Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Abstract

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slightly in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated. (J. Clin. Invest. 1995. 96:2719-2726

Changes in Nitric Oxide Synthase Activity in the Ovary of Gonadotropin Treated Rats. The Role of Nitric Oxide during Ovulation.

“…Therefore, the main isoform increasing during ovulation would seem to be eNOS. These results agree well with previous reports that eNOS increases after hCG injection and is localized to blood vessels in hilum and stroma [1] or thecal layer and mural granulosa cells [2] . It is well known that the LH surge induces ovarian hyperemia and vasodilation [18], which may be mediated by eNOS…”

“…As shown in the present results with iNOS Western blot analysis, Van Voohris et al have shown that iNOS mRNA levels are highest in the unstimulated ovary and decline with follicle growth, and also that iNOS mRNA is localized to the granulosa cells of secondary follicles and small antral follicles [1]. On the other hand, Jablonka-Shariff and Olson have reported that iNOS protein increases after hCG injection and is localized to the thecal layer and stroma [2].…”

“…NADPH-diaphorase positive cells after the fixation and incubation made in this study are equal to NOS positive cells [16] and the time course changes of their localizations after hCG injection coincided with the changes of eNOS localization. bNOS protein could not be detected in the ovary by Western blot analysis or immunohistochemistry (unpublished data; Nakamura et al) as reported for bNOS mRNA expression [1]. Therefore, the main isoform increasing during ovulation would seem to be eNOS.…”

“…NITRIC oxide synthase (NOS) expression and NO generation have been recently reported in ovaries of rats [1,2], guinea pigs [3] and humans [4,5]. NO is known to play several roles in ovarian steroidogenesis [4,6], ovulation [7,8] and follicular apoptosis [5,9].…”

“…NO is produced from arginine by NO synthase (NOS) which has three isoforms, brain NOS (bNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) [10]. Van Voorhis et al have shown that eNOS mRNA increases after human CG (hCG) injection and its protein is localized to blood vessels in hilum and stroma, and that iNOS mRNA declines after hCG injection and its expression is localized mainly in granulosa cells of small follicles but not large follicles [1]. Jablonka-Shariff and Olson have reported that eNOS protein increases after hCGlnjection and is localized to the thecal layer and mural granulosa cells, and that iNOS protein also increases after hCG injection and is localized to the thecal layer and stroma [2].…”