Identification Of The Interaction Between RATIZUMO1and PDIA3and The Localization Of IZUMO1in Testes And Sperm Cells

Mammalian fertilization is a multistep process.The sperm-egg fusion is a crucial step in fertilization,but its mechanism remains not well-known.Based on the previous results of our lab and of other research groups, especially the limited data about the structural characteristics of sperm-egg fusion protein IZUMO1and protein disulfide isomerase family member PDIA3,we speculated that PDIA3may interact with IZUMO1and facilitate IZUMO1function. Therefore we choose to identify the interaction between IZUMO1and PDIA3in the present study.To further understand Izumo1function, we also determine the localization of IZUMO1in rat testes and sperm cells.The rat IZUMO1cDNA was subcloned into prokaryotic expression vector pET-32a to construct the pET-IZUMO1for expressing IZUMO1in E. coli BL21. HIS-tagged IZUMO1protein was successfully harvested and purified by slicing the gel to immune KM mice,generating mouse anti-rat IZUMO1ascites polyclonal antibody. Then, localizations of IZUMO1in rat testes and sperm cells were observed by immunohistochemistry and immunocytochemistry methods, respectively.The results indicate that IZUMO1was expressed widely in rat testes,stored in the acrosome of the sperm head, and was moved to the equatorial region after acrosome reaction.In addition,we also identified the interaction between PDIA3and IZUMO1by yeast two hybrid(Y2H) system and co-immunoprecipitation(Co-IP). The yeast two hybrid expression vectors pGBK-IZUMO1, pGBK-IZUMO1(A) and pGBK-IZUMO1(B),as well as the corresponding recombinant vectors pGAD-PDIA3, pGAD-PDIA3(A) and pGAD-PDIA3(B) were constructed and were transformed into the competent yeast cells which then bring the recombinant pGBK and pGAD vectors together through hybridization. Our results show that IZUMO1and PDIA3can interact with each other and the extracellular fragment of IZUMO1interacts with PDIA3.The existence of interaction between PDIA3and IZUMO1were confirmed by co-immunoprecipitation with the recombinant pcDNA vectors which express the HA-tagged IZUMO1and HIS-tagged PDIA3co-transfecting the293T cells followed by Western blotting.