Identification of, and development of radioimmunoassays for 17α,21-dihydroxy-4-pregnene-3,20-dione and 3α,17α,21-trihydroxy-5β-pregnan-20-one in the ovaries of mature plaice (Pleuronectes

GENERAL AND COMPARATIVE ENDOCRINOLOGY T&273-28.5 (1990)

Identification of, and Development of Radioimmunoassays for 170~,21 -Dihydroxy-dpregnene-3,20-dione and

3~~,17~~,21-Trihydroxy-5p-pregnan-20-one in the Ovaries of Mature Plaice (Pleuronectes platessa)’

A. V. M. CANARIO* AND A. P. SCOTT?

*Universidade do Algarve, Camp0 de Gambelas, Apartado 322, 8004 Faro Codex, Portugal; and tMinistry of Agriculture Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft,

Suffolk NR33 OHT, England

Accepted June 26, 1989

Ovaries from a female plaice (Pleuronectes platessa) that had been injected with human chorionic gonadotrophin were incubated in vitro with l’la-hydroxy[ 1 ,2,6,7-3H]progesterone. The major steroids produced by the ovaries were tentatively identified as 17a,21-dihydroxy&pregnene-3,2Odi-one (11-deoxycortisol; 17,21-P), 17a,21-dihydroxy-5R-pregnane- 3,20-dione (3a,17,21-P-5@. A high proportion of these steroids was found in a conjugated form (sulphates or glucuronides). Radioimmunoassays were developed for 1 l-deoxycortisol and for 3a,17,21-P-5g and were applied to fractions of mature male and female plaice plasmas and plaice ovarian incubates that had been separated on thin-layer chromatography. The presence of all three steroids, in vivo and in vitro, was confimxd. Particularly high amounts of conjugated 3a,17,21-P-5p were found in the plasma of mature females (2M ng ml-‘). The 3a,17,21-P-5g radioimmunoassay also identified 3a,l7a-dihydroxy-Sg- pregnane-20-dione in all three fluids, despite the fact that this steroid was not among the radioactive incubation products of the ovary. These findings are compared with those from another flatfish, the dab (Limanda limanda), where the major gonadal steroids have been shown to be 17a,20a-dihydroxy-4-pregnen-3-one and its Sg-pregnane (3-keto and 3g-hydroxyl) metabolites.

It has been demonstrated in teleosts that tion of oocyte maturation in a wide variety the process of oocyte final maturation is un- of teleosts (Scott and Canario, 1987). This der the control of a gonadotrophin(s) se- steroid is released into the plasma of some creted by the pituitary and is mediated by fish in very high amounts (100-800 ng ml-‘) steroids secreted by the follicular cells at the time of oocyte maturation (e.g., rain- (Goetz, 1983). One particular steroid, bow trout, Oncorhynchus mykiss Scott et 17,2013-P,2 has been implicated in the induc-

r The reference to proprietary products in this paper should not be construed as an offtcial endorsement of these products, nor is any criticism implied of similar products which have not been mentioned.

’ Steroids abbreviation: 17,20@P, 17a,20g-dihydroxy-4-pregnen-3-one; 17,2Og,21-P, 17a,20fi, 21-trihydroxy4pregnen-3-one; 17,2Oa-P, 17a,2Oudi- hydroxy-4-pregnen-3-one; 17,2Oa-P-5g, 17a,20a-dihydroxy-5p-pregnan-3-one; 3g,17,20a-P-Sg, 3g,17a,

2Oa-trihydroxy-5g-pregnane; 3a,17,2Oa-P-5p, 3a,17a, 2Oa-trihydroxy-5R-pregnane; 1 I-deoxycortisol, 17,21- dihydroxy-4-pregnene-3,20-dione; 17,21-P-5R, 17a,21-trihydroxy-5g-pregnane-3,20-dione; 3a, 17,21-P-5g,3a,17a,2l-trihydroxy-5~-pregnan-2O-one; 11-deoxycorticosterone, 21-hydroxy-4-pregnene- 3,2Odione; 17-P, 17a-hydroxy4pregnene-3,2O-dione; 3a,17-P-5g, 3a,17a-dihydroxy-5g-pregnan-2O-one; testosterone, 17g-hydroxy4androsten-3-one; androstenedione, 4-androstene-3,lFdione.

273 0016-6480/90 $1.50

274 CANARIO AND SCOTT

al., 1983; and carp, Cyprinus carpio, San- tos et al., 1986). In many other species that have been studied, however (e.g., Atlantic croaker, Micropogonias undulatus, Trant et al., 1986; and dab, Limanda limanda, Canario and Scott, 1989, 199Oa), 17,20@P is a relatively minor product of the ovaries and is released into the plasma in only very low amounts (4 ng ml-‘). Instead, the ovaries of these fish manufacture large amounts of other steroids. In the Atlantic croaker, the major steroid produced by the ovary is 17,2Op,21-P, which is as active as 17,2Op-P in inducing oocyte maturation (Trant et al., 1986; Trant and Thomas, 1988). In the dab, however, the main steroids produced by the ovary are 17,2ti-P, 3P,17,20a-P-5P, and 17,2O~P-5p, none of which is very active in inducing oocyte maturation (Canario and Scott, 1989, 199Ob).

