The effects of FSH and a GnRH agonist on adenylate cyclase activity and FSH receptor content were studied in granulosa cells cultured from the ovaries of diethylstilbestrol-treated hypophysectomized immature rats. In freshly prepared cells incubated with [[3[H]adenine to label intracellular ATP, FSH stimulated a 25-fold increase in the conversion of [[3[H] ATP to [[3[H]cAMP. FSH-sensitive adenylate cyclase activity then decreased by approximately 50% after 8 and 16 h of culture, but returned to the levels seen in freshly prepared cells by 48 h of culture. The GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa) partially inhibited FSH-stimulated cAMP production during the first hour of culture, but addition of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine prevented inhibition of cAMP synthesis, suggesting that GnRHa initially acts by stimulating phosphodiesterase activity. In contrast, GnRHa progressively impaired FSH-stimulate adenylate cyclase activity from 16–48 h of culture in the absence or presence of 3-isobutyl-l-methylxanthine with no detectable cAMP synthesis observed by 48 h of culture. Also, GnRHa inhibited cAMP synthesis after pretreatment of cells with FSH alone for 16, 24, or 48 h of culture, indicating that the presence of GnRHa in the initial hours of culture was not required for the subsequent expression of GnRHa action. Uptake of radiolabeled adenine into cells and formation of [3[H]ATP was not inhibited by GnRHa. In the presence of FSH, specific binding sites for FSH decreased at 8 h of culture, concomitant with the loss in adenylate cyclase activity at that time, to less than 15% of the receptor levels in freshly prepared cells. However, FSH receptors then rose to 25% of the initial value at 24 and 48 h simultaneously with the secondary rise in adenylate cyclase activity. The increase in available FSH-binding sites at 24 and 48 h of culture was inhibited by GnRHa. These results indicate that adenylate cyclase responsiveness to FSH varies during granulosa cell culture, and that the progressive inhibition of FSH-stimulated cAMP synthesis by GnRHa is partially due to a decline in FSH receptors.
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