Identification Of Interactions Among SPACA3,SPACA1,PDIA3 And CRISP2 On Sperm

Fertilization is an essential process of mammalian sexual reproduction.Sperm-egg fusion is a key step in the fertilization process.It is a process in which multiple molecules participate and assist in completion.Researches had found that mammalian sperm-egg fusion requires not only the interaction of sperm protein IZUMO1 with the egg protein JUNO,but also interactions among other sperm or egg protein such as PDIA3,CRISP2,SPACA3,and SPACA1.In this experiment,the interaction between rat SPACA1 and SPACA3 was identified at the molecular level using yeast two-hybrid?Y2H?and Co-immunoprecipitation?Co-IP?techniques.And co-localization of SPACA1 with SPACA3,PDIA3 with SPACA3,PDIA3 with CRISP2 on sperm was also investigated using indirect immunofluorescence technology.The results provided new insights into comprehensive understanding of the molecular mechanism of mammalian sperm-egg fusion.First,we cloned rat spaca1 and spaca3 cDNAs and sheep pdia3,crisp2 and spaca3 cDNAs to construct yeast expression vestors pGBK-spaca1 and pGAD-spaca3 for Y2H and eukaryote expression vectors pCMV-C-HA-spaca1 and pCMV-C-Flag-spaca3 for Co-IP.The results of Y2H and Co-IP showed that there was an interaction between rat SPACA1 and SPACA3.Antibodies are required to identify the cellular and subcellular localization of proteins.To prepare rat SPACA1,SPACA3 and sheep PDIA3,CRISP2,SPACA3antibodies,we constructed prokaryotic expression vectors of pET-28a-spaca1,pET-28a-spaca3 to express rat SPACA1-HIS and SPACA3-HIS His-taged proteins,pGEX-pdia3,pGEX-crisp2,and pGEX-spaca3 to express sheep PDIA3-GST,CRISP2-GST and SPACA3-GST GST-fusion proteins in E.coli.Rabbits were immunized with SPACA3-HIS and PDIA3-GST to generate rabbit anti-rat SPACA3and rabbit anti-sheep PDIA3 serum,respectively.Mouse were immunized with SPACA1-HIS,CRISP2-GST and SPACA3-GST to generate mouse anti-rat SPACA1,mouse anti-sheep CRISP2,mouse anti-sheep SPACA3 ascites polyclonal antibody,respectively.Then,using the rabbit anti-rat SPACA3 serum and mouse anti-rat SPACA1ascites polyclonal antibody as the primary antibody,the location of SPACA1 and SPACA3 on rat testis tissue and sperm was identified.The results showed that SPACA1 is present on mature sperm in sperm cells and seminiferous tubules of testis,but is not expressed in sertoli and interstitial cells;SPACA3 is expressed in interstitial cells,spermatogonia and spermatocytes of rat testis,mainly in mature sperm.After acrosome reaction,SPACA1 and SPACA3 can co-localize in the equatorial segment of rat sperm head.Using rabbit anti-sheep PDIA3 serum,mouse anti-sheep CRISP2 and mouse anti-sheep SPACA3 ascites polyclonal antibodies as primary antibodies,the co-localization of PDIA3,CRISP2 and SPACA3 on sheep sperm was identified.The results showed that after Ca²⁺-induced sperm acrosome reaction,SPACA3 and PDIA3 could co-localize in the equatorial segment of sheep sperm head,while PDIA3 and CRISP2 could not co-localize.The results above indicated that the two sperm acrosome-associated protein SPACA3 may participate in the sperm-egg binding or fusion process jointly with SPACA1 and PDIA3 after sperm acrosome reaction.CRISP2 and PDIA3 may independently participate in the sperm-egg binding or fusion process.The interaction between CRISP2 and PDIA3 did not occur on sperm,but might occur in other processes of cell biology.