Like the dab, plaice have low 17,2Op-P levels and although 17,2Op-P and 17,20& 21-P are equally effective in inducing in vitro oocyte final maturation, we have not been able to detect 17,2Op,21-P in either species (Scott and Canario, 1987; Scott and Canario, 1990). Both species belong to the same order (Pleuronectiformes) and although, on the basis of phylogeny, similar steroidogenic enzymes should perhaps be expected, preliminary studies indicated that plaice plasma contained only very tiny amounts of 20a-hydroxylated steroids (Scott and Canario, 1990). In addition, the ovaries of another pleuronectiform, the sole (Solea impar), produce mainly ll- deoxycortisol (Colombo and Belvedere, 1977). The present study was therefore un- dertaken to identify, and to develop specific radioimmunoassays for, the main steroidal products of plaice ovaries.

MATERIALS AND METHODS

Solvenrs and reagents. Dioxane, activated alumina, chloramine-T, sodium metabisulphite, and histamine were. obtained from BDH Limited, Dagenham, Essex, England. Sodium azide (NaN,) and EDTA were ob-

tained from Sigma Chemical Co., Poole, Dorset, En- gland. [1251]sodium iodide was obtained from Amer- sham International, Little Chalfont, Buckinghamshire, England. The origins and grades of other solvents, steroids, and of the radioimmunoassay, oocyte incuba-

tion, and steroid identification reagents have been published elsewhere (Canario and Scott, 1988, 1989; Canario et al., 1989).

Chromatography and steroid derivative formation. Details about the thin-layer chromatography (TLC) plates, chemical reactions, and methodology for steroid identification have already been reported by Ca- nario and Scott (1989). The TLC plates were developed at room temperature in one of the following solvent systems: (I) ethyl acetate:petroleum ether (50:50); (II) chloroform:methanol(47.5:2.5); (III) chloroform: methanol (49.5:0.5); (IV) chloroform:methanol:water (45:5:0.25).

Fish. Adult plaice were caught either by commercial trawlers or by MAFF research vessels in the southern North Sea and kept in the laboratory for several months before being used.

Incubation of ovarian tissue with radioactive steroids. One plaice female, weighing 370 g, was injected intramuscularly with a dose of 140 IU human chorionic gonadotrophin (HCG) dissolved in 500 pl of 0.8% NaCl. Forty hours later, the fish was bled, decapi- tated, and the ovaries removed. Some oocytes had already undergone germinal vesicle breakdown. About 350 follicles were dissected out and incubated with 10 $i 17a-hydroxy[1,2,6,7-3H]progesterone in 2 ml of medium for 6 hr at 12”, as described by Canario and Scott (1989). The steroids were also extracted as described previously (Canario and Scott, 1989). How- ever, the aqueous phase (after extraction) was mixed with 2 ml 0.1 M acetate buffer, pH 5.0, containing 50 ~1 snail (Helix pomatia) juice, and incubated for 24 hr at 37” in order to hydrolyse steroid conjugates. These

steroids were treated in the same way as those extracted from the “free” steroid pool. Prior to extraction, 10 bg each of 17,208,21-P, 17,21-P, 17-P, and androstenedione were added to the free steroid fraction, and 10 pg each of cortisol, 17,2Oa-P, 17,2Of3-P, and testosterone to the conjugated steroid fraction. These were used as reference standards and were detected on the TLC plates by their uv absorption. The chromatograms were developed fmt in solvent system I to separate nonpolar material and then in solvent system II. They were divided into 0.5-cm strips which were scraped off and eluted with ethanol (3 x 1 ml). A 20-pl aliquot of each eluate was counted for radioactivity. The main radioactive peaks were stored at 4” for subsequent chemical analyses.

The radioactive products were tentatively identified by their isopolarity with cold standards and by their isopolarity with at least one chemical derivative.

Production ofantisera and ‘%labelled 3a,l7-P-5& The 11-deoxycortisol and 3a,17,21-P-5fi antisera were

STEROIDS IN PLAICE OVARY 275

raised in rabbits against 1 l-deoxycortisol- 3-(OcarboxymethyBoxime (ll-deoxycortisol-3-CMO) and 3a,17-P-SB-20-(O-carboxymethyl)oxime (3a,17-P-SB-2OCMO) linked to bovine serum albumin (BSA). The l l-deoxycortisol-3-CM0 was synthesised according to the procedure of Janoski et al. (1974) using 200 mg of steroid and the 3a,17-P-5B-2O-CMO was synthesised according to the procedure of Kohen et al.

(1975) using 50 mg of steroid. The CM0 derivatives were conjugated to BSA (400 and 200 mg amounts, respectively) according to the procedure of Kellie et al. (1975). The degree of incorporation of ll- deoxycortisol-CM0 to BSA was checked using a uv spectrophotometer and was found to be 18 mol of steroid mol-i of protein. The degree of incorporation of 3a,17-P-58-2OCMO was not checked because it did not absorb uv.

