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

我们发现MHC-II类抗原和CD4/p561ck复合体分别表达在精子后膜和卵细胞质膜上。此外，我们在转AcNPV-CD4cDNA的Sf9细胞上产生了CD4蛋白，然后Sf9-CD4细胞与精子细胞之间的相互作用显示了物种特异性的黏附反应。另一方面，我们证明了Fas-Fas配体(FasL)系统的表达，并揭示了其在小鼠卵巢闭锁形成中的作用。通过原位杂交和RT-PCR方法，Fas主要在卵母细胞、超排卵子和部分颗粒细胞上表达，而FasL主要在颗粒细胞上表达。通过IIF、EIA和免疫沉淀法在翻译水平上进一步证实了这些结果。促性腺激素调节Fas-FasL系统在卵巢中的表达。我们还构建了AcNPV-FasL基因在Sf9(Sf9-FasL)细胞中的表达载体。TUNEL法检测颗粒细胞/Sf9-FasL细胞与无ZP卵细胞的相互作用可诱导卵细胞发生凋亡。相反，在该系统中ZP完整的卵子中没有发生细胞凋亡。综合我们的实验结果和其他研究人员的报道，我们强烈认为，在卵子发生过程中，FasL阳性的颗粒细胞与表达Fas的卵母细胞/卵子之间的相互作用可能导致了卵巢闭锁的形成，直到ZP完成。

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.