PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/μg cell protein) in response to an acute FSH stimulus (5 μg/ml NIH-FSH-S11, 2 h). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/μg cell protein). Cells cultured with prostaglandin E₂ (PGE₂, 1 μg/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/μg cell protein, respectively) than that of control cultures. Therefore the presence of PGE₂ or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH.Paradoxically, granulosa cells cultured with PGE₂ produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 250.3 fm/μg cell protein). This may be due to a suppressive effect of prior exposure to PGE₂ on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

R5020 enhances PGE2 stimulated steroidogenesis in cultured rat granulosa cells.

Progesterone biosynthesis and metabolization to 20 alpha-hydroxyprogesterone was stimulated in granulosa cells cultured in the presence of 20 ng/ml of follicle stimulating hormone (FSH) or increasing concentrations of PGE2 (10(-9)-10(-7)M). Concurrent treatment with the synthetic progestin R5020 (10(-6) M) enhanced the FSH or PGE2 stimulated progesterone and 20 alpha-hydroxyprogesterone accumulation in culture media, as well as delta 5-3 beta-hydroxysteroid dehydrogenase activity in granulosa cell homogenates. These findings may represent another example of an autocrine control mechanism in which the steroidogenic product of the granulosa cell exerts an ultra-short loop regulation of its own production.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Changes in responsiveness of rat granulosa cells to prostaglandin E2 and follicle-stimulating hormone during culture

The changes in responsiveness of granulosa cells to either FSH or prostaglandin E2 (PGE2), during culture of the cells, have been examined. In freshly isolated cells, FSH and PGE2 stimulated both cyclic AMP and progesterone production in a dose-dependent manner. In these cells, FSH stimulated cyclic AMP production to a greater extent than did PGE2. After the cells had been cultured for 2 days, neither FSH nor PGE2 stimulated progesterone production to any detectable extent. In these cells the ability of FSH to stimulate cyclic AMP was decreased, and that of PGE2 was increased markedly, such that PGE2 was far more effective than FSH in stimulating cyclic AMP. After culture of the cells for a further 2 days (4 days total), the FSH stimulation of cyclic AMP returned to that seen in freshly isolated cells, whereas the stimulation by PGE2 remained elevated. The acute stimulation of progesterone production could be restored by chronic exposure of the cells to either FSH or PGE2. These results demonstrate that dramatic changes in responsiveness of granulosa cells take place during culture. The results also suggest that some stimulating factor must be present to maintain the steroidogenic capabilities of the cells. Without this, although the cells are able to produce cyclic AMP in response to FSH, they cannot produce progesterone.     

Hormone-stimulated secretion of luteinization factor in porcine granulosa cells.

Luteinization stimulator (LS) is an intrafollicular compound which was shown to be released by granulosa cells under in vitro conditions with stimulatory effects on immature granulosa cell differentiation. This study was undertaken to determine the effects of various endocrine agents which are involved in the regulation of ovarian function on LS secretion by porcine granulosa cells isolated from 5-8-mm follicles (LGC). Cell conditioned media (CM) obtained after the 4-day culture of LGC were tested in the culture of immature (small) granulosa cells (SGC). The activity of LS released into the LGC conditioned medium was estimated by measuring progesterone (P4) produced by SGC in the presence of CM. Stimulation of P4 secretion was observed after addition of media from cultures treated by LHRH (10(-4) mol.l-1), epinephrine (10(-5) mol.l-1), LH (1 microgram.ml-1), dbcAMP (0.5 and 2.0 micrograms.ml-1) or insulin (1.0-5.0 micrograms.ml-1). Norepinephrine (10(-5) and 10(-7) mol.l-1), estradiol (0.1 and 1.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml-1) did not change steroidogenic activity of CM. Epinephrine and norepinephrine (10(-5) and 10(-7) mol.l-1), LH (1 microgram.ml-1), dbcAMP (2.0 microgram.ml-1) and estradiol (1 microgram.ml-1) alone enhanced P4 production by SGC, whereas LHRH (10(-3) and 10(-4) mol.l-1), insulin (1.0-5.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml.-1) did not have any effect. These observations suggest that the process of LS secretion in developing follicles is subject to a specific hormonal control.     

