Roles And Regulatory Mechanisms Of The No/Cgmp Pathway In The Follicular Formation And Development Of Neonatal And Immature Rats

The primordial follicle formation and development in the rat ovary initiates after birth. Originally from egg nests, most follicles go atresia during postnatal days at different developmental stages, the other follicles grow into antral follicles. There are two important phenomenons attract attentions of scientists:1. How the primordial follicle formation from egg nests and grows as antral follicle?2. What impact factors and regulation mechanisms of follicular atresia are contained in the ovary before puberty? The NO/cGMP pathway regulates significantly the biologic and physiologic processes of the reproductive system and is an important modulator of folliculogenesis, atresia, steroidogenesis, prostaglandin biosynthesis, ovulation, luteolysis, oocyte growth, and maturation. However, there is no report about biological functions and regulatory mechanisms of the NO/cGMP pathway during the follicular formation and development in the neonatal and immature rat. Therefore, my present study uses the histological observation, immunohistochemistry (IHC), RT-PCR, western blot to provide related data. The mainly findings and conclusions are as follows.1. During postnatal days1,5,7,10, and19, all three isoforms of NOS were mainly localized to the oocytes and expressed as a gradual increase in granulosa cells and theca cells within the growing follicle. The ovarian total NOS activities and NO levels were increased at postnatal days7and10compared with other days. Our findings suggest that the locally produced NO and the NO/cGMP signaling systems are involved in the follicular development before puberty.2. RT-PCR analysis is to detected the expression and contents of PDEs during the follicle development in the female Sprague-Dawley rats at postnatal days1,5,7,10, and19. The results finds that PDE1A, PDE3A, PDE3B, PDE5A, PDE9A expressed among11kind of cGMP related PDEs. Furthermore, semiquantitative analysis shown that PDE3A and PDE5A are mainly PDEs at different postnatal days and their expression at PND1are lower compared to the other days. And then the expressions are stabilizing. Therefore, our data suggest that PDE3A and PDE5A are involved in the follicular development before puberty by hydrolaseintra-cellular cGMP. 3. We establish the model of inhibition and activation NOS on neonatal and immature rats. The follicular development dynimics have shown that the percentage of antral follicle is markly decreased in the L-NAME+SMT treated group (p<0.05), but increased significantly in the L-Arg group (p<0.01). Moreover, the detection of total NOS, iNOS, eNOS, nNOS have shown that their activities are markly decreased (p<0.05) in the L-NAME+SMT treated group, but increased significantly (p<0.01) in the L-Arg group. However, there are no statistics difference between the single treated with L-NAME or SMT groups and control group. Therefore, our findings suggest that the NOS involved in the follicular development before puberty result and enhance the follicle development.4. Immunohistochemistry (IHC) and Western Blotting (WB) analysis are used to detect the PI3K/AKT/Foxo3a pathway in the neonatal and immature female rat ovary treated with different NOS inhibition and acitivtion. The IHC shown that PTEN, AKT and FoxO3a mainly localized in the oocytes at postnatal days5,10, and19, and expressed as a gradual increase in granulosa cells at postnatal day19. Furthermore, WB analysis of the PI3K/AKT/Foxo3a pathway in the neonatal and immature female rat ovary treated with different NOS inhibition and acitivtion have been used. The expresstion of PTEN is increased and phosphorylatcd AKT and FoxO3a decreased in the L-NAME+SMT treated group at5,10and19days. However, the expresstion of PTEN is decreased and phosphorylated AKT and FoxO3a increased in the L-Arg treated group. But, there are no statistics differences between the single treated with L-NAME or SMT groups and control group. Therefore, our results suggest that NOS involved in the follicular development of neonatal and immature rats through PI3K/AKT/Foxo3a pathway.5. Immunohistochemistry (IHC) and WB analysis are to detected wheather NOS involved in the follicular development of neonatal and immature rats by autophagy and apoptosis. Our results have shown that Caspase-3(marker of cell apoptosis) and LC3(marker of cell autophagy) are common localized at granulosa cells, oocytes and theca cells. Furthermore, WB analysis of the LC3-Ⅱ and Cleaved-Caspase-3changing in the neonatal and immature female rat ovary treated with different NOS inhibition and acitivtion. The expresstion of Cleaved-Caspase-3of is increased and LC3-Ⅱ decreased in the L-NAME+SMT treated group at5,10and19days. However, the expression of LC3-Ⅱ is decreased and Cleaved-Caspase-3increased in the L-Arg treated group. But, there are no statistics changing between the single treated with L-NAME or SMT groups and control group. Therefore, our results suggest that NOS involved in the follicular development of neonatal and immature rats by autophagy and apoptosis.