The Interaction Between EFEMP1 And IZUMO1 And Its Effect On Fertilization

Fertilization occurs in the process of sexual reproduction in mammals,in which the most critical step is the fusion of sperm and egg cell membrane.Sperm-egg membrane fusion is based on the interaction of multiple protein molecules on the surface of sperm and egg.At present,among the known protein interactions required for sperm-egg membrane fusion,the most important one is the interaction between the IZUMO1 on the sperm surface and the JUNO on the egg surface.However,the interaction between IZUMO1 and JUNO alone is not enough to promote the membrane fusion of sperm and egg.Their should be other proteins required in the form of protein-protein interaction with IZUMO1 or JUNO.Here we found that the EFEMP1 interacted with IZUMO1.Therefor,we prepared anti-EFEMP1 ascites polyclonal antibody,and invested the localization of EFEMP1 in mouse testis,ovary,sperm and egg,and the co-localization of EFEMP1 with IZUMO1 in rat sperm and mouse sperm and finally the effect of EFEMP1 antibody on the in vitro fertilization rate of mouse sperm and eggs to evaluate the role of EFEMP1 in the process of sperm and egg membrane fusion.Firstly,the cDNA of rat efemp1 was cloned to construct the pGAD-efemp1 yeast expression vector and the p CMV-Flag-efemp1 eukaryotic expression vector.The yeast two-hybrid assay was performed with the p GBK-Izumo1 and p GBK-juno,and the co-immunoprecipitation assay was carried out with p CMV-Flag-efemp1 and p CMV-HA-Izumo1.Both the results showed that EFEMP1 interacted with IZUMO1,but not with JUNO.For generation of anti-EFEMP1 antibody,pET-28a-efemp1 prokaryotic expression vector was constructed,and transformed into E.coli BL21(DE3)competent cells.The fusion protein of EFEMP1-HIS was expressed and purified,and then mouse anti-EFEMP1-HIS ascitic polyclonal antibodies were obtained by immunizing Kunming mice followed by intraperitoneal injection of S180 cells.The localization of EFEMP1 in mouse testis,ovary,sperm,egg,and co-localization of EFEMP1 and IZUMO1 in rat and mouse sperm were investigated by indirect immunofluorescence assay with mouse anti-EFEMP1-HIS ascitic polyclonal antibodies as the primary antibody.The results showed that EFEMP1 was expressed in the cytoplasm of muscle like cells in mouse testicular tissue and spermatozoa at different developmental stages,the follicular cells in mouse ovarian tissue,as well as in the cytoplasm and on the cytoplasmic membrane of mouse eggs;EFEMP1 was localized in the head and tail of rat and mouse sperm before and after acrosome reaction,and on the cell membrane and in cytoplasm of mouse MII oocytes.EFEMP1 and IZUMO1 were co-localized on the head of rat sperm after acrosome reaction and the head of mouse sperm before acrosome reaction.The in vitro fertilization rate of the mouse sperm and eggs treated with mouse anti-EFEMP1 ascites was insignificantly lower than that of mouse sperm and eggs treated with ascites,and that of untreated mouse sperm and eggs.We found that the EFEMP1 interacted with IZUMO1,but not with JUNO.The localization of EFEMP1 in mouse testis,ovary,sperm,egg,and co-localization of EFEMP1 and IZUMO1 in rat and mouse sperm;the in vitro fertilization rate of the mouse sperm and eggs treated with mouse anti-EFEMP1 ascites was insignificantly lower than that of mouse sperm and eggs treated with ascites,and that of untreated mouse sperm and eggs.