Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the 48-h culture period, 4-hydroxy-4-androstene-3,17-dione (4-OHA; greater than or equal to 100 microM) and 1,4,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by 40% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-48 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with 4-OHA and 95% with 1,4,6-androstatriene-3,17-dione). Addition of estradiol, but not androstenedione, reversed the reduction of LH receptor formation by 4-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with androstenedione (80-fold) during the 48-h culture period was prevented by 4-OHA. FSH-stimulated cAMP production was initially enhanced by 4-OHA from 0-20 h of culture, but was reduced from 20-48 h. Lower concentrations of 4-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that 4-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of 4-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.     

Estrogens enhance the adenosine 3',5'-monophosphate-mediated induction of follicle-stimulating hormone and luteinizing hormone receptors in rat granulosa cells

The effects of estrogens on cAMP-induced FSH and LH receptor expression were studied in granulosa cells isolated from immature diethylstilbestrol-implanted rats. Although estradiol alone had negligible effects on granulosa cell maturation, estradiol concentrations from 10(-11)-10(-8) M progressively enhanced cAMP production and gonadotropin receptor formation in choleragen-stimulated cells. During 48 h of culture, estradiol augmented cAMP levels by 2-fold, LH receptors by 4- to 6-fold, and FSH receptors by 20-40%. Estradiol also enhanced the extent of LH and FSH receptor formation by other cAMP-inducing ligands, including FSH, prostaglandin E2, and forskolin. The stimulatory action of 8-bromo-cAMP on gonadotropin receptors was also increased by estradiol, indicating that part of the estrogenic effect was exerted on cAMP-activated processes. Scatchard analyses indicated that estradiol increased the number of choleragen-induced FSH receptors from 2,600 to 3,200/cell and of LH receptors from 13,000 to 86,000/cell with no changes in receptor binding affinity. Choleragen-stimulated cAMP accumulation was enhanced by estradiol during the later stages of culture (after 30 h), while increased LH receptors were detected by 30 h and FSH receptors by 43 h. The stimulatory effects of estradiol were not due to increased cellular proliferation and were also exerted by other estrogens, including estrone and diethylstilbestrol. Androgens, including testosterone and androstenedione, also amplified choleragen action. This effect was largely through conversion to estrogens, since dihydrotestosterone, a nonaromatizable androgen, did not markedly enhance LH receptor formation by choleragen. In contrast, progestins and pregnenelone had no facilitative effect on choleragen-induced responses. Although cortisol and dexamethasone increased choleragen-induced cAMP accumulation, only cortisol elevated LH receptors, and dexamethasone inhibited FSH receptor formation. These results demonstrate that estrogens enhance both ligand-induced cAMP production and cAMP-activated responses during granulosa cell differentiation. In particular, estrogens exert a major effect on the levels of gonadotropin receptors expressed in response to FSH and other cAMP-inducing ligands.     

Induction of granulosa cell differentiation by forskolin: stimulation of adenosine 3',5'-monophosphate production, progesterone synthesis, and luteinizing hormone receptor expression

The effects of forskolin on the acquisition of differentiated functions in cultured ovarian granulosa cells were compared with the actions of FSH and prostaglandin E2 (PGE2). In 48-h granulosa cell cultures from immature diethylstilbestrol-treated rats, 100 microM forskolin caused a 45-fold increase in cAMP accumulation and stimulated progesterone production from undetectable levels (less than 0.2 ng/ml) to 80 ng/ml. The forskolin-induced increase in cAMP was similar to the maximum response to FSH, and progesterone production was about 50% of that elicited by FSH. PGE2 also enhanced cAMP and progesterone production in a concentration-dependent manner, with a maximum 8-fold increase in cAMP accumulation and an increase in progesterone to 5.6 ng/ml when the PGE2 concentration was 10 micrograms/ml. The time course of forskolin-stimulated cAMP production was notable for its rapid rise to the maximum level during the first 24 h of culture, followed by a plateau for up to 72 h. This contrasted with FSH-stimulated cAMP production, which increased progressively for up to 72 h when measured at 24-h intervals. LH receptor levels were low in untreated cells and after exposure to the various stimuli for 24 h, but increased 9- to 11-fold after culture with FSH or forskolin for 48-72 h. PGE2-induced LH receptor formation was about 20% of that seen after FSH stimulation. Forskolin enhanced cAMP and progesterone production in response to FSH and choleragen, but impaired the effects of these ligands on LH receptor formation. Exposure of the cultured cells to a potent GnRH agonist inhibited forskolin-induced progesterone and LH receptor synthesis, but did not influence forskolin-stimulated cAMP production. These results demonstrate the ability of forskolin to serve as a nonhormonal stimulator of granulosa cell differentiation and indicate the importance of cAMP in this process, as well as the ability of GnRH agonists to exert inhibitory effects on post-cAMP steps in cellular maturation.     

