Ovarian action of leptin: effects on insulin-like growth factor-I-stimulated function of granulosa and thecal cells

To test the hypothesis that leptin signals metabolic information to the reproductive system in cattle by directly affecting IGF-I-induced ovarian cell function, granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in serum-free medium with added hormones. Recombinant human leptin at 30 and 300 ng/mL had no effect on basal thecal cell steroidogenesis or thecal cell numbers. However, 300 but not 30 ng/mL of leptin attenuated (p<0.05) luteinizing hormone-induced androstenedione production by 24% in the absence of IGF-I and by 16% in the presence of IGF-I. Leptin had no effect on IGF-I-induced estradiol production in the presence of follicle-stimulating hormone (FSH), but at 100 ng/mL, leptin inhibited (p<0.05) FSH plus IGF-I-induced progesterone production and granulosa cell proliferation by 29 and 31%, respectively. Leptin did not compete for ¹²⁵I-IGF-I binding to granulosa or thecal cells, whereas unlabeled IGF-I did. In conclusion, leptin has weak inhibitory effects on gonadotropin-and/or IGF-I-induced steroidogenesis of thecal and granulosa cells.     

Effects of oxytocin on steroidogenesis by bovine theca and granulosa cells

Oxytocin (OT) is secreted during the final stages of bovine follicular development. To test OT's potential role as a regulator of follicular steroidogenesis, theca and granulosa cells were isolated from bovine preovulatory follicles 48 h after initiation of luteolysis with prostaglandin F2 alpha, and cultured with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml). Granulosa cells were cultured with testosterone (0.5 microM) in either defined medium or medium containing 10% fetal bovine serum in the presence or absence of FSH (300 ng/ml); medium was collected and replaced daily for 5 days. In defined medium, oxytocin alone significantly increased progesterone production by granulosa cells (P less than 0.001) in a dose-dependent manner; over 5 days, doses of 0.5, 5, 50, and 500 mIU/ml OT caused 1.7-, 2.0-, 2.2-, and 2.6-fold increases. FSH enhanced progesterone 5-fold, but no dose of OT increased progesterone in the presence of FSH. OT also elevated progesterone in serum-containing medium (P less than 0.005), but the magnitude of its effects was lower (1.07-, 1.1-, 1.2-, and 1.4-fold increases with 0.5, 5, 50, and 500 mIU/ml OT). OT had little effect on estradiol secretion by granulosa cells cultured with or without FSH. To test the specificity of OT's effects on progesterone production by granulosa cells, granulosa cells were treated with graded doses of an OT antagonist (0, 1, 10, 100, and 1000 ng/ml) in the presence or absence of OT (5 and 50 mIU/ml). Progesterone production by granulosa cells in the presence of the antagonist alone was similar to production in control cultures. The stimulatory effects of 5 and 50 mIU OT were completely abolished in the presence of 100 or 1000 ng antagonist, respectively (P less than 0.01). Preparations of theca interna were cultured in defined medium with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml) in the presence or absence of LH (300 ng/ml), with collection and replacement of medium at 3, 6, 12, 24, 48, and 72 h. LH alone increased both progesterone (12-fold) and androstenedione (4-fold) production over controls. However, no dose of OT significantly affected either progesterone or androstenedione production. These results show that OT stimulates progesterone production by granulosa cells, and thus, suggest that OT regulates steroidogenesis in bovine granulosa cells in vivo.     

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.     

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

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

Establishment of a human nonluteinized granulosa cell line that transitions from the gonadotropin-independent to the gonadotropin-dependent status

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.     

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125-1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17 alpha-hydroxylase (17 alpha-OH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.     

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.     

Effects of growth hormone-releasing factor and vasoactive intestinal peptide on proliferation and steroidogenesis of bovine granulosa cells.

Recent studies have suggested that growth hormone-releasing factor (GRF), like vasoactive intestinal peptide (VIP), may enhance follicle-stimulating hormone (FSH)-stimulated steroidogenesis in cultured rat granulosa cells (GC). Because effects of GRF or VIP on GC proliferation have not been reported, we evaluated and compared the effect of GRF to that of VIP using cultured bovine GC. Undifferentiated GC from 1-5 mm bovine follicles were established for 2 days in medium containing 10% fetal calf serum, washed and then cultured in chemically defined medium for an additional 2 days. Two-day treatment with 2.5-1000 ng/ml of VIP had no effect (P greater than 0.05) on proliferation or progesterone production of bovine GC in the presence or absence of 200 ng/ml FSH. In comparison, 100, 250, 500, 1000 or 2000 pg/ml of human [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 analog caused a dose-dependent stimulation (P less than 0.05) of GC proliferation in the absence and presence of 5 micrograms/ml insulin. However, the GRF analog had no effect (P greater than 0.05) on GC progesterone production (expressed as ng/10(5) cells/24 h) in the absence or presence of 5 micrograms/ml insulin. The effects of GRF analog on progesterone production and cell proliferation were not influenced by co-culture with 200 ng/ml FSH. GRF(1-44)-NH2 also stimulated cell proliferation but had no effect on basal or FSH-induced progesterone production. These results suggest that GRF may play a role in GC proliferation during follicular development in the bovine.     

