Molecular Cloning And Characterization Of PDIA3 From Sheep And Cashmere Goat, Identification Of Its Expression In Testis And Binding To IZUMO1

Gamete fusion is a complex process which mediated by many molecules in the sperm and egg membrane. It has been established in knockout mice that IZUMO1, a novel member of immunoglobulin superfamily (IgSF) located on the sperm, is crucial for sperm-egg fusion. Recent studies found that inhibitors of protein disulfide isomerases (PDIs) and specific antibodies against PDIs were able to inhibit sperm-egg fusion in vitro, suggesting that PDIA3 plays an important role in gamete fusion, but the mechanism of IZUMO1 and PDIA3 in the gamete fusion is still unkown. Our laboratory had cloned sheep and cashmere goat Izumo1 gene, now we cloned the Pdia3 cDNA from sheep and cashmere goat for identification of the potential interaction between PDIA3 and IZUMO1.We first extracted the total RNA from sheep and cashmere goat testis tissue and designed primers according to the cDNA sequence of bovine Pdia3 gene. The Pdia3 cDNA of sheep and goat were amplified successfully by RT-PCR. The cloned sheep and cashmere goat Pdia3 cDNA are 1660 bp and 1591 bp, respectively. Bioinformatics analysis showed that sheep and goat Pdia3 cDNA have the same nucleotide sequence encoding the same 505 amino acides, with high homologies to those of bovine (99.21%), human (95.05%), rat (89.50%) and mouse (90.89%). We aligned PDIA3 amino acids sequences from 31 species animals released in GeneBank and Ensembl databases with our cloned sheep and cashmere goat PDIA3, the result showed that the PDIA3 of different species share high homology, some mitifs, including the CGHC, are absolutely conserved. Then we inserted the Pdia3 CDS into the prokaryotic expression vector pGEX-4T-1, and expressed GST-PDIA3 fusion protein in E. coli BL21(DE3). The GST-PDIA3 fusion protein was purified by Glutathione-Sepharose 4B column, and was used as an antigen to immunize the Kunming (KM) mouse. The ascites polyclonal antibody against sheep GST-PDIA3 was harvested after four times immunization followed by celiacly injection of SI80 cells. Western blot analysis showed that the polyclonal antibody can specifically recognize both PDIA3 and the GST tag. Then the antibody was purified by glutaraldehyde cross linking method to remove the anti-GST component, leaving the polyclonal antibody with much higher specificity to PDIA3.The PDIA3 was detected to be expressed in cytoplasm of spermatogenic cells from spermatocytes to the prespermatid phases as well as in leydig cells of sheep testis by immunohistochemistry. It is also found in the equatorial segment of sperm. The results of RNA in situ hybrization showed that the Pdia3 gene transcribes in the spermatogonium,spermiocytes and prespermatid. Finally, we transiently cotransfected eukaryotic expression vectors pBiFC-VC155-PDIA3 and pBiFC-VN173-IZUMO1 into the human HeLa cells, and the binding of PDIA3 to IZUMO1 was proved by Co-immunoprecipitation and Western blot analysis.