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.     

Regulation of inhibin production by rat granulosa cells

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

Hormonal regulation of the growth and steroidogenic function of human granulosa cells.

The aim of this study was to assess development-related interactions between gonadotropins and insulin-like growth factor (IGF-I) on DNA synthesis and steroidogenesis in human granulosa cells. "Immature" granulosa cells were obtained from follicles during the late luteal phase or first half of the follicular phase; "mature" granulosa cells came from follicles during the second half of the follicular phase but before the midcycle LH surge; and granulosa-lutein cells were obtained as a by-product of in vitro fertilization. Granulosa cells were cultured for 96 h in serum-free medium 199 with and without LH or FSH, and in the presence and absence of IGF-I. The cell monolayers were then incubated with [3H]methyl thymidine to assess DNA synthesis. Spent culture medium was assayed for progesterone and estradiol content. Immature granulosa cells: Tritiated thymidine uptake in granulosa cell cultures from immature follicles were significantly increased by IGF-I. FSH was able to maintain or increase basal and IGF-I stimulated growth whereas LH had no effect. Basal progesterone production was low and not increased by either FSH or LH. However, treatment with FSH, but not LH, increased aromatase activity. Mature granulosa cells: IGF-I also stimulated thymidine uptake. However, whereas FSH either maintained or increased thymidine uptake by these cells, LH dose dependently suppressed thymidine uptake. This inhibitory action of LH was accentuated by the presence of IGF-I. Despite the inhibitory effect of LH on thymidine uptake, the gonadotropin markedly stimulated steroid production and the maximal steroidogenic response to LH was equivalent to 3-fold greater than that to FSH. Granulosa-lutein cells: Patterns of basal and IGF-I- and gonadotropin-stimulated steroid synthesis were similar to those observed for mature granulosa cells but steroid production rates were higher. Suppression of basal and IGF-I-stimulated thymidine uptake by LH was even more pronounced. These results suggest that the granulosa cell LH receptor, once expressed, negatively regulates cell growth and, simultaneously, positively regulates steroid synthesis. This development related event could be crucial to the mechanism whereby granulosa cells cease to divide and commence maximal rates of steroid synthesis in response to the LH surge.     

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.     

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.     

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.     

Independence of steroidogenic capacity and luteinizing hormone receptor induction in developing granulosa cells.

The relationship between FSH-induced acquisition of LH/hCG receptors and the steroidogenic capacity of granulosa cells from estrogen-primed hypophysectomized rat ovaries has been examined. Granulosa cells harvested from the immature preantral follicles of animals not treated with FSH (controls) displayed negligible specific human [125I]iodo-hCG binding and produced only minimal amounts of progesterone during 48 h of culture in vitro. Addition of highly purified hFSH or prostaglandin-E2 (PGE2) to the culture medium elicited substantial increases in progesterone production which were not accompanied by measurable increases in [125I]iodo-hCG binding. Treatment with oFSH in vivo for 24 h led to the initiation of antrum formation in many follicles and was accompanied by an 8-10-fold increase in hCG binding by freshly isolated granulosa cells. Basal, hFSH-, and PGE2-stimulated progesterone production during culture was also greater than controls. In contrast, cells from animals receiving oFSH in vivo for only 12 h showed no increase in hCG binding either before or after culture, yet basal and stimulated progesterone production in vitro was significantly greater than controls, indicating that the initiation of steroidogenesis was antecedent to LH/hCG receptor induction. Only those cells obtained after the 24-h in vivo treatment with oFSH produced elevated amounts of progesterone when incubated in the presence of hCG, thereby showing that the observed increases in [125I]iodo-hCG binding reflected the induction of functionally active LH/hCG receptors. Pharmacological stimulation of steroidogenesis by cell suspensions with N,O'-dibutyryl cAMP resulted in consistently high levels of progesterone production irrespective of previous treatment with FSH in vivo. This uniform expression of in vitro steroidogenic capacity occurred in the complete absence of measurable increases in LH/hCG receptors, suggesting that these two fundamental developmental processes are independent phenomena which may be under separate regulation in vivo.     

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

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

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.     

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.     

Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells

The influence of estrogens on gonadotropin-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) was examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of FSH in the presence or absence of either diethylstilbestrol (DES) or estradiol. FSH treatment increased progestin production in a dose-dependent manner, whereas treatment with estrogens alone were ineffective. In contrast, concomitant addition of either DES or estradiol augmented FSH-stimulated production of progesterone and 20 alpha-OH-P. Increasing concentrations of estradiol (10(-10) - 10(-7) M) augmented the stimulatory effect of FSH (30 ng/ml) on progesterone production in a dose-dependent manner with ED50 values of approximately 3 X 10(-9) M. The facilitatory action of estradiol was time-related, becoming significant after 36 h of treatment. Granulosa cells were also cultured for 2 days with FSH to induce functional LH receptors. The FSH-primed cells were treated for an additional 3 days with increasing concentrations of LH (0.3-30 ng/ml) in the absence or presence of DES (10(-7) M). LH stimulated progesterone and 20 alpha-OH-P production in a dose-dependent manner, whereas concomitant addition of DES further enhanced LH-induced progestin biosynthesis. (Bu)2cAMP also increased progesterone and 20 alpha-OH-P production by the granulosa cells; however, concurrent addition of DES did not augment the actions of (Bu)2cAMP. The effect of estrogens on gonadotropin-stimulated cAMP accumulation was also examined. FSH treatment dose-dependently increased cAMP accumulation, whereas concomitant treatment with estradiol further increased the FSH action. Similarly, LH treatment also stimulated cAMP accumulation in FSH-primed cells, whereas concurrent addition of DES further augmented LH action. Thus, the stimulatory effect of estrogens upon gonadotropin-stimulated progestin production may be related to the augmentation of cAMP biosynthesis. The present observations suggest that intraovarian estrogens may act locally to enhance the sensitivity of granulosa cells to FSH and LH, thereby increasing the biosynthesis of progestins and cAMP by the granulosa cells.     

