Interleukin-1 inhibits follicle stimulating hormone-induced differentiation in rat granulosa cells in vitro

Increasing evidence suggests that factors secreted from cells of the immune system can affect endocrine function. In this report we show that the monokine, interleukin-1, inhibits follicle stimulating hormone-induced development of luteinizing hormone receptors and reduces progesterone secreted from cultured rat granulosa cells. These effects of interleukin-1 were observed in the physiological range of 10(-9) M. The ability of sex steroids to influence the immune response together with our results support the hypothesis that there is a bidirectional communication network which links the immune and reproductive endocrine systems.     

Gamma-interferon inhibits rat granulosa cell differentiation in culture

A growing body of evidence indicates that factors secreted by cells of the immune system can directly affect a variety of endocrine phenomena. In the present study we examined the direct effects of the cytokine, interferon (IFN), on FSH-stimulated steroidogenesis and LH/hCG receptor induction in rat granulosa cells. We show that gamma-IFN, but not alpha-IFN, inhibits FSH-stimulated progesterone, 20 alpha-hydroxypregn-4-en-3-one and estrogen production as well as gonadotropin-induced LH/hCG receptor formation in a dose-dependent manner. The results suggest that gamma-IFN may play a role in the maturation and differentiation of granulosa cells and thus may serve as a regulatory link between the immune and reproductive endocrine systems.     

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.     

Effect of follicular fluid and oestradiol on the luteinization of rat granulosa cells in vitro.

The effect of pig follicular fluid (FF), total or oestrogen-free, and of oestradiol-17 beta on the luteinization and progesterone secretion of rat granulosa cells was investigated during 4 days in culture. Both FF and oestrogen-free FF modified the differentiation of the granulosa cells, particularly their size and the appearance of their nucleoli. Addition of total FF or oestradiol-17 beta (50, 100 or 500 ng/ml) to the control medium markedly increased the progesterone secretion of the granulosa cells, but oestrogen-free FF and dialysed fetal calf serum had no effect. It was concluded that (1) FF could modify the morphological changes of the rat granulosa cells in vitro, but could not inhibit their secretion of progesterone, (2) the granulosa cells were able to synthesize progesterone regardless of their stage of differentiation, (3) oestradiol 17 beta had a direct stimulatory effect on progesterone secretion by granulosa cells in vitro. The possible mode of action of FF upon luteinization is discussed.     

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate. When granulosa cells were treated with glycosaminoglycan-degrading enzymes before or after addition of follistatin to the cultures, heparinase and heparitinase treatments resulted in significant suppression of the binding, whereas treatment with chondroitinase ABC had no effect. A competition study of the binding using heparin derivatives demonstrated that follistatin seemed to recognize O-sulfate groups in the heparin molecule. Heparitinase-treated granulosa cells exhibited almost full responsiveness to activin, indicating that the enzyme treatment had no effect on activin and receptor interaction. These results suggest that follistatin/activin-binding protein binds to heparan sulfate side chains of proteoglycans on the granulosa cell surface to regulate the various actions of activin.     

Fibronectin stimulates growth but not follicle-stimulating hormone-dependent differentiation of rat granulosa cells in vitro

Since fibronectin is a secretory product of immature rat granulosa cells in culture and may contribute to the follicular microenvironment in vivo, we have studied the effects of this adhesion factor on follicle-stimulating hormone (FSH)-dependent differentiation in short-term (2-3-day) cultures and on growth and protein synthesis in long-term (12-day) cultures. In comparison with cells plated on tissue culture plastic, those plated on an optimal fibronectin-coated substratum showed much greater cell spreading. There were no short-term effects of this morphological change on FSH-stimulation of cyclic AMP production, apparent activities of aromatase or cholesterol side-chain cleavage enzymes, or acquisition of luteinizing hormone (LH) responsiveness in cultured cells. However, progesterone metabolism to 20 alpha-hydroxypregnan-4-en-3-one was increased. Only cultures on fibronectin showed increases between days 3 and 9 in protein (2.5-fold) and DNA (1.4-fold) contents. Cells cultured on fibronectin also showed greater uptake and incorporation of [3H]leucine in comparison with cells cultured on plastic. FSH treatment caused cell aggregation and rounding and delayed the increase in protein content of cells cultured on fibronectin. The results presented demonstrate that the principal direct effect of fibronectin-mediated adhesion on rat granulosa cells is to enhance cell maintenance and growth, while having no generalized action on FSH-dependent differentiation.     

Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro.

