Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

Follicular fluid effects on progesterone secretion are not due to follicle-stimulating hormone or steroids

An antiserum raised against porcine follicle-stimulating hormone (FSH) was unable to eliminate the stimulatory action of fluid from large antral porcine follicles on progesterone secretion by granulosa cells from small antral porcine follicles. The same titers of the antiserum were completely effective at eliminating the effect of 2 micrograms of NIH-FSH-P12, whereas maximal stimulation of progesterone secretion was observed with 0.5 micrograms FSH/ml. The androgen and estrogen concentrations measured in charcoal-treated inhibitory follicular fluid from small porcine antral follicles and from stimulatory follicular fluid from large follicles were added separately and together to culture media supplemented with serum to determine if these low concentrations (5 X 10(-11) to 5 X 10(-10) M) of steroids could mimic the actions of follicular fluid on progesterone secretion. Neither the inhibitory nor the stimulatory actions of the follicular fluids could be mimicked by these low concentrations of steroids. Higher concentration of steroids (10(-8) to 10(-7) M range) did stimulate progesterone secretion as reported by others. Our data indicate that the actions of charcoal-treated follicular fluids on granulosa cell progesterone secretion cannot be explained by difference in FSH or steroid contents between the inhibitory and stimulatory fluids and serum.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Stimulatory action of follicular fluid components on maturation of granulosa cells from small porcine follicles

Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.     

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.     

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.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the granulosa cells of mouse ovarian follicles by follicular fluid peptide

A protein fraction (GF2) was purified from sheep ovarian follicular fluid. Its action on 3 beta-HSD activity in the mouse granulosa cells was measured in an in vitro system. The fraction (GF2) caused dose-dependent depletion of the 3 beta-HSD activity in granulosa cells and progesterone in the spent medium. A maximum inhibition of the activity was achieved after 30 min incubation of the granulosa cells with the GF2 fraction. Further purified HPLC fraction (Fr1) also inhibited 3 beta-HSD activity. In vivo administration of the GF2 fraction in normal cycling female mice also decreased the 3 beta-HSD activity in the granulosa cells of the ovarian follicles and plasma progesterone levels indicating the GF2 fraction to be a 3 beta-HSD inhibitor.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts

In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.     

Insulin-like growth factors (IGFs) as autocrine/paracrine regulators of granulosa cell differentiation and growth: studies with a neutralizing monoclonal antibody to IGF-I

Evidence that granulosa cells secrete and respond to insulin-like growth factors (IGFs) suggests, but does not prove, the importance of IGFs as intraovarian regulators. To further assess the role of these peptides in ovarian function, a neutralizing monoclonal antibody to IGF-I was employed to block the actions of IGFs in porcine follicular fluid and in granulosa cell-conditioned medium. In one series of experiments, granulosa cells from immature porcine follicles were cultured in medium containing porcine follicular fluid that had been charcoal-treated to remove steroids. As noted before, fluid from large follicles (LFF) stimulated progesterone production in a dose-dependent manner. The stimulatory effect of LFF (30% v/v) could be inhibited by greater than 50% by the anti-IGF monoclonal antibody. This inhibitory action was specific for the anti-IGF antibody and could be overcome by the addition of excess exogenous IGFs. In another series of experiments, granulosa cells were made dependent on endogenously produced IGFs by culturing them in a serum-free medium without exogenous growth factors. The effects of follicle-stimulating hormone (FSH), estradiol (E2), growth hormone (GH), and combinations thereof on progesterone production were inhibited by approximately 50% by the anti-IGF antibody. The effects of IGFs on indices of cell growth (judged by the criterion of being inhibited by the anti-IGF antibody) were less dramatic. A modest 18% increase in cell number was observed with FSH and E2 treatment in serum-free medium; this effect was virtually abolished by the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the actions of stimulatory follicular fluid and gonadotropin-releasing hormone analogs on progesterone secretion by porcine granulosa cells

