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)

Tumor necrosis factor a (TNF) inhibits follicle-stimulating hormone- (FSH)i nduced estradiol secretion by granulosa cells in several species, including humans. One major inhibitory effect of TNF in rat granulosa cells is at th e level of stimulatable adenylyl cyclase, resulting in reduced cAMP concent rations. The purpose of the present study was to investigate whether a redu ction 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 unr elated to ovarian pathology. Women in this study were in various stages of the menstrual cycle or exhibited irregular cycles, Granulosa cells from fol licles ranging from 5 to 10 mm diameter were subjected to culture for 48 an d 96 h, Granulosa cells were cultured with human FSH (2 ng/ml) and testoste rone (1 mu M) in the presence and absence of human TNF (20 ng/mL). Media we re collected at 48 h, fresh media and hormones added, and cultures continue d for an additional 48 h. Accumulation of cAMP, progesterone, and estradiol in media were determined by radioimmunoassay (RIA). FSH induced significan t increases in cAMP, progesterone, and estradiol by 96 h of culture. TNF in hibited the secretion of estradiol at 96 h without reducing the accumulatio n of cAMP and progesterone in media. Similar results were observed in the p resence of 0.1 mM isobutylmethylxanthine (D3MX), a phosphodiesterase inhibi tor that would prevent metabolism of cAMP to AMP, To determine whether TNF would inhibit the ability of cAMP to induce estradiol and progesterone secr etion, 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 inhibiti on of FSH- or cAMP-induced estradiol secretion in human granulosa cells is at post-cAMP sites rather than inhibition of FSH-stimulatable adenylyl cycl ase.