Molecular and Cellular Endocrinolow, 65 (1989) 195-201 Elsevier Scientific Publishers Ireland, Ltd.

195

MOLCEL 02121

Effects of prolactin on steroidogenesis and CAMP accumulation in rat luteal cell cultures

Hela Gitay-Goren 1,2, Ella S. Lindenbaum 2 and Zaki Kraiem ‘v3 ’ Endocrine Research Unit, Carmel Hospital, 34362 Haifa, Israel, ’ Morphological Sciences Unit, Faculty of Medicine, Technion - Israel

Institute of Technologv, 31096 Haifa, Israel, and 3 Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel

(Received 29 March 1989; accepted 16 May 1989)

Key worh: Prolactin; Cyclic AMP; Steroidogenesis; Progesterone; Luteal cell

The effects of prolactin (PRL), alone and as a modulator of human chorionic gonadotropin (hCG) action, on steroidogenesis and CAMP accumulation in rat luteal cell cultures were examined. Cultured rat luteal cells were prepared from immature rats primed with pregnant mare serum gonadotropin and hCG. In vitro treatment was performed with 0.1 and 0.2 IU/ml hCG and l-100 ng/ml PRL. Cultures were incubated for 48 h for evaluation of progesterone (P4) secretion and for 1 h for measurement of CAMP accumulation. The same doses of hormones and incubation periods were also used in preovulatory rat granulosa cell cultures and found to cause a significant, dose-dependent inhibition in estradiol, P4 and CAMP accumulation. In luteal cell cultures, on the other hand, P4 secretion was significantly elevated, in a dose-dependent manner, by PRL. Moreover, identical doses of PRL caused a significant, dose-dependent stimulation of CAMP accumulation. Basal levels of P4 were also significantly elevated by PRL alone, but no such stimulation by PRL was detected in basal levels of CAMP. Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, increased the stimulation of P4 and CAMP by hCG + PRL in a manner dependent on PRL concentrations. The overall data therefore demonstrate divergent effects of PRL on CAMP accumulation and steroidogenesis in the ovary: inhibitory in the preovulatory and stimulatory in the postovulatory state. Moreover, these findings suggest a possible common mechanism linking the effects of PRL before and after ovulation: inhibition of CAMP accumulation via enhanced breakdown of the nucleotide.

Introduction

Prolactin (PRL) is well established tropic agent in the rat, functioning

as a luteo- as it does

Address for correspondence: Zaki Kraiem, Ph.D., Endo- crine Research Unit, Carmel Hospital, 7 Michal Street, 34362 Haifa, Israel.

A preliminary report of this work was presented at the International Workshop on Maternal Recognition of Preg- nancy, March 1988, Jerusalem, Israel.

synergistically with luteinizing hormone (LH) (Rothchild, 1981). The role of PRL in maintaining a functional corpus luteum is reported to be either indirect, via induction and maintenance of LH receptors (Casper and Erickson, 1981; Van Straa- len and Zeilmaker, 1982) and inhibition of pros- taglandin production during the early stages of the corpus luteum (Rothchild, 1981), or direct, via a luteotropic influence of PRL on the steroidogenic pathway (Murphy and Rajkumar, 1985). The two luteotropic effects of PRL, stimulation of pro-

0303-7207/89/$03.50 6 1989 Elsevier Scientific Publishers Ireland, Ltd.

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gesterone (P4) secretion and of LH receptors, are considered two separate, independent events (Gibori and Richards, 1978). Some authors also believe that in luteal cell cultures the luteotropic effect of PRL is mainly via inhibition of P4 deg- radation (Eckstein and N&rod, 1979; Jones et al., 1983).

In contrast to its inhibitory effect in preovula- tory mouse and human ovaries (McNatty et al., 1974,1976), high PRL levels stimulate P4 secretion in postovulatory mouse ovaries (McNatty et al., 1976). PRL administered in vivo or in vitro has been reported to cause a dose-dependent stimula- tion of basal and human chorionic gonadotropin (hCG)-induced P4 secretion in cultured rat luteal cells (Tesone et al., 1984).

