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M Knecht

Publications and source records attributed to M Knecht.

At least 55 records · Page 3Linked to original sources
PubMed1985

Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Enhanced follicle-stimulating hormone activity of deglycosylated human chorionic gonadotropin in ovarian granulosa cells.

The receptor binding properties and biological actions of chemically deglycosylated asialo human CG (AHF-hCG) were studied in ovarian granulosa cells from diethylstilbestrol (DES)-treated immature rats. In ovarian homogenates from DES- and FSH-treated rats, the relative binding affinity of AHF-hCG was 2-fold higher than that of native hCG and 14-fold higher than that of ovine LH. When assayed for LH-like activity in granulosa cells from DES plus FSH-treated animals, the deglycosylated hormone behaved as a partial agonist in terms of cAMP formation, but fully stimulated progesterone production to the same extent as that elicited by LH. When added with LH to FSH-treated cells, AHF-hCG inhibited LH-stimulated cAMP formation by 70% but did not alter the elevated level of progesterone production. These findings are consistent with the presence of excess or spare LH receptors in the maturing granulosa cell. When added to freshly prepared granulosa cells which have minimal LH receptors, AHF-hCG decreased FSH-stimulated cAMP production by 20% and reduced progesterone production by 50% and increased cGMP formation by 100% during 48 h of culture. The ability of AHF-hCG to decrease the progesterone response to FSH suggests that no spare FSH receptors are present during granulosa cell differentiation. In contrast, native hCG did not alter FSH-induced cAMP or progesterone production but reduced the cGMP responses to FSH and choleragen. Whereas native hCG displayed negligible binding potency when compared with that of ovine FSH in competition with [125I]iodo-human FSH for ovarian receptors, AHF-hCG bound to FSH receptors with about 5% of the binding affinity of ovine FSH. In choleragen-treated granulosa cells, the increases in cAMP and progesterone synthesis were enhanced by addition of both hCG and AHF-hCG, and cGMP production was increased by AHF-hCG but slightly decreased by hCG. These results indicate that the enhanced LH receptor affinity caused by removal of the sugar moieties from hCG is accompanied by a relatively greater increase in FSH receptor affinity, and that deglycosylated hCG acts as a partial agonist with the ability to modify granulosa cell responses to both LH and FSH.

Animals↗
PubMed1985

Estrogen dependence of luteinizing hormone receptor expression in cultured rat granulosa cells. Inhibition of granulosa cell development by the antiestrogens tamoxifen and keoxifene.

In rat ovarian granulosa cells cultured for 48 h, addition of 10(-8) M estradiol (E2) enhanced choleragen-induced cAMP formation and LH receptor content by 2-fold and 6-fold, respectively. Two potent antiestrogens, tamoxifen and keoxifene, inhibited these effects of E2 in a concentration-dependent manner and significantly reduced cAMP production and LH receptors below the levels induced by choleragen. Both antiestrogens (greater than or equal to 1 microM) also reduced the effects of choleragen on cAMP levels and LH receptor content in the absence of exogenous E2. In addition, the antiestrogens (1 microM) inhibited the stimulatory effects of FSH and forskolin on granulosa cell maturation, as well as the enhancement of their actions by exogenous E2. FSH caused a concentration-dependent rise in endogenous E2 accumulation during the 48-h culture period, suggesting that antiestrogens may prevent FSH-stimulated increases in LH receptors by inhibiting the actions of newly formed E2. Tamoxifen prevented the induction of LH receptors by 8-bromo-cAMP, indicating that its effects were on both cAMP production and cAMP action, whereas keoxifene predominantly altered granulosa cell development by its inhibition of estrogen effects on cAMP production. Although both exogenous E2 and the antiestrogens modified cAMP accumulation and LH receptor expression largely during the second 24 h of culture, their actions commenced during the first day. The antiestrogens had no effect alone and did not reduce the DNA content of granulosa cells. Also, they could be washed from the cells after 48 h of culture with complete recovery of forskolin-stimulated cAMP responsiveness by 72-96 h of culture. At a lower concentration (0.4 microM), tamoxifen, but not keoxifene, acted as a partial estrogen agonist since it enhanced choleragen action. These results indicate that the cAMP-mediated induction of LH receptors in cultured granulosa cells is dependent upon the continued actions of estrogen throughout the maturation process.

