The Interaction Between ENO1 And IZUMO1 And Its Effect On Fertilization

The interaction between IZUMO1 on the surface of the sperm and JUNO on the surface of the egg is essential to mammalian sperm-egg fusion,but not enough to cause membrane fusion.So there must be some other sperm or egg proteins involved in sperm egg membrane fusion.In order to find more sperm and egg proteins that may have an interaction with JUNO and IZUMO1,the laboratory conducted a preliminary screening using yeast two-hybrid(Y2H)technology.The sequencing results after screening show that a protein named ENO1 may interact with IZUMO1 and JUNO.We identified the interaction between rat ENO1 and IZUMO1 using yeast two-hybrid and co-immunoprecipitation(Co-IP)techniques,and observed localization of ENO1 and colocalization with IZUMO1 on sperm and eggs of rats using indirect immunofluorescence(IIF)technique.Finally,we measured the effects of ENO1 on fertilization of mouse eggs using antibody suppression and in vitro fertilization(IVF)techniques to identify whether ENO1 is involved in sperm-egg fusion.The cDNA of rat eno1 was cloned at first,and yeast expression vector pGAD-eno1 was constructed.Y2 H experiments were carried out with pGAD-eno1,pGBK-Juno and pGBK-Izumo1 which had been constructed in the laboratory.The results suggested that ENO1 interacts with IZUMO1,but does not interact with JUNO.Then pCMV-Flag-eno1 eukaryotic expression vector was constructed,and Co-IP experiment was performed by co-transfecting pCMV-Flag-eno1 into HEK293 T cells with pCMV-HA-Izumo1 which had been stored in the laboratory.The results confirmed that ENO1 interacts with IZUMO1.To generate antibodies aganist rat ENO1,pET-28a-eno1 prokaryotic expression vector was constructed to express His tagged ENO1 protein in E.coli.,His-ENO1 recombinant protein was purified immunized rabbits.Rabbit anti-rat ENO1 serum polyclonal antibodies were obtained and purified.A pGEX-eno1 prokaryotic expression vector was also constructed to express GST tagged ENO1-GST fusion protein.Mouse anti-rat ENO1 ascites polyclonal antibody was obtained after immunizing mice with ENO1-GST fusion protein followed by inoculating S180 cells.The purified ENO1 antibodies and the IZUMO1 antibody were used to carry out IIF experiments to demonstrate the subcellular localization of ENO1 in rat sperm and mouse eggs and its co-localization with IZUMO1 in rat sperm.The results showed that ENO1 locates in the cell membrane and cytoplasm of mouse MII ovum,and in the tail of intact rat sperm,and both tail and head of the acrosome reacted(AR)rat sperm.ENO1 and IZUMO1 co-localize in the head of the AR rat sperm.After treatment of mouse sperm and eggs with purified ENO1 antibodies,IVF experiments were carried out and results showed that the fertilization rate was lower than that of untreated sperm and eggs,but not statistically significant.