In previous studies, we reported that PRL in preovulatory rat granulosa cell cultures inhibits hCG-induced steroidogenesis as well as CAMP accumulation (Gitay-Goren et al., 1989), biochem- ical findings which were also correlated with mor- phological changes (Gitay-Goren et al., 1988). The inhibition by PRL was abolished by the phos- phodiesterase (PDE) inhibitor 3-isobutyl-l-meth- ylxanthine (MIX), indicating stimulation of PDE as a possible mode of action of PRL in inhibiting steroidogenesis. The aim of the present investi- gation was to examine, in the same experimental system, the effects of PRL following ovulation.

Materials and methods

Animals Immature female rats (Sprague-Dawley, 25 days

old) were given a superovulatory treatment con- sisting of a subcutaneous injection of 50 IU preg- nant mare serum gonadotropin (Gestyl, Organon, Oss, The Netherlands) and 65 h later a subcuta- neous injection of 25 IU hCG (Pregnyl, Organon). To obtain corpora lutea, the animals were sacrificed by cervical dislocation 4 days following hCG administration.

Cell culture The culture method for rat luteal cells was

based on a combination of several in vitro systems (Behrman et al., 1980; Azhar and Menon, 1981; Massicotte et al., 1984). The medium consisted of a serum-free mixture (1 : 1, v/v) of Dulbecco’s modified Eagle’s medium and Ham’s nutrient

mixture F-12 (Biological Industries, Beth Haemek, Israel) buffered with 20 mM Hepes buffer, pH 7.4, and supplemented with 100 units/ml penicillin, 100 pg/ml streptomycin, 50 pg/ml neomycin and 2.5 pg/ml amphotericin B (Sigma, St. Louis, MO, U.S.A.).

The ovaries were excised, sliced, and red blood cells removed by repeated washings with the medium. Thereafter, medium (1 ml/ovary) con- taining 0.2% collagenase (Boehringer, Mannheim, F.R.G.), 0.2% bovine serum albumin (BSA; Sigma) and 10 pg/rnl deoxyribonuclease I (Sigma) was added. The sliced ovaries were incubated for 90 min in a humidified 5% CO,-95% air incubator at 37°C and the incubation was stopped by centri- fugation (250 X g for 5 min) every 30 min. Luteal cells were recovered from the supernatants after further washings in medium. After counting with trypan blue (40-6096 viable cells), luteal cells were plated onto 16-mm multiwell plates (4 X lo5 via- ble cells/well) in 0.5 ml culture medium supple- mented with 15 ~1 of a mixture yielding a final concentration of 2 pg/ml insulin, 40 ng/ml hy- drocortisone and 5 pg/ml transferrin (Sigma). The wells were precoated with 0.2 ml/well fetal calf serum to facilitate cell attachment; excess serum was removed 3 h later, and the wells were then washed twice with culture medium (May et al., 1982). Before in vitro incubation, the cells were cultured for 24 h in a humidified atmosphere of 5% CO,-95% air at 37 o C to form a monolayer.

In vitro incubation Several doses of hCG (1 IU = 100 ng) (Pregnyl,

Organon) and ovine PRL (NIADDK-o-16, kindly provided by the National Hormone and Pituitary Program, NIADDK, Bethesda, MD, U.S.A.) were added to the culture medium and incubated at 37°C in a humidified 5% CO,-95% air incubator. Incubation periods were 1 h for CAMP measure- ments and 48 h for P4 determinations. In some experiments, MIX (Sigma) was added at a dose of 0.1 mM, an amount in which the action of MIX is restricted to PDE inhibition. BSA (O.l%, final concentration) was added to each of the treatment mixtures as well as to controls.

Incubations were stopped by the addition of 1 ml cold absolute ethanol to the aspirated incuba- tion medium. Cold absolute ethanol (1 ml) was

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then added to each well, and the multiwell plate was stored overnight at -20” C. The cells were then scraped, combined with the medium, and centrifuged at 800 X g for 10 min. The super- natants were evaporated, and the samples were diluted in the assay buffer (0.05 M Tris-HCl buffer, pH 8.0, for the P4 radioimmunoassay (RIA) and 0.05 M sodium acetate buffer, pH 6.2, for the CAMP RIA) (May and Schomberg, 1984).

