Interactions between activin and follicle-stimulating hormone-suppressing protein and their mechanisms of action on cultured rat granulosa cells

Direct roles of follicle-stimulating hormone (FSH)-suppressing protein (FSP) and activin in regulation of ovarian granulosa cell differentiation have been reported recently. The present study further investigated the effects of these peptides on steroidogenesis and inhibin production as well as cAMP generation in cultured granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of FSH (20 ng/ml) and activin (30 ng/ml), which enhanced FSH-induced aromatase activity, progesterone production and inhibin production, FSP (1-100 ng/ml) reversed the stimulating activities of activin in a dose-dependent manner. In addition, activin reversed the inhibitory effects of FSP on FSH-induced aromatase activity and inhibin production. In the presence of FSH, activin enhanced FSH-stimulated extracellular cAMP accumulation, and FSP caused a reduction in extracellular cAMP. Activin but not FSP also stimulated basal cAMP level. In the presence of forskolin, a potent stimulant of adenyl cyclase activity which stimulated extracellular cAMP, aromatase activity, progesterone production and inhibin production, activin augmented the effect of forskolin on all four parameters, whereas FSP significantly enhanced progesterone production without changing the other three parameters. Our findings suggest that activin action on rat granulosa cells may be mediated via regulation of cAMP generation. The action of FSP and FSH and/or activin-dependent, consistent with either an action as an activin binding protein or by a direct action of FSP on the granulosa cells.     

The effect of bovine activin and follicle-stimulating hormone (FSH) suppressing protein/follistatin on FSH-induced differentiation of rat granulosa cells in vitro

The time- and dose-dependent effects of bovine activin A and bovine follicle stimulating hormone (FSH) suppressing protein (FSP) or follistatin on basal and FSH-induced steroidogenesis and inhibin production were studied in granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of rat FSH (20 ng/ml) which stimulates aromatase activity and the production of progesterone and inhibin, activin (0.3-100 ng/ml) augmented all three parameters, whereas FSP (0.3-100 ng/ml) enhanced progesterone production and attenuated the other two parameters. In the absence of FSH, the basal parameters were unaffected by treatment with either activin or FSP alone, except for a statistically significant increase in basal inhibin in the presence of activin alone (P less than 0.05, at doses of 30 and 100 ng/ml). Neither activin nor FSP influenced the timing of the maxima of FSH-induced activities over 5 days. These findings suggest that activin and FSP, both present in follicular fluid, may play an important role in the local regulation of granulosa cell differentiation.     

Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.     

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.     

An inhibitory effect of transferrin on differentiation of rat granulosa cells in vitro

The effects of human transferrin (TRF) on granulosa cell function were examined using serum-free cultures of rat granulosa cells obtained from immature, diethylstilbestrol-treated rats. The results show that TRF had dose- and time-dependent inhibitory effects on FSH-induced inhibin and progesterone production with the half-maximal inhibitory dose of 6.1-6.3 micrograms/ml. The inhibitory effect of TRF on FSH-induced inhibin and progesterone production was not reversed by removing TRF and changing medium after 48 h of treatment. TRF also inhibited insulin- and insulin-like growth factor-I (IGF-I)-induced inhibin production in a dose-dependent manner. TRF did not inhibit forskolin- and 8-bromo-cAMP-induced progesterone production but did inhibit inhibin production induced by these agents. TRF had no effect on basal production of inhibin and progesterone. On the other hand, high concentrations of insulin and cortisol completely counteracted the inhibitory effect of TRF on FSH-induced progesterone production but only partially counteracted the inhibitory effect of TRF on FSH-induced inhibin production. Our data suggest that: 1) TRF may be an important negative modulator of the stimulatory actions of FSH or IGF-I and other factors acting on granulosa cells; 2) the inhibitory effects of TRF require the presence of FSH or other factors such as IGF-I or insulin, which facilitate granulosa cell differentiation; and 3) different mechanisms are involved in the modulating effects of TRF on inhibin and progesterone production.     