Enhanced follicle-stimulating hormone activity of deglycosylated human chorionic gonadotropin in ovarian granulosa cells

The receptor binding properties and biological actions of chemically deglycosylated asialo human CG (AHF-hCG) were studied in ovarian granulosa cells from diethylstilbestrol (DES)-treated immature rats. In ovarian homogenates from DES- and FSH-treated rats, the relative binding affinity of AHF-hCG was 2-fold higher than that of native hCG and 14-fold higher than that of ovine LH. When assayed for LH-like activity in granulosa cells from DES plus FSH-treated animals, the deglycosylated hormone behaved as a partial agonist in terms of cAMP formation, but fully stimulated progesterone production to the same extent as that elicited by LH. When added with LH to FSH-treated cells, AHF-hCG inhibited LH-stimulated cAMP formation by 70% but did not alter the elevated level of progesterone production. These findings are consistent with the presence of excess or spare LH receptors in the maturing granulosa cell. When added to freshly prepared granulosa cells which have minimal LH receptors, AHF-hCG decreased FSH-stimulated cAMP production by 20% and reduced progesterone production by 50% and increased cGMP formation by 100% during 48 h of culture. The ability of AHF-hCG to decrease the progesterone response to FSH suggests that no spare FSH receptors are present during granulosa cell differentiation. In contrast, native hCG did not alter FSH-induced cAMP or progesterone production but reduced the cGMP responses to FSH and choleragen. Whereas native hCG displayed negligible binding potency when compared with that of ovine FSH in competition with [125I]iodo-human FSH for ovarian receptors, AHF-hCG bound to FSH receptors with about 5% of the binding affinity of ovine FSH. In choleragen-treated granulosa cells, the increases in cAMP and progesterone synthesis were enhanced by addition of both hCG and AHF-hCG, and cGMP production was increased by AHF-hCG but slightly decreased by hCG. These results indicate that the enhanced LH receptor affinity caused by removal of the sugar moieties from hCG is accompanied by a relatively greater increase in FSH receptor affinity, and that deglycosylated hCG acts as a partial agonist with the ability to modify granulosa cell responses to both LH and FSH.     

Inhibition of granulosa cell differentiation by dioctanoylglycerol--a novel activator of protein kinase C

The actions of dioctanoylglycerol, a novel and specific activator of protein kinase C, on granulosa cell steroidogenesis and LH/hCG receptor induction were examined in vitro. Granulosa cells were cultured for 2 days in media containing 100 nM androstenedione. FSH treatment stimulated progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-P) and estrogen production by 60- to 80-fold over basal levels. Treatment with dioctanoylglycerol increased progesterone production 3-fold over basal levels but failed to affect 20 alpha-OH-P or estrogen production. Concomitant treatment of cells with FSH and increasing amounts of dioctanoylglycerol produced dose-dependent inhibition of FSH-stimulated progesterone, 20 alpha-OH-P and estrogen production with IC50 values of 75, 40 and 50 microM, respectively. Dioctanoylglycerol also inhibited production of all three steroids induced by forskolin, an activator of adenylate cyclase, and dibutyryl cAMP. The effects of dioctanoylglycerol on cAMP responses to FSH were also examined. The increase in both intracellular and extracellular cAMP at 1 h following treatment with FSH was suppressed in the presence of dioctanoylglycerol as was the extracellular accumulation of cAMP at 48 h of culture. FSH induction of LH/hCG receptors, known to be a cAMP-mediated event, was also inhibited in the presence of dioctanoylglycerol. In contrast, a thio-derivative of dioctanoylglycerol (3-thio-1,2-dioctanoylglycerol), which does not activate protein kinase C, failed to inhibit both steroidogenesis and LH/hCG receptor induction in response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Granulosa cell differentiation in vitro: induction and maintenance of follicle-stimulating hormone receptors by adenosine 3',5'-monophosphate

