Nitric oxide mediates inhibitory effect of leptin on insulin-like growth factor I augmentation of 17beta-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 microM 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 (E2) production in human GCs, respectively. FSH plus IGF-I induced a further increase in E2 production. Leptin did not significantly alter basal or FSH-dependent E2 production, but it inhibited the effect of IGF-I on E2 production and the synergistic effect of IGF-I on FSH-stimulated E2 production. The inhibitory effect of leptin on IGF-I argumentation of E2 production was attenuated by pretreatment of human GCs with 100 microM L-NAME. In conclusion, leptin could induce NO production in human GCs. The inhibitory effect of leptin on IGF-I augmentation of E2 production in human GCs was attenuated by L-NAME, strongly suggesting that NO may mediate the action of leptin in human GCs.
Huang
He-Feng
HF
Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310006, China. huanghefg@hotmail.com
Wang
Bo
B
Yang
Xiao-Fu
XF
Luo
Qiong
Q
Sheng
Jian-Zhong
JZ
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D019793:Fluorescein / Q000032:analysis / Q000378:metabolism; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D020738:Leptin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D054411:Receptors, Leptin
C111205:4,5-diaminofluorescein; D004791:Enzyme Inhibitors; C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D009569:Nitric Oxide; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D019001:Nitric Oxide Synthase; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester