Expression of nitric oxide synthase and effect of substrate manipulation of the nitric oxide pathway in mouse ovarian follicles

BACKGROUND: Nitric oxide (NO) is a cell messenger with multiple actions in different biological systems, implicated in the control of follicle and oocyte function. NO is formed from L-arginine by isoforms of nitric oxide synthase (NOS) via NG-hydroxy-L-arginine, with L-citrulline as a byproduct. This study aimed to show how modulation of NO by manipulating NOS substrates would affect mouse follicle growth and ovulation in vitro, where vascular effects of NO are attenuated. METHODS: Immunohistochemistry [endothelial (eNOS) and inducible (iNOS)] and in situ hybridization (iNOS) were applied on mouse ovaries. Cultured follicles were also stained for iNOS by immunohistochemistry. For follicles cultured in the presence or absence of L-arginine, the ability of L-citrulline or NG-hydroxy-L-arginine to substitute for L-arginine was assessed in terms of follicle growth and ovulation. RESULTS: iNOS and eNOS were localized in oocytes and theca, with some staining in granulosa. iNOS mRNA occurred predominantly in granulosa and oocyte. Omission of L-arginine significantly reduced follicle survival and ovulation. Partial compensation for L-arginine withdrawal was achieved with L-citrulline and NG-hydroxy-L- arginine. Specific abnormalities of follicle growth were noted. CONCLUSIONS: NOS is present in mouse follicles, and its action is necessary at a local level for normal follicle development in vitro. Reduced growth, persistent basement membranes and reduced ovulation were associated with in vitro disruption of NO.