Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells

Roles of follicle-stimulating hormone (FSH) and sex steroids in regulating the expression of mRNA species encoding the alpha-, beta A- and beta B-subunits of inhibin were studied in cultured granulosa cells from immature rat ovaries. Inhibin subunit mRNAs were detected by Northern blot analysis of total RNA extracted from granulosa cell monolayers which had been incubated for 48 h in serum-free medium containing FSH (100 ng/ml) and/or a steroid (10(-6) M): estradiol (E), testosterone (T) or 5 alpha-dihydrotestosterone (DHT). Levels of mRNA encoding each inhibin subunit in untreated (control) cultures were low. In cultures treated with FSH alone, levels of inhibin alpha-, beta A- and beta B-subunit mRNA were approximately 60-fold, 70-fold and 66-fold greater than control, respectively. In cultures treated with E alone, levels of inhibin alpha- and beta B-subunit mRNA were elevated approximately 4-fold and 2-fold, respectively, but the level of inhibin beta A-subunit mRNA was not measurably affected. Treatment with T or DHT alone had no consistent effect on the levels of any inhibin subunit mRNA. The stimulatory effects of FSH were not consistently altered by the presence of either androgen or estrogen. These results confirm the role of FSH in regulating inhibin alpha-subunit gene expression and provide direct evidence that both inhibin beta-subunit genes are inducible by FSH in granulosa cells. All three inhibin subunit mRNAs followed the same pattern, suggesting that their expression is coordinately regulated by FSH during granulosa cell differentiation.     

Androgens augment FSH-induced progesterone secretion by cultured rat granulosa cells.

Granulosa cells have been isolated from ovaries of estrogen-treated immature intact and hypophysectomized rats, and have been maintained in culture in a chemically-defined medium. Progesterone secretion by these cells was testosterone or 17beta-OH-5alpha-androstan-3-one (DHT), progesterone secretion was low or undetectable. However, the addition of testosterone or DHT together with FSH caused a dramatic 8- to 19-fold increase over that caused by FSH alone. On the other hand, luteinizing hormone (LH) alone had no effect on progesterone secretion, but produced a small stimulation when added together with testosterone. These results demonstrate synergism between androgens and FSH in the control of progesterone secretion by granulosa cells in culture.     

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.     

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.     

Comparison of the facilitative roles of insulin and insulin-like growth factor I in the functional differentiation of granulosa cells: in vitro studies with the porcine model

The facilitative effects of insulin and IGF-I were compared in vitro with regard to induction of differentiated functions of porcine granulosa cells. The monolayers were maintained under serum-free conditions in the absence or presence of porcine FSH (20 micrograms/l), with or without graded doses of insulin or IGF-I. Concurrent treatment with IGF-I and FSH produced morphological differentiation and augmented LH/hCG receptor binding together with an enhancement in progesterone and estradiol secretion relative to treatment with FSH alone. IGF-I alone was incapable of exhibiting these effects. Insulin synergized with FSH to facilitate the granulosa cell functions except estradiol secretion. Maximal effective dose of IGF-I was 100 micrograms/l which is within the physiological concentration in vivo, whereas that of insulin was 1.0 mg/l, which is 1000-fold higher than the physiological level. Although the maximal effective doses of IGF-I and insulin produced a comparable increment in progesterone secretion and LH/hCG receptor induction, combined treatment with IGF-I and insulin did not prove additive. [125I]IGF-I binding revealed that specific IGF-I receptors with two classes of binding sites are present on porcine granulosa cells. No distinct differences were detected between IGF-I receptors of granulosa cells from small, medium and large follicles. Insulin was approximately 100-fold less active than IGF-I in competing for [125I]IGF-I binding. These findings suggest that porcine granulosa cells possess specific IGF-I binding sites which may mediate the cytodifferentiative actions of insulin-like peptides. Since IGF-I is more potent than insulin in amplifying the actions of FSH and maximally exerts the cytodifferentiative effects at the physiological concentration, it is likely that IGF-I plays the more important role in granulosa cell differentiation in synergy with FSH.     

Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone.

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.     

Luteinizing hormone receptor appearance in cultured porcine granulosa cells requires continual presence of follicle-stimulating hormone.

During the differentiation of ovarian granulosa cells, follicle-stimulating hormone (follitropin; FSH) mediates the induction of cell surface receptors for luteinizing hormone (lutropin; LH). Using primary cultures of porcine granulosa cells, we demonstrate that both the induction and maintenance of LH receptors are critically dependent upon the continual presence of FSH. The termination of FSH-promoted LH receptor induction is paralleled by the termination of FSH-induced intracellular cAMP accumulation. Changing the medium is without effect on FSH-induced appearance of LH receptors. Furthermore, 1 mM aminoglutethimide, which completely blocks FSH-stimulated progesterone biosynthesis, does not decrease the induction of LH receptors by FSH. Thus, the induction of LH receptors by FSH does not appear to require the accumulation of a factor in the medium, nor does it appear to be mediated via FSH-stimulated progesterone synthesis. These data suggest that intracellular cAMP, produced while FSH remains bound to the cell surface, mediates the induction of LH receptors and that the continual presence of FSH is required for both the induction and maintenance of cell surface LH receptors.     

Serotonin may alter the pattern of gonadotropin-induced progesterone release of human granulosa cells in superfusion system

Serotonin plays a hormonal function in several nonneuronal peripheral tissues, such as the ovaries. Our aim was to investigate whether there is a modulatory action of serotonin on gonadotropin-induced steroid secretion of human granulosa cells. In granulosa cell culture, serotonin was administered alone or in combination with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Also, granulosa cells were transferred into a dynamic superfusion apparatus and challenged by FSH and LH alone or along with serotonin. Estradiol and progesterone concentrations of samples were measured by radioimmunoassay. As expected, administration of FSH, LH, and serotonin alone resulted in a significant estradiol and progesterone release in cell culture, as well as a significant increase in progesterone release in dynamic superfusion system. In cell culture, co-administration of serotonin with gonadotropins had no additive effect on gonadotropin-induced secretion of progesterone, while it further augmented that of estradiol. In superfusion system, when gonadotropins were added along with serotonin, the increase in progesterone release was markedly less, while peaks of hormone response were remarkably prolonged compared to challenges by LH and FSH alone. The observed effects of serotonin on gonadotropin-induced steroid release of granulosa cells may reveal further details about the regulation of granulosa cell function.     

Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation

Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human G(s)alpha Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active G(s)alpha Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and G(s)alpha signaling pathways are required.     

The influence of chronic morphine treatment on the negative feedback regulation of gonadotropin secretion by gonadal steroids

The influence of continuous stimulation of opiate receptors with morphine (M) on the negative feedback effects of testosterone (T), 5 alpha-dihydrotestosterone (DHT), and 17 beta-estradiol (E2) on LH and FSH secretion was studied in rats that had been castrated 2 weeks previously. In the absence of gonadal steroids, 4 days of continuous M exposure did not alter LH or FSH levels. Similarly, Silastic capsules containing crystalline T (5 mm) or E2 [5 mm long (75 micrograms E2/ml) to 7.5 mm long (300 micrograms E2/ml)] alone had little effect on LH or FSH release. However, in M-exposed rats, T reduced serum LH by greater than 90%, and E2 reduced LH by more than 75%. Among the doses of DHT evaluated, only the highest dose (7.5-mm Silastic capsules packed with crystalline DHT) reduced LH secretion, and M exposure only slightly enhanced this suppression. M or gonadal steroids alone produced little change in FSH levels in castrated rats. However, the combination of M plus E2 or DHT further reduced FSH levels. Evaluation of pituitary responses to LHRH revealed that when administered alone, T did not alter, DHT reduced, and E2 enhanced the LH response to the decapeptide. Neither M treatment alone nor M plus T or DHT altered the pituitary LH response to LHRH. On the other hand, M appeared to enhance the stimulatory effects of E2 on pituitary responsiveness to LHRH. These findings suggest that the interaction of M and gonadal steroids at the level of the pituitary could not explain the observed marked suppression of gonadotropin secretion by suboptimal T or E2 during opiate receptor stimulation with M. Collectively, these observations are in accord with the view that endogenous opioid peptides may play a role in modulating the sensitivity of the hypothalamus to the negative feedback effects of gonadal steroids.     