Animals↗
PubMed1985

Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression.

To determine the role of newly synthesized estrogens in LH receptor expression, granulosa cells from diethylstilbestrol-implanted immature rats were cultured with FSH plus aromatase inhibitors. When present throughout the 48-h culture period, 4-hydroxy-4-androstene-3,17-dione (4-OHA; greater than or equal to 100 microM) and 1,4,6-androstatriene-3,17-dione (greater than or equal to 5 microM) inhibited FSH-induced LH receptor formation by 40% and 90%, respectively. Both aromatase inhibitors caused relatively greater inhibition of LH receptor formation when added from 20-48 h of culture, the period during which FSH-stimulated estrogen synthesis occurs (85% maximal inhibition with 4-OHA and 95% with 1,4,6-androstatriene-3,17-dione). Addition of estradiol, but not androstenedione, reversed the reduction of LH receptor formation by 4-OHA, indicating that the effects of the aromatase inhibitors were specifically related to their blockade of estradiol synthesis. The stimulation of estrogen production by FSH alone (8-fold) or with androstenedione (80-fold) during the 48-h culture period was prevented by 4-OHA. FSH-stimulated cAMP production was initially enhanced by 4-OHA from 0-20 h of culture, but was reduced from 20-48 h. Lower concentrations of 4-OHA (less than or equal to 50 microM) amplified FSH-stimulated cAMP production and LH receptor formation. However, these responses were blocked by the antiestrogen keoxifene or the antiandrogen flutamide, indicating that 4-OHA or a metabolite may have partial estrogenic or androgenic properties. The inhibitory effects of higher concentrations of 4-OHA on LH receptor expression were potentiated by keoxifene or flutamide. These results indicate that estrogen production and action are necessary for LH receptor expression in the granulosa cell.

Androstenedione↗
PubMed1984

Hormonal regulation of cyclic AMP-dependent protein kinase in cultured ovarian granulosa cells. Effects of follicle-stimulating hormone and gonadotropin-releasing hormone.

The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.

Animals↗
PubMed1984

[Insulin and C-peptide in chronic liver diseases during oral glucose tolerance testing].

Insulin and C-peptide in venous blood were determined during oral glucose tolerance testing in 59 non-manifest diabetics with histologically established chronic liver disease (fatty degeneration, chronic aggressive hepatitis, cirrhosis). Glucose tolerance was pathologic in 60-80% of patients. When compared to a control group patients with chronic liver disease showed significantly increased values of blood glucose (after glucose intake), of insulin and of C-peptide (fasting and after glucose intake). The C-peptide/insulin ratio, a measure of hepatic insulin degradation, was significantly decreased after glucose uptake. There were no significant differences of blood sugar, insulin and C-peptide among the various liver diseases. In chronic aggressive hepatitis and in cirrhosis the C-peptide/insulin ratio was partly significantly lower than in fatty degeneration. From the increased C-peptide values increased insulin secretion in chronic liver diseases can be deducted. In addition, the decreased C-peptide/insulin ratios show an impairment of insulin degradation in liver cirrhosis and other chronic hepatic diseases. However, in fatty liver degeneration this is clearly less pronounced than in more serious liver diseases.

C-Peptide↗
PubMed1984

GnRH agonist-induced inhibitory and stimulatory effects during ovarian follicular maturation.