A 3a,17-P-5B-CMG-histamine-1*51 conjugate was used as the label in the 3a,17,21-P-58 radioimmunoassay. It was prepared exactly as described by Cook and Beastall (1987).

RIA procedures. The 1 I-deoxycortisol IUA procedure was the same as for other tritiated label assays already described (Scott et al., 1982; Canario et al., 1989). The 3a,17,21-P-58 procedure diiered from the 1 I-deoxycortisol procedure in the following ways:

(a) the assay buffer consisted of 2 g liter-’ BSA (instead of gelatine), 8 g liter-’ NaCl, 0.3 g liter-’ EDTA, and 0.1 g liter-’ sodium azide. BSA effec- tively prevented nonspecific binding of label to the surface of the borosilicate culture tubes (L.I.P. Ltd, Shipley, England) used for the assays;

(b) ca. lo4 cpm of tracer were added to each tube (instead of ca. 1.5 x ld cpm in the 11-deoxycortisol assay);

(c) the antiserum and steroid mixture (labelled and unlabelled) was allowed to incubate for either 2 hr at room temperature or overnight at 4”; the dilution of antiserum to obtain ca. 60% binding in the absence of unlabelled steroid was adjusted accordingly. Both antisera operated at final dilutions of between l/70,008 and 1/200,080;

(d) bound and unbound label was separated with dextran-coated charcoal suspended in phosphate/ gelatine buffer (Scott et al., 1982) but the supematant was aspirated and discarded and the charcoal pellet (which retained the unbound label fraction) was counted.

Collection of plasmas and ovarian incubates for RIA. Plasmas were collected from three mature males and three mature females which had been injected 36 hr and then 12 hr previously with HCG (100 and 300 IU kg-*). The fish were bled through the caudal vein using heparinized syringes fitted with 22-gauge needles. Blood cells were separated by centrifugation and plasmas were stored at -20”.

The incubate was obtained from 5 g of ovarian tissue, which was incubated for 72 hr at 10-15” in 30 ml

of ringer containing an homogenate of freshly collected plaice pituitaries (0.5 pituitary ml- l), The iucu- bation procedure is described in more detail in Canario and Scott (1988).

Extraction and TLC of samples for RIAs. The extraction and TLC procedures were the same as those described in Canario and Scott (199Oa), except that the extractions were carried out with 5 ml of diethyl ether: heptane (4:1, v/v) and that the dried-down extracts were partitioned between 90% methanol (1.5 ml) and heptane (1.5 ml). These procedures prevented fatty polar substances from being carried through to the TLCs. The procedures were carried out on 250 ul of plaice female plasma, 250 pl of plaice male plasma, and 1 ml of ovarian incubation medium.

Testing for the presence of a IO-keto group. The two major peaks of immunoreactivity in the plaice plasma and incubate were tentatively identified as 3a,17, 21-P-58 and 3a,17-P-58. In order to conhn that these steroids were unreduced at the 20 position, a further l-ml aliquot of the ovarian incubate was extracted, hydrolysed with snail juice, and separated on TLC. The areas on the TLC plate corresponding to the po sitions of these steroids (both free and conjugated fractions 5 and 10) were eluted with 1 ml dichlo- romethane:ethanol (8:2) and then split between two glass tubes. After evaporation of the solvents, 250 ~1 of Tris buffer, 0.05 M, pH 7.6, was added to one of the tubes (“control”), and 250 p,l of the same buffer, containing 0.4 units 208-HSD, to the other (“208-HSD”). All tubes received a further 250 pl buffer containing NADH (2 mg ml-‘). Standards (3a,17,21-P-58 and 3a,17-P-58) were treated in the same way. The tubes were incubated for 3 hr at room temperature with gen- tle shaking and then extracted and recbromatographed on TLC system II. Fractions (0.6 cm) were eluted with 1 ml BSA assay buffer and reassayed for 3a,17,21-P-5g.

RESULTS

Incubations of Ovarian Tissue with Z7-[3HJP

A high percentage of the radioactive Czl steroid metabolites produced by the plaice ovary (Table 1) were 21-hydroxylated (73%)) 57% were conjugated, and 43% were SP-reduced. Less than 5% of the metabolites were in the form of C19 steroids. The unidentified steroids comprised a number of small peaks, each with less than 1% of the total radioactivity. The main peak in the free fraction (14%) comigrated with the 17- P standard and was not studied further.


TABLE 1 PERCENTAGEOFYIELD,DER~VATIZATIONREACTIONS,AND TLC SYSTEMS USEDINTHE~DENTIFICATIONOF

THEFREESTEROIDSPRODUCEDBYAGONADOTROPHIN-STIMULATEDPLAICEOVARYINCUBATEDWITH I%-HYDROXY[I,2,6,%'H]PROGESTERONE

Metabolite Free Conjugated

3a,17,21-P-5B 4.8 23.4 17,21-P 9.4 20.1 17,21-P-5B 1.0 14.1 Testosterone 1.9 1.3 17-P 13.8 - Androstenedione 1.6 - Unidentified 5.6 3.0 Total 38.1 61.9

TLC systems0 and reaction@

1,II; ac+, III; bis’, III; 20B-HSD,,+, IV; 3a-HSD,,+, II. 1,II; ac+, III; his+, III; 20B-HSD,d+, IV. 1,II; acf, III; his+, III; 20B-HSDsd+, IV; 3a-HSDd+, IV. IJI; ac+, III. 1,II. IJI; ac-, III.

