Tumor necrosis factor alpha inhibition of follicle-stimulating hormone-induced granulosa cell estradiol secretion in the human does not involve reduction of cAMP secretion but inhibition at post-cAMP site(s).

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Tumor necrosis factor alpha (TNF) inhibits follicle-stimulating hormone- (FSH)induced estradiol secretion by granulosa cells in several species, including humans. One major inhibitory effect of TNF in rat granulosa cells is at the level of stimulatable adenylyl cyclase, resulting in reduced cAMP concentrations. The purpose of the present study was to investigate whether a reduction in cAMP secretion could account for the inhibitory effects of TNF on FSH-induced estradiol in human granulosa cells. Granulosa cells were taken from ovaries of premenopausal women undergoing oophorectomy for reasons unrelated to ovarian pathology. Women in this study were in various stages of the menstrual cycle or exhibited irregular cycles. Granulosa cells from follicles ranging from 5 to 10 mm diameter were subjected to culture for 48 and 96 h. Granulosa cells were cultured with human FSH (2 ng/mL) and testosterone (1 microM) in the presence and absence of human TNF (20 ng/mL). Media were collected at 48 h, fresh media and hormones added, and cultures continued for an additional 48 h. Accumulation of cAMP, progesterone, and estradiol in media were determined by radioimmunoassay (RIA). FSH induced significant increases in cAMP, progesterone, and estradiol by 96 h of culture. TNF inhibited the secretion of estradiol at 96 h without reducing the accumulation of cAMP and progesterone in media. Similar results were observed in the presence of 0.1 mM isobutylmethylxanthine (D3MX), a phosphodiesterase inhibitor that would prevent metabolism of cAMP to AMP. To determine whether TNF would inhibit the ability of cAMP to induce estradiol and progesterone secretion, granulosa cells were incubated with 0.1 mM cAMP in the presence and absence of TNF. TNF consistently inhibited the ability of cAMP to increase estradiol secretion. These results indicate that a pathway for TNF inhibition of FSH- or cAMP-induced estradiol secretion in human granulosa cells is at post-cAMP sites rather than inhibition of FSH-stimulatable adenylyl cyclase.