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The synthesis of estradiol by the granulosa cells is a prominent event in ovarian physiology and depends on the expression of P450(AROM). FSH induces the expression of P450(AROM) in granulosa cells as a result of the presence in the ovarian promoter of a CRE (cAMP response element)-like sequence (CLS). In rodents, LH downregulates aromatase expression during luteinization by an as yet undescribed mechanism. In granulosa cells, LH increases the expression of the inducible cAMP early repressor (ICER), an isoform of CREM (cAMP-responsive element modulator) that represses cAMP-induced transcription. The possibility that ICER represses the activity of the aromatase ovarian promoter, thus being part of the mechanism underlying the effects of LH was investigated. We have found that: (1) nuclear proteins from forskolin-stimulated granulosa cells were specifically bound to an oligonucleotide containing the CLS sequence of the CYP19 ovarian promoter and one out of the two protein-DNA complexes formed was supershifted by an anti-CREM antibody; (2) in granulosa cells, forskolin-induced increases in P450(AROM) promoter luciferase reporter gene activity were prevented by the transient overexpression of ICER; (3) similar results were obtained in 8-Br-cAMP-stimulated R2C cells, a Leydig tumor cell line routinely used for the study of P450(AROM) promoter activity; (4) both ICER mRNA levels and P450(AROM) promoter-driven luciferase activity were elevated 6 and 12 h after stimulation of R2C cells with 8-Br-cAMP and were decreased 24 and 48 h later; (5) in an R2C polyclonal line overexpressing ICER, the promoter activity at early stages of stimulation was completely attenuated, while 24 and 48 h downregulation was prevented in another R2C line stably transfected with an antisense ICER construct. These results suggest that ICER represses CYP19 ovarian promoter and that LH-induced expression of ICER may serve to downregulate P450(AROM) transcription in granulosa cells during luteinization.

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Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase Cy (PLCgamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however observed after TPA-induced activation of PKC. In addition EGF-stimulated PLCgamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced 3H-InsP3 generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T 17)-induced cell proliferation. Treatment of EGFR-TI7 cells with C2-cer (0.1-10 microM) did not affect cell viability, but prevented EGF-induced 3H-thymidine incorporation in a dose-dependent manner. In contrast, 3H-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 microM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.

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In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.

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To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

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Stimulation of [3H]serine-labeled A431 cells with tumor necrosis factor-alpha (TNFalpha) or bacterial sphingomyelinase (SMase) resulted in a rapid decrease (approximately 50% by 15 min) in cellular [3H]sphingomyelin content and generation of the lipid moiety [3H]ceramide, which remained elevated 60 min later. Sphingomyelin hydrolysis in response to TNFalpha or bacterial SMase resulted in a time-dependent decrease in the phosphorylation state of c-Jun protein, an effect that was also observed in cells treated with the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-ceramide). The rapid dephosphorylation of the c-Jun gene product in response to TNFalpha, SMase, or C6-ceramide was not observed in A431 cells treated with the serine-threonine phosphatase inhibitor okadaic acid. After the initial steps of previously described methods for the purification of a ceramide-activated protein phosphatase termed CAPP (Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530), we obtained a cytosolic fraction from A431 cells that specifically dephosphorylated 32Pi-labeled c-Jun protein used as substrate in an immunocomplex phosphatase assay. Phosphatase activity in vitro was apparent only in the presence of ceramide (5 micro) and was specifically abrogated when okadaic acid (1 n) was included in the immunocomplex phosphatase assay. These results provide strong evidence for c-Jun as a downstream target for CAPP activated in response to post-TNF signaling in A431 cells.

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In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

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In [3H]serine-labelled granulosa cells treatment with TNF alpha (10 ng/ml) resulted in a transient decrease in cellular [3H]sphingomyelin and generation of [3H]ceramide that remained elevated 60 min later. In cells labelled with [methyl-14C]choline, TNF alpha induced a similar reduction in [14C]sphingomyelin content that was accompanied by a sustained elevation in [14C]phosphorylcholine levels. In FSH-primed cells, TNF alpha inhibited P450-AROM activity in a dose-dependent manner, an effect that was also observed in cells treated with bacterial sphingomyelinase (SMase 0.003-0.3 U/ml) or increasing concentrations (0.1-10 microM) of N-acetylsphingosine (C2-cer) a membrane-permeable analogue of ceramide. These results support the notion that sphingomyelin degradation to a bioeffector molecule ceramide, may be an early event involved in TNF alpha-induced signal transduction in granulosa cells.

