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

我们发现MHC i类抗原和CD4/p561ck复合物分别在精子后膜和卵子质膜上表达。此外，我们在转染AcNPV-CD4 cDNA的Sf9细胞上生成CD4蛋白。然后，Sf9-CD4细胞与精子细胞之间的细胞间相互作用以物种特异性的方式展示了粘附反应。另一方面，我们证实了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检测，Fas主要在卵母细胞、高排卵卵和部分颗粒细胞上的转录水平表达，而FasL则定位在颗粒细胞上。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。促性腺激素调节卵巢Fas-FasL系统的表达。我们还构建了AcNPV-FasL cDNA转染Sf9 （Sf9- fasl）细胞。TUNEL法检测的颗粒/Sf9-FasL细胞与体外无ZP卵子相互作用诱导卵子凋亡。相比之下，该系统中ZP完整卵未发生凋亡。结合我们的实验结果和其他研究者的报道，强烈建议卵巢闭锁的形成是由FasL阳性颗粒细胞和表达Fas的卵母细胞/卵子在卵发生过程中相互作用诱导的，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.