D Roman numerals refer to the different TLC systems described under Materials and Methods. b ac, acetylation; bis, bismuthate; HSD, hydroxysteroid dehydrogenase; red, reduction; ox, oxidation. A plus

indicates that a derivative with the same mobility as the expected product was formed; a negative indicates that no modification occurred.

The three main metabolites, which were more abundant in the conjugated fraction, had Rp of 0.23,0.47, and 0.52 on TLC system II. Acetylation of the steroid with Rf 0.23 yielded a diacetate. Acetylation of the other two steroids yielded monoacetates. All three compounds could be oxidized with sodium bismuthate, indicating the presence of a 17@-side chain, and at least an extra hydroxyl group, in addition to the one (already present in the precursor) attached to carbon 17 (Bush, 1961). The relative polarities of the three oxidation products were the same as in the intact steroids. The derivative from the steroid with R,-0.23 comigrated with 3ol-hydroxy-SP-androstan- 17-one; the derivative from the steroid with Rf 0.47 comigrated with androstenedione; the derivative from the steroid with Rf 0.52 comigrated with SP-androstane-3,17-dione.

All three steroids could be reduced with %OP-HSD, indicating that the extra hydroxyl on the side chain must be attached to the carbon 2 1.

From the results with the sodium bismuthate and 20@-HSD, it could be inferred that the differences among the three steroids lay only in the steroid nucleus. Using the enzyme 3a-HSD, it was possible to ox- idize the steroid with Rf 0.23, obtaining a

steroid with Rf0.52, and to reduce the steroid with Rf 0.52 to a steroid with Rf 0.23. The steroid with Rf0.47 could not be mod- itied by the enzyme. This indicated that there was only a single (reduced) bond between carbons 4 and 5 in the Rf 0.23 and 0.52 steroids but a double (A’) bond in the Rf0.47 steroid. It also indicated that the Rf 0.52 steroid was an oxidized form of the Rf 0.23 steroid (3-keto vs 3a-hydroxyl).

The polarities of the Rf0.23 and 0.52 steroids suggested that they were unlikely to be 5a-reduced. The weight of evidence suggested that the Rf 0.23 and 0.52 steroids were most likely to be 3a,17,21-P-5p and 17,21-P-5p. The chromatographic properties of the Rf0.47 steroid and its derivatives suggested that it was 1 I-deoxycortisol (17,21-P).

Fragments of ovarian tissue were also incubated with [4,7-3H]pregnenolone (10 t&i). The main peaks coincided with those from the 17-r3H]P incubation. Up to 25% of precursor remained, however. The products were not further identified.

Antisera Cross-Reactions

The cross-reactions of the ll-deoxycortisol antiserum and of the two 3a,17-P-


5p antisera (RIA 1 and RIA 2) with syn- significantly with cortisone (2%) and corti- thetic steroids are shown in Table 2. sol (0.8%).

The 1 1-deoxycortisol antiserum tended to cross-react with steroids containing 17- and/or 21-hydroxyl groups, but not with those with a 20-hydroxyl group. It also had a moderate to high cross-reaction with 5a- or SP-reduced derivatives of 1 l-deoxycortisol, except for 3a,17,21-P-5P (only 0.38%). The antiserum also cross-reacted

The two antisera raised against 3a,17- P-5p had slightly different patterns of cross- reactions. As expected, both antisera cross-reacted more than 100% with 3a,17- P-5p. The RIA 2 antiserum had a lower cross-reaction with steroids with a 4-ene structure and a higher cross-reaction with steroids with a 20-hydroxyl group (a or l3)

TABLE 2 CROSS-REACTIONS OF VAIUOUS STEROIDS WITH THE 11-DEOXYCORTISOL (S) AND 3a-17,21-P-5B (RIA 1 AND

RIA 2) ANTISERA

Cross-reaction’