The in vivo regulation of ovarian gonadotropin and prolactin receptors and adenylate cyclase activity by FSH, and the potent GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), was studied in immature hypophysectomized diethylstilbestrol-implanted rats. During FSH treatment over a 48 h period, FSH receptors increased 2-fold with the maximum response during the first 12 h, whereas LH and prolactin receptors increased by 10-fold and 6-fold with the maximum response from 12 to 48 h. Administration of GnRHa at any time during the 48 h period of FSH treatment inhibited the subsequent development of gonadotropin and PRL receptors. In contrast, administration of a single dose of 10 micrograms GnRHa after 48 h of FSH treatment stimulated follicular luteinization and caused increases in basal adenylate cyclase activity, ovarian weight and PRL receptor content, and concomitant decreases in gonadotropin receptors and adenylate cyclase responses. In the immature follicles of animals not primed with FSH, GnRHa caused progressive inhibition of FSH-sensitive adenylate cyclase activity, with a decrease in FSH receptors, but increased both basal and GMP-P(NH)P-stimulated adenylate cyclase activities. These results demonstrate that GnRHa causes marked inhibition of gonadotropin receptor expression in the basal and FSH-stimulated ovary. This decrease in gonadotropin receptors is an important component of the mechanism by which GnRH agonists inhibit ovarian gonadotropin-sensitive adenylate cyclase activity. In addition, these peptides exert stimulatory effects upon ovarian weight and basal adenylate cyclase activity, and cause an increase in PRL receptors and luteinization of mature ovarian follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Inhibitory actions of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

To determine the effects of adenosine on follicle-stimulating hormone (FSH)-induced differentiation, granulosa cells isolated from the ovaries of diethylstilbestrol-treated immature rats were cultured with increasing concentrations of the nucleoside and modulators of adenosine action. Although adenosine had no effect on basal granulosa cell function during 48 h of culture, concentrations of the nucleoside from 10 microM to 1 mM progressively inhibited FSH-induced responses, including progesterone production and expression of FSH and luteinizing hormone (LH) receptors. Adenosine had biphasic effects on FSH-stimulated cAMP accumulation, causing inhibition of cAMP production at 10 to 100 microM and stimulation at higher concentrations. The enhancement of cAMP production by 1 mM adenosine occurred during the first 24 h of culture, while both 100 microM and 1 mM adenosine reduced FSH-stimulated cAMP production from 24 to 48 h. The inhibitory effects of adenosine were prevented by adenosine deaminase and dipyridamole, an inhibitor of adenosine transport, and were antagonized by 1-methyl-3-isobutylxanthine. The inhibition of cAMP and progesterone production by adenosine was partially overcome when cells were washed and reincubated with forskolin, but not with FSH. Adenine, guanosine, and inosine at concentrations of 100 microM did not modify FSH-induced cAMP formation or LH receptor induction. These results indicate that adenosine exerts predominantly inhibitory actions on hormone-induced granulosa cell differentiation, as manifested by prominent reductions in steroidogenesis and gonadotropin receptor expression.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Induction of granulosa cell differentiation by forskolin: stimulation of adenosine 3',5'-monophosphate production, progesterone synthesis, and luteinizing hormone receptor expression.

The effects of forskolin on the acquisition of differentiated functions in cultured ovarian granulosa cells were compared with the actions of FSH and prostaglandin E2 (PGE2). In 48-h granulosa cell cultures from immature diethylstilbestrol-treated rats, 100 microM forskolin caused a 45-fold increase in cAMP accumulation and stimulated progesterone production from undetectable levels (less than 0.2 ng/ml) to 80 ng/ml. The forskolin-induced increase in cAMP was similar to the maximum response to FSH, and progesterone production was about 50% of that elicited by FSH. PGE2 also enhanced cAMP and progesterone production in a concentration-dependent manner, with a maximum 8-fold increase in cAMP accumulation and an increase in progesterone to 5.6 ng/ml when the PGE2 concentration was 10 micrograms/ml. The time course of forskolin-stimulated cAMP production was notable for its rapid rise to the maximum level during the first 24 h of culture, followed by a plateau for up to 72 h. This contrasted with FSH-stimulated cAMP production, which increased progressively for up to 72 h when measured at 24-h intervals. LH receptor levels were low in untreated cells and after exposure to the various stimuli for 24 h, but increased 9- to 11-fold after culture with FSH or forskolin for 48-72 h. PGE2-induced LH receptor formation was about 20% of that seen after FSH stimulation. Forskolin enhanced cAMP and progesterone production in response to FSH and choleragen, but impaired the effects of these ligands on LH receptor formation. Exposure of the cultured cells to a potent GnRH agonist inhibited forskolin-induced progesterone and LH receptor synthesis, but did not influence forskolin-stimulated cAMP production. These results demonstrate the ability of forskolin to serve as a nonhormonal stimulator of granulosa cell differentiation and indicate the importance of cAMP in this process, as well as the ability of GnRH agonists to exert inhibitory effects on post-cAMP steps in cellular maturation.

