Inhibitory effects of nitric oxide on steroidogenesis by buffalo granulosa cells cultured in vitro

Abstract

This study examined the effects of S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO) donor and Nw-nitro-L-arginine
methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor on estradiol-17β and
progesterone production by buffalo granulosa cells. Granulosa cells (3×105) from small (<5 mm diameter) or large (> 9
mm diameter) follicles were cultured for 24 h under completely serum-free conditions in DMEM: nutrient mixture F-12
Ham (1:1 ratio) supplemented with 10–7 M androstenedione, 5 mg/ml human apo-transferrin, 0.1% BSA, in the presence
or absence of FSH (8 ng/ml). Granulosa cells from large follicles produced higher estradiol-17β and progesterone than
those from small follicles. SNAP reduced estradiol-17β and progesterone production at concentrations of 0.1 and 1 mM
when used alone and in the presence of FSH by granulosa cells from follicles of both size categories. L-NAME (0.2, 1
and 5 mM) had, however, no effect on estradiol-17β or progesterone production, alone or in the presence of FSH. Our
results demonstrated strong inhibitory effects of NO on estradiol-17β and progesterone production by buffalo granulosa
cells from small and large follicles and indicated that it may be an autocrine/paracrine regulator of steroidogenesis by
granulosa cells in buffalo.