Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.     

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.     

A shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation

Gonadotropins (GTHs; FSH and LH) require two major steroidal mediators, estradiol-17 beta (E(2)) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP) to act as critical hormones to execute oocyte growth and maturation, respectively. A two-cell type model has been proposed, where the theca cells provide the precursor steroids, and the granulosa cells produce the two steroidal mediators under the direct influence of FSH and LH. A distinct shift in steroidogenesis, i.e. from E(2) to 17 alpha,20 beta-DP as well as the steroidogenic enzyme genes from ovarian cytochrome P450 aromatase (oP450arom) to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD), occurs in the granulosa layers of ovarian follicles prior to oocyte maturation. The triggering of the steroidogenic shift by GTHs in granulosa cells occurs through the subjugation of Ad4BP/SF-1 expression in respect of oP450arom, followed by an over-expression of 20 beta-HSD probably through the CREB.     

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.     

Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells

Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.     

Factors influencing follicle-stimulating hormone-responsive steroidogenesis in marmoset granulosa cells: effects of androgens and the stage of follicular maturity

Preovulatory changes in the steroidogenic function of primate granulosa cells were studied using the cyclic marmoset (Callithrix jacchus) as a model. Antral follicles (greater than or equal to 0.5 mm diameter) were dissected from mid-late follicular phase ovaries (7 days after prostaglandin-induced luteolysis) and classified by diameter as small (0.5-1.0 mm), medium (1.1-1.9 mm) or large (greater than or equal to 2.0 mm). Granulosa cells from follicles in each size category were isolated and pooled to assess steroid biosynthesis. The aromatase activity of freshly isolated granulosa cells from large follicles was 200 times greater than that of small follicles, confirming their relatively advanced preovulatory status. Granulosa cells were cultured for 48 h in the presence and absence of human (h) FSH (0.1 ng/ml), with and without 0.1 microM androgen (testosterone or 5 alpha-dihydrotestosterone), to assess basal and hormone-responsive steroidogenesis (progesterone accumulation in culture medium and aromatase activity in washed granulosa cell monolayers). Basal granulosa cell steroidogenesis increased with follicular size, and there was a development-related pattern of response to hFSH and androgen. hFSH responsiveness (maximum fold-stimulation induced by hFSH) declined with follicular size, being 2-6 times greater for granulosa cells from small vs. large follicles. On the other hand, hFSH sensitivity increased with follicular size; the dose of hFSH giving 50% of the maximum response (ED50) for cells from large follicles being 10-20 times less than that of cells from small follicles. For granulosa cells from small follicles, treatment with 0.1 microM androgen in the presence of hFSH led to dramatic (up to 16-fold) enhancement of steroidogenic responses to hFSH. In contrast, for granulosa cells from large follicles, the presence of androgen substantially inhibited aromatase activity stimulated by hFSH and had weak inhibitory effects on progesterone accumulation. These results show that granulosa cell steroidogenesis becomes increasingly sensitive to hFSH during preovulatory follicular development in marmosets. The marked ability of androgen to directly augment hFSH-responsive steroidogenesis in vitro is lost during preovulatory development, such that androgen acts in mature granulosa cells to suppress hFSH-stimulated aromatase activity. These observations are evidence of development-dependent changes in granulosa cell responses to FSH and androgens which may contribute to the control of preovulatory follicular development in primates.     

Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17alpha-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated.     

Essential role of the oocyte in estrogen amplification of follicle-stimulating hormone signaling in granulosa cells

The establishment of dominant ovarian follicles that are capable of ovulating fertilizable oocytes is a fundamental determinant of female fertility. This process is governed by pituitary gonadotropins as well as local ovarian factors. Within the follicle, estrogen acts in an autocrine/paracrine manner to enhance FSH action in the granulosa cells. These effects include the augmentation of P450aromatase expression and estradiol production. This feed-forward effect of estrogen is believed to play a key role in follicle dominance. Here we found the essential role of the oocyte in this physiological process using primary cultures of rat granulosa cells. In the presence, but not absence, of oocytes, estrogen amplified FSH-stimulated increases in mRNA expression of P450aromatase, FSH receptor, LH receptor, and inhibin alpha-, betaA-, and betaB-subunits as well as cAMP production. Thus, oocytes mediate the estrogen enhancement of FSH action in the granulosa cells. In comparison with FSH, cotreatment with estrogen and oocytes failed to amplify the stimulatory effects of forskolin or 8-bromoadenosine-cAMP on granulosa cell responses including P450aromatase mRNA expression and cAMP production, indicating that estrogen/oocytes amplify FSH action at a site upstream of adenylate cyclase. These findings support the novel conclusion that communication between the oocyte and granulosa cells plays a crucial role in mediating estrogen action during FSH-dependent folliculogenesis.     