Granulosa cell differentiation is stimulated in vitro by FSH and other agents that increase cAMP production. To determine if alterations in FSH receptors are associated with cAMP-mediated granulosa cell maturation, FSH-binding sites were measured during culture of undifferentiated granulosa cells from hypophysectomized diethylstilbestrol-implanted rats. FSH receptors decreased rapidly in the absence of stimulatory ligands, with loss of 75% and 90% of the FSH-binding activity from freshly prepared cells after 24 and 48 h, respectively. The decline in FSH receptors during culture was accompanied by a corresponding decrease in cAMP responses to added FSH. In cells cultured with FSH, the available FSH receptor content fell to 15% after 8 h, then rose to 25% of the initial receptor levels from 24-48 h of culture. Cells treated with 8-bromo-cAMP or choleragen for 48 h retained about 40% and 90%, respectively, of their initial FSH-binding activity. Although choleragen did not prevent the 60-70% fall in FSH binding during the first 6 h of culture, it increased receptors 2.5-fold from 12-48 h. Also, the addition of choleragen after 3, 6, or 12 h of culture elevated FSH receptors at 48 h in proportion to the amount of cAMP produced. The FSH receptors induced by choleragent treatment were functionally active, as shown by their ability to mediate FSH-stimulated cAMP production. A GnRH agonist prevented the choleragen-induced rise in FSH receptors and cAMP accumulation from 24-48 h of culture. Scatchard analysis revealed a single population of FSH receptors after all treatments, with association constant of 5 X 10(9) M-1. Freshly prepared cells contained approximately 1600 FSH binding sites/cell, while treatment with choleragen or FSH for 48 h restored receptor levels to 1150 and 500 sites/cell, respectively. These results indicate that the expression of functional FSH receptors during granulosa cell maturation is mediated by cAMP.     

Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.     

Bifunctional role of transforming growth factor-beta during granulosa cell development

Regulatory actions of transforming growth factor-beta (TGF beta) on granulosa cell function were analyzed in cells cultured from the ovaries of diethylstilbestrol-implanted rats. In the presence of a suboptimal concentration of FSH (5 ng/ml) that increased LH receptors by 100-fold during a 72-h culture, TGF beta augmented this response in a dose-dependent manner with a maximal effect at 16 pM. In contrast, the growth factor inhibited the LH receptor response to an optimal dose of FSH (50 ng) by up to 50% and was inactive in the absence of gonadotropin. TGF beta also enhanced the formation of cAMP by 5 ng FSH and partially inhibited the effects of higher FSH concentrations. However, the actions of TGF beta were more prominent on LH receptor induction than on cAMP production with either low or high amounts of FSH. In addition, TGF beta had little effect on cAMP production stimulated by cholera toxin or forskolin, but amplified the actions of these ligands as well as that of 8-bromo-cAMP on LH receptor expression. TGF beta also modulated the steroidogenic activity of the granulosa cells, with increased production of progesterone in response to 5-100 ng FSH. The bifunctional actions of TGF beta on FSH-induced LH receptor formation were observed throughout a 96-h culture period. However, the presence of the growth factor was not required for the first 24 h of culture, indicating that TGF beta alters the later events involved in LH receptor formation. TGF beta augmented the stimulatory actions of 5 ng FSH on LH receptors in the absence or presence of insulin, but its inhibitory effect on these receptors was only observed in cells treated with insulin. These results indicate that TGF beta modifies FSH action during granulosa cell development in a biphasic manner. TGF beta can exert stimulatory or inhibitory effects depending upon the concentration of FSH and the presence of insulin, and these are due to alterations in cAMP action as well as cAMP production. Autocrine and/or endocrine actions of TGF beta during granulosa cell differentiation may be involved in the processes of follicle selection and development.     

Regulation of the content and phosphorylation of RII by adenosine 3',5'-monophosphate, follicle-stimulating hormone, and estradiol in cultured granulosa cells

The mechanisms by which FSH and cAMP induce receptors for LH (RLH) and increase progesterone (P) production in estradiol (E)-primed ovarian granulosa cells remain unclear, but may involve increases in the regulatory subunit of cAMP-dependent protein kinase II (RII) and the phosphorylation of specific cellular proteins. To examine the relationship of these events, primary cultures of granulosa cells (10(6) cells/ml) from E-treated (1.5 mg/day for 3 days) immature female rats were incubated with 10 nM E with or without FSH (25 ng/ml) for 0-120 h. The cytosolic content of RII was analyzed by four techniques: 1) immunoblotting using an antibody to bovine heart RII; 2) photoaffinity labeling with [32P]8-azido-cAMP; 3) phosphorylation with [gamma-32P]ATP with or without 2 microM cAMP or with the catalytic subunit of cAMP-dependent protein kinase; and 4) phosphorylation of intact cells with [32P] orthophosphate. All approaches revealed a time-dependent 5- to 6-fold increase in RII content in granulosa cells cultured for 48 h with E and FSH compared to that in cells treated with E alone. The content of RI, the regulatory subunit of protein kinase type I, remained low throughout the culture period regardless of hormone treatment. Granulosa cells were also cultured with E (10 nM) and 8-bromo-cAMP (8-Br-cAMP; 0.25-3 mM) or forskolin (0.5-100 microM), agents that increase intracellular cAMP, for 48 or 72 h. The cytosolic content and phosphorylation of RII were increased by culturing granulosa cells in E and 8-Br-cAMP (1 mM) or forskolin (50 microM) for 48 h. The increase in RII was associated with a FSH-mediated increase in the content and phosphorylation of other cAMP-dependent phosphoproteins. The increases in RII and cAMP-dependent phosphoproteins were associated with specific alterations in granulosa cell function: a FSH-mediated rise in 1) RLH [59.3 +/- 7.4 cpm/micrograms DNA (without FSH) to 1171.5 +/- 157 cpm/micrograms DNA (with FSH]) and 2) P accumulation in the medium [0.05 +/- 0.03 ng/ml (without FSH) to 25.3 +/- 4.6 ng/ml (with FSH]) at 48 h. A dose-dependent increase in the RLH and P accumulation in the medium was observed at 48 h of culture with E and 8-Br-cAMP or E and forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-induced expression of receptors for growth hormone releasing factor in cultured granulosa cells.

The hypothalamic neuropeptide, GRF, is formed in the ovary and acts via specific receptors in granulosa cells to enhance cAMP production and steroidogenic responses to the pituitary gonadotropin, FSH. Granulosa cells cultured without hormonal treatment displayed low levels of binding sites for GRF and the related neuropeptide, vasoactive intestinal peptide. However, treatment with increasing concentrations (50-500 ng/ml) of FSH caused dose-dependent increases in cAMP production and expression of binding sites measured with radioiodinated [His1, Nle27]human GRF(1-32)NH2, with no change in binding affinity. The maximum increase in GRF binding sites (2.2-fold) was elicited by 250 ng/ml FSH after 72 h incubation. GRF binding sites were also increased by agents that elevate intracellular cAMP, including choleragen, vasoactive intestinal peptide, dibutyryl cAMP, and forskolin. Low doses of forskolin that did not alone increase [125I] [His1, Nle27] human GRF(1-32)NH2 binding potentiated the action of FSH on GRF binding sites, but the effects of maximal stimulatory doses of both agents were not additive. These findings demonstrate that FSH promotes the expression of GRF receptors in maturing granulosa cells through cAMP-dependent mechanisms. Since GRF enhances the actions of FSH on cAMP production and granulosa cell differentiation, and GRF receptors are increased by the cAMP-mediated actions of FSH, locally produced GRF could exert a positive autoregulatory action to accelerate follicular maturation by amplifying the granulosa cell response to FSH.     

Calcium dependence of the inhibitory effect of gonadotropin-releasing hormone on luteinizing hormone-induced cyclic AMP production in rat granulosa cells

The role of calcium in the inhibitory action of the GnRH superagonist, [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), on LH-stimulated cAMP production was studied in cultured granulosa cells obtained from immature hypophysectomized diethylstilbestrol (DES)-implanted rats. After culture for 48 h with FSH to induce LH receptors, cells were incubated for 2 h with LH or LH plus GnRHa. In this system, the cAMP response to LH was independent of extracellular calcium. In the presence of 0.25 to 1 mM extracellular calcium, 10(-8) M GnRHa caused a 15 to 40% inhibition of LH-stimulated cAMP production. Omission of extracellular calcium completely abolished the inhibitory effect of GnRHa upon LH-induced cAMP production. The inhibitory effects of GnRHa on prostaglandin E2 (PGE2) and isoproterenol-induced cAMP productions were also markedly reduced in the absence of extracellular calcium. Addition of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), reversed the inhibitory action of GnRHa on LH-induced cAMP production. These results demonstrate that extracellular calcium is necessary for the acute inhibitory action of GnRHa upon LH-induced cAMP production in cultured rat granulosa cells, and indicate that increased degradation of cAMP may be a contributing factor for this effect of GnRHa.     

Direct actions of 17 beta-estradiol on progesterone production by highly differentiated porcine granulosa cells in vitro. II. Regulatory interactions of estradiol with luteinizing hormone and cyclic nucleotides

We investigated direct actions of 17 beta-estradiol and LH in the coordinate control of progesterone production by highly differentiated porcine granulosa cells maintained in monolayer culture. The administration of estradiol acutely suppressed both basal and LH-stimulated progesterone synthesis in vitro, i.e within the first 24-36 h of estrogen treatment. In contrast, continuation of estradiol administration alone beyond 48 h significantly augmented progesterone production per 10(5) granulosa cells. Among 12 independent experiments, the absolute stimulatory effects of estradiol were highly correlated (r = 0.991) with basal progesterone production by granulosa cells at the outset of culture, i.e. when steroid synthesis presumably reflected the degree of prior cytodifferentiation attained in vivo. Notably, estrogens also facilitated the dose-dependent actions of LH in a synergistic fashion. Synergism occurred during periods of both maximal and spontaneously declining steroidogenesis in vitro, and could be impeded by specific inhibitors of steroid biosynthesis (10 microM cyanoketone and 50 microM trilostane). In experiments designed to assess granulosa cell responsivity to delayed hormone rechallenge, there was a critical bihormonal requirement for both estradiol and LH in order to sustain maximal long term progesterone secretion. Further investigation of the biochemical mechanisms subserving synergistic effects demonstrated that estradiol was capable of augmenting the stimulatory actions of either exogenously supplied or endogenously generated cAMP. In particular, estradiol markedly enhanced the effects of potent phosphodiesterase resistant analogs of cAMP, 8-bromo-cAMP (0.1 mM), dibutyryl cAMP (2 mM) or 8-thio-cAMP (1 mM). Estradiol also significantly facilitated the stimulatory effects of agents that putatively increase or sustain intracellular pools of cAMP by various well defined mechanisms, i.e. choleratoxin (10 microgram/ml), guanyl-5'-imido-diphosphate (1.0 mM) or 3-isobutyl-1-methylxanthine (0.25 mM). Thus, the current in vitro studies delineate directly major interactions between estradiol and LH in the control of progesterone synthesis by highly differentiated granulosa cells. The present data further indicate that the synergistic stimulation of progesterone production by LH and estradiol is mediated in part by intracellular mechanisms operating distal to LH-stimulated cAMP production. These in vitro observations using physiological concentrations of hormones suggest a critically bihormonal role for estradiol and LH in the facilitation of progesterone secretion in vivo during late follicular phase differentiation of granulosa cells.     

The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells

We describe an in vitro system of swine granulosa cells which remain responsive to estradiol (E2) and FSH. In this system, we examined mechanisms by which E2 amplifies the stimulatory actions of FSH. E2 stimulated progesterone production in a dose-dependent manner and enhanced the tropic effects of FSH. The facilitative interaction between E2 and FSH could not be accounted for by mitogenic effects, by a leftward shift in the dose-response curves for FSH or E2, or by catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. E3 also enhanced stimulatory actions of 8-bromo-cAMP, choleratoxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Lipoproteins were required for maximal interactive effects between E2 and FSH. However, estrogen significantly increased the effects of FSH even in serum- and lipoprotein-free medium. In addition, when de novo cholesterol biosynthesis by granulosa cells was suppressed by Compactin, the actions of E2 and/or FSH were not impeded. In contrast, E2 alone and FSH alone significantly augmented progesterone production in response to the oxygenated sterol 5-cholesten-3 beta, 25-diol, which is an effective substrate for cholesterol side-chain cleavage. In the presence of 5-cholesten-3 beta, 25-diol, the magnitude of the synergism between E2 and FSH was also increased markedly. We conclude that E2 augments the stimulatory actions of FSH or cAMP on steroidogenesis. This facilitative interaction is amplified by exogenous lipoproteins and does not seem to depend upon endogenous cholesterol biosynthesis. Studies with 5-cholesten-3 beta, 25-diol suggest that estrogen and FSH exert significant stimulatory effects at the level of cholesterol side-chain cleavage. These observations reveal important interactions of E2 and fSH in preparing granulosa cells for the high rates of steroidogenesis they ultimately express in the luteinized state.     

Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation

The hormonal induction of epidermal growth factor (EGF) receptor formation was analyzed during the maturation of granulosa cells obtained from diethylstilbestrol-implanted immature rats. In the absence of FSH, EGF receptors (as measured by the binding of [125I]iodo-EGF to the intact cells) rose by 50% at 6 h of culture, but then declined to about 25-40% of their initial levels at 24-96 h of culture. Scatchard analyses demonstrated the presence of high affinity EGF-binding sites in both freshly prepared cells and after FSH treatment. FSH stimulated a dose-dependent increase in the EGF receptor content of granulosa cells during a 96-h culture period. Concentrations of FSH as low as 2.5-5 ng/ml elevated EGF receptor levels 2- to 3-fold compared to those in untreated control cells, and 30 ng/ml FSH caused a maximal 15-fold rise. FSH increased EGF receptor levels approximately 2-fold in the first 6 h of culture and by up to 7-fold at 96 h compared to levels in freshly prepared cells. FSH treatment did not change the binding affinity (Kd = 5-6 X 10(-11) M) of the EGF receptor, but increased the total number of EGF-binding sites. The stimulatory effects of FSH on EGF receptor expression were mimicked by other cAMP-inducing ligands, including 8-bromo-cAMP, forskolin, and choleragen. Ligands known to inhibit granulosa cell function, including GnRH agonists and the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate, reduced the stimulation of EGF receptors by FSH. However, only 12-O-tetradecanoyl-phorbol 13-acetate suppressed the induction of EGF receptors by 8-bromo-cAMP. In granulosa cells cultured for 48 h with FSH, subsequent treatment with hCG for 24 h reduced EGF receptor content by 25%. Autoradiographic studies with [125I]iodo-EGF in ovarian thin sections demonstrated that EGF-binding sites were uniformly dispersed throughout the ovaries of diethylstilbestrol-implanted rats. Treatment with PMSG markedly increased EGF receptors in the outer walls of the growing follicles, while hCG treatment after PMSG caused a general decline in ovarian labeling. These results indicate that FSH maintains and increases the number of EGF receptors during granulosa cell differentiation, while LH/hCG reduces EGF-binding sites. Such changes in EGF receptors in the presence of endogenous growth factors may influence the number and selection of follicles destined for ovulation.     

LH stimulation of estrogen secretion by cultured rat granulosa cells

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for 4 additional days in media containing aromatase substrate (10(-7) M androstenedione) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and androstenedione, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (400-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a 4-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was 45 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for 4 days in medium containing 10(-7) M androstenedione and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.     

CI628 inhibits follicle-stimulating hormone (FSH)-induced increases in FSH receptors of the rat ovary: requirement of estradiol for FSH action

Although estradiol (E2) alone does not increase receptors for FSH in granulosa cells, E2 priming before administration of FSH increases numbers of FSH receptors significantly compared with FSH alone. We hypothesized that if E2 is required for FSH to increase its own receptor, blocking estrogen action should prevent FSH-induced increases in FSH receptors. Five groups of hypophysectomized rats were injected sc with: saline at 0 h; the antiestrogen CI628 (1 mg) at -6 h; human FSH (hFSH, 2 micrograms) at 0 h; CI628 at -6 h, then hFSH at 0 h; and CI628 plus E2 (2 mg) at -6 h, then FSH at 0 h. Animals were decapitated at 0, 6, 12, or 24 h, and granulosa membrane receptors for FSH, LH, and nuclear receptors for E2 were measured. LH receptor levels increased only after administration of E2 before hFSH. Treatment with hFSH for 6 h increased numbers of FSH receptors 3-fold (P less than 0.01) without any increase in numbers of E2 receptors. At 12 and 24 h, hFSH increased numbers of FSH and E2 receptors 6- and 7-fold (P less than 0.01) over controls. CI628 prevented the hFSH-induced increases in FSH receptors at 6, 12, and 24 h. Administration of E2 concomitant with CI628 before hFSH significantly reversed the inhibitory effects of CI628 on hFSH-induced increases in FSH receptors. There were no changes in affinity of FSH or E2 receptors from 0 to 24 h. To determine whether E2 was acting on the adenylate cyclase system, the ability of hFSH to increase the content of cAMP in granulosa cells in each treatment group was determined. After an iv injection of hFSH, cAMP levels were similar in CI628- and saline-treated rats but had increased 6-fold (P less than 0.01) in hFSH or CI628 plus hFSH-treated animals. Thus, blocking hFSH-induced increases in FSH and E2 receptor appeared to have no effect on FSH stimulation of cAMP. In conclusion E2 appears to be required for FSH action, perhaps by acting within granulosa cells distal to the cAMP-adenylate cyclase system.     