Animals↗
PubMed1984

Estrogens enhance the adenosine 3',5'-monophosphate-mediated induction of follicle-stimulating hormone and luteinizing hormone receptors in rat granulosa cells.

The effects of estrogens on cAMP-induced FSH and LH receptor expression were studied in granulosa cells isolated from immature diethylstilbestrol-implanted rats. Although estradiol alone had negligible effects on granulosa cell maturation, estradiol concentrations from 10(-11)-10(-8) M progressively enhanced cAMP production and gonadotropin receptor formation in choleragen-stimulated cells. During 48 h of culture, estradiol augmented cAMP levels by 2-fold, LH receptors by 4- to 6-fold, and FSH receptors by 20-40%. Estradiol also enhanced the extent of LH and FSH receptor formation by other cAMP-inducing ligands, including FSH, prostaglandin E2, and forskolin. The stimulatory action of 8-bromo-cAMP on gonadotropin receptors was also increased by estradiol, indicating that part of the estrogenic effect was exerted on cAMP-activated processes. Scatchard analyses indicated that estradiol increased the number of choleragen-induced FSH receptors from 2,600 to 3,200/cell and of LH receptors from 13,000 to 86,000/cell with no changes in receptor binding affinity. Choleragen-stimulated cAMP accumulation was enhanced by estradiol during the later stages of culture (after 30 h), while increased LH receptors were detected by 30 h and FSH receptors by 43 h. The stimulatory effects of estradiol were not due to increased cellular proliferation and were also exerted by other estrogens, including estrone and diethylstilbestrol. Androgens, including testosterone and androstenedione, also amplified choleragen action. This effect was largely through conversion to estrogens, since dihydrotestosterone, a nonaromatizable androgen, did not markedly enhance LH receptor formation by choleragen. In contrast, progestins and pregnenelone had no facilitative effect on choleragen-induced responses. Although cortisol and dexamethasone increased choleragen-induced cAMP accumulation, only cortisol elevated LH receptors, and dexamethasone inhibited FSH receptor formation. These results demonstrate that estrogens enhance both ligand-induced cAMP production and cAMP-activated responses during granulosa cell differentiation. In particular, estrogens exert a major effect on the levels of gonadotropin receptors expressed in response to FSH and other cAMP-inducing ligands.

Androgens↗
PubMed1984

Dependence of cyclic nucleotide production and luteinizing hormone receptor formation upon ribonucleic acid synthesis in cultured rat granulosa cells.

The role of newly synthesized RNA in the differentiation of granulosa cells isolated from diethylstilbestrol-treated immature rats was studied during culture with actinomycin D. Choleragen-induced LH receptor formation and cGMP production at 48 h were completely inhibited by actinomycin D (greater than or equal to 100 ng/ml) added as late as 20 h after the initiation of culture and were partially reduced by addition of the antibiotic from 30-48 h. In contrast, addition of actinomycin D to freshly prepared cells enhanced choleragen-stimulated cAMP accumulation during the 48-h culture period. This effect was caused by both elevation of adenylate cyclase activity at 3 and 6 h of culture and inhibition of choleragen-induced phosphodiesterase activity during culture. The increase in cAMP production by actinomycin D was confined to the first few hours of culture, since the antibiotic did not enhance cAMP levels when added after 3 h and significantly reduced cAMP accumulation when added from 20-48 h of culture. Actinomycin D inhibited choleragen-stimulated incorporation of [3H]uridine into RNA of freshly prepared cells by 65% and reduced both RNA synthesis and incorporation of [3H]leucine into protein at 20 and 48 h of culture by approximately 90%. In untreated cells, RNA and protein synthesis and phosphodiesterase activity increased to a larger extent from 20-48 h than after choleragen treatment, but did not lead to elevated cAMP levels or LH receptors. These results suggest that the cAMP-induced syntheses of RNA and protein that are specific for increases in cGMP production and LH receptor formation occur predominantly during the second day of granulosa cell culture. In contrast, cAMP production can be markedly altered by changes in RNA and protein syntheses during the first hours of culture.