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Metabolic labelling experiments performed with cultured pituitary lactotrophes revealed the presence of a glycosyl-phosphatidylinositol (GPtdIns) structurally related to GPtdIns lipids isolated from other cell types as demonstrated by: (i) metabolic incorporation of [3H]galactose, [3H]glucosamine and [3H]inositol into the polar inositolphosphoglycan moiety (InsPG) and [3H]myristate and [3H]palmitate into the diacylglycerol (DAG) backbone of GPtdIns; (ii) sensitivity of the [3H]labelled GPtdIns to nitrous acid deamination and; (iii) sensitivity of GPtdIns to phosphatidylinositol (PtdIns)-specific phospholipase C (PLC) hydrolysis. In cultured pituitary cells labelled to isotopic steady state with 10 microCi/ml of [3H]glucosamine, treatment with hypothalamic TRH (10(-6) M) induced a rapid and transient hydrolysis (ca. 50%) of the labelled GPtdIns. Moreover, as demonstrated in [3H]inositol labelled cells, treatment with thyrotropin releasing hormone (TRH) elicited the cleavage of [3H]GPtdIns in a similar manner, and this effect was followed by the phosphoinositide (PtdIns, PtdInsP and PtdInsP2) hydrolysis 30 s later. These results suggest that the phosphodiesterase cleavage of GPtdIns could be an early event implicated in TRH action in pituitary lactotrophes.

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Treatment of cultured testicular cells from adult rats with 5 alpha-dihydrotestosterone (DHT; 10(-6) M) or the synthetic androgen methyltrienolone (R1881; 10(-6) M) inhibited Leydig cell 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas no effect of both androgens on cultured cells derived from neonatal animals could be observed. The inhibitory effect of DHT or R1881 on Leydig cell 3 beta-HSD enzyme activity, however, was abolished when adult cells were cultured in the presence of the anti-androgen cyproterone acetate (CPA; 10(-6) M) or the protein synthesis inhibitor cycloheximide (CX; 1 microgram/ml). Testicular cells from adult animals were also cultured in the presence of the different treatments described above, and the spent media was collected and thereafter used as conditioned culture medium (CCM) in subsequent experiments performed with neonatal cells. Dispersed testicular cells from neonatal rats were cultured for 12 days in McCoy's 5a medium of in CCM derived from R1881-treated adult cells, and fresh culture medium or CCM was replaced every 2 days. The human chorionic gonadotropin (hCG)-stimulated testosterone production of neonatal cells was abolished in the presence of CCM derived from R1881-treated adult cells. Nevertheless, the steroidogenic response to hCG recovered when neonatal cells were cultured for two additional days in McCoy's 5a medium. Treatment of neonatal cells with increasing concentrations of hCG (0.1-10 ng/ml) resulted in a dose-dependent augmentation in Leydig cell 3 beta-HSD enzyme activity and testosterone production.(ABSTRACT TRUNCATED AT 250 WORDS)

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Initial biosynthetic radiolabelling experiments with cultured granulosa cells revealed the presence of an oligosaccharide-phosphatidylinositol (glycosyl-phosphatidylinositol; (Ose)nPtdIns) structurally related to (Ose)nPtdIns-lipids isolated from other cell types. Prolactin (PRL) stimulated [3H]glucosamine-(Ose)nPtdIns turnover and the rapid generation of [3H]myristoyl-diacylglycerol in cultured follicle-stimulating hormone-(FSH)-primed granulosa cells endowed with PRL receptors. In parallel experiments performed with [3H]myo-inositol-labelled granulosa cells, treatment with PRL stimulated (Ose)nPtdIns hydrolysis in a similar manner, whereas no effect on phosphoinositide (PtdIns, PtdInsP and PtdInsP2) turnover could be observed. These results strongly suggest that the cleavage of (Ose)nPtdIns by phosphodiesterase followed by the subsequent generation of diacylglycerol and a soluble phosphoinositol-oligosaccharide (inositol-phosphoglycan; (Ose)nInsP) moiety could be part of the signal-transduction mechanism linking PRL receptors to their biological effects in granulosa cells. To test this hypothesis, we examined the effect of PRL and purified (Ose)nInsP moiety (from rat liver membranes) on granulosa cell 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity. Results presented show that, in FSH-primed granulosa cells, PRL (40 nM) and (Ose)nInsP (5 microM) prevented gonadotropin-stimulated 3 beta-HSD activity. Furthermore, in undifferentiated granulosa cells where PRL receptors are absent, no effect of the hormone on 3 beta-HSD activity could be observed, whereas (Ose)nInsP (1-10 microM) inhibited enzyme activity in a dose-dependent manner.