Radioimmunoassays The secretion of P4 by luteal cells was de-

termined by RIA using [1,2,6,7-3H(N)]P, (New England Nuclear, Boston, MA, U.S.A.) and an antibody obtained from Bio-Yeda, Rehovot, Israel. Cross-reactivity at 50% replacement was as fol- lows: 5cY-pregnane-3,20-dione, 6%; 3/?-hydroxy-5- pregnane-20-one, 0.5%; 17a-hydroxyprogesterone, 2.5%; 20a-hydroxy-4-pregnane-3-one, 0.3%; 20/I- hydroxy-4-pregnane-3-one, 1.2%; 11/3-hydroxypro- gesterone, 1.8%; llcY-hydroxyprogesterone, 31%; corticosterone, 0.3%; and 17B-estradiol, < 0.1%. Assay sensitivity was 5 pg/O.l ml. Intra- and interassay coefficients of variation were 5 and 9%, respectively.

CAMP was assayed by RIA using [‘251]cAMP (Amersham Radiochemical Centre, Amersham, U.K.) and an antibody obtained from Bio-Yeda. Cross-reactivity at 50% replacement was as fol- lows: cyclic GMP, < 7 X 10K6%; AMP, < 3 X

10-4%; ADP, < 10-4%; and ATP, < 2.5 X

10p5%. Assay sensitivity was 5 fmol/O.l ml. In- tra- and interassay coefficients of variation were 4 and 7%, respectively.

Data analysis Experimental data are presented as the mean k

SEM of measurements from 2-3 experiments, each carried out in triplicate wells. Data were subjected to one-way analysis of variance (ANOVA), and individual comparisons between means were made using unpaired Student’s t-test. Values of P < 0.05 were considered significant.

Results

Effects of PRL on basal and hCG-induced P4 secre- tion

A dose-response relationship for P4 secretion by cultured luteal cells was obtained in response

to increasing concentrations of hCG (Fig. 1). 0.1 and 0.2 IU/ml were chosen as optimal submaxi- mal working hCG doses. Luteal cells cultures were incubated for 48 h with either 0.1 and 0.2 IU/ml hCG or l-100 ng/ml PRL, or with combinations of both hormones (Fig. 2). The results indicated that PRL induced a significant rise in basal P4 secretion. Addition of the lactogenic hormone to hCG significantly increased P4 levels beyond those obtained with hCG alone. One-way ANOVA showed a significant stimulation of P4 secretion by PRL alone (P < 0.01) and by PRL added to 0.1 IU/ml hCG (P < 0.01). When MIX (0.1 mM) was added to 0.1 IU/rnl hCG combined with 1, 10 and 100 ng/ml PRL, the stimulation of P4 was enhanced 1.2-, 1.3- and 1.7-fold, respectively (P < 0.01; data not shown).

Effects of PRL on basal and hCG-induced CAMP accumulation in the absence and presence of MIX

A dose-response curve was performed in terms of CAMP response of luteal cells to hCG stimula- tion (Fig. 3). Concentrations of 0.1 and 0.2 III/ml hCG, i.e., the same as those used for P4 response, were chosen as submaximal working doses. Luteal cell cultures were incubated for 1 h with hCG (0.1 and 0.2 IU/ml) and increasing concentrations of PRL (l-100 mg/ml) in the absence and presence of MIX. In the absence of MIX, PRL alone after 1

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Fig. 1. Dose-response curve for hCG-induced P., secretion in luteal cell culture. Luteal cells (4 x 105/well) were cultured for 48 h with increasing concentrations of hCG (0.1-1.0 IU/ml). Data represent the mean& SEM of triplicate wells of two

separate experiments.

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Fig. 4. Effect of PRL on basal and hCG-induced CAMP accumulation in luteal cell cultures. Luteal cells (4 X lOs/well)

were incubated for 1 h with increasing concentrations of PRL

(1, 10, and 100 ng/ml) alone and in the presence of 0.1 and 0.2

IV/ml hCG. CAMP accumulation was expressed as per-

centage, 100% being designated as CAMP accumulation in the

presence of each hCG concentration alone. Absolute values of

100% CAMP accumulation were 7.9 4.0.9 and 10.1 kO.7

pmol/well with 0.1 and 0.2 IU/ml hCG, respectively. Data

represent the mean + SEM of triplicate wells of three separate

experiments.

h incubation did not significantly affect basal CAMP levels (Fig. 4). When combined with hCG (0.1 and 0.2 IU/ml), however, PRL caused a significant dose-dependent increase in CAMP ac- cumulation (Fig. 4). In the presence of MIX, PRL alone failed to significantly alter basal CAMP levels (Fig. 5). In contrast, PRL significantly increased, in a dose-dependent manner, hCG (0.1 and 0.2 IU/~)-indu~d CAMP accum~ation (Fig. 5).

