Molecular and Celhdar Endocrinology, 72 (1990) R7- R13

Elsevier Scientific Publishers Ireland. Ltd. R?

MOLCEL 02360

Effects of human chorionic gonadotropin (hCG) and prolactin (PRL) on 3/I-hydroxy-5-ene-steroid dehydrogenase/A5-A4 isomerase (3&HSD)

expression and activity in the rat ovary

Rapid Paper

Celine Martel, Claude Labrie, Jacques Co&t, Eric DuPont, Claude Trudel, Van Luu-The, Masakazu Takahashi, Georges Pelletier and Fernand Labrie

Medical Research Councii Croup in ~o~ee~~ar Endocrinofo~, CHUL Research Center and Lmal University. Q&bee Gf V 4G2, Canadu

(Received 2 July 1990; accepted 5 July 1990)

Key lords: Steroidogenesis; 3p-Hydroxysteroid dehydrogenase/A5-A4 isomerase mRNA; Ovary; Luteinizing hormone/human chorionic gonadotropin; Prolactin; (Rat)

Summary

Using a recently cloned rat ovary 3&HSD cDNA and antibodies raised against purified human placental 3&HSD, we have studied the effects of treatment with human chorionic gonadotropin (hCG) and hyperprolactinemia achieved by pituitary implants, alone or in combination, on the expression and activity of ovarian 3/3-hydroxysteroid dehydrogenase/A5-A4 isomerase (3/3-HSD) in intact adult rats. 32P- and “S-labeled cDNA probes were used to evaluate the effects of treatments on 3/?-HSD mRNA levels by dot blot and in situ hybridization, respectively, while enzymatic activity was measured by the conversion of [*4C]dehydroepiandrosterone into [14C]androstenedione. The present data show that hCG exerts a marked trophic effect on rat corpora lutea with an increase in total ovarian 3/3-HSD mRNA levels, 3j2-HSD protein content as well as enzymatic activity, resulting in an increase in serum progesterone levels. Prolactin-secreting pituitary implants alone, on the other hand, while exerting small effects on 3&HSD expression and activity, led to a marked potentiation of the stimulatory effect of hCG on all parameters. The present data show that hCG and PRL act synergistically to stimulate ovarian progesterone secretion via an increase in 3P-HSD mRNA levels, protein content and enzymatic activity.

Introduction

The biosynthesis of progesterone and estradiol by specific ovarian cell types involves several

Address for correspondence: Professor Femand Labrie,

Laboratory of Moiecular Endocrinology, CHUL Research

Centre, 2705 Laurier Blvd., Quebec ClV 4G2, Canada.

This work was supported by grants from the Medical

Research Council of Canada, Le Fonds de la Recherche en

Sante du Quebec (FRSQ), and Le Fonds pour la Formation de

Chercheurs et I’Aide a la Recherche. C.L., C.T., CM. and J.C.

are supported by Fellowships from the FRSQ.

steroidogenic enzymes whose expression is regu- lated in a timely and cell-specific manner by luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL), as well as other factors (Hsueh et al., 1984; Erickson et al., 1985; Richards et al., 1987). Among these en-

zymes, 3~-hydroxysteroid dehydrogenase (EC

1.1.1.145)/A5-A4 isomerase (EC 5.3.3.1), hereafter referred to as 3/3-HSD, plays a key role in ovarian steroidogenesis since it catalyzes the obligatory conversion of pregnenolone and other 5-ene-3/3- hydroxysteroids into progesterone as well as pre- cursors of all androgens and estrogens.

0303-7207/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland, Ltd.

RX

In view of its crucial role in ovarian steroido- genesis, we have investigated the possible modula- tion of ovarian 3P-HSD expression and activity by human chorionic gonadotropin (hCG) and pro- lactin (PRL) hypersecretion in intact adult rats. The recently cloned rat ovary 3fl-HSD cDNA

(Zhao et al., 1990) was used to measure 3P-HSD mRNA levels by dot blot as well as by in situ hybridization while ovarian 3P-HSD protein con- tent was measured by immunoblotting using rab- bit antibodies raised against purified human

placental 3P-HSD (Luu-The et al., 1989). Correla- tion of 3/3-HSD mRNA and protein levels was made with enzymatic activity and serum pro-

gesterone levels.

