Molecular Cloning, Localization Of IZUMO2 In Testes And Sperm Cells And Interaction Of IZUMO2 With Other Sperm-Egg Fusion Related Proteins In Sheep

Fertilization is the sum of the cellular mechanisms that passes the genome from one generation to the next and initiates development of a new organism. In mammals, sperm-egg fusion is a process mediated by interactions among multiple proteins on sperm and egg. Multiple sperm molecules had been identified in sperm and egg to be involved in sperm-egg fusion, such as CRISPs, SLLP1, PDIA3, IZUMO family, TSSK6, SPESP1, EQUATORIN, etc. However the molecular mechanisms of sperm-egg fusion remain largely unknown. To elucidate the potential interaction between IZUM02 and other sperm proteins, we first cloned the sheep Izumo2 cDNA sequence and expressed GST-IZUM02 fusion protein in E. coli, then generated ascitic polyclonal antibodies against the sheep IZUM02 by immunizing mouse with the purified GST-IZUM02 fusion protein. We examined the localization of IZUM02 in testis and sperm cells by immunohistochemistry and immunocytochemistry with our purified ascitic polyclonal antibodies against the sheep IZUMO2 protein. Finally, we explored the interactions between IZUM02 and IZUMO1, IZUMO3, IZUM04, PDIA3, TSSK6, SPESP1, SLLP1, CRISP1, MN9 by yeast two-hybrid technology.We designed the primers according to the predicted Izumo2 cDNA sequence released in GenBank （XM₀04015399.1） for cloning the Izumo2 cDNA with the sheep testicular total cDNA as template. The cloned sheep Izumo2 cDNA was 813 bp in length with a CDS region of 666 bp encoding a putative peptide of 222 amino acids with a putative signal peptide of 15 aa at its N terminus and a transmembrane domain （186-206 aa）. The results of BLAST showed that the cloned Izumo2 shared 98.6% identity with the predicted sheep Izumo2 cDNA sequence in GenBank.We constructed prokaryotic expression vector pGEX-Izumo2 and transformed it into competent E. coli BL21（DE3） cells. An 50 kDa GST-IZUMO2 fusion protein was expressed in E. coli BL21（DE3） after induction with IPTG. Recombinant GST-IZUMO2 fusion protein was purified by gel extraction and was injected into mouse hypodermically to generate ascitic polyclonal antibodies against the sheep IZUMO2 protein. Immunohistochemistry analysis showed that IZUM02 was expressed widely in testis sections. IZUMO2 was mainly detected in the sperm head before acrosome reaction, but disappeared after the acrosome reaction.The results of yeast two-hybrid did not demonstrate any interaction between IZUMO2 and other sperm-egg fusion related proteins such as IZUMO1, IZUMO3, IZUMO4, PDIA3, TSSK6, SPESP1, SLLP1, CRISP1, MN9.