Bone morphogenetic proteins (BMP) -4, -6, and -7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6, and -7 potently suppress both basal (P < 0.0001; respective IC(50) values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC(50) values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3beta-hydroxy- steroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7, respectively, and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.     

Effect of thyroid hormone on steroidogenic enzyme induction in porcine granulosa cells cultured in vitro

In order to elucidate the mechanism of thyroid hormone action on the ovary, direct effects of L-thyroxine (T4) or L-triiodothyronine (T3) on steroidogenic enzyme induction were investigated in vitro using a monolayer culture system of porcine granulosa cells obtained from small follicles. The cells were cultured in the absence or presence of porcine FSH (20ng/ml) for 6 days, with or without T4 or T3, under sparsely (4%) serum supplemented condition. The mechanism of thyroid hormone action on the granulosa cells was studied by testing the capability of thyroid hormone to enhance the steroidogenesis in response to exogenously provided substrates. Concomitant treatment with FSH (20ng/ml) and T4 (10(-7) M) caused a further increased production of progesterone in response to the addition of pregnenolone compared to that in the absence of pregnenolone. The same treatment with FSH and T4 also caused a further increased production of estrone in response to the addition of androstenedione. Concomitant treatment with 10(-9) MT3 demonstrated similar stimulatory effects on the steroidogenesis in cultured granulosa cells. T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. Furthermore, aromatase activity in cultured granulosa cells assessed by the release of tritiated water from [1 beta-3H, 4-14C] androstenedione was significantly higher in the cells treated concomitantly with FSH (20ng/ml) and T4 (10(-7) M) than that in the cells treated with FSH alone. These results suggest that thyroid hormone synergizes with FSH and increases FSH-mediated induction of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and aromatase activity in immature granulosa cells. Since the effective dose of T4 and T3 observed in our studies is in the physiological range of circulating total levels of T4 and T3, it can be concluded that the synergism between FSH and thyroid hormone is of physiological importance to the full expression of FSH actions in the functional differentiation of immature granulosa cells.     

Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro.

The mechanism of action of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) upon various cell types of the mammalian ovary is reviewed. Emphasis is placed upon in vitro studies using organ and cell culture as well as short-term incubations. FSH and LH actions upon the following ovarian functions are discussed: steroidogenesis and metabolism of the ovary as a whole and of the isolated follicle and its component cell types, the granulosa and thecal cells, as well as folliculogenesis and follicular growth, oocyte maturation, follicular rupture, and corpus luteum maintenance and steroidogenesis. The roles of gonadotropin receptors, AMP, prostaglandins, protein kinase, and protein synthesis in these LH and FSH actions are discussed. Intra-ovarian regulation of LH and FSH action is reviewed, including a discussion of the possible roles of follicular fluid inhibitors upon oocyte maturation and granulosa cell luteinization.     

The orphan nuclear receptors NURR1 and NGFI-B modulate aromatase gene expression in ovarian granulosa cells: a possible mechanism for repression of aromatase expression upon luteinizing hormone surge

Ovarian granulosa cells play pivotal roles in many aspects of ovary functions including folliculogenesis and steroidogenesis. In response to FSH and LH, the elevation of intracellular cAMP level in granulosa cells leads to activation of multiple ovarian genes. Here, we report findings from a genome-wide study of the cAMP-responsive gene expression profiles in a human granulosa-like tumor cell line, KGN. The study identified 140 genes that are either activated or repressed by 2-fold or greater after stimulation by the adenylyl cyclase activator forskolin. The induction patterns of some cAMP-responsive genes were further analyzed by quantitative real-time PCR. Consistent with previous observations, the LH-responsive genes, such as the nuclear receptor 4A subfamily (NURR1, NGFI-B, and NOR-1), were rapidly but transiently induced, whereas the FSH-responsive gene CYP19 encoding aromatase was induced in a delayed fashion. Interestingly, ectopic expression of NURR1 or NGFI-B severely attenuated the cAMP-responsive activation of the ovary-specific aromatase promoter. Reduction of the endogenous NURR1 or NGFI-B by small interfering RNA significantly elevated aromatase gene expression. The cis-elements responsible for NURR1/NGFI-B-mediated repression were mapped to the minimal aromatase promoter sequence that confers camp responsiveness. Furthermore, the DNA-binding domain of NURR1 was required for the repression. Taken together, these results strongly suggest a causal relationship between the rapid decline of aromatase mRNA and induction of nuclear receptor subfamily 4A expression, which concomitantly occur upon LH surge at the later stages of ovarian follicular development.     

Activin signaling through type IB activin receptor stimulates aromatase activity in the ovarian granulosa cell-like human granulosa (KGN) cells

In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.     