A comparison of absolute values of CAMP ac- cumulation with and without MIX revealed not only that basal and hCG-induced values were higher in the presence of MIX, but that the degree of stimulation with MIX was dependent on PRL ~n~ntration (Fig. 6). Thus, with a constant dose of 0.1 III/ml hCG, MIX enhanced CAMP levels 1.5-, 1.8- and 2.1-fold when combined with 1, 10 and 100 ng/ml PRL, respectively. Similarly, MIX enhanced cAMP levels 1.8-, 2.0- and 2.5fold when

- hCQ MJ/ml) 0 PRL @g/ml) 6

Fig. 2. Effect of PRL on basal and hCG-induced P4 secretions

in luteal cell cultures. Luteal cells (44x105/well) were in-

cubated for 48 h with increasing concentrations of PRL (1, 10

and 100 q/ml) alone and in the presence of 0.1 and 0.2

IU/ml hCG. P., secretion was expressed as percentage, 100%

being designated as P4 secretion in the presence of each hCG

concentration alone. Absolute values of 100% P4 secretion were

19.0 i0.4 and 21.4i 0.8 ng/well with 0.1 and 0.2 IU/ml hCG,

respectively. Data represent the mean f SEM of triplicate wells

of two separate experiments.

Fig. 3. Dose-response curve for hCG-induced CAMP accumu- lation in luteal cell cultures. Luteal cells (4X10s/well) were

incubated for 1 h with increasing concentrations of hCG

(0.01-1.0 IU/ml). Data represent the meanf SEM of triplicate wells of the three separate experiments.

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Fig. 5. Effect of PRL on basal and hCG-induced CAMP accumulation in the presence of MIX in luteal cell cultures. Conditions were the same as those described in the legend to Fig. 4, except for the addition of 0.1 mM MIX. CAMP accumu- lation was expressed as percentage, 100% being designated as CAMP accumulation in the presence of each hCG concentra- tion alone. Absolute values of 100% CAMP accumulation were 11.4* 1.1 and 16.2kO.7 pmol/well with 0.1 and 0.2 W/ml hCG, respectively. Data represent the meanf SEM of triplicate

wells of the two separate experiments.

given together with the same PRL doses in the presence of a constant dose of 0.2 IU/ml hCG. One-way ANOVA showed significant stimulation of CAMP by PRL with 0.1 and 0.2 IU/ml hCG (P -z 0.01) as well as with MIX (P < 0.01).

Discussion

This study demonstrates a stimulatory effect of PRL on rat luteal cells in monolayer culture. Basal and hCG-induced levels of P4 were significantly stimulated by PRL in a dose-dependent manner. This is in keeping with the results of in vitro systems of suspended rat luteal cells (Sala et al., 1979; Behrman et al., 1980; Tesone et al., 1984)

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and with the luteotropic function of PRL in ro- dents observed in in vivo studies (Rothchild, 1981; Rajkumar et al., 1985). The parallel dose-depen- dent increase in hCG-induced CAMP accumu- lation by PRL observed in this study is consistent with the role of CAMP as mediator of P4 secretion in luteal cells (Sala et al., 1979; Behrman et al., 1980; Massicotte et al., 1984). Tesone et al. (1984) did not show an increase of hCG-induced CAMP levels upon addition of PRL to cultured rat luteal cells, despite an increase in P4 levels. The dis- crepancy with our data may be due to the dif- ference in age of the corpora lutea (2 days in our study as compared to 5 days in that of Tesone et al. (1984)). In this regard, it is worth noting that CAMP levels start decreasing on day l-2 of the corpus luteum (Norjavaara et al., 1982).