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In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5-96 h) with [3H]galactose, [3H]glucosamine, or [3H]myoinositol and treatment of the purified [3H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([3H]galactose and [3H]myoinositol) or 72 h ([3H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([3H]galactosamine, [3H]mannose, and [3H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5-72 hr) with [14C]linoleate, [3H]myristate, [3H]oleate, [3H]palmitate, or [3H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [3H]palmitate and [3H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA2 treatment of the dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [3H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 microgram/ml), or the membrane permeable cAMP analog (but)2cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)2cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [3H]glucosamine in the presence of (but)2cAMP (1 mM), TPA (10(-7) M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [3H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone.(ABSTRACT TRUNCATED AT 400 WORDS)

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Treatment of cultured granulosa cells with PLC or GnRH stimulated the rapid generation of DAG and phosphoinositide turnover. The PKC activators PLC (3 mU/ml) and TPA (10(-7)M) or the decapeptide GnRH (10(-6)M) elicited similar inhibitory responses on FSH or cAMP stimulated granulosa cell steroidogenesis. Mobilization of intracellular Ca2+ with A23187 (10(-8)M) was followed by a slight increase in the steroidogenic activity of cultured granulosa cells, whereas elevation of extracellular K+ (50 mM) largely augmented the steroid biosynthetic activity of the granulosa cells. These results suggest that the inhibitory effect of GnRH on granulosa cell steroidogenesis is mediated by generation of DAG, rather than by increases in intracellular Ca2+ concentrations.

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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.

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The in-vivo regulatory effect of androgens on steroidogenesis was investigated. Adult (2 to 3 months old) hypophysectomized rats were treated intratesticularly with increasing doses of 5 alpha-dihydrotestosterone (DHT; 10-200 micrograms/100 g body weight) or vehicle (50 microliters dimethyl sulphoxide; DMSO) in the contralateral testis. Intratesticular testosterone concentrations were extremely low in hypophysectomized rats 15-20 days after surgery. Treatment with DHT caused a dose-dependent inhibition of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) 2 h later, and this effect was apparent at the dose of 20 micrograms/100 g body weight (P less than 0.01). The inhibitory effect of 3 beta-HSD was not due to a possible interference of DHT in the enzyme assay, since various concentrations of the androgen (0.1-100 mumol/l) were ineffective as inhibitors of 3 beta-HSD. The highest dose of DHT used in this study (200 micrograms/100 g body weight) resulted in a rapid (1-2 h) and transient (4-6 h) inhibition (approximately 80%) of 3 beta-HSD activity. Pretreatment of rats with the antiandrogen cyproterone acetate (5 mg/rat) or the protein synthesis inhibitor cycloheximide (10 mg/rat) did not affect the enzyme activity of testes injected with DMSO, but counteracted the inhibitory effect of DHT on 3 beta-HSD activity in the contralateral testis. The results presented suggest that the inhibitory effect of the non-aromatizable androgen DHT is receptor-mediated and involves the synthesis of a factor(s) that modulates 3 beta-HSD activity.

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In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.

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The influence of a synthetic androgen R1881 upon hCG stimulated steroidogenesis in cultured rat testicular cells was investigated. Testicular cells were cultured for 8 days in medium alone and thereafter reincubated for 48 h with appropriate treatments before the collection of media for steroid RIA. Addition of R1881 (10(-6) M) resulted in an overall decrease of hCG (0.3-10 ng/ml) stimulated pregnenolone and progesterone production by cultured cells. The conversion of exogenous steroids of the delta 4 pathway (progesterone,17 alpha-OH-P and delta 4-A) was also studied in cultures supplemented with cyanoketone (10(-5) M) and/or spironolactone (10(-5) M) to prevent endogenous testosterone production. R1881 inhibited progesterone and 17 alpha-OH-P conversion to testosterone (T) and was ineffective when delta 4-A served as precursor for T biosynthesis. The inhibitory effect of R1881 upon Testosterone production was prevented by concomitant treatment with CPA. These observations suggest that R1881 decreases the hCG stimulated testosterone production via inhibition of CSCCE,3 beta-HSD,C17-20 Lyase and likely 17 alpha-Hydroxylase, whereas no effect on 17 beta-HSD could be observed.

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Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.

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