Co-Localization Of TSSK6 With IZUMO1 And IZUMO4 On Sheep Sperm

Mammalian sperm-egg fusion is mediated by multiple protein interactions between sperm and eggs.However,the specific roles of those proteins in the process of sperm-egg fusion are still unclear.The knockout mouse model identified that IZUMO1,a member of the IZUMO family,on sperm and TSSK6,a member of the testis-specific serine/threonine-protein kinase（TSSK）family,are essential proteins for sperm in sperm-egg fusion.There are four members in the IZUMO family,of which IZUMO1,IZUMO2 and IZUMO3 are testicular-specific membrane proteins,while IZUMO4 is a soluble protein expressed in testis and other tissues.Previously,TSSK6 can bind to both IZUMO1 and IZUMO4 at the molecular level was identified in our lab by using yeast two-hybrid,mammalian two-hybrid and co-immunoprecipitation techniques in sheep.But do the three proteins can bind on the sheep sperm cells?And when and to what extent they bind?Further identification is needed to answer these questions.In this experiment,the colocalization of TSSK6with IZUMO1 and IZUMO4 on sheep sperm was studied by indirect immunofluorescence technique to further determine whether these three proteins bind at the cellular level.cDNA coding sequences of sheep Tssk6,Izumo1 and Izumo4 were cloned and inserted into pGEX-4T-1 plasmid to construct prokaryotic expression vectors of pGEX-TSSk6,pGEX-IZUMO1 and pGEX-IZUMO4,respectively.Then the fusion protein of GST-TSSK6,GST-IZUMO1 and GST-IZUMO4 were expressed in E.coli and were isolated and purified.The rabbits were immunized with the GST-TSSK6fusion protein to generate rabbit anti-TSSK6 serum polyclonal antibody,while the mice were immunized with the GST-IZUMO1 and GST-IZUMO4,respectively,to generate mouse anti-IZUMO1 and mouse anti-IZUMO4 ascites polyclonal antibody.With the rabbit anti-sheep TSSK6 serum as the primary antibody,donkey anti-rabbit IgG-Alex Flour^TM 568 as the secondary antibody,mouse anti-sheep IZUMO1 ascites as the primary antibody and goat anti-mouse IgG-Alex Flour^TM 488as the secondary antibody,indirect immunofluorescence experiments were performed on the surface of the competent sperm,IZUMO1 is located in the equatorial region of the acrosome,and TSSK6 is distributed on the equator and tail,but the two are not colocated.With the mouse anti-sheep IZUMO4 ascites as the primary antibody and the goat anti-mouse IgG-Alex Flour^TM 488 as the secondary antibody,indirectimmunofluorescence technique was used to observe that on the surface of the competent sperm,IZUMO4 was distributed in the head of the sperm,and TSSK6 was distributed on the equator and tail.