In contrast to the hCG-induced rise in P4 and CAMP levels, the PRL stimulatory effect on basal levels of P4 was not accompanied by any detect- able rise in basal CAMP accumulation. This was also noted by Tesone et al. (1984) in rat luteal cultures treated in vitro with PRL, in contrast to the in vivo PRL treatment that gave rise to a parallel increase in P4 and CAMP accumulation (Tesone et al., 1984). A rise in progestin secretion without a concomitant rise in CAMP accumulation has also been described in rat luteal cells stimu- lated in vitro by hCG (Conti et al., 1977; Sala et al., 1979). Sala et al. (1979) seem to have resolved the apparent discrepancy regarding the effect of hCG: they demonstrated an increase in binding of CAMP to the regulatory subunit of protein kinase during stimulation of P4 production by hCG, con- sistent with the intermediate role of CAMP in gonadotropin-induced steroidogenesis. Experi- mental proof is lacking to confirm or deny whether this is also the case regarding PRL.

Upon addition of MIX to PRL, the stimulation of hCG-induced CAMP accumulation by the lacto- genie hormone increased more than in the absence of the PDE inhibitor and in a fashion dependent on PRL concentration. Taken together with our previously reported data on rat granulosa cell cultures, which showed that PRL inhibition of steroidogenesis and CAMP formation are abolished by MIX (Gitay-Goren et al., 1989), it is reason- able to reach the following conclusions. Our stud- ies on granulosa (Gitay-Goren et al., 1988, 1989)

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0 WITHOUT MIX

a WITH MIX (0.1 mM)

hCG (NJ/ml) 0 0 0 0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 a1 0.2020202 020.202~ PRL(ngIml) 0 0 loo 100 0 0 1 1 lo lo im 100 0 0 1 1 10 10 100 100

Fig. 6. Comparison between the effects of PRL on basal and hCG-induced CAMP accumulation in the absence and presence of MIX in luteal cell cultures. Absolute values of the data in Figs. 4 and 5 are presented.

and luteal (present report) cell cultures indicate that PRL exerts divergent effects on steroidogene- sis in the ovary: inhibitory in the preovulatory and stimulatory in the postovulatory state. The use of the same experimental system in these studies, including the same rat strain, the same doses and source of PRL, hCG and MIX, and the same incubation periods, makes the divergency even sharper. The inhibition exerted by PRL is prob- ably an enhancement of PDE activity that could be overcome by MIX: This inhibitory effect, which we showed in the preovulatory ovary (Gitay-Goren et al., 1989), can be hypothesized to exist in the postovulatory ovary as well. The difference in the net overall effect of PRL on the pre- and postovu- latory phases (i.e., inhibitory and stimulatory in- fluences, respectively) may lie in the existence, as referred to in the Introduction, of an additional action of PRL after ovulation: a luteotropic stimu- latory effect. The addition of graded doses of PRL to constant concentrations of hCG resulted in a

dose-dependent increase of CAMP and P4 accumu- lation, reflecting the luteotropic effect of PRL. This luteotropic action may mask the restraining influence of PRL on CAMP accumulation in the postovulatory ovary. By inhibiting PDE activity, MIX abolishes the reduction in CAMP accumula- tion, hence our observation of a clearer expression of the luteotropic action of PRL when MIX was added. Obviously, PDE activity would have to be measured directly to substantiate this hypothesis. It is worth noting that insulin has been shown to stimulate PDE activity in rat luteal cells (Tellez- Inon et al., 1987).

Other studies have demonstrated divergent ef- fects of PRL on steroidogenesis (McNatty et al., 1974, 1976; Kalison et al., 1985) in the preovula- tory in contrast to the postovulatory ovary, i.e., inhibitory prior to ovulation and stimulatory fol- lowing ovulation. The present report, together with our previous studies (Gitay-Goren et al., 1988, 1989), demonstrates such divergent effects of PRL

201

measuring CAMP accumulation as well as ste- roidogenesis, drawing such conclusions using the same experimental system and conditions in granulosa and luteal cell cultures. Moreover, this is the first report providing data suggesting a possible common mechanism linking the effect of PRL before and after ovulation: inhibition of CAMP accumulation via enhanced breakdown of the nucleotide.

Acknowledgements

This study was supported in part by the Tech- nion V.P.R. Fund - New York Metropolitan Re- search Fund.

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