Materials and methods

Adult female Sprague-Dawley rats (CrL: CD(SD)Br) (Charles River, St-Constant, Canada) received twice daily (b.i.d.) subcutaneous injec- tions of vehicle (1% gelatin (w/v) in 0.9% NaCl solution) or vehicle containing 10 IU of hCG (Profasi HP, Pharmascience, Montreal, Canada). An equal number of animals (8-9/group) re- ceived, 2 days before the start of the experiment, three pituitary implants under the left kidney capsule in order to achieve hyperprolactinemia.

The animals were treated for 15 days before sacrifice and blood collection for measurement of serum pituitary and steroid hormones. Serum

pregnenolone and progesterone were measured by RIA in duplicate after diethyl ether extraction followed by chromatography on LH-20 columns using antisera developed and characterized in our laboratory (Belanger et al., 1980). Serum LH and PRL were measured by double-antibody RIA using rat LH-I-5 and rat PRL-I-1 for iodination,

rat LH-RP-22 and PRL-RP-1 as standards, and rabbit antiserum (anti-LH-S8 and anti-PRL-Sl), kindly supplied by the National Pituitary Program (Baltimore, MD, U.S.A.).

For 3P-HSD mRNA dot blot analysis, the right ovary from each rat was promptly weighed and frozen in liquid nitrogen. RNA was extracted in a TL 100.2 rotor essentially as described (Chirgwin et al., 1979; Kingston, 1987) with the addition of phenol, phenol/ chloroform (1 : 1) and chloroform extractions. RNA was denatured by heating for 15

min at 65°C in 10 x SSC (1 X SSC being 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing

6% formaldehyde. Serial 2-fold dilutions of RNA in 10 X SSC were blotted onto nylon membranes (Hybond-N, Amersham, Arlington Heights, IL, U.S.A.) using a 96-well Hybri-Dot Manifold (Be- thesda Research Laboratories, Gaithersburg, MD, U.S.A.).

The RNA blots were prehybridized at 42°C for 4 h in buffer consisting of 50% formamide, 5 x SSPE, 5 X Denhardt’s, 0.1% sodium dodecyl

sulfate (SDS), 0.2 mg/ml yeast tRNA, 0.2 mg/ml denatured salmon testis DNA, and 2 pg/ml poly(A). The random primer-labeled (Feinberg and

Vogelstein, 1983) [a-32P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL, U.S.A.) full- length 3b-HSD cDNA probe (ro3&HSD56, Zhao et al., 1990) was heat-denatured and added to hybridization buffer (prehybridization buffer con-

taining 4% dextran sulfate). After 16 h of hybridi- zation at 42 o C, the blots were washed 2 x 30 min at 25°C in 0.1 X SSC/O.l% SDS and 2 x 30 min

at 65°C in 0.1 X SSC/O.l% SDS. The blots were autoradiographed at - 80°C with intensifying screens before quantification of the intensities of the autoradiographic spots with an Amersham RAS Image Analyzer System. The slopes of the dot intensities of each dilution series were calcu- lated by linear regression using Cricket Graph (Cricket Software, Malvern, PA, U.S.A.) and 3p-

HSD mRNA levels expressed as means k SEM (n = 6) relative to 3P-HSD mRNA levels in con- trol intact rat ovaries. Total RNA from intact adult rat ovaries was used as internal control.

The contralateral ovary from each rat was ho- mogenized with a Polytron in phosphate buffer (50 mM KH,PO,, 20% glycerol, 1 mM EDTA, pH 7.5) containing protease inhibitors (1 mM phenyl- methylsulphuryl-fluoride and 5 pg/ml each of pepstatin A, antipain and leupeptin) and centri- fuged for 30 min at 1000 X g. Five-fold dilutions of aliquots of the supernatant were incubated for 20 min at 37°C in 0.5 ml phosphate buffer con- taining 10 PM unlabeled dehydroepiandrosterone (DHEA), 1 PM [4-r4C]DHEA and 0.8 mM NAD+. [4-14C]DHEA (51 mCi/mmol) was purchased from DuPont (Markham, Ont., Canada) and purified by thin-layer chromatography (TLC) on