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and then maintained in culture. Proteoglycans were labeled using [35S]sulfate, D-[3h]glucosamine, or L-[3H]serine as precursors. 35S-labeled proteoglycans in the medium increased linearly up to 72 h after a 6- to 8-h lag period, and those in a 4 M guanidine HCl extract of the cell layer increased for about 16 h and then reached a plateau and stayed fairly constant up to 72 h. Two distinct sizes of proteoglycans were observed in the medium. The smaller (Kav = 0.60 on Sepharose CL-2B) had lower buoyant densities in dissociative gradients (rho less than 1.4 g/ml). The larger (Kav = 0.26 on Sepharose CL-2B) had high buoyant densities (recovered mainly in the bottom (D1) fraction of the dissociative gradient). More than 90% of the D1 proteoglycans contained dermatan sulfate chains (average Mr = 38,000) which yielded 84% 4-sulfated and 15% disulfated disaccharides after digestion with chondroitinase ABC. About 8% of the 35S-label in D1 was present as a heparan sulfate proteoglycan. When [3H]-glucosamine was used as a precursor, 28% of the 3H activity in the D1 proteoglycans was located in three major oligosaccharide components, two of which were similar or identical with those observed previously in D1 proteoglycans isolated from porcine follicular fluid. These results plus similar susceptibility of the labeled proteoglycans to proteolytic enzymes, especially plasmin, suggest that the granulosa cells synthesize the predominant follicular fluid proteoglycans.     

Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro.

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.     

Androgen-induced changes in rat ovarian granulosa cells in vitro

Ovarian granulosa cells collected from small antral follicles from immature rats were cultured in McCoy's 5A medium, for 1-6 days in the presence of delta 4-androstenedione, testosterone, dihydrotestosterone, and dehydroepiandrosterone (10(-5) M and 10(-7) M). Granulosa cells examined by electron microscopy demonstrated many lipid droplets, mitochondria with tubular cristae and profiles of smooth endoplasmic reticulum, all suggestive of active metabolism in the cell. Cells cultured in androstenedione, testosterone, dihydrotestosterone and dehydroepiandrosterone produced estrogen and progesterone as measured by radioimmunoassay. By day 4, cells cultured in androgen had almost completely degenerated. The control cells acquired none of the aforementioned characteristics and survived up to beyond 6 days, at which time the experiments were terminated. This study supports the hypothesis that high concentrations of androgens in cultured granulosa cells contribute to their degeneration through altered structure, which is associated with functional change.     

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.     

Gonadotropin-releasing hormone inhibits cyclic nucleotide accumulation in cultured rat granulosa cells

Ovarian granulosa cells obtained from hypophysectomized, diethylstilbestrol-treated rats were cultured in the presence of ovine follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH). FSH stimulated the production and accumulation of both cAMP and cGMP, as well as progesterone, during a 48-h incubation period. Addition of GnRH or an agonist analog, [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), did not influence the cyclic nucleotide response to FSH in the first 6 h of incubation, but caused dose-dependent inhibition of the FSH-induced rise in cyclic nucleotide production from 24 to 48 h of incubation. Cellular production of both cyclic nucleotides and progesterone was decreased by GnRHa concentrations as low as 10(-12) M, with maximum inhibition at 10(-9) M GnRHa. These results suggest that the in vitro antigonadal actions of GnRH and related peptides are expressed through inhibition of cyclic nucleotide production.     

Histochemical studies of rat ovarian follicular cells in vitro

Summary Membrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δ^p5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation. This research was supported in part by a Faculty Research Grant from the Horace H. Rackham School of Graduate Studies, and by United States Public Health Service Grants RR-05383-09 and AM-06918-06.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Production of activin-binding protein by rat granulosa cells in vitro

We have developed an assay method for activin-binding protein, which exploits its high affinity for sulfated polysaccharides. We used this method to investigate the production of activin-binding protein by rat ovarian granulosa cells, in vitro. The production of activin-binding protein by granulosa cells was dependent on the cell density; the maximum was observed at 6 x 10(5) cells/ml. Follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), enhanced production significantly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting analyses of the activin-binding protein secreted by rat granulosa cells demonstrated it was the same protein molecule as that purified from rat ovaries. It is inferred from these results that the granulosa cell is a source of ovarian activin-binding protein and that its secretion is regulated by FSH.     

Erythroid differentiation factor can modulate follicular granulosa cell functions

The action of human erythroid differentiation factor (EDF) on the functions of rat granulosa cells cultured in a chemically defined medium was investigated. In the presence of FSH that induced LH receptor expression and progesterone synthesis during culture of the cells, EDF augmented both responses in a dose- and time-dependent manner. Unlike FSH, EDF itself did not have such an inducing effect at all. Furthermore, in the absence of FSH, EDF was found to strongly enhance the ability of granulosa cells to produce inhibin. Thus, EDF may play an important role in the regulation of granulosa cell function and differentiation during follicle development.     

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20 alpha-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20 alpha-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was x 2 and x 1.6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.