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.     

Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP–dependent luteinizing hormone receptor formation in ovarian granulosa cells

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (³H)-ATP to (³H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibition of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.     

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.     

Action of gonadotropin-releasing hormone II on the baboon ovary

The content, binding affinity, and bioactivity of chicken II GnRH (GnRH II) and a stable analogue of GnRH II (GnRH II analogue) in the baboon ovary were studied. Although mammalian GnRH is rapidly degraded by baboon ovarian extracts, we designed a GnRH II analogue that is stable to ovarian enzymatic degradation. This analogue binds to the ovarian membranes with high affinity (41 +/- 3 nM), having 20-fold the affinity of a potent mammalian GnRH analogue. The bioactivity of GnRH II and this GnRH II analogue on the regulation of ovarian progesterone release was compared with that for a potent mammalian GnRH analogue using a baboon granulosa cell culture system. Both GnRH II and GnRH II analogue produced significant inhibition of progesterone release from the granulosa cells (P < 0.03 and P < 0.005, respectively), with a greater reduction observed using the GnRH II analogue. After 24 h in culture, this GnRH II analogue produced a 59% +/- 5% inhibition of progesterone with a concentration as low as 1 nM. Maximal inhibition of 75% +/- 1% was attained with 10 nM GnRH II analogue. The endogenous GnRH II content in the baboon ovary was 5-14 pmoles/g protein. The release of endogenous GnRH II from granulosa cells was observed throughout the 48 h in culture. These studies demonstrated the presence of high enzymatic activity for the degradation of mammalian GnRH in the ovary, whereas this GnRH II analogue was stable. High-affinity binding sites for this GnRH II analogue were also found. GnRH II and this GnRH II analogue can regulate progesterone production from baboon granulosa cells, suggesting that GnRH II is a potent regulator of ovarian function.     

Hormonal regulation of the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells

These studies were undertaken to determine the molecular events by which estradiol and follicle-stimulating hormone (FSH) stimulate in ovarian granulosa cells the increase in the content of one of the regulatory subunits of cAMP-dependent protein kinase type II, RII51 (Mr = 51,000), and its electrophoretic variants RII51.5 (Mr = 51,500) and RII52 (Mr = 52,000). To analyze the de novo synthesis of RII51/52, granulosa cells were cultured (10(6) cells/ml) for 0, 12, 24, or 48 h with estradiol (10 nM) +/- FSH (12.5, 25, and 50 ng/ml), 8-bromo-cAMP (0.25-3 mM), or forskolin (0.5-100 microM) and then pulse-labeled with [35S]methionine (300 microCi/ml; 4 h). Labeled RII51, present either in urea extracts of total cellular protein or after partial purification from a soluble cell extract by cAMP-Sepharose chromatography, was quantitated by autoradiography of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and by excision of the silver-stained spots of the RII51 variants from the gels and counting. Synthesis of RII51 and its electrophoretic variants was low in cells cultured with estradiol alone for 48 h, whereas it was increased 4-5-fold in cells cultured with estradiol and FSH. Changes in the synthesis of actin were minor throughout the culture period regardless of hormone treatment. Pulse-chase experiments using [35S]methionine provided evidence that the isoelectric variants RII51.5 and RII52 may be derived from RII51 by post-translation modification, such as phosphorylation. Labelling with [32P]orthophosphate showed that RII52 contained more radioactivity than RII51.5 and RII51. Northern and filter hybridization assays demonstrated a 6-10-fold dose- and time-dependent increase in the amount of RII51 mRNA in granulosa cells exposed to estradiol and FSH or estradiol and forskolin compared to those cultured with estradiol alone. In vitro translation of poly(A)+ mRNA of granulosa cells from estradiol- and FSH-treated hypophysectomized rats also demonstrated an increase in the content of translatable RII51 mRNA. These studies indicate that in cultured rat granulosa cells the synthesis of RII51 and the content of its mRNA are selectively increased by estradiol and cAMP in a time- and dose-dependent manner. Based on these observations, RII51 appears to be a useful marker to determine the molecular (genomic?) sites of estradiol and FSH action in differentiating rat granulosa cells.     

Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells

The effect of an agonistic gonadotropin releasing hormone (GnRH)-analog (D-Ala6, des-Gly10-NH2-GnRH-ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare's serum gonadotropin (PMSG)-treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20 alpha-dihydro-progesterone (20 alpha-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH-stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20 alpha-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Furthermore, the response is also to some extent determined by the maturational stage of the granulosa cells.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone

FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.     