Inhibition by estradiol-17 beta of porcine thecal androgen production in vitro.

Thecal preparations from medium-sized procine ovarian follicles (3.5-5 mm diameter) were incubated for 4 h in a chemically defined medium in the presence or absence of highly purified luteinizing hormone (LH) and/or estradiol-17 beta (estradiol). LH (1 microgram/ml) stimulated the thecal production of testosterone (T) and dihydrotestosterone (DHT) by 2- to 3-fold. Although estradiol (10 microgram/ml) alone had only a slight but non-significant inhibitory effect on basal testosterone production, it significantly inhibited the production of both T and DHT as well as decreasing the DHT/T ratio in a dose-related manner in the presence of LH. Production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone by the thecal cells was stimulated 50-to 200-fold and 2.5-fold, respectively, by LH. Estradiol had no significant effect on thecal cAMP and progesterone production in the presence or absence of the gonadotropin. These findings are consistent with the concept that estradiol produced by granulosa cells following hormonal stimulation may serve as a local negative feedback mechanism to control thecal androgen 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.     

Estrogen dependence of luteinizing hormone receptor expression in cultured rat granulosa cells. Inhibition of granulosa cell development by the antiestrogens tamoxifen and keoxifene

In rat ovarian granulosa cells cultured for 48 h, addition of 10(-8) M estradiol (E2) enhanced choleragen-induced cAMP formation and LH receptor content by 2-fold and 6-fold, respectively. Two potent antiestrogens, tamoxifen and keoxifene, inhibited these effects of E2 in a concentration-dependent manner and significantly reduced cAMP production and LH receptors below the levels induced by choleragen. Both antiestrogens (greater than or equal to 1 microM) also reduced the effects of choleragen on cAMP levels and LH receptor content in the absence of exogenous E2. In addition, the antiestrogens (1 microM) inhibited the stimulatory effects of FSH and forskolin on granulosa cell maturation, as well as the enhancement of their actions by exogenous E2. FSH caused a concentration-dependent rise in endogenous E2 accumulation during the 48-h culture period, suggesting that antiestrogens may prevent FSH-stimulated increases in LH receptors by inhibiting the actions of newly formed E2. Tamoxifen prevented the induction of LH receptors by 8-bromo-cAMP, indicating that its effects were on both cAMP production and cAMP action, whereas keoxifene predominantly altered granulosa cell development by its inhibition of estrogen effects on cAMP production. Although both exogenous E2 and the antiestrogens modified cAMP accumulation and LH receptor expression largely during the second 24 h of culture, their actions commenced during the first day. The antiestrogens had no effect alone and did not reduce the DNA content of granulosa cells. Also, they could be washed from the cells after 48 h of culture with complete recovery of forskolin-stimulated cAMP responsiveness by 72-96 h of culture. At a lower concentration (0.4 microM), tamoxifen, but not keoxifene, acted as a partial estrogen agonist since it enhanced choleragen action. These results indicate that the cAMP-mediated induction of LH receptors in cultured granulosa cells is dependent upon the continued actions of estrogen throughout the maturation process.     

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration, respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24-48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin aslo had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and t 1/2 of 0.078 h-1 and 8.9 h for FSH receptors and 0.086 h-1 and 8.0 h for the LH receptors, respectively.     