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.     

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.     

Effect of insulin-like growth factor I on steroidogenesis in cultured human granulosa cells

The effect of IGF-I on steroidogenesis in human granulosa cells was studied. Granulosa cells were obtained from follicles of both natural and stimulated cycles. The cells were cultured 4 to 6 days and the effect of IGF-I (1 to 100 micrograms/l) on basal, LH- and FSH-stimulated steroidogenesis was studied. It was found that in granulosa cells from follicles of natural cycles, FSH as well as IGF-I significantly stimulated progesterone and estradiol production in a majority of the experiments. A synergistic effect of FSH and IGF-I could be seen when low (1 and 10 micrograms/l) concentrations of the two hormones were used. Also in granulosa luteal cells from stimulated cycles a stimulatory effect of IGF-I on estradiol as well as progesterone production was observed. The present results suggest that IGF-I in combination with gonadotropins has a physiological role in the human follicle in controlling differentiation of the granulosa cells.     

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

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

The role of mitogen-activated protein kinase in insulin and insulin-like growth factor I (IGF-I) signaling cascades for progesterone and IGF-binding protein-1 production in human granulosa cells

Insulin and IGF-I participate in the regulation of ovulation, steroidogenesis, and IGF-binding protein (IGFBP) production in the ovary. Insulin and IGF-I actions in the ovary are closely related. For example, insulin may amplify IGF-I action in the ovary by up-regulating type I IGF receptors and inhibiting IGFBP-1 production, thus increasing the bioavailability of IGF-I. It is hypothesized that ovarian effects of insulin in insulin-resistant states are mediated via an insulin action pathway(s) distinct from those involved in glucose transport. We previously reported that insulin-induced stimulation of progesterone and inhibition of IGFBP-1 production in the human ovary are mediated by signaling pathways that are independent of phosphatidylinositol 3-kinase, the enzyme whose activation is crucial for glucose transport. We now examined whether activation of MAPK is necessary to mediate insulin-induced or IGF-I-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells. Human granulosa cells were obtained during in vitro fertilization. Cells (0.5-1 x 10(5)) were incubated for 24 h in the presence of 0, 10, 10(2), or 10(3) ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 micro M PD98059, a specific inhibitor of ERK1/2 MAPK. The progesterone concentration in the tissue culture medium was measured by RIA (Pantex, Santa Monica, CA), and the IGFBP-1 concentration was measured by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX). MAPK activity was assessed using the MAPK IP-Kinase assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). ANOVA was used to compare mean values of progesterone or IGFBP-1 concentrations. MAPK was stimulated by insulin up to 350% of the baseline value. Progesterone production in human granulosa cells was stimulated by insulin in a dose-related manner to 123% of the control value (P < 0.001), and IGFBP-1 production was inhibited to 25% of the baseline value (P < 0.001). Despite inhibiting MAPK activity by 99%, PD98059 (1 micro M) did not interfere with insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production. MAPK was stimulated by IGF-I to 730% of the baseline value, with maximal stimulation achieved at 0.5 ng/ml IGF-I. Progesterone production in granulosa cells was stimulated by IGF-I to 130% of the control value (P < 0.001), whereas IGFBP-1 production was inhibited to 44% of the control value (P < 0.001). PD98059 (1 micro M) inhibited IGF-I-induced MAPK activity by 94%. In the presence of 1 micro M PD98059, IGF-I-induced stimulation of progesterone production was inhibited by 96% (P < 0.001). The inhibitory effect of IGF-I on IGFBP-1 production was reduced in the presence of 1 micro M PD98059 by 45% at 5 ng/ml IGF-I and was completely abolished in the presence of 1 micro M PD98059 at concentrations of IGF-I ranging from 0.5-2.5 ng/ml (P < 0.001). We conclude that, under conditions of our experiments, insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells does not require MAPK activation, whereas similar effects of IGF-I are largely MAPK dependent.     

A functional bone morphogenetic protein system in the ovary

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells.

FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of rat ovarian thecal cells: evidence for functional luteinization

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.