Systematic name S RIAl RIA2

4-Pregnene-3,20-dione 17a-Hydroxy4pregnene-3,20-dione 2Oa-Hydroxy4pregnen-3-one 21-Hydroxy4pregnene-3,20-dione 17a,2Oa-Dihydroxy-4-pregnen-3-one 17a,20B-Dihydroxy-4-pregnen-3-one 17a,21-Dihydroxy-4-pregnen-3-one 17a,21-Dihydroxy4pregnene-3,1l-dione 2Oa,2l-Dihydroxy-4-pregnene-3,20-dione 20g,21-Dihydroxy-4-pregnene-3,20-dione 11/3,17a,21-Trihydroxy4pregnene-3,2O-dione 17a,2Oa,2l-Trihydroxy-4-pregnen-3-one 17a,208,21-Trihydroxy&pregnen-3-one 38,17a,2Oa-Trihydroxy-5-pregnene 3g,17a,20g-Trihydroxy-S-pregnene 3a,17a,208-Trihydroxy-5-pregnane 3g,17a,20B-Trihydroxy-Sa-pregnane 3B,17a,21-Trihydroxy-5a-pregnan-2O-one 17a,21-Dihydroxy-5B-pregnane-3,2O-dione 3a-Hydroxy-Sg-pregnan-20-one 38-Hydroxy-5B-pregnan-20-one 17a-Hydroxy-5B-pregnane-3,2Odione 3a,l7a-Dihydroxy-5B-pregnan-2O-one 3a,17a,2Oa-Trihydroxy-5g-pregnane 3a,17a,20B-Trihydroxy-Sg-pregnane 3a,17a,2l-Trihydroxy-5g-pregnan2O-one 3p,l7a,21-Trihydroxy-5~-pregnan-20-one 3a$a,17a,2Oa-Tetrahydroxy-58-pregnsne 3a$a,17a,208-Tetmhydroxy-5g-pregnsne 3a,11~,17a,21-Tetrahydroxy-5g-pregnan-2O-one 3a,17a,20B,21-Tetrahydroxy-SB-pregnane 178-Hydroxy4androstene-3-one 3a-Hydroxy-SB-a&o&n-17-one

0.27 2.2

co.01 6.7 0.03

CO.01 100

2.0 CO.01

0.06

0.84 CO.01

CO.01 CO.01

CO.01 -

X0.01

13.0* 10.3 - -

0.13 CO.01

-

CO.01 0.38 1.9

- - -

CO.01 co.01

-

- 1.3 - 0.11 0.14 0.05 1.1 -

- 0.04 -

CO.01 CO.01 <O.Ol

co.05 -

- - -

0.06

- CO.01

- - -

CO.01 CO.01

0.01 CO.01 CO.01

13.76 5.2

<0.08 14.36

200 6.8 1.8

100 0.87

<O.Ol -Co.01

CO.01 3.0

-

CO.01

<O.Ol 0.13

<O.Ol -

0.26 5.6

co.05 0.63

150 19.7 7.3

100

0.70 co.01 <O.Ol

7.96 6.4

- - 0.25 0.76

LI Cross-reactions = 100 x mass of S needed to displace 50% of the label/mass of X needed to displace 50% of the label, in which S is the authentic steroid and X is the competing steroid.

* Steroid dilution curve is not parallel to authentic steroid standard curve.


in comparison to the RIA 1 antiserum. Un- like RIA 2, RIA 1 had a substantial (though nonparallel) cross-reaction with 17,21- P-5p. The cross-reactions of both antisera with 5-ene or So-reduced steroids was low.

Immunoreactivity with the ll-Deoxycortisol RIA

Female plasma (Fig. I top). In both the free and conjugated fractions, three peaks of 1 1-deoxycortisol immunoreactivity were found. The least polar peak corresponded to the elution position of the tritiated ll- deoxycortisol standard and overlapped to some extent the position of the 17,21-P-@ standard. The most polar peak fell across the elution positions of 3a,17,21-P-5P and cortisol (both of which cross-react to a small extent with the antiserum; see above). The middle peak corresponded to the elution position of cortisone. The positions of cortisol and cortisone were established by means of a cortisol-binding globulin assay (Wingfield and Grimm, 1977). The immunoreactivity in the ll-deoxycortisol peak was equivalent to a plasma level of 4 ng ml- ’ in the free fraction and 15 ng ml- ’ in the conjugated fraction.

Ovarian incubate (Fig. 2 top). Almost no 1 l-deoxycortisol immunoreactivity could be detected in the free fraction of the ovarian incubate. However, there was a substantial amount in the conjugated fraction, the bulk of it being coincident with the ll- deoxycortisol standard. The immunoreactivity in the ll-deoxycortisol peak was equivalent to cit. 0.1 ng ml- ’ in the free fraction and 12 ng ml- ’ in the conjugated fraction.

Male plasma (Fig. 3 top). The three peaks found in female plasma were also present in male plasma. However, the over- all immunoreactivity was lower than in the females, and the percentage of nonspecific immunoreactivity was higher. The immunoreactivity in the 1 l-deoxycortisol peak

300. A pa,17,2w--5pRyIy

200. ‘I : : : :

loo- / :

/ 0, o- -----AA -_ . . . . __-__

-!- 2 31 -- I ~,~,.,~,~,.,.I. I ,I.,

0 2 4 6 6 10 12 14 16 18 20

fraction number (0.6 cm)

FIG. 1. TLC scan of steroid immunoreactivity in a pool of plasmas from three female plaice. Free steroid levels are shown a factor of five times greater than conjugated steroids in order to emphasise the peaks, which were smaller. Top graph: 1 1-deoxycortisol immunoreactivity. Middle and bottom graphs: 3a,17, 21-P-5g immunoreactivities using two different antisera @IA 1 and 2). Note differences in the ordinate scales. Fraction 0 is the origin of the chromatogram. Horizontal bars correspond to the migration areas on TLC of (1) 3a,17,21-P-5f3; (2) 3a,17-P-5& (3) ll- deoxycortisol; (4) 17,21-P-5f3.

was equivalent to a plasma level of 1.4 ng ml-‘inthefreefractionand6.2ngml-1in the conjugated fraction.

