牛原始卵泡体外培养和发育机理的研究

By using immunohistochemical staining, western blot and ndirect.immunoflorescence, the expression and phosphorylation of mitogen-activated protein kinases(MAPK) and the expression of p90rsk, one of the substrate of MAPK, in immature and mature rat follicle growth, the expression and phosphorylation of MAPK cascade in rabbit oocyte.maturation in vitro, the interaction of MAPK with microtubule organization and chromatin in rabbit oocyte during meiosis, and the cross-talking between PKC, cAMP and MAPK in oocyte maturation were studied. The results above showed that.(1) MAPK was expressed in both granulosa cells and oocytes at all stages of developmental follicles, whereas phosphorylated MAPK was detected only in some granulosa cells, which were in proliferation. Meanwhile, p90rsk was exclusively expressed in oocytes, and could not be detected in granulosa cells, it maybe represented that the differences of the action pattern of MAPK cascade in oocyte and granulosa cell. The phosphorylation of MAPK was also detected in oogonia from fetal rat ovaries, suggesting that MAPK play a role in the.mitosis of oogonia. (2) MAPK activation was coincidence with GVBD in rabbit oocyte, but is not necessary for oocyte to resumption of meiosis. OA can induce oocyte to complete GVBD without MAPK activation; this showed activation of MAPK is not a requisite for the resumption of oocyte meiosis in rabbit and was possibly.activated by ways other than MAPK. (3) One substrate of MAPK was activated later than MAPK phosphorylation, and the results from indirect immunofluoresence show that p90rsk may play a role in microtubule reorganization and spi ndle assembly. But there exist some other way to active p90rsk which may independent with MAPK phosphorylation. (4) Increased cAMP concentration and inhibition of PKC in oocytes entrains a cascade of protein phosphorylation and dephosphorylation reactions and finally inhibited GVBD, while protein phosphatases act as an intermediate molecular.

建立牛原始卵泡体外培养体系，观察原始卵泡体外激活和生长的全过程，探讨颗粒细胞和卵母细胞被激活的时序性和相互作用。利用原位反转录聚合酶链式反应技术(in situ RT-PCR)，以增殖细胞核抗原（PCNA）基因转录的mRNA为卵子发育启动的标志物，研究不同激素和生长因子对于卵泡发育的影响，探讨原始卵泡发育启动的机理和相关物质的作用机制。