Divergent effects of prolactin on estrogen and progesterone production by granulosa cells of rat Graafian follicles

The effect of PRL on ovarian steroidogenesis was studied in cultured granulosa cells isolated from follicles of mature cycling rats on the morning of proestrus. Ovine PRL 10-1000 ng/ml) inhibited estradiol production but stimulated progesterone biosynthesis in a dose-dependent manner. The effect of PRL was most prominent after 4 days of culture: 1000 ng/ml PRL suppressed estradiol production by 80% but increased progesterone synthesis by 290%, whereas the lower dose of 10 ng/ml inhibited estrogen secretion by 20% without altering progesterone synthesis. The divergent effect of PRL was not shown to be species specific, since ovine, rat and human PRL had similar effects. Using increasing concentrations of androstenedione (the aromatase substrate), estrogen secretion remained suppressed and progesterone production was stimulated by PRL. FSH stimulated both estrogen and progesterone production. The FSH-induced increased in estrogen production was inhibited by concomitant treatment with PRL. In contrast, PRL and FSH had an additive action in stimulating progesterone production. Although LH alone had no effect on steroidogenesis, concomitant treatment with LH and PRL resulted in a stimulation of progesterone production that was additive. This study demonstrates that PRL acts directly on granulosa cells of Graafian follicles of adult cycling rats to stimulate the secretion of progesterone and to suppress estradiol production.     

The effect of insulin on inhibin production in isolated seminiferous tubule segments from adult rats cultured in vitro

The effect of insulin and its interaction with intracellular messenger systems on in vitro inhibin production by adult rat isolated seminiferous tubules has been investigated using a recently developed inhibin radioimmunoassay (RIA). Seminiferous tubule segments (5 cm) from intact adult rats were exposed to insulin (0.05-5000 ng/ml) for 2 days of culture. Insulin caused a dose-dependent inhibition of basal inhibin secretion with reversal of this inhibition at very high doses (5000 ng/ml). The ability of follicle-stimulating hormone (FSH) to induce inhibin secretion was also inhibited by insulin (50 ng/ml). Insulin reduced the stimulation of inhibin production by dibutyryl cyclic AMP (dbcAMP) and this effect was prevented by the addition of theophylline (0.4 mM), while theophylline alone was unable to prevent the effect of insulin on basal inhibin secretion. Phorbol 12-myristate 13-acetate (PMA) mimicked the effect of insulin reducing basal and FSH-induced secretion of inhibin. No additive effects on basal inhibin secretion were observed with a combination of PMA and insulin. Ethylenediaminetetraacetic acid (EDTA, 2 mM) significantly reduced basal and FSH-induced inhibin production, while the combined effects of EDTA and insulin on basal and FSH-induced inhibin production were additive. These data demonstrate an inhibitory effect of insulin on inhibin production by isolated seminiferous tubules mediated via at least two mechanisms namely the inhibition of the cAMP-protein kinase A system and stimulation of protein kinase C activity.     

Regulation of inhibin production by rat granulosa cells

Inhibin production by cultured granulosa cells from immature diethylstilbestrol (DES)-primed rats was studied in relation to estradiol and progesterone production. The inhibin content in culture media was assayed with a specific radioimmunoassay (RIA) using an antibody to porcine 32 kDa inhibin that recognizes rat inhibin as well. Inhibin production was about 10 ng/ml/2 X 10(4) cells/72 h at the basal levels and was maximally stimulated with 25 ng/ml of follicle stimulating hormone (FSH) to 45 ng/ml which was 4.5 times the basal levels, with an ED50 value of 2.0 ng/ml. A cyclic AMP analog (dibutyryl cyclic AMP) or reagents that promote cAMP production were also effective in inhibin production, indicating that FSH stimulates inhibin production through a cAMP-dependent pathway. Luteinizing hormone (LH) was not effective in producing inhibin from freshly prepared granulosa cells, whereas granulosa cells pre-incubated with FSH for 48 h because responsive to LH regarding inhibin production. Testosterone sensitized the granulosa cells to the FSH stimulation, whereas hydrocortisone (4 ng/ml) decreased the sensitivity of granulosa cells by increasing the ED50 value for inhibin production by FSH about 10 times. A similar effect was observed regarding estradiol production, while progesterone production due to stimulation by FSH was enhanced by the hydrocortisone treatment. Insulin and platelet extract both stimulated inhibin production and enhanced the maximal response of inhibin production due to stimulation by FSH without altering, or even increasing the ED50 values. Epidermal growth factor (EGF), (D-Leu6)Des-Gly10-LHRH N-ethylamide (GnRH agonist) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator, inhibited both inhibin production and estradiol or progesterone production. Consequently, the regulation of inhibin production was similar to that of estradiol production, but markedly different from that of progesterone. However, inhibin and estradiol production were modulated differently by various growth factors and hormones. These phenomena might account for possible discrete changes in the plasma levels of inhibin and estradiol in vivo.     