Control of immunoactive inhibin production by human granulosa cells

OBJECTIVE: The aim was to determine the relation between stage of antral follicular development and granulosa cell production of immunoactive inhibin. DESIGN: Primary granulosa cell cultures in serum-free Medium 199 were incubated at 37 degrees C for 96 hours with a change of medium at 48 hours. Inhibin and steroid levels in culture medium were determined by radioimmunoassay. The inhibin assay was based on the N-terminal 1-26 amino acid sequence of the alpha-chain of porcine 32 kDa inhibin using pl alpha 1-26-GLY27-TYR28 as the immunogen, tracer and standard. PATIENTS: Granulosa cells were obtained from the ovaries of women with regular menstrual cycles undergoing hysterectomy with unilateral or bilateral oophorectomy to treat non-malignant gynaecological disease. RESULTS: Basal production of immunoactive inhibin by granulosa cells from presumptive preovulatory follicles (greater than 15 mm diameter) was 5-13 times higher than that by granulosa cells from immature (less than 10 mm diameter) or intermediately mature (10-15 mm diameter) follicles. Basal production of progesterone and oestradiol followed a qualitatively similar pattern, establishing a positive relation between functional granulosa cell maturity and inhibin production. Treatment of granulosa cell cultures from immature follicles with follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), increased inhibin production, time and dose dependently. FSH, but not LH, also brought about similar increases in steroid hormone synthesis by granulosa cells from immature follicles. The stimulatory effect of FSH on granulosa cell inhibin production was augmented at least twofold by the presence of testosterone or 5 alpha-dihydrotestosterone (1.0 mumol/l) but was unaffected by oestradiol. Granulosa cells from intermediately mature follicles undertook variable degrees of both FSH and LH-responsive inhibin production which generally corresponded with gonadotrophin-responsive steroid production. Granulosa cells from presumptive preovulatory follicles showed inconsistent inhibin responses to FSH. However, LH caused marked (at least twofold) increases in inhibin production, paralleling LH-responsive steroid production. CONCLUSION: These results show that for human beings, granulosa cell capacity to produce immunoactive inhibin in vitro increases with follicular maturity. FSH, but not LH, stimulates inhibin production by immature granulosa cells and this response to FSH is subject to modulation by androgen. During preovulatory follicular development, production of inhibin, like steroids, becomes increasingly responsive to LH. Such a development-related pattern of granulosa cell inhibin production helps explain how, post-ovulation, the corpus luteum is able to secrete inhibin as well as steroids. It is also compatible with the concept that locally produced inhibin could participate in the paracrine control of follicular development during the human menstrual cycle.     

Adenovirus-directed expression of functional luteinizing hormone (LH) receptors in undifferentiated rat granulosa cells: evidence for differential signaling through follicle-stimulating hormone and LH receptors

This study was conducted to determine the feasibility of using replication-defective adenovirus vectors to express receptors for LH. Two vectors were constructed, one that directs the expression of wild-type human LH receptor (LHr; AdRSVLHrwt) and another that directs the expression of the constitutively activated D578H mutant human LH receptor (AdRSVD578HLHr). When infected with AdRSVwtLHr and AdRSVD578HLHr, COS-1 cells expressed LH/hCG-binding sites as reflected by specific binding of [(125)I]hCG. To determine the ability of the vectors to confer LH responsiveness, undifferentiated rat granulosa cells, which possess only FSH receptors, were infected with AdRSVwtLHr and AdRSVD578HLHR: Expression of the constitutively activated D578H LHr increased basal (gonadotropin-independent) estrogen and progesterone production. Expression of the wild-type LHr in granulosa cells did not stimulate basal steroid production, but conferred responsiveness to exogenous LH. For both wild-type LHr and D578HLHr, the absolute levels of steroid production were dependent upon the input of viral titers. Using these vectors, we compared effects of FSH and LH receptor activation in undifferentiated granulosa cells. Stimulation of undifferentiated granulosa cells by FSH and D578HLHr, as well as activation of wild-type LHr with LH resulted in comparable production of progesterone. In contrast, estradiol production in cells stimulated with FSH was greater than that in cells that expressed either D578H receptors or wild-type LHr in the presence of LH. Analysis of messenger RNAs (mRNAs) revealed that activations of FSH and the LH receptors were comparable in the induction of alpha-inhibin and 3betahydroxysteroid dehydrogenase mRNAS: However, activation of FSH receptor led to significantly greater expression of P450 aromatase and LHr mRNAs than did activation of LHR: These results suggest that activation of FSH and LH receptors in granulosa cells may differ with respect to activating intracellular signaling pathways and stimulating gene expression.     