Mechanism of action of 2-hydroxyestradiol on steroidogenesis in ovarian granulosa cells: interactions with catecholamines and gonadotropins involve cyclic adenosine monophosphate

Previously we have shown that 2-hydroxyestradiol (2-OH-E2) synergizes with catecholamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2 and catecholamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum-free medium for periods of 6-9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 micrograms/ml) but significantly reduced the effect of 4 micrograms/ml 2-OH-E2 on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2 and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as "-fold of controls") stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2 was 1.4 +/- 0.2, 8.2 +/- 2.2, and 10.7 +/- 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 +/- 0.1, 7.8 +/- 1.4, and 10.6 +/- 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2 significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Lack of effect of oocytectomy on expansion of the porcine cumulus.

Oocyte-cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0.01-1.0 micrograms/ml) or forskolin (50-100 mumol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0.1 microgram/ml) or forskolin (100 mumol/l) increased the contents of intracellular adenosine 3',5'-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0.1 microgram/ml) or forskolin (100 mumol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P less than 0.01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Granulosa cells from pig follicles of different sizes demonstrate maturational differences in their steroidogenic responses to FSH, calcium ionophore A23187, and phorbol diester

Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Discordance in the effects of interleukin-1 on rat granulosa cell differentiation induced by follicle-stimulating hormone or activators of adenylate cyclase

Recombinant human interleukin-1 (IL-1) inhibits the follicle-stimulating hormone (FSH)-induced development of luteinizing hormone (LH) receptors and suppresses progesterone secretion in cultured rat granulosa cells. Since activation of adenylate cyclase by FSH is considered to be the primary second messenger system responsible for differentiation of granulosa cells, we examined whether IL-1 could alter the FSH, cholera toxin, or forskolin-induced accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) from these cells. In addition, we sought to determine if IL-1 could influence differentiation induced by the cAMP analog, 8-bromo cAMP. Cells collected from ovaries of immature, diethylstilbestrol-treated rats were stimulated to differentiate by addition of FSH, cholera toxin, forskolin, or 8-bromo cAMP to the cultures. IL-1 or interleukin-2 (IL-2) was added to some of the tubes, and the primary cultures were incubated for various periods of time. At the end of the culture, the tubes were centrifuged, the medium was saved for progesterone and cAMP radioimmunoassay, and the cells were assayed for specific 125I-human chorionic gonadotropin (hCG) binding to determine the number of LH receptors. In the presence of FSH, IL-1, at a dose as small as 5 ng/ml, but not IL-2, significantly inhibited LH receptor formation and suppressed progesterone secretion in a dose-related manner. IL-1 also significantly suppressed FSH-induced cAMP accumulation after 72 h of incubation but did not appear to do so in a dose-related fashion. In the presence of FSH, IL-1 did not significantly alter the protein content of granulosa cells at the end of culture. During stimulation of granulosa cells with cholera toxin, forskolin, or 8-bromo cAMP, IL-1 significantly reduced LH receptor formation compared to that observed in the absence of IL-1. However, in contrast to IL-1 in the presence of FSH, IL-1 significantly augmented the forskolin-induced secretion of progesterone and accumulation of cAMP after 72 h at subsaturating doses of forskolin. Thus, IL-1 appeared to inhibit forskolin-induced and cholera toxin-induced formation of LH receptors even when cAMP levels were elevated. Similar to forskolin, 8-bromo cAMP-stimulated progesterone secretion was significantly enhanced by IL-1, but LH receptor formation was inhibited. Over a 72-h time course at single doses of FSH or forskolin, IL-1 did not affect cAMP accumulation until 48 h of culture, at which time IL-1 significantly suppressed FSH-induced, but augmented forskolin-induced, accumulation of cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of follicle-regulatory protein by porcine granulosa cells

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

Cultivation of rat granulosa cells in a serum-free chemically defined medium--a useful model to study lipoprotein metabolism

