Herbimycin, a tyrosine kinase inhibitor with Src selectivity, reduces progesterone and estradiol secretion by human granulosa cells

The purpose of the present study was to investigate whether the tyrosine kinase inhibitor herbimycin with some selectivity to block Src would alter the stimulatory effects of follicle-stimulating hormone (FSH) and cyclic adenosine monophosphate (cAMP) on estradiol secretion by human granulosa cells. Granulosa cells were taken from ovaries of premenopausal women undergoing oophorectomy for reasons unrelated to ovarian pathology. Granulosa cells from follicles ranging from 5–20 mm in diameter were subjected to culture. Granulosa cells were cultured with human FSH (2 ng/mL) or cAMP (0–1 mM) and testosterone (1 µM) in the presence and absence of herbimycin (0–2 µM). Media were collected at 24, 48, and 72 h. Accumulation of cAMP, progesterone, and estradiol in the media was determined by radioimmunoassay. Herbimycin dose dependently inhibited the ability of FSH to induce increases in progesterone and estradiol secretion. Although herbimycin increased (p < 0.0001) the accumulation of cAMP in response to FSH, this was evident only at the high concentrations of herbimycin (2 µM). To determine whether herbimycin would inhibit the ability of exogenous cAMP to induce estradiol and progesterone secretion, granulosa cells were incubated with 0–1 mM cAMP in the presence and absence of various doses of herbimycin. Herbimycin inhibited cAMP-induced estradiol and progesterone secretion in granulosa cells. The results from seven experiments indicate that herbimycin inhibits FSH stimulation of estradiol and progesterone secretion and that this inhibition may be, in part, at post-cAMP site(s).