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.     

Inhibitory effects of nitric oxide on estrogen production and cAMP levels in rat granulosa cell cultures

Previous studies demonstrated inhibitory effects of nitric oxide (NO) and cGMP on ovarian steroidogenesis. This study examined the effects of NO on estrogen levels and cAMP accumulation from immature cultured rat granulosa cells. Granulosa cells were incubated with media alone (control), FSH or FSH plus increasing concentrations of the NO generator, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO). While FSH increased estrogen levels 15-fold compared with controls, DETA/NO inhibited FSH-stimulated aromatase activity in a dose-dependent manner. Time-course studies revealed that the inhibitory effects of DETA/NO on aromatase activity persisted throughout the 72 h culture period. Treatment with DETA/NO also inhibited the stimulatory effects of forskolin on estrogen production, indicating that NO can influence steroidogenesis by actions downstream of the FSH receptor. Incubation of cells with FSH plus DETA/NO increased cGMP accumulation over 100-fold, compared with cells treated with media or FSH alone. In this regard, a cGMP analog mimicked the inhibitory effects of NO on FSH- and forskolin-stimulated estrogen production, indicating a potential mechanism of NO action. NO also decreased FSH-stimulated (cAMP) accumulation from cultured cells, indicating an antagonistic effect of NO on the second messenger mediating FSH actions. These findings demonstrate that NO inhibits estrogen production from rat granulosa cells, potentially reflecting actions on the second messengers cGMP and cAMP.     

Control of FSH receptor mRNA expression in rat granulosa cells by 3',5'-cyclic adenosine monophosphate, activin, and follistatin.

FSH is required to maintain FSH and LH/hCG receptors at elevated steady-state levels after receptor induction. Although this function of FSH is mediated by cAMP, how cAMP level is related to the maintenance of gonadotropin receptors is unknown. To investigate cAMP's effect on changes in the levels of FSH receptor mRNAs in rat granulosa cells, total RNA from cells was prepared and analyzed by Northern blots. Incubation with 8-Br-cAMP for 24 h produced a dose-related increase in FSH receptor mRNA in granulosa cells of DES-primed immature rats. On the other hand, 8-Br-cAMP, washed at 24 h, exerted inverse dose-related effects on FSH receptor mRNA levels at 96 h. The addition of 1 mM cAMP resulted in higher levels of FSH receptor mRNA than that induced by 0.2 mM cAMP at 24 h, while 0.2 mM cAMP is as effective as 1-2 mM cAMP for the induction of FSH receptor mRNA at 96 h. To further analyze cAMP's role in the production of activin in granulosa cells, we measured activin levels in the culture medium after the addition of 8-Br-cAMP. The levels of activin A were suppressed by the addition of 8-Br-cAMP in a dose-dependent manner. In addition, the procedure by which 8-Br-cAMP was removed after 24 h incubation showed that the level of activin in the medium increased after medium change. With regard to the actions of activin A on gonadotropin receptors, our laboratory has demonstrated that activin A increases the levels of FSH receptor mRNAs. Therefore, cAMP has a negative effect on FSH receptor expression by suppressing the activin level. Since follistatin production is up-regulated by cAMP in this system, we examined the effect of follistatin on FSH receptor mRNA level, which is induced by activin and FSH. Cotreatment with follistatin (0-100 ng/ml) and activin (50 ng/ml) in the presence of FSH (30 ng/ml) caused a significant reduction in FSH receptor mRNA levels induced by activin. Based on these observations, it is possible that cAMP has both stimulatory and inhibitory effects on the expression of gonadotropin receptors, and the overall influence of cAMP on their expression might be determined by the integration of such opposing effects.