Regulation of Sertoli cell activin A and inhibin B by tumour necrosis factor α and interleukin 1α: interaction with follicle-stimulating hormone/adenosine 3',5'-cyclic phosphate signalling

Regulation of crucial events during spermatogenesis involves dynamic changes in cytokine production and interactions across the cycle of the seminiferous epithelium. Regulation of activin A and inhibin B production by the inflammatory cytokines, tumour necrosis factor α (TNFα) and interleukin 1α (IL1α), alone and in conjunction with FSH or a cAMP analogue (dibutyryl cAMP), was examined in cultures of Sertoli cells from 20-day old rats. Both TNFα and IL1α stimulated activin A secretion and expression of its subunit (β(A)) mRNA, and suppressed inhibin B secretion and expression of its subunit (α and β(B)) mRNAs. The actions of TNFα and IL1α were opposed by FSH and dibutyryl cAMP. Both cytokines inhibited FSH/dibutyryl cAMP-stimulated inhibin B secretion and mRNA expression as well as stem cell factor mRNA expression. Both cytokines also inhibited FSH-induced cAMP production, and reduced baseline FSH receptor mRNA expression. These data highlight the reciprocal relationship that exists between FSH/cAMP signalling and inflammatory cytokine signalling pathways in the control of Sertoli cell function, and production of activin A/inhibin B in particular. It is anticipated that these interactions play important roles in the fine control of events during the cycle of the seminiferous epithelium and in the inhibition of spermatogenesis during inflammation.     

In vitro synthesis and release of inhibin in response to FSH stimulation by isolated segments of seminiferous tubules from normal adult male rats

The production of inhibin by isolated segments of seminiferous tubules from adult male rats cultured in vitro was investigated using a heterologous specific radioimmunoassay. Increasing lengths of tubules (5, 10, 20 and 40 cm) maintained in culture for 4 or 5 days produced increasing amounts of inhibin in vitro. A dose-dependent increase in inhibin production was observed after stimulation with ovine follicle-stimulating hormone (FSH)-s17 (0.1-1000 ng/ml). The tubule segments remain sensitive to FSH stimulation for up to 20 days of culture despite a progressive decline in basal inhibin production, resulting in an increase in the magnitude of the response to FSH stimulation between 0-5, 5-10 and 10-20 days of culture. In the presence of the protein synthesis inhibitor, cycloheximide (50 micrograms/ml), both basal and FSH-stimulated inhibin secretion are inhibited. Testosterone (10(-8)-10(-5) M) does not affect basal inhibin production, although inhibition of the FSH-induced production of inhibin occurred at only the highest dose of testosterone used (10(-5) M). These data demonstrate that the production of inhibin by segments of seminiferous tubules from adult male rats can be used to study the control of inhibin secretion.     

Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 microm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin alpha- and beta B-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell alpha-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced beta B-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin beta B-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.     

Site of action of androgens on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

This paper describes experiments on cultured granulosa cells isolated from ovaries of immature rats designed to locate the site of action of androgens on FSH-induced aromatase activity. Treatment of cells during a 36-h induction period with (Bu)2cAMP, 8- BrcAMP , FSH, prostaglandin E2, or cholera toxin resulted in induction of aromatase activity measured as 17 beta-estradiol accumulation during a 6-h test period with testosterone (5 X 10(-7) M) added to medium as substrate. Presence of testosterone (5 X 10(-7) M) during the induction period enhanced the effects of FSH, cholera toxin, and prostaglandin E2 on aromatase activity, but not those of the cAMP analogs. The effects of culturing and steroids on responsiveness of granulosa cells to FSH (measured as FSH-stimulated cAMP production during a 1-h test period) were examined. The data showed that culturing in medium alone for 36 h resulted in a decrease in the ability of FSH to stimulate cAMP production when compared to that of freshly isolated cells. After culture with testosterone (5 X 10(-7) M), dihydrotestosterone (DHT) (5 X 10(-7) M), or 17 beta-estradiol (5 X 10(-7) M), responsiveness was at least partially restored. After treatment with progesterone (5 X 10(-7) M), FSH stimulation of cAMP production was not significantly different from that of cells cultured in medium alone. Hydroxyflutamide (5 X 10(-5) M), an antiandrogen known to block androgen-receptor interaction, abolished the effect of DHT and depressed the effect of testosterone on responsiveness of granulosa cells to FSH. Cells treated for 36 h with testosterone (5 X 10(-7) M) bound significantly more [125I]iodo-FSH than cells cultured in medium alone. Although DHT (5 X 10(-7) M) slightly increased FSH binding, the effect was not statistically significant. These results suggested that androgens regulate granulosa cell aromatase activity not only as substrates, but also by acting at a site before cAMP production (possibly at the level of the FSH receptor) in the control of FSH-induced enzyme activity.     