Increase in follicle stimulating hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates FSH production in the human fetus, dispersed pituitary cells from second trimester human fetuses were cultured on surface-modified plates. Exposure of cells to GnRH [(10(-8) and 10(-7) mol/L), study I] or [D-Ala6]des-Gly10-GnRH ethylamide (DALA) [(10(-11) to 10(-7) mol/L), study II] for 48 h resulted in an elevation of total FSH which correlated with an increase in releasable, but not nonreleasable, FSH. When pituitary cells were incubated for 24, 48 and 72 h with and without 10(-8) mol/L GnRH (study III), total FSH was significantly increased in cells cultured for 48-72 h without GnRH compared to cells lysed at the beginning of the incubation (p less than 0.001). At all intervals, GnRH significantly enhanced total FSH compared to respective controls (p less than 0.05).     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

Differential androgenic control of prostatic cytosolic cAMP-dependent and -independent protein kinases

Whether or not various cytosolic protein kinases (and especially the type I cAMP-dependent protein kinase) of rat ventral prostate are specifically regulated with respect to total activity or specific activity by androgen has been investigated. Following androgen deprivation, the total activity per prostate of cAMP-dependent protein kinase (with histone as substrate) changed little at 24 h, declining by about 20% at 96 h. Under these conditions, its specific activity remained unaltered at 24 h, but was markedly enhanced at 96 h postorchiectomy. Type II cAMP-dependent protein kinase in rat ventral prostate cytosol was the only form of cAMP-dependent protein kinases present as determined by measurement of catalytic activity as well as [32P]-8-N3-cAMP binding to the regulatory subunits. There was no alteration in the distribution of the isoenzymes of cAMP-dependent protein kinases or the response of these kinase activities to cAMP owing to castration of animals. The prostatic cytosol also contains free regulatory subunit (with molecular weight similar to that of regulatory subunit R1) which coelutes with type II cAMP-dependent protein kinase. This finding was confirmed by using [32P]-8-N3-cAMP photoaffinity labeling of cAMP-binding proteins. With respect to cAMP-independent protein kinase (measured with dephosphophosvitin as substrate), a decline of 31% in its specific activity was observed in cytosol of prostates from rats castrated for a period of 24 h without significant further change at later periods following castration. However, there was a marked progressive reduction in total activity of this enzyme per prostate (loss of 72% at 96 h postorchiectomy). The increase in specific activity of cAMP-dependent, but not cAMP-independent, protein kinase in the face of decreasing total activity in the cytosol at later periods of castration (e.g., at 96 h) may reflect a slower loss of the former enzyme protein than the bulk of the cytosolic proteins. Administration of testosterone to castrated animals prevented these changes. These data do not indicate a specific regulation by steroid of the type I cAMP-dependent protein kinase in the prostate. Rather, the cAMP-independent protein kinase (with dephosphophosvitin as substrate) appears to be modulated by the androgenic status of the animal.     

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.     

Regulation of tissue-type plasminogen activator activity and messenger RNA levels by gonadotropin-releasing hormone in cultured rat granulosa cells and cumulus-oocyte complexes

Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels.     

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.     

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells.

A major concept in mammalian ovarian physiology is that follicle-stimulating hormone (FSH) activates the granulosa cells (GCs) in the Graafian follicle to selectively produce estradiol, but not progesterone, during the follicular phase of the menstrual or estrous cycle. However, given the fact that FSH can induce production of both estradiol and progesterone by GCs cultured in vitro, it has been postulated for a long time that there is a factor present in the ovary that selectively prevents FSH-induced progesterone production. Here, we provide evidence that two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase-1 and -2 (ERK1/2) can differentially regulate FSH-stimulated estradiol and progesterone production. Using primary rat GCs from early antral follicles cultured in serum-free medium for 48 h, we found that the addition of a specific inhibitor of ERK1/2 activation, U0126, caused the attenuation or enhancement of FSH-induced progesterone or estradiol production, respectively, in a dose-dependent manner. Throughout the 48-h culture period in this culture system ERK1/2 molecules in their activated state (phospho-ERK1/2) were clearly detectable in GCs exposed to FSH. The addition of U0126 caused a decrease in the levels of phosphorylated but not unphosphorylated ERK1/2 which was maintained throughout the 48-h culture, suggesting that U0126 was continuously active to inhibit the phosphorylation of ERK1/2. The divergent regulation of FSH-induced progesterone and estradiol synthesis by U0126 was further supported by demonstrating that U0126 inhibits and stimulates FSH-induced mRNA levels of steroidogenic acute regulatory protein and P450 aromatase, respectively. Collectively, this study clearly identified ERK1/2 as the first intracellular signaling molecules that differentially regulate FSH-induced progesterone and estradiol synthesis in GCs.     

Use of monoclonal antibodies for purifying the regulatory subunit of cAMP-dependent protein kinase

Monoclonal antibodies against regulatory subunit of cAMP-dependent protein kinase, type II, were obtained from pig brain (R II). The immune-affinity sorbent has been synthesized on the basis of monoclonal antibodies against R II. The method was proposed for the purification of homogeneous R II with high cAMP-binding activity using immune-affinity sorbent.     

促乳素抑制培养的大鼠颗粒细胞中卵泡素介导的组织纤溶酶原激活因子的表达

本文主要是观察促乳素（ＰＲＬ）是否曩体外培养的大鼠颗粒细胞中,组织纤溶酶原激活因子（ｔＰＡ）和Ｉ型纤溶酶原激活因子抑制因子（ＰＡＩ－Ｉ）基因表达间的协调作用。我们采用了多种方法,例如ＳＤＳ－ＰＡＧＥ、免疫印迹等,来检测ＰＲＬ对ｔＰＡ和ＰＡＩ－Ｉ基因表达的作用。结果证实：（１）在离体条件下促乳素（ＰＲＬ）能刺激颗粒细胞（ＧＣ）中ＰＡＩ－Ｉ ｍＲＮＡ的合成,而ＦＳＨ无此作用。但ＦＳＨ可与ＰＲＬ协同增加     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.