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Biological studies on expression and function of Ig related molecules and zona pellucida in mammalian gametes

哺乳动物配子中Ig相关分子及透明带表达及功能的生物学研究

基本信息

批准号：
07457381
负责人：
MORI Tsuneatsu
金额：
$ 3.97万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

We have found that MHC classII antigen and CD4/p561ck complex were expressed on sperm posterior membrane and egg plasma membrane respectively. Furthermore, we generated the CD4 proteins on Sf9 cells transfected with AcNPV-CD4 cDNA.Then, the intercellular interactions between Sf9-CD4 cells and sperm cells demonstrated the adhesive reactions in the species specific manner.On the other hand, we have demonstrated the expression of Fas-Fas ligand (FasL) system and revealed its roles for the formation of atresia in murine ovary. By means of in situ hybridization and RT-PCR,the expression of Fas was mainly detected at transcriptional levels on oocytes, hyperovulated eggs and some granulosa cells, whereas, that of FasL was localized on granulosa cells. These results were further confirmed at translational levels by IIF,EIA and immunoprecipitation method. The expression of Fas-FasL system in ovary was regulated by gonadotropins. We also constructed the transfectants of AcNPV-FasL cDNA to Sf9 (Sf9-FasL) cells. The interaction between granulosa/Sf9-FasL cells and ZP free eggs in vitro resulted in the induction of apoptosis in eggs detecting by TUNEL method. In contrast, the apoptosis in ZP intact eggs in this system did not occur. Taken together with our experimental results and other investigator's reports, the formation of ovarian atresia was strongly suggested to be induced by the interaction between FasL positive granulosa cells and Fas expressing oocytes/eggs during oogenesis until the accomplishment of ZP.

结果表明，精子后膜和卵细胞质膜上分别表达MHC-II类抗原和CD 4/p561 ck复合物。在此基础上，我们利用AcNPV-CD 4 cDNA转染Sf 9细胞，在细胞内产生了CD 4蛋白，并通过细胞间相互作用研究了Sf 9-CD 4细胞与精子细胞间的粘附反应，同时也研究了Fas-Fas配体（FasL）系统在小鼠卵巢闭锁形成中的作用。原位杂交和RT-PCR检测结果表明，Fas主要在转录水平表达于卵母细胞、超排卵卵和部分颗粒细胞，而FasL则定位于颗粒细胞。这些结果进一步证实了在翻译水平上的IIF，EIA和免疫沉淀法。卵巢中Fas FasL系统的表达受促性腺激素的调节。构建了AcNPV-FasL cDNA转染Sf 9细胞（Sf 9-FasL）。TUNEL法检测颗粒细胞/Sf 9-FasL细胞与去ZP卵细胞的相互作用可诱导卵细胞凋亡。相反，在该系统中，ZP完整卵中未发生凋亡。结合我们的实验结果和其他研究者的报告，卵巢闭锁的形成强烈建议在卵子发生过程中，直到ZP完成之间的FasL阳性颗粒细胞和Fas表达的卵母细胞/卵子之间的相互作用诱导。