Stimulatory and inhibitory influences of serum from pregnant women on aromatase activity of immature rat Sertoli cells

Effects of serum from pregnant women on basal and FSH (or cAMP) stimulated aromatase activity of immature rat Sertoli cells in primary culture were studied. Pregnancy serum caused a dose-dependent stimulation of Sertoli cell aromatase activity and the response curves were parallel to those obtained with human FSH. This stimulatory (FSH-like) activity increased progressively during pregnancy, with a sharp drop immediately after delivery. However, the FSH-like bioactivity was not associated with immunoreactive FSH when a specific radioimmunoassay was employed. On the other hand, serum from pregnant women caused a dose-dependent inhibition of FSH and dibutyryl-cAMP-stimulated aromatase activity. These data suggest that human pregnancy serum contains factor(s) which may stimulate basal aromatase activity of Sertoli cells and may inhibit FSH-induced aromatase activity. These factors, most probably of placental origin, may play a role in the regulation of estrogen production during gestation.     

Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells.

Estradiol (E) biosynthesis by the cytochrome P450 aromatase (P450arom) enzyme system increases as preovulatory follicles develop and is subsequently reduced by the ovulatory LH surge. To determine the specific effects of gonadotropins and steroids on expression of P450arom in rat granulosa cells, steady state levels of messenger (m) RNA were examined in vivo and in vitro, with the latter also being related to aromatase enzyme activity and cAMP production. P450arom mRNA and activity were induced in granulosa cells by FSH alone in a dose-, time-, and stage-dependent manner. E enhanced the effects of FSH in vivo and in vitro. The synergistic effect of E with FSH (50 ng/ml) was observed in the absence/presence of serum and was mimicked by a similar concentration (20 nM) of testosterone, dihydrotestosterone, or dexamethasone. In contrast, ovulatory doses of LH (500 ng/ml) or forskolin (10 microM) but not concentrations of progesterone reached in preovulatory follicles (100-1000 nM) acted on differentiated (FSH + E) granulosa cells to cause a rapid loss of P450arom mRNA. Whereas cycloheximide prevented the LH/cAMP-mediated decrease in P450arom mRNA in the differentiated cells, enzyme activity remained unaltered during the same 6-h period. Thus, expression of aromatase mRNA in rat granulosa cells is induced primarily by low FSH/cAMP, enhanced by physiological doses of several steroids (except progesterone), and, once induced, can be rapidly inhibited by elevated gonadotropin/cAMP via a pathway requiring protein synthesis.     

Hormonal regulation of granulosa cell inhibin biosynthesis

The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolactin inhibition of estrogen production by cultured rat granulosa cells

The effect of prolactin (PRL) treatment on estrogen production by rat granulosa cells was investigated in vitro. Immature, hypophysectomized, DES-treated rats were injected for 2 days with FSH to induce aromatase enzymes and receptors for PRL and LH. After FSH priming, the granulosa cells were cultured for 4 days in serum-free medium containing 10(-7) M androstenedione and purified FSH, LH and/or PRL. A dose-related inhibition of estrogen production from control cells was observed following PRL treatment in which 1 micrograms/ml of PRL inhibited estrogen formation by > 90%. In these same cultures, PRL caused a dose-related increase in progesterone and 20 alpha-dihydroprogesterone secretion. Treatment with purified FSH or LH stimulated estrogen synthesis by 3-10-fold. Concomitant treatment with PRL suppressed the FSH- and LH-induced increases in estrogen production in a dose-dependent manner; 1 micrograms/ml PRL suppressed estrogen production by > 80% during days 2-4 of culture. In these same cultures, PRL did not alter the stimulatory effects of FSH and LH on progesterone and 20 alpha-dihydroprogesterone production. These experiments demonstrate that PRL acts directly on rat granulosa cells in vitro to suppress basal and gonadotropin-induced increases in estrogen production.     

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.