Changes in nitric oxide (NO) synthase isoforms and NO in the ovary of Heteropneustes fossilis (Bloch.) during the reproductive cycle

Abstract

The purpose of the study was to demonstrate the presence of nitric oxide (NO) synthase (NOS) isoforms (neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS)) and the role of NO in the ovary of Heteropneustes fossilis. In one half of the ovary collected during different reproductive stages, NOS isoforms were localized immunohistochemically in paraffin sections whereas the other half was processed for NOS and NO quantification using western blot followed by densitometry and nitrate/nitrite assay respectively. The role of NO on oocyte maturation was studied by examining the effect of NO donor (sodium nitroprusside; SNP) and NOS inhibitor (Nomega-nitro-l-arginine methyl ester) on 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P)-induced germinal vesicle breakdown (GVBD) in the cultured oocyte collected during prespawning phase. NOS immunostaining was predominantly localized in previtellogenic follicles, with nNOS detected in the nucleus and cytoplasm of oocytes whereas iNOS and eNOS localized in granulosa, theca cells, and cytoplasm of oocytes. The NOS expression was higher in previtellogenic phase when compared with vitellogenic phase. The nitrate/nitrite concentrations in ovary showed gradual increase from recrudescence (4.9+/-0.19 nM/mg protein) to late previtellogenic phase (7.02+/-0.53 nM/mg protein), but showed a sharp decline during the vitellogenic phase (0.41+/-0.053 nM/mg protein). Serum and ovarian nitrate/nitrite level showed a close association during the reproductive cycle. The results showed an increase in NOS activity and nitrate/nitrite concentrations as the follicle grow suggesting involvement of NO in follicular development. SNP significantly inhibited 17alpha,20beta-P-induced GVBD in fish oocytes. Thus, it is concluded that the fish ovary possesses NOS/NO system and a possibility that NO has a role in follicular development and regulation of oocyte maturation in fish, H. fossilis.