sa-pregnanes were

STEROID

METABOLISM

1.4.Pregnadlene-3.20.dione

POa-Hydroxypregn-4-en-3-one

3BHydroxy-Sa-pregnan-20one

Boa-Hydroxy-Su-pregnan-3-one

il Sa-Pregnan-3B,ZOa-dlol FIG. 6. Proposed pathway for the metabolism of [4-‘4C]progesterone in vitro by stage II ovary of P. monodon, showing the nature of the enzymes involved. Enzymes: 1, Sa-reductase; 2, 20-oxo-steroid reductase; 3, 3-oxo-steroid reductase; 4, A’desaturase

detected, the most abundant being 3phydroxy-5o-pregnan-20-one. This contrasted with the stage II ovary, in which 20o-hydroxy-5a-pregnan-3-one was the most abundant metabolite. It appears that the relative activities of the 20-oxo-steroid and 3-oxo-steroid reductase enzymes are markedly different, with the activity of the 20-oxo-steroid reductase relative to the 3-oxo-steroid reductase increased during ovariandevelopment, as is the overall metabolic capacity of the tissue. Although 201~ hydroxypregn-4-en-3-one was identified as a metabolite of progesterone in this study, its presence as an endogenous steroid in this species has not been observed (Fairs et

IN P. monodon

3Q9

al., 1990). Also, the presence of endogenous Sol-pregnanes in ovary of P. monodon remains to be demonstrated. In a parallel study of progesterone metabolism in stage II ovary of N. florvegicH,r only one metabolite, 2Oa-hydroxypregn-en-3-one, was produced in low yield (2.6% of the starting material) indicating the presence of a 20-oxo-steroid reductase enzyme in the maturing ovary. The relatively low level of activity of this enzyme compared to that in the ovary of P. monodon at the same stage of development may be due to species differences or the lower incubation temperature to which N. norvegicus was exposed. 20a-Hydroxypregn-4-en-3-one has been shown to be an endogenous steroid in stage II ovary of N. norvegicus (Fairs et aE., 1989). The time-course study of progesterone metabolism by stage 0 ovary of P. rnonodon supported the observation that 3@-hydroxy5ol-pregnan-20-one is the more abundant metabolite in this tissue, accounting for 24.7% of the activity after 8 hr incubation and the results were consistent with the proposed metabolic pathway (Fig. 6). A small amount of 2Ocu-hydroxy-5ol-pregnan3-one was produced during the incubation, accounting for 3.8% of the activity after 8 hr incubation. Also, 5cw-pregnan-3@-20ol-diol was detected after 8 hr incubatian; It appears that the reduction of the A4-bond and the 0x0 group at the C-3 position of progesterone occurs rapidly, as these metabohtes could be observed after 30 min incubation, whereas the reduction at C-20 occurs much more slowly, presumably a reflection of the low activity of 20-oxo-Steroid reducrase in this tissue. Lack of adequate ovary tissue precluded a similar time-course study of the more mature stage II ovary. Of the other P. monodon tissues studied, by far the most active was the hepatopancress, metabolising 65.3% of theadded progesterone during the incubation This is perhaps not surprising as the hepatopancress is the major metabolic and digestive

310

YOUNG,

QUINLAN,

organ of the decapod crustacean (Gibson and Barker, 1979). In addition to a range of 5ol-pregnanes, fatty acyl esters of steroids were also detected. The production of such fatty acyl esters in vivo in the hepatopancress of Car&us maenas has also been reported (Hazel, 1986), and esterification may be related to steroid storage and/or inactivation. The gill was also relatively active in terms of steroid metabolism, converting 39.7% of the added progesterone to a variety of So-pregnanes. A low level of metabolism was observed in the muscle, with only 7.3% of the added progesterone reduced to Sex-pregnanes. Of the three tissues examined, only the hepatopancreas appeared to possess an active 20-0x0steroid reductase. The actions of enzymes such as 17~ hydroxylase, C,,-C,, lyase, 1701hydroxysteroid dehydrogenase, and aromatase, which catalyse the conversion of progesterone to androgens and estrogens in vertebrates, were not demonstrated in the crustacean tissues studied. As both androgens and estrogens occur in crustacean tissues (Nikitina et al., 1977; Jeng et al., 1978; Burns et al., 1984b; Ollevier et al., 1984, 1986; Couch et al., 1987; Van Beek and De Loof, 1988; Fairs et al., 1989, 1990) their origin is a topic which requires further investigation, particularly as Swevers et al. (1991) suggested that they may be of dietary origin. The significance of the production of both the Sol-pregnanes, and the two A4pregnenes in stage II ovary of P. monodon, has yet to be determined. It is difficult to assign a specific role to the Sew-pregnanes, particularly as they were formed in all tissues studied. The A4-pregnenes, which were observed in the vitellogenic ovary only, may be of more importance in terms of hormonal control of ovarian development and their functions certainly warrant further investigation. The role of 20ahydroxypregn-4-en-3-one could be of particular importance as it has been observed

AND

GOAD

as a metabolite of progesterone in vitellogenie ovary of both P. monodon and N. nowegicus. It may be relevant that vitellogenesis in prawns is stimulated by exogenous hydroxylated progesterone derivatives such as 17a-hydroxyprogesterone (Nagabhushanam et al., 1980; Yano, 1987). ACKNOWLEDGMENTS The authors thank Unilever Research for providing the Penaeus monodon. This work was supported by the Science and Engineering Research Council, Great Britain, and Unilever Research, Colworth House, Bedford.

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