Dentification Of The Interaction Between Goat Crisp1and Pdia3and The Localization Of Crisp1in Testes And Sperm Cells

Mammalian fertilization refers to the process that a sperm binds and fuses with an egg, forming a zygote. It is not only the beginning of embryo development, but also the starting point of life.However, the mechanism of sperm-egg fusion is not well-known. Based on the previous results of our lab and of other research groups, we speculated that the CRISP1, which is one of the several sperm-egg fusion related molocles and characterized with enriched cysteines, may interact with another sperm-egg fusion related molocle, PDIA3, which is a disulfide isomerase family member.Therefore we choosed to identify the interaction between CRISP1and PDIA3in the present study. To further understand CRISP1function, we also determined the localization of CRISP1in rat testes and sperm cells.We used pET-32a to construct prokaryotic expression vector pET-CRISP1for expressing CRISP1in E.coli BL21by induction of IPTG. We obtained the HIS-tagged CRISP1seccessfully. Through slicing the gel, we purified the HIS-CRISP1and immuned KM mice, generating mouse anti-goat CRISP1ascites polyclonal antibody. Then, localizations of CRISP1in goat testes and sperm cells were observed by immunohistochemistry and immunocytochemistry methods, respectively. The results indicated that CRISP1is expressed widely in goat testes, and is mainly stored in the sperm tail, and was moved to the equatorial region after acrosome reaction.We also identified the interaction between CRISPl and PDIA3by yeast two hybrid(Y2H) system and co-immunoprecipitation(Co-IP). We constructed yeast two hybrid expression vectors pGAD-CRISP1、pGAD-CRISP1(A)、pGAD-CRISP1(B) and pGBK-PDIA3, as well as the corresponding recombinant vectors pGBK-PDIA3(A) and pGBK-PDIA3(B). Then the vectors were transformed into the competent yeast cells which then bring the recombinant pGBK and pGAD vectors together through hybridization. The results suggested that CRISP1interates with PDIA3(A). The existence of interaction between CRISP1and PDIA3was confirmed by co-immunoprecipitation with the recombinant pcDNA vectors which express the HIS-tagged CRISP1and HA-tagged PDIA3and co-transfect the293T cells followed by Western blotting.In addition, we also selected goat CRISP1stably transformed293T cell line in which goat CRISP1is constitutively expressed.