Investigation on a Possible Involvement of Nitric Oxide in the Follicular Development

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Both in situ hybridization and immunohistochemistry revealed that inducible nitric oxide synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary, whereas granulosa cells of either healthy mature follicles or follicles destined to be atretic are devoid of iNOS.These findings lead us to suggest that the presence of iNOS is pivotal for immature follicles to remain dormant. To test this hypothesis, we examined the effects of a GnRH agonist (buserelin), a proapoptotic substance, and epidermal growth factor (EGF), a mitogenic and consequently antiapoptotic factor, on the amount of iNOS mRNA in rat granulosa cells. The administration of buserelin in immature female rats transiently diminished iNOS mRNA levels in the ovaries as determined by Northern blot analysis. In cultured rat granulosa cells, buserelin and EGF increased the incidence of apoptosis and DNA synthesis, respectively, while both reduced iNOS mRNA levels as determined by RT-PCR.The concomitant addition of S-Nitroso-N-acetyl-DL-penicillamine (SNAP), an NO donor, together with buserelin or EGF eliminated the observed effects of these substances, i.e. induction of apoptosis and stimulation of DNA synthesis, respectively. These results suggest that the changes of developmental status of immature follicles either into development or atresia are associated with a reduction in iNOS levels in granulosa cells, thus reinforcing the notion of the role of NO as a cytostatic factor in ovarian follicles.