Testosterone and dihydrotestosterone, but not estradiol, selectively maintain pituitary and serum follicle-stimulating hormone in gonadotropin-releasing hormone antagonist treated male rats

Recently it has been found that testosterone can maintain and restimulate serum and pituitary follicle-stimulating hormone (FSH) in the gonadotropin-releasing hormone (GnRH) antagonist treated adult male rat. The present investigation was undertaken to determine (1) which metabolite of testosterone, dihydrotestosterone (DHT), or estradiol accounts for the effects of testosterone in GnRH antagonist suppressed rats and (2) whether these effects of testosterone are influenced by other testicular factors. Eight groups of 6-8 adult male Sprague-Dawley rats were subjected to the following treatments: vehicle, GnRH antagonist (75 micrograms/day s.c.), testosterone-filled Silastic implants (3 x 5 cm, s.c.), DHT-filled Silastic implants (3 x 5 cm, s.c.), estradiol benzoate (15 micrograms/day s.c.), and combined administration of GnRH antagonist with either steroid. In addition, the GnRH antagonist/testosterone treatment regimen was applied to rats orchidectomized 72 h prior to initiation of treatments. After 3 weeks of treatment, serum was analyzed for concentrations of luteinizing-hormone (LH), FSH, testosterone, DHT, and estradiol. Pituitary extracts were analyzed for LH and FSH content. Except for the vehicle-treated groups, serum and pituitary LH concentrations were markedly suppressed by all treatments. In intact rats treated with GnRH antagonist alone and/or estradiol, the pituitary FSH level was reduced by more than 70% relative to controls, while both testosterone and DHT maintained pituitary FSH. Similarly, testosterone and DHT, but not estradiol, delayed the decline of serum FSH induced with GnRH antagonist alone. In orchidectomized animals, testosterone was also capable of preventing a reduction of pituitary FSH despite concomitant GnRH antagonist administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation

FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and p450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSH-stimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for p450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with Ad-LRH-1 did not stimulate estradiol production or increases in mRNA for p450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation.     

Multihormone regulation of steroidogenesis in cultured porcine granulosa cells: studies in serum-free medium

FSH, LH, and estradiol are known to modulate ovarian follicular differentiation. However, the cellular site of action and relative importance of the three hormones have remained uncertain. The recent development of a serum-free system for the culture of immature porcine granulosa cells has enabled us to reinvestigate these issues with better control of pituitary peptides and gonadal steroids. Progesterone production in response to FSH was higher in cells cultured in serum-free complete medium than in those grown in the presence of 10% fetal calf serum [10-fold vs. 1.5-2 fold (control)]. Ovine LH alone was also able to stimulate progesterone production in serum-free free complete medium (6-fold); this effect could not be accounted for by FSH contamination. The LH stimulation, however, was enhanced by FSH. Insulin was required for both FSH and LH stimulation of progesterone production. Estradiol stimulated progesterone production per se (2- to 3-fold) and also enhanced FSH and LH actions. The estimated ED50 for estradiol in FSH-treated cells was 20 ng/ml. Maximal levels of progesterone after 6 days were observed when the combination of FSH, LH, and estradiol was present from the onset of the culture. Incubations carried out in the presence of 5-cholesten-3 beta-25-diol indicated that the hormonal interactions take place, at least in part, at the level of the side-chain cleavage enzyme. These results indicate that FSH is the most important hormonal stimulus for progesterone synthesis in immature granulosa cells. However, LH, estradiol, and insulin (or insulin-like growth factors) exert direct actions on the granulosa cell that may be required for the development of optimal steroidogenic potential.