Adenosine Triphosphate↗
PubMed1983

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

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 inhibiton 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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

Inhibitory actions of a gonadotropin-releasing hormone agonist on ovarian follicle-stimulating hormone receptors and adenylate cyclase in vivo.

The regulation of ovarian gonadotropin-sensitive adenylate cyclase and FSH receptors was studied in hypophysectomized diethylstilbestrol-primed rats treated with FSH and/or the potent GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). The animals were treated with 7.5 micrograms ovine FSH twice daily for 2 days, either alone or with 10 micrograms GnRHa. FSH-stimulated adenylate cyclase activity was augmented by 2.5- to 3.5-fold in the presence of 5'-guanyl-imidodiphosphate. Adenylate cyclase responses to FSH were almost completely abolished by GnRHa treatment in ovarian homogenates from control animals and rats treated with FSH. This inhibition was receptor specific, since GnRHa did not block adenylate cyclase stimulation by prostaglandin E2 or isoproterenol. No inhibition of 5'-guanyl-imidodiphosphate- or sodium fluoride-stimulated adenylate cyclase activity was noted after any hormone treatment. When GnRHa treatment was initiated at 12, 24, or 36 h during the 2-day period of FSH treatment, inhibition of both FSH- and LH-stimulated adenylate cyclase was observed in ovaries collected at 48 h. Whereas FSH treatment increased the ovarian FSH receptor concentration by more than 100%, concomitant treatment with GnRHa prevented this increase and reduced FSH receptors to 60% of the control level. Treatment with GnRHa alone caused a 65% decrease in FSH receptor levels below the untreated control values. Histological analysis of hormone-treated ovaries indicated that FSH stimulated follicle growth and antrum formation, but caused little luteinization. GnRHa did not completely prevent the effects of FSH on follicle growth, but did induce degeneration and premature cleavage of ova. GnRHa alone suppressed the diethylstilbestrol-stimulated mitotic activity, slightly increased degenerative changes in granulosa cells, and caused oocyte cleavage and premature antrum formation. These findings demonstrate that GnRHa inhibits FSH-dependent adenylate cyclase by a mechanism involving the loss of binding sites for FSH. It is also evident that only short term exposure to GnRHa is necessary for expression of the inhibitory action of the peptide upon FSH- and LH-stimulated adenylate cyclase.

Adenylyl Cyclases↗
PubMed1983

Calcium dependence of the inhibitory effect of gonadotropin-releasing hormone on luteinizing hormone-induced cyclic AMP production in rat granulosa cells.

The role of calcium in the inhibitory action of the GnRH superagonist, [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), on LH-stimulated cAMP production was studied in cultured granulosa cells obtained from immature hypophysectomized diethylstilbestrol (DES)-implanted rats. After culture for 48 h with FSH to induce LH receptors, cells were incubated for 2 h with LH or LH plus GnRHa. In this system, the cAMP response to LH was independent of extracellular calcium. In the presence of 0.25 to 1 mM extracellular calcium, 10(-8) M GnRHa caused a 15 to 40% inhibition of LH-stimulated cAMP production. Omission of extracellular calcium completely abolished the inhibitory effect of GnRHa upon LH-induced cAMP production. The inhibitory effects of GnRHa on prostaglandin E2 (PGE2) and isoproterenol-induced cAMP productions were also markedly reduced in the absence of extracellular calcium. Addition of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), reversed the inhibitory action of GnRHa on LH-induced cAMP production. These results demonstrate that extracellular calcium is necessary for the acute inhibitory action of GnRHa upon LH-induced cAMP production in cultured rat granulosa cells, and indicate that increased degradation of cAMP may be a contributing factor for this effect of GnRHa.

1-Methyl-3-isobutylxanthine↗