Interactions Between PDIA3 And CRISP2 And Their Localization In Rat Testes And Sperm

Mammalian sperm-egg fusion including many procedures is one kind of the cell membrane fusions. Lot kinds of interaction among specific proteins or lipids and other molecules may be involved in this process. It had been reported several proteins on egg and sperm were identified to be associated with sperm-egg fusion, such as JUNO, CD9, CD81 and GPI-Aps on egg; PDIA3, CRISP2, CRISP1, SLLP1,Equatorin and Izumol on sperm. However, how these proteins mediate the sperm-egg fusion is still unclear. In this study, we selected rat CRISP2 and PDIA3 to study the interaction of sperm surface proteins, to provide clues for unvealing the underneath molecular mechanism of mammalian sperm-egg fusion.We designed primers according to the rat Crisp2 (ENSRNOG00000013225) and Pdia3 (ENSRNOG00000015018) cDNA sequence downloaded from Ensembl database, and cloned these sequences by RT-PCR, then constructed prokaryotic expression vector for expressing recombinant CRISP2 and PDIA3 proteins in E. coli followed by immunizing mouse to get our anti-CRISP2 and anti-PDIA3 polyclonal antibodies. With these antibodies, we studied localization of CRISP2 and PDIA3 proteins in the rat testis and sperm by immunohistochemistry and immunocytochemistry methods.The results showed that CRISP2 and PDIA3 proteins appeared in large number of cells inside of the seminiferous tubules and the central district area where mature sperms arises from. These results showed that CRISP2 protein localizes throughout the sperm; and PDIA3 protein localizes mainly in the sperm head and body parts, with only a small amount in the sperm tail of rat sperm cells before acrosome reaction. After acrosome reaction, CRISP2 protein exists in the body and tail parts of rat sperm, whereas PDIA3 disappears from the sperm.The cloned rat Crisp2 and Pdia3 cDNA were 730 bp and 1497 bp, respectively, which codes 243 aa of CRISP2 and 499 aa od PDIA3 with 22 aa and 24 aa of signal peptide, respectively. We divided CRISP2 and PDIA3 into two segments according to their structural and functional domains, and constructed pGAD-Crisp2A, pGAD-Crisp2B, pGBKT-Pdia3A and pGBKT-Pdia3B yeast two hybrid vectors of their two segments, and pGAD-Crisp2 and pGBKT-Pdia3 yeast two hybrid vectors of their full length mature peptide, to screen the interaction between CRISP2 and PDIA3. Then we constructed pcDNA-Crisp2A, pcDNA-Crisp2B, pcDNA-Pdia3A, pcDNA-Pdia3B eukaryotic expression vectors of their two segments and pcDNA-Crisp2, pcDNA-Pdia3 of their full length mature peptide to verify their protein interactions by Co-immunoprecipitation and Western blot methods between rat CRISP2 and PDIA3 in 293T cells.The results showed rat PDIA3 could interacted with CRISP2, and the segments of CRISP2A-PDIA3A and CRISP2A-PDIA3B also existed interactions, but there was no interaction between CRISP2B-PDIA3A and CRISP2B-PDIA3B.