60 f254 silica gel plates (E. Merck, Darmstadt,

F.R.G.) in a benzene/acetone (4: 1, v/v) system before use. The enzymatic reaction was stopped by chilling the incubation mixture in an ice-water slurry and adding 2 ml dichloromethane. The organic phase was then evaporated to dryness under a stream of nitrogen. 14C-labeled 4-andros- tene-3,17-dione (A4-dione) produced from [4- 14C]DHEA by 3/3-HSD was then separated on TLC plates using a 4: 1 mixture of benzene and acetone, and then autoradiographed for 48 h. The TLC areas corresponding to A4-dione and DHEA

were identified on the autoradiographs, scraped and transferred to scintillation vials containing 0.5 ml ethanol to which 10 ml scintillation fluid were added for radioactivity measurement. Ovarian protein content was measured by the method of Bradford (1976) using bovine serum albumin as standard.

Ovarian proteins were subsequently size-sep- arated on a 1.5 mm-thick 5-15s polyacrylamide gel and transferred to nitrocellulose filters. Puri- fied human placental 3P-HSD and BRL protein weight markers were used as positive control and for estimation of molecular size, respectively. The

blots were treated with wash-solution (5% fat-free milk (Carnation)/O.l% Nonidet P-40 in PBS) (3 X

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30 min) and incubated with a 1 : 2000 dilution of rabbit antiserum raised against purified human placental 3P-HSD for 18 h at 4°C. The blots were washed 3 times (30 mm/wash) and incubated for an additional 4 h at 4°C in a 1 : 1000 dilution of

‘251-labeled goat anti-rabbit immunoglobulin G. After three washes in the same solution, autoradi- ography was performed at - 80°C with intensify- ing screens and XAR-5 films. The intensity of the signal emitted by “‘I-antibodies recognizing the

42 kDa protein was quantified using an Amersham

RAS Image Analyzer System (Amersham, Arling- ton Heights, IL, U.S.A.). In situ hybridization was performed as described (DuPont et al., 1990) on two rat ovaries from each treatment group.

All results are expressed as means + SEM. Sta- tistical significance was determined according to the multiple-range test of Duncan-Kramer

(Kramer, 1956).

Results

Intact or pituitary implant-bearing rats were treated with vehicle or hCG (10 IU, b.i.d.) for 15 days. At the end of the treatment period, serum LH levels were 80% inhibited in all hCG-treated

A q ICM

1.5 a hCG

B

200-

P

5 loo-

2 B

Intact Pit. Impl.

C

5-l l *

Fig. 1. Effect of treatment with hCG on ovarian 3/?-HSD mRNA concentration (panel A), ovarian weight (panel B) and total ovarian 3b-HSD mRNA content (corrected for ovarian weight) (panel C) in intact and pituitary implant-bearing adult female rats.

The animals received twice-daily injections of hCG (10 IU) or vehicle for 15 days. Ovarian 3P-HSD mRNA levels were measured by

dot blot hybridization using the 32P-labeled rat ovary 3P-HSD cDNA probe. Data are expressed as means+ SEM (RNA

measurements, n = 6; ovarian weight, n = 18). * P < 0.05, * * P c 0.01 vs. intact control.

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animals while pituitary implants alone caused a 56% decrease in serum LH concentrations as com- pared to control intact female rats (data not shown). On the other hand, serum PRL concentra- tion rose from 2.0 f 0.3 to 6.8 + 2.9 and 39.5 + 14.7 ng/ml in pituitary implant- and hCG + pituitary implant-treated animals, respectively,

with a value of 22.8 + 9.0 ng/ml in hCG-treated animals (data not shown).