Developmental and hormonal regulation of bovine granulosa cell function in the preovulatory follicle

Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Role of estradiol as a biological amplifier of gonadotropin action in the ovary: in vitro studies using swine granulosa cells and homologous lipoproteins

Swine granulosa cells cultured under serum-free conditions in vitro exhibit significant responsivity to the stimulatory actions of estradiol (E2) and FSH or LH. Under these conditions, granulosa cells harvested from either immature or mature Graafian follicles synthesized significantly increased quantities of progesterone in response to homologous low density lipoprotein (LDL) and, to a lesser degree, homologous high density lipoprotein (HDL) particles. The effects of LDL and HDL were dose dependent and saturable. The stimulatory influence of E2, FSH, or LH alone was significantly enhanced in the presence of pig LDL or HDL. Moreover, the synergism between E2 and FSH or between E2 and LH was significantly augmented by porcine LDL and, to a lesser degree, porcine HDL. To assess the physiological relevance of these observations, the lipoprotein contents of swine blood and follicular fluid were determined by heparin-manganese precipitation and after differential ultracentrifugation. The majority (greater than 70%) of cholesterol in pig blood resided in the LDL fraction, but follicular fluid was essentially devoid of LDL. On the other hand, follicular fluid contained large quantities of a presumptive HDL species with a density between 1.063-1.210. The HDL particle in follicular fluid was further characterized by agarose gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses, which demonstrated an alpha-migrating species whose major apoprotein exhibited an apparent mol wt of 28,000 and comigrated with human apoprotein A-1. Analytical ultracentrifugation of the pig follicular fluid HDL revealed a sedimentation coefficient (S20,w) of 4.93, similar to that of serum HDL (S20,w = 5.0). The physiological relevance of the HDL particle purified from follicular fluid was further demonstrated by its ability to significantly increase progesterone production by granulosa cells cultured under serum-free conditions in vitro. In summary, we have demonstrated striking responsivity of cultured pig granulosa cells to exogenously supplied LDL and, to a lesser degree, HDL, with further stimulation when cells are treated with estrogen and/or LH and FSH. Although LDL is the predominant lipoprotein in swine blood, it is essentially undetectable in the antral fluid of the intact Graafian follicle. Thus, the unambiguous in vitro responsiveness of granulosa cells to LDL that we observe suggests that the marked increase in availability of blood-borne LDL to granulosa-luteal cells that presumptively occurs at ovulation would contribute significantly to augmented rates of progesterone biosynthesis by luteal tissues in the pig.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of prolactin receptors in cultured rat granulosa cells by FSH,LH and GnRH

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by ~ 4–6-fold in a dose-dependent manner. Concomitant treatment with 10^?8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10^?6 M of a GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6]GnRH. PRL-binding capacity was also increased (~2-fold) by in vitro treatment with cholera toxin (10 μg/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (~ 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by ~ 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.     

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.     

The biocellular effect of thyroid hormone on functional differentiation of porcine granulosa cells in culture

To elucidate if the thyroid hormone acts directly on the ovary, the biocellular effect of L-thyroxine (T4) on porcine granulosa cells cultured in vitro was investigated. Monolayer cultures of porcine granulosa cells obtained from small (1 approximately 2 mm), medium (3 approximately 5 mm) or large (6 approximately 11 mm) follicles were carried out in the presence of porcine FSH (100 ng/ml). Concomitant treatment with T4 promoted FSH-dependent morphological luteinization, i.e. alteration of immature granulosa cells obtained from small follicles to epithelioid form. T4 also increased FSH-stimulated induction of hCG/LH receptor on immature granulosa cells. Furthermore, T4 augmented FSH-mediated production of progesterone and estradiol by immature granulosa cells cultured in vitro. The concentration of T4 to produce the maximal stimulatory effect was 10-7 M, demonstrating that optimal concentration of thyroid hormone is required for the expression of this stimulatory action. Since T4 alone demonstrated no effect on the differentiation of porcine granulosa cells and all the stimulatory effect of T4 seems to have a permissive action on FSH-induced granulosa cell luteinization. Although insulin showed a similar effect on porcine granulosa cells, no stimulation of estradiol production by porcine granulosa cells was observed with insulin in the culture system used in this study. These results suggest that the thyroid hormone acts directly on the ovary and plays an important role in modifying the FSH-dependent cellular differentiation of immature granulosa cells.