Studies On The Interaction Between Sperm Specific Proteins Between TSSK6 And IZUMO1 Of Sheep

Mammalian fertilization is a sophisticated and complex physiological process that includes three major processes:sperm capacitation,sperm and egg zona pellucida(ZP)combination and triggering acrosome reaction(AR),sperm and egg membrane fusion.The interactions between the proteins on the sperm membrane create a structural basis for the subsequent sperm fusion.Recent studies have shownthat the movement of the IZUMO1 protein on the sperm membrane is associated with the TSSK6 protein,but whether there is a direct interaction between the two proteins has not been verified yet.Therefore,we checked the interaction between the two proteins by yeast two-hybrid,co-immunoprecipitation(co-IP)and mammalian two-hybrid techniques.Firstly,we cloned the Tssk6 cDNA from the testis tissue of male sheep.The length of cloned Tssk6 cDNA is 985 bp with full ORF in which two bases in the CDS region are different from the predicted sequence in GeneBank,but without amino acid alteration.We upload the sequence of Tssk6 cDNA to NCBI,the accession number is KY914489.Then we constructed prokarotic expression vectors of pGEX-Tssk6 and pGEX-Izumol,immediately expressed and purified the recombinant fusion proteins TSSK6-GST and IZUMO1-GST to immunize mice and rabbit respectively to generate rabbit anti-IZUMO1 serum and mouse anti-TSSK6 serum.In the yeast two-hybrid experiment,we hybridized yeast Y2H transformed with pGAD-Tssk6 and yeast Y187 transformed with pGBK-Izumol.There were blue colonies growing on SD/-Trp-Leu and SD/Ade-His-Trp-Leu medium.The results showed that TSSK6 and IZUMOl interacted with each other in yeast cells.In the Mammalian Two-hybrid experiment,we detected the results of double luciferase reporter genes after co-transfecting pFN-10A-Izumo1/pFN-11 A-Tssk6 and reporter gene vector pGL4.31 into human 293T cells and analyzed the varances.The results showed that the active-control group was p<0.05,while the experimental group was p<0.01,indicating that IZUMOl and TSSK6 take strong interaction in 293T cells.In immunoprecipitation experiment,we co-transfected pCMV-Flag-Tssk6 and TpCMV-HA-Izumo1 vectors into 293T cells,and used the anti-HA antibody to precipitate the IZUMO1-HA protein,then blotted the TSSK6 with the anti-Flag antibody.The Western-blot results showed that the TSSK6-Flag protein was pulled by the IZUMO1-HA protein,indicating that the TSSK6 and the IZUMO1 bind in the 293T cells.Taken together,we conclude that there are direct interactions between IZUMO1 and TSSK6 in cells.