When expressed as amount of 3P-HSD mRNA relative to total ovarian RNA, treatment with hCG, pituitary implants or hCG + pituitary im- plants had no significant effect on the level of

ovarian 3/3-HSD mRNA (Fig. 1A). However, as shown in Fig. IB, treatment of intact and pitui- tary implant-bearing rats with hCG caused respec- tive 2.7- (P < 0.01) and 4.5-fold (P < 0.01) in-

creases in ovarian weight from 37.8 k 1.1 to 103.2 -t 14.1 and 171.2 k 18.7 mg, respectively. When relative levels of 3P-HSD mRNA (Fig. 1A) were corrected for changes in ovarian size (Fig. lB), a highly significant stimulatory effect of hCG alone (P < 0.05) and in combination with pituitary im- plants (P < O.Ol), was observed on ovarian 3p- HSD mRNA, while pituitary implants alone

caused a 25% (P < 0.05) increase in 3P-HSD mRNA content. Such a measure provides an as- sessment of the whole ovarian content in 3/I-HSD mRNA and, subsequently, of the ovary’s capacity for steroidogenesis.

As illustrated by X-ray autoradiographs of in situ hybridization performed on ovarian sections

with the 35S-labeled 3&HSD cDNA probe (Fig. 2) the increase in ovarian weight associated with

Fig. 2. X-ray autoradiographs showing the in situ hybridization of ovarian sections with the 35S-labeled rat ovary 3P-HSD cDNA probe. Treatments correspond to those described in legend to Fig. 1. Note that the probe hybridizes mostly to corpora lutea which

markedly increase in size following treatment with hCG and hCG + pituitary implants. Magnification: x 8. Exposure time: 3 days.

Rll

Intact Pii Impl.

B

6 1

Fig. 3. Effect of treatment with hCG on ovarian 3/3-HSD protein, activity and serum progesterone levels in intact and pituitary

implant-bearing adult female rats. The rabbit antibody raised against purified human placental 3/3-HSD was used to measure rat

ovary 3/3-HSD by immunoblotting (panel A), while ovarian 3/3-HSD activity was measured by the formation of [‘4C]A4-dione from

[14C]DHEA by ovarian homogenates and is expressed in nmol formed/mg protein/mm (panel B). Serum progesterone levels (panel

C) were measured by double-antibody RIA. Data are expressed as means+ SEM (3P-HSD measurements, n = 6; serum progesterone,

n = 9). * * P < 0.01 vs. intact control.

hCG treatment mainly results from an increase in the size of the corpora lutea (CL), with no signifi- cant change in the intensity of the labeling.

The stimulatory effect of hCG on 3B-HSD ex- pression and activity is well illustrated in Fig. 3. Ovarian 3B-HSD protein content was measured by immunoblotting using rabbit antibodies raised

against purified human placental 3B-HSD (Luu- The et al., 1989) which also recognize rat ovary 3B-HSD which possesses electrophoretic proper- ties similar to the human placental enzyme. It can

be seen that hCG alone caused 100% (P < 0.01) and 50% (P < 0.05) increases above control in ovarian 3B-HSD protein concentration (Fig. 3A) and specific enzymatic activity (Fig. 3B), respec- tively. While pituitary implants alone did not af- fect the above-mentioned parameters, treatment of pituitary implant-bearing animals with hCG caused 3.2- (P -c 0.01) and 3.5-fold (P < 0.01) in- creases in ovarian 3B-HSD protein content (Fig. 3A) and enzymatic activity (Fig. 3B), respectively, confirming the synergistic effects of LH/hCG and PRL on ovarian 3B-HSD expression and activity. In fact, treatment with hCG + pituitary implants increased the formation of [i4C]A4-dione from

[14C]DHEA from 1.89 + 0.17 to 4.85 + 0.46 nmol/mg protein/mm. The increased steroido- genie capacity of the hCG-treated rat ovaries is manifested by a marked increase in the levels of serum progesterone (Fig. 3C). In fact, treatment

with hCG, pituitary implants and hCG + pituitary implants caused 4.9- (P -c 0.01) 3.7-, and lo-fold (P -c 0.01) increases in serum progesterone levels.

Although of smaller amplitude than the effects on serum progesterone, hCG and hCG + pituitary implants caused 80% (P < 0.05) and 200% (P -c 0.01) increases above control in serum pregnen- olone concentrations, from 3.1 + 0.3 nmol/l to 5.6 f 1.1 and 9.4 f 1.1 nmol/l, respectively, while pituitary implants alone had no significant effect (data not shown).

