Nitric oxide mediates inhibitory effect of leptin on insulin-like growth factor I augmentation of 17β-estradiol production in human granulosa cells

In the present study the authors investigated if the inhibitory effect of leptin in the ovary was mediated via nitric oxide (NO) using human granulosa cells (GCs). Human GCs were obtained from preovulatory follicles of women who underwent IVF. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that human GCs expressed mRNA of leptin and mRNA of isoforms of leptin receptor, including one long form and two types of short forms. Exposure of human GCs to leptin at concentrations of 3-30 ng/ml for 60 min dose-dependently increased the fluorescence of 4,5-diaminofluorescein (DAF-2), an NO-sensitive dye. The effect of leptin on DAF-2 fluorescence was inhibited by pretreatment of human GCs with 100 muM nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS), indicating that the increase in DAF-2 fluorescence properly reflected the intracellular NO production. FSH (1 ng/ml) and IGF-I (30 ng/ml) stimulated 17beta-estradiol (E) production in human GCs, respectively. FSH plus IGF-I induced a further increase in E, production. Leptin did not significantly alter basal or FSH-dependent E-2 production, but it inhibited the effect of IGF-I on E, production and the synergistic effect of IGF-I on FSH-stimulated E-2 production. The inhibitory effect of leptin on IGF-I argumentation of E-2 production was attenuated by pre-treatment of human GCs with 100 muM L-NAME. In conclusion, leptin could induce NO production in human GCs. The inhibitory effect of leptin on IGF-I augmentation of E-2 production in human GCs was attenuated by L-NAME, strongly suggesting that NO may mediate the action of leptin in human GCs.