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

原位杂交和免疫组化结果表明，诱导型一氧化氮合酶（iNOS）主要定位于颗粒细胞的健康的未成熟卵泡在大鼠卵巢，而颗粒细胞的健康成熟卵泡或卵泡注定要闭锁是缺乏的iNOS。这些研究结果使我们建议，iNOS的存在是关键的未成熟卵泡保持休眠。为了验证这一假设，我们研究了促凋亡物质GnRH激动剂（布舍瑞林）和促有丝分裂和抗凋亡因子表皮生长因子（EGF）对大鼠颗粒细胞iNOS mRNA量的影响。通过北方印迹分析测定，未成熟雌性大鼠中布舍瑞林给药可一过性降低卵巢中的iNOS mRNA水平。在培养的大鼠颗粒细胞中，布舍瑞林和EGF分别增加了细胞凋亡和DNA合成的发生率，同时通过RT-PCR测定两者均降低了iNOS mRNA水平。同时加入S-亚硝基-N-乙酰基-DL-青霉胺（SNAP）（一种NO供体）与布舍瑞林或EGF一起消除了观察到的这些物质的作用，即分别诱导细胞凋亡和刺激DNA合成。这些结果表明，未成熟卵泡的发育状态的变化，无论是发展或闭锁与颗粒细胞中的iNOS水平的降低，从而加强了概念的作用，NO作为一种细胞生长抑制因子在卵巢卵泡。

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.