Discussion

The present data demonstrate the stimulatory effects of hCG on 3B-HSD expression and activity in the intact rat ovary as well as the potent poten- tiating effect of PRL on the same parameters. In fact, a synergistic stimulatory effect of hCG and PRL is clearly observed on corpora lutea size,

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total 3/3-HSD mRNA content, protein level as well as enzymatic activity, which translate into marked increases in serum progesterone levels.

The augmentation of LH’s stimulatory effect on progesterone secretion by PRL has been ob-

served previously (Armstrong et al., 1969). In fact, LH is known to increase luteal cell PRL receptors (Richards and Williams, 1976). Conversely, PRL treatment enhances the LH binding capacity of

corpora lutea and consequently increases pro- gesterone formation (Holt et al., 1976). Interest- ingly, the present data show that hCG treatment was accompanied by elevated serum PRL con-

centrations. In fact, these were higher in hCG - and hCG + pituitary implant-treated animals than in animals bearing pituitary implants alone. In addition, combined treatment with hCG + pituitary implants produced increased estradiol (E,) levels as indicated by the observed increase in uterine weight. As a possible explanation, E, is well known to stimulate PRL expression and secretion (Nicoll and Meites, 1962; Labrie et al., 1980; Tong et al., 1989) and hCG-induced ovarian E, formation may thus be responsible for the observed increases in serum PRL concentrations which, in turn, could further stimulate ovarian function. The relatively small stimulatory effect of

pituitary implants alone on 3P-HSD mRNA, pro- tein and activity can result from the decreased

serum LH levels accompanying hyperprolactin- emia (Marchetti and Labrie, 1982).

While the effects of hCG/LH on ovarian steroidogenic enzyme gene expression and activity are exclusively stimulatory, PRL exerts both stimulatory and inhibitory effects on the expres- sion of these enzymes, depending on the endocrine status of the animals studied. In granulosa cells, PRL has been shown to stimulate progesterone production by increasing pregnenolone formation and 3P-HSD activity (Jones et al., 1983). On the other hand, PRL has been shown to inhibit luteal

cell P-450,,,, mRNA and protein (Krasnow et al., 1990). In addition, PRL exerts both luteotrophic and luteolytic effects, depending on the time at which it is administered and the delay after hy- pophysectomy (Malven and Sawyer, 1966).

While 3/?-HSD is not the only enzyme targeted for regulation by hCG/LH and PRL (Gibori et al., 1979; Jones et al., 1983), the close parallelism

observed between ovarian 3/3-HSD protein con- tent, enzymatic activity and serum progesterone concentrations indicates that 3P-HSD could well play a major role in the activation of ovarian progesterone formation. While these measure- ments were made on whole ovarian RNA and

protein samples, we believe that the effects of hCG and PRL which we have observed reflect changes occurring in corpora lutea since they rep- resent the most abundant ovarian cell population and the labeling observed with the 35S-labeled 3P-HSD cDNA probe was most intense in these

cells. The present results suggest that the stimulatory

effects of hCG and PRL on ovarian corpora lutea 3P-HSD protein content and activity and subse- quently on serum progesterone levels are media- ted, at least in part, via post-translational mecha- nisms since the effects on 3/3-HSD protein con-

centration and activity are greater than those on ovarian 3/3-HSD mRNA levels. An analogous

situation prevails for the P-450,,, and P-450,,,,,, enzymes. Indeed, while P-450,,, mRNA may be induced by hCG and FSH in rat and human granulosa cells during and prior to luteinization (Trzeciak et al., 1986; Voutilainen et al., 1986; Goldring et al., 1987), it is constitutively expressed in corpora lutea. Similarly, P-450,,,, mRNA, pro- tein and activities are constitutively expressed in

luteinized granulosa cells (Hickey et al., 1988). Finally, the present results, taken with those ob-

tained for the P-450,,, and P-450,,,, enzymes, tend to indicate that PRL plays a major role in controlling the expression and activity of steroido- genie enzymes in luteal cells in association with

LH.

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