Androgen/antiandrogen modulation of cyclic AMP-induced steroidogenesis during granulosa cell differentiation in tissue culture

FSH stimulation of granulosa cell differentiation is believed to be mediated by the intracellular cyclic AMP (cAMP) level. However, steroidogenic enzyme induction in the differentiating granulosa cell is subject to direct modulation by androgenic steroid: in granulosa cell cultures established from ovaries of oestrogen-pretreated, prepubertal rats, potentiating effects of testosterone (T) on FSH induction of oestrogen synthetase (aromatase) and progesterone (P) biosynthesis can be blocked by including a stoichiometric excess of antiandrogen (hydroxyflutamide, SCH 16423) in the culture medium. In this study we used the same experimental model to determine effects of T and SCH 16423 on the induction of steroidogenesis by endogenous cAMP and an exogenous cAMP analogue, 8-bromo-cAMP (8brcAMP). Granulosa cells were cultured in medium containing variable FSH concentration (3–300 ng/ml) with a fixed (100 μm) dose of 3-isobutylmethylxanthine (MIX), or containing a fixed (minimally effective: 10–15 ng/ml) dose of FSH with MIX concentration variable (50–800 μM). By relating steroidogenic endpoints at 48 h to the acute cAMP response (accumulations in the medium) at 1 h, it was deduced that aromatase induction was saturable under conditions where FSH-sensitive cAMP production and the induction of P biosynthesis showed further, proportionate increases.Although T (0.1μM) did not alter acute FSH-responsive cAMP production, its presence throughout the 48 h culture was required for full expression of FSH-induced steroidogenesis in the cell monolayers. When the aromatase response (but not the P response) was ‘supersaturated’ by endogenous cAMP (i.e. culture with FSH plus MIX), SCH16423 was unable to antagonize the potentiating effect of T on aromatase induction while it continued to block T-potentiated P biosynthesis. Steroidogenic induction by cholera toxin (100 ng/ml) was also subject to similar modulation by T and SCH16423. However, the phosphodiesterase-resistant cAMP analogue 8brcAMP (3 mM) not only induced each response (albeit submaximally in the case of aromatase) in the absence of T, but its effects tended to be refractory to androgen/antiandrogen modulation. Accumulations of cAMP in the medium from 48 h cultures which had been incubated with FSH (100 ng/ml) were increased 2–3-fold by the additional presence of T (0.1 μM). This long-term stimulatory effect of T on FSH-dependent cAMP accumulation was blocked by culture in the presence of SCH16423 (10 μM). Thus, androgen potentiation of steroidogenic enzyme induction during FSH-stimulated granulosa cell differentiation may involve a suppression of cAMP catabolism exerted by way of the androgen-receptor system.     

Activation of the bone morphogenetic protein signaling pathway induces inhibin beta(B)-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells

During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin alpha- and beta(B)-subunits, fluctuate in a fashion distinct from that of inhibin A, the alpha-beta(A)-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin alpha- and beta(A)-subunits, but not those of the beta(B)-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGF beta. We recently identified the TGF beta gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin beta(B)-subunit mRNAs in hGL cells without affecting the levels of alpha- or beta(A)-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on beta(B)-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin beta(B)-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGF beta-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.     

Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor

We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.     

Modulation of FSH-controlled steroidogenesis in rat granulosa cells: direct in-vitro effects of LHRH and ICI-118630

Direct inhibitory effects of LHRH and an LHRH agonist (ICI-118630) on FSH-controlled steroidogenic processes in ovarian granulosa cells were characterized in vitro. Over a 2-day culture period in the presence of testosterone (10(-7) M), FSH (3-3 000 ng/ml) caused dose-dependent increases in the aromatase activity of granulosa cells isolated from oestrogen-pretreated immature rats. Progestogen biosynthesis was stimulated in a similar manner. The presence of LHRH (10(-9) - 10(-7) M) in the culture medium inhibited these responses by right-shifting the dose-response curves. Thus the net effect was one of reduced sensitivity to FSH. ICI-118630 was approximately 10 times more effective than LHRH as an inhibitor of aromatase induction and progestogen biosynthesis in response to FSH. Over a 1-h incubation at concentrations up to 10(-7) M, neither decapeptide had a consistent inhibitory effect on FSH-stimulated granulosa cell cAMP formation either in the presence or absence of 1-methyl-3-isobutyl-xanthine (MIX); but during the 2-day culture, ICI-118630 and occasionally LHRH significantly inhibited aromatase induction by cholera toxin and 2 different cAMP analogues. Over the same range of concentrations, each peptide progressively inhibited the stimulatory effect of MIX on FSH-induced aromatase activity and progestogen biosynthesis. Thus LHRH/ICI-118630 can directly modulate FSH-controlled granulosa cell steroidogenesis in vitro via effects on one or more biochemical loci distal to the FSH-receptor coupled adenylate cyclase system. These experiments have implications for the role of a putative LHRH-like ovarian substance(s) in the local co-ordination of follicular development and function.