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

原位杂交和免疫组化结果显示，诱导型一氧化氮合酶（iNOS）主要存在于大鼠卵巢健康未成熟卵泡的颗粒细胞中，而健康成熟卵泡或闭锁卵泡的颗粒细胞则缺乏iNOS。这些发现使我们认为iNOS的存在对未成熟卵泡保持休眠至关重要。为了验证这一假设，我们检测了促凋亡物质GnRH激动剂（buserelin）和表皮生长因子（EGF）对大鼠颗粒细胞中iNOS mRNA数量的影响，EGF是一种促有丝分裂和抗凋亡因子。Northern blot检测发现，未成熟雌性大鼠给予buserelin可瞬间降低卵巢内iNOS mRNA水平。RT-PCR结果显示，在培养的大鼠颗粒细胞中，buserelin和EGF分别增加了细胞凋亡和DNA合成的发生率，同时降低了iNOS mRNA的水平。同时加入NO供体S-Nitroso-N-acetyl-DL-penicillamine （SNAP）与buserelin或EGF可以消除这些物质的作用，即诱导细胞凋亡和刺激DNA合成。这些结果表明，未成熟卵泡发育或闭锁状态的变化与颗粒细胞中iNOS水平的降低有关，从而加强了NO在卵泡中作为细胞抑制因子的作用。

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.