Zmmunoreactivity with the 301,17,21 -P-5$ RIAs

Female plasma (Fig. 1 middle and bottom). Both 3a,17,21-P-5@ RIAs picked up two peaks of immunoreactivity in the free steroid fraction. These corresponded to the elution positions of 301,17,21-P-5l3 and 3a,17-P-5B (the latter steroid, surprisingly, not having been found among the radioac-


200. 84,

150. -free -----conjugoled

/ : ' ;/ 8, Ill -d.oysoltl~l RLq

loo- ,/ : ; :

50, ! ; :

1800

1350 1 pu,17.21-P-56m4p

:,: jJ&

1 r’,‘, . I’,. I, I ,,.I. I ., 0 2 4 6 6 10 12 14 16 16 20

fraction number(0.6 cm)

FIG. 2. TLC scan of (top graph) 11-deoxycortisol and (middle and bottom graphs) 3a,17,21-P-5g steroid immunoreactivities in an extract of an in vitro incubate of plaice oocytes. Germinal vesicle breakdown was induced in the oocytes using a pituitary homogenate (0.5 pit * set ml-‘). Free and conjugated fractions were assayed at the same dilution. Note differences in the ordinate scales. See Fig. 1 for further details.

tive metabolites of the ovary). RIA 1 picked up two peaks of immunoreactivity in the conjugated steroid fraction-one corresponding to the elution position of 3a, 17,21-P-5(3 and the other to the elution position of 17,21-P-5& RIA 2, which does not cross-react with 17,21-P-5p (Table 2), detected only the first peak and not the sec- ond. The amount of immunoreactivity in the 3a,17,21-P-5p peak was equivalent to 8 ng ml-’ in the free fraction and ca. 200 ng ml- ’ in the conjugated fraction. The esti- mated plasma level of 3a, 17-P-5(3 in the free fraction, taking into account the degree of cross-reaction with the antisera, was 7 ng ml-‘.

Ovarian incubate (Fig. 2 middle and bot-

150.

100.

50.

1 -- 2 ti ,' I I, I. I I, I, I., . I. I

0 2 4 6 6 10 12 14 16 16 20


FIG. 3. TLC scan of (top graph) 11-deoxycortisol and (middle and bottom graphs) 3a,17,21-P-Sg steroid immunoreactivities in a pool of plasmas from three male plaice. Free and conjugated fractions were assayed at the same dilution. Note diierences in the ordinate scales. See Fig. 1 for further details.

tom). The same peaks of immunoreactivity were detected in the ovarian incubate as in the female plasma. However, the relative amounts of 3a,17-P-5P appeared to be much higher in the incubate, and some of this steroid was also found in the conjugated fraction. The amount of immunoreactivity in the 3a,17,21-P-5p peak corresponded to a level of 21 ng ml- ’ in the free fraction and 80 ng ml- ’ in the conjugated fraction. The amounts of immunoreactivity in the 3a,17-P-5P peak corresponded to a level of 30 ng ml- ’ in the free fraction and 16 ng ml-’ in the conjugated fraction.

Male plasma (Fig. 3 middle and bottom). The pattern of immunoreactivity in the male plasma was similar to that in the female plasma. Steroid levels were, however,


much lower in the male plasma, to the extent that 3a,17,21-P-5P was barely detect- able in the free fraction (less than 2 ng ml-‘). The level in the conjugated fraction was ca. 34 ng ml- ‘. The level of 3a, 17-P-5p in the free fraction was ca. 5 ng ml- ‘.

20P-Reduction of the Main Immunoreactive Peaks from the Ovarian Incubate

The two major immunoreactive steroids (from both free and conjugated fractions) were incubated with 20l3-HSD and NADH to verify that they had unreduced 20-keto groups. The reaction products were chro- matographed alongside controls (one-half of the same sample incubated without 20@-HSD) and assayed with RIA 2 (because it had a higher cross-reaction with 2Op- hydroxylated steroids than RIA 1). Figures 4 and 5 show that 2OP-HSD treatment re- sulted in the disappearance of the major peak and the appearance of a much smaller, more polar, peak. The 3a, 17-P-5p extracted from the incubate was converted to the same extent by the enzyme and the product had a similar polarity to synthetic 3a,17-P- Sp, which had been subjected to the same treatment. These results confirm that both of the plaice steroids have 20-keto groups and that the less polar one is almost certainly 3a, 17-P-5l3.

DISCUSSION

We have shown in this paper that mature plaice ovaries contain 21-hydroxylase, Sp-reductase, 3a-HSD, and conjugating enzyme activities, the main products of the 17-[3H]P incubations being free and conjugated ll-deoxycortisol (17,21-P), 17,21-P- Sp, and 3a,17,21-P-5@. According to the classification scheme of Sandor and Idler (1972), the identities of these steroids can only be described as tentative. However, they are strongly supported by the radioimmunoassay data.