We have developed a chemically defined, serum-free medium for the culture of rat granulosa cells. This medium contains Dulbecco's modified Eagle's medium/Ham's nutrient F12 (DME:F12) (1:1) plus insulin (2 micrograms/ml), hydrocortisone (100 ng/ml), transferrin (5 micrograms/ml) and fibronectin (2 micrograms/cm2). Granulosa cells grown in this medium have an absolute requirement for added cholesterol-rich lipoproteins for steroidogenesis. When cells are cultured in basal medium, progestin production is low; when cells are cultured in the presence of follicle-stimulating hormone (FSH) or dibutyryl cAMP [Bu)2 cAMP), progestin secretion is increased 10-100-fold. Both heterologous and homologous lipoproteins synergistically increased the effects of (Bu)2 cAMP or FSH: e.g., addition to the medium of human (h)-HDL3 produced a significant increase in both basal (approx. 15-fold) and (Bu)2 cAMP-stimulated (approx. 1000-2000-fold) progestin production. LDL were less effective than HDL at equivalent concentrations of lipoprotein cholesterol. FSH invoked changes similar to that of (Bu)2 cAMP, although the magnitude of the FSH-induced change was less dramatic than that seen with (Bu)2 cAMP. The effect of h-HDL3 and h-LDL on both basal and hormone-stimulated progestin production was concentration- and time-dependent. The maximum effect of h-HDL3 was achieved at a protein concentration of 500 micrograms/ml, with an ED50 of approx. 90 micrograms/ml. In contrast, h-LDL was most effective at a concentration of 30-40 micrograms protein/ml. Likewise, rat (r-)HDL and r-LDL supported steroidogenesis in a concentration-dependent manner. Maximal responses to all additions were observed after 72 h of treatment. Granulosa cells secreted 20 alpha-hydroxypregn-4-ene-3-one as the predominant steroid in response to (Bu)2 cAMP. However, with the addition of h-HDL3, the major secreted product was progesterone. In conclusion, rat granulosa cells maintained in the described serum-free medium are exquisitely sensitive to supplied cholesterol-rich lipoproteins. When cultured in the presence of both lipoproteins and stimulatory agents, they produce from 1000-2000-times the progestins made by comparable cells maintained in medium alone. This responsiveness of the cells to both lipoprotein and hormone stimulation makes them uniquely suitable for studies involving the uptake and metabolism of lipoproteins during steroidogenesis.     

Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH

Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.     

Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs

In addition to increasing cyclic adenosine 3',5'-monophosphate (cAMP) levels, luteinizing hormone (LH) stimulation of granulosa results in phosphoinositide hydrolysis producing inositol trisphosphate (IP3) and diacylglycerol. The roles of these putative second messengers were investigated by measuring production of progesterone and inositol phosphates by granulosa from medium-sized porcine follicles (3-7 mm) after 15 min incubation with or without LH (1 microgram/ml), 5 microM dibutyryl cAMP (dbcAMP), or 5 microM 1-oleoyl,2-acetylglycerol (OAG). Compared to a control rate of 5.4 pmoles/10(7) cells/15 min, LH and dbcAMP stimulated progesterone production to 12.8 and 15.9 pmoles, respectively, and OAG decreased progesterone production to 3.7 pmoles. LH also stimulated inositol phosphate (IP) and bisphosphate (IP2) accumulations by approximately 5-fold and IP3 accumulation by 20-fold. In experiments where granulosa were premeabilized with saponin, LH, dbcAMP, and IP3 stimulated progesterone production from 1.3 pmol in control cells to 5.2, 3.2, and 5.1 pmol, respectively, and OAG decreased progesterone production to 1.0 pmol. LH stimulated accumulation of all inositol phosphates in permeabilized cells, whereas the addition of IP3 only increased IP2 and IP3 accumulations. In granulosa preincubated with 0.9 mM [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, A23187 increased progesterone production from 3.7 to 5.8 pmol. Addition of 1-20 nmoles IP3 to 10(7) granulosa incubated in a Ca2+-free medium increased Ca2+ efflux linearly. These data suggest that IP3 may have a role in regulating steroid production in granulosa by regulating intracellular Ca2+.