Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development

Our own recent studies have demonstrated that inducible nitric oxide syntha se (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary, whereas granulosa cells of either healthy matu re follicles or follicles destined to be atretic are devoid of iNOS. These findings suggest that iNOS is pivotal for immature follicles to remain dorm ant. To test this hypothesis, we examined the effects of a GnRH agonist (bu serelin), a proapoptotic substance, and epidermal growth factor (EGF), a mi togenic and, consequently, antiapoptotic factor, on the amount of iNOS mRNA in rat granulosa cells. Administration of buserelin in immature female rat s transiently diminished iNOS mRNA levels in the ovaries as determined by N orthern blot analysis. In cultured rat granulosa cells, buserelin and EGF i ncreased the incidence of apoptosis and DNA synthesis, respectively, wherea s both reduced iNOS mRNA levels as determined by reverse transcription-coup led polymerase chain reaction. The concomitant addition of S-nitroso-N-acet yl-DL-penicillamine, 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 th e changes in developmental status of immature follicles either into develop ment or atresia are associated with reduced iNOS levels in granulosa cells, thus reinforcing the notion of NO as a cytostatic factor in ovarian follic les.