The 11-deoxycortisol RIA detected two

100

fraction number (0.6 cm)

FIG. 4. Effect of reduction with 20lGHSD on the immunoreactivity of putative 3a,17,21-P-5& free (top) and conjugated (bottom), derived from the ovarian incubate. The fractions containing the steroid were sep arated into two equal aliquots, one of them was incubated with enzyme and NADH (“20~-HSD”) and the other without enzyme (“control”). The products were run on TLC and immunoreactivity was assayed with RIA 2. In both graphs the ordinate scales indicate the amount of immunoreactivity per fraction as a percentage of the total immunoreactivity per fraction in controls. (1) 3a,17,21-P-5g standard.

nonspecific immunoreactive peaks in the TLCs of male and female plasma, but not in the ovarian incubate. We have shown, using a cortisol-binding globulin assay, that these were due to a low (0.8 to 2%) but significant cross-reaction of the antiserum with cortisol and cortisone. Cortisol levels of 2OOXKl ng ml - ’ have been found in stressed female plaice (White and Fletcher, 1986). Cortisol levels have also been shown to vary seasonally in female plaice (range 20 to 150 ng ml-‘), peaking at, or about, the time of spawning (Wingfield and Grimm, 1977). Clearly, in order to measure 1 l- deoxycortisol accurately at low levels (< 10 ng ml-‘), it would be necessary to carry out some sort of separation prior to assay.

Both 3a,17,21-P-5P antisera detected an


loo-

--SO@-HSD 75. ---control

50. ,

25.

o- L

r*,-,.I.,.,.I.,

0 2 4 6 6 10 12 14


FIG. 5. Effect of reduction with 2Op-HSD on immunoreactivity of putative 3a,17-P-Sp, free (top) and conjugated (bottom) derived from the ovarian incubate. Further details as in Fig. 4. (2) 3a,17-P-5P standard.

immunoreactive steroid in the position of 3a,l7-P-5p. The identity of this steroid was further confirmed by 208-HSD reduction. Although relatively high concentrations of radioimmunoassayable 3a,l7-P-S@ (free and conjugated) were found in the ovarian incubate which we studied, it was not found among the radioactive products of the 17-[H3]P incubation. Possibly, the addition of an exogenous precursor might have af- fected the steroidogenic pathway. The most likely explanation, however, is that the ovaries used in the two separate studies were probably at different maturational stages and had different relative enzymatic activities. Further studies are needed to confirm this.

The reasons for using 3a,l7-P-5P, rather than 3a,l7,21-P-5& to generate the antibodies were twofold. First, 3a,l7-P-5l3 was much cheaper than the latter steroid. Sec- ond, because the steroid was linked to the CM0 at position 20, on the side chain, we expected the antibodies to be unable to dis- tinguish between steroids such as 3a,l7-

P-5l3, 3a,l7,21-P-58, 3a,l7,2Op-P-5l3, and 3a,l7,20a-P-5P. Indeed, the antibodies cross-reacted very well with 3a,l7,21-P-5P. Surprisingly, however, they cross-reacted very poorly with the 20-reduced steroids (all less than 10%). This result implies that a reduction of the keto group on the side chain of the steroid molecule can have a radical effect on the way in which the rest of the molecule binds to the antibodies.

Colombo and co-workers (Colombo and Belvedere, 1977; Colombo et al., 1973, 1978) were the first to demonstrate the presence of 21-hydroxylase in the ovaries of teleosts, the main products being ll- deoxycortisol and/or 1 l-deoxycorticosterone. The species in which they found the enzyme were Leptocottus armatus, Micro- gadus proximus, Gobius jozo, Diplodus an- nularis, Dicentrarchus labrax, and Solea impar. The same steroids were also identified in the ovaries and testis of the viviparous fish (Jenynsia line&z; Tesone and Charreau, 1980). Another 21-hydroxylated steroid, 17,2Op,21-P, has been identified in the ovaries of the Atlantic croaker (Micro- pogonias undulatus; Tram et al., 1986).

5P-reduced steroids have been found in ovaries of Spicara maena and Serranus ca- brilla (Reinboth, 1979), ayu (Plecoglossus altivelis; Suzuki et al., 1981), Indian catfish (Heteropneustes fossilis; Ungar et al., 1977), African catfish (Clarias gariepinus; Lambert et al., 1986; Schoonen et al., 1988), sailfin molly (Poecilia lattipina; Kime and Groves, 1986), dab (Canario and Scott, 1989), and goldfish (Curassius auru- tus; Lessman, 1987).

Conjugated (glucuronidated and/or sul- phated) steroids have been reported in sailfin molly (Kime and Groves, 1986), African catfish (Schoonen et al., 1989), carp (Kime et al., 1987), zebrafish (Bruchydanio rerio; Van den Hurk and Lambert, 1983), and dab (Canario and Scott, 1989).

Howell and Scott (1984) found a very significant positive correlation between plasma levels of 11-deoxycortisol and the stage


of maturation of turbot (Scophthalmus maximus). However, in the winter flounder (Pseudopleuronectes americanus), Camp- bell et al. (1976) found no differences in concentrations of 1 1-deoxycortisol or 1 l- deoxycorticosterone in plasmas from maturing and nonmaturing fish. Canario and Scott (199Oa) also failed to find any changes in 1 1-deoxycortisol levels in maturing/ ovulating female dabs.

The levels of conjugated 3u,17,21-P-5p were 200 ng ml-‘, and up to 600 ng ml-’ have been measured in individual fish (Scott and Canario, 1990). These levels are similar to those of 17,20@P in salmonids (see review by Goetz et al., 1987) and carp (Santos et al., 1986; Levavi-Zermonski and Yaron, 1986), 17-P in winter flounder (Campbell et al., 1976), and 3p,17,2Oo-P-5p in dab (Ca- nario and Scott, 1990a). These results sug- gest that the capacity for steroid production is probably very strong in most teleost ovaries. The major products, however, appear to differ widely.

There seems to be a far higher percentage of conjugated steroids in plaice than in the dab (Canario and Scott, 199Oa). The reason for this may be that the 21-hydroxyl group, being on a primary carbon atom, may be more readily accessible to glucuronyl or sulphate transferases than the 20a-hydroxyl group, which is on a secondary carbon atom position. We observed in the dab, both in vivo and in vitro, that the glucuronyl or sulphate transferase(s) appeared to have a higher affinity for the 4-pregnene as op- posed to SP-pregnane steroids. This implied that the steroids were probably conjugated through the 2Oa-hydroxyl group (Canario and Scott, 199Oa). In the plaice, there was also some evidence that different types of steroid may have different amities for the conjugating enzyme(s). In the incubate that was studied by radioimmunoassay, for ex- ample, 3a,17-P-5P was found to be conjugated. 17-r3H]P, which was added to the other incubate, was not found to be conju-

gated. This suggests that, in vitro at least, the 301 position is susceptible to conjugation. In vivo, however, 3a,17-P-5P was found only in the free form, so the signifi- cance of this finding is difficult to assess. The ratio of conjugated:free 1 l-deoxycortisol in the incubates was much higher than that of 3a,17-P-5P, which suggests that the 21-hydroxyl is a “preferred” site of conjugation and that conjugation through the 3a-hydroxyl group in vitro may be artefac- tual .

The function of these steroids has not yet been established. In vitro studies on oocyte final maturation in plaice have shown that, for maximal biological activity, Czl steroids must have a 20P-hydroxyl group (Canario and Scott, 199Ob). 5@reduced steroids are generally the least potent in inducing oocyte maturation. The picture is similar to that in the dab in which the major ovarian steroids have also been found to have very low oocyte maturation-inducing activities (Canario and Scott, 199Ob). Increasingly, there is evidence that, during the spawning period, many fish produce pheromones in order to synchronise the reproductive pro- cesses of males and females. Several sex pheromones have been identified and shown to be steroids of gonadal origin and to have one or several of the following features: pluri-hydroxylation, conjugation, 5@-reduction (see review by Stacey et al., 1987). These features are also exhibited by the major ovarian steroids which we have identified in the plaice and the dab.

If, as we suspect, some, or all, of these steroids in the plaice and dab do have a pheromonal role, then the interesting fact emerges that steroidal pheromones, although having similar physical properties (i.e., high solubility in water), are structur- ally different between species. This is perhaps not surprising, as many species tend to spawn in the same area and at the same time as each other (the dab and the plaice being good examples), and in order for their


pheromones to be specific only to individ- uals of their own species, they must, by definition, be different.

Although 2Olkeduction would appear to be a structural requirement for maximum oocyte maturation-inducing activity in plaice (Canario and Scott, 199Ob), the ovarian incubations containing labelled precursor failed to produce any 20@reduced steroids. However, very small amounts of 17,2Op-P (but not 17,2Ol3,21-P) have been detected by RIA in ovarian incubates (own unpub- lished results) and the blood plasmas of maturing female plaice (Scott and Canario, 1987, 1990). The failure to detect 20@HSD in this study could have been due to an in- appropriate stage of the follicles, as there are rapid steroidogenic changes around the period of oocyte maturation. It is possible that in plaice, as we have suggested in the dab (Canario and Scott, 1989), 17,2Op-P still might be the maturation-inducing steroid, but that the triggering process re- quires only very small amounts, over only a very brief period (cf. Jalabert, 1976; only 1 min contact with 17,2Op-P was sufficient to trigger maturation in rainbow trout oocytes). Certainly there has been no indica- tion in any of our studies (see also Canario and Scott, 1990b) of any other steroid which might fulfil this role in the plaice.

ACKNOWLEDGMENTS

A.V.M.C. gratefully acknowledges receipt of Grant 33/A/85/PO from the NATO Programme for Scientific Studies, Portugal. Both authors would like to thank Keith Way of the MAFF Fish Diseases Laboratory, Weymouth; Dr. John Sumpter of Brunel University, London; and Professor Philip Lowry of Reading Uni- versity for their help in raising the antisera.

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Documents

Identification of, and development of radioimmunoassays for 17α,21-dihydroxy-4-pregnene-3,20-dione and 3α,17α,21-trihydroxy-5β-pregnan-20-one in the ovaries of mature plaice (Pleuronectes