Granulosa cells from small and medium porcine follicles (1-5 mm) were incubated with charcoal-treated follicular fluid from large (6-10 mm) follicles or porcine serum in the presence and absence of gonadotropin-releasing hormone (GnRH) analog and luteinizing hormone (LH) or follicle-stimulating hormone (FSH). A GnRH agonist inhibited follicular fluid's enhancement of basal and LH-stimulated progesterone secretion but did not block follicular fluid's enhancement of FSH-stimulated progesterone secretion. A GnRH antagonist mimicked follicular fluid's enhancement of basal and LH-stimulated progesterone secretion but did not mimic follicular fluid's action on FSH-stimulated progesterone secretion. When the GnRH antagonist and follicular fluid were added together, they acted synergistically in stimulating basal progesterone secretion, and were additive in enhancing LH-stimulated progesterone secretion. These observations suggest that separate follicular fluid molecules are responsible for its influence on LH and FSH actions on granulosa cells and that a GnRH-antagonist-like molecule could be responsible for some of follicular fluid's "luteinization stimulatory" action. Alternatively, the stimulatory follicular fluid molecule may not resemble GnRH but may act via a mechanism that is opposed by GnRH.     

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles

The purpose of this study was to establish a culture model for isolated intact porcine antral follicles and investigate the relationship between granulosa cell apoptosis and follicular atresia. Small (<3 mm), medium (3–5 mm) and large (>5 mm) healthy porcine follicles were isolated and cultured in serum‐free TCM199 with or without follicular stimulating hormone (FSH). Microscopic identification of healthy follicles was confirmed by histology. A spontaneous onset of apoptotic cell death in granulosa cells was observed from cultured antral follicles. The apoptotic rate of granulosa cells from small follicles cultured for 24 hr was higher than those of large and medium follicles, accompanied with high FasL mRNA abundance in granulosa cells. Supplementation with 3 or 5 IU/ml FSH significantly inhibited the percentage of granulosa cells that became apoptotic. FSH did not significantly alter estradiol secretion from cultured follicles. Progesterone secretion significantly decreased after culture for 48 hr, coinciding with the morphological changes observed. FasL and Fas mRNA were expressed in the healthy, early atretic, and progressed atretic porcine follicles regardless of follicular size. However, FasL but not Fas mRNA levels increased during follicular atresia. Addition of FSH significantly decreased FasL rather than Fas mRNA levels in granulosa cells and could attenuate apoptosis. Small follicles seemed to be more susceptible to atresia as compared to medium and large follicles. Mol. Reprod. Dev. 77: 670–678, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.     

The glycosaminoglycan chondroitin-4-sulfate alters progesterone secretion by porcine granulosa cells

Porcine granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) antral follicles were cultured in monolayer for 2 to 3 days with 0 to 3 mg of chondroitin-4-sulfate (C-4-S)/ml in the presence or absence of 0.5 microgram follicle-stimulating hormone (NIH-FSH-S13)/ml. Testosterone (1.4 microgram/ml) was added to some cultures as substrate for estrogen synthesis. Progesterone and estrogen secreted into the media were measured by radioimmunoassay. Concentrations of C-4-S similar to concentrations of chondroitin sulfates (CS) reported for small antral or atretic follicles inhibited both basal and FSH-stimulated progesterone secretion. Progesterone secretion was not inhibited by C-4-S when pregnenolone was added to the media. Thus 3 beta-hydroxysteroid dehydrogenase activity was not inhibited by C-4-S. Estrogen secretion was also not inhibited by even the highest concentration of C-4-S tested. Testosterone did not influence C-4-S inhibition of progesterone secretion. Granulosa cells from medium-sized follicles were more sensitive to C-4-S than cells from small follicles. Granulosa cells from large follicles were completely resistant to C-4-S inhibition of progesterone secretion. These observations suggest that C-4-S may play a role in altering gonadotrophin-stimulated and basal progesterone secretion in follicles during differentiation of granulosa cells.     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Inhibitory effects of the porcine follicular fluid on estradiol and progesterone secretion by cultured rat granulosa cells

The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.     

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.     

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)