| Objective: to study and explore the expression and regulatory function of sperm-associated antigen 6(SPAG6)in the formation of mouse sperm acrosome.Methods: The Spag6 and Spink2 coding region was amplified by PCR based on cDNA from mice testis tissue to construct recombinant plasmids of pGBKT7/SPAG6,pGADT7/SPINK2,pEGFP-N2/SPAG6 and pCS3+FLT/SPINK2.The expression of SPAG6 during the first wave of spermatogenesis(postnatal day 8,12,16,20,24,28,30,35)was examined by Western blotting.Localization of SPAG6 in testicular germ cells was analyzed by immunofluorescence.Mammalian SPAG6 and SPINK2 expression plasmids were constructed and the interaction between SPAG6 and SPINK2 was examined by yeast two-hybrid assays,co-localization assays in CHO cells and Co-Immunoprecipitation in COS-1 cells.Localization of SPINK2 in testicular germ cells of spag6 knockout mice was also examined by immunofluorescence.Western blot analysis of SPINK2 expression in testis tissue of spag6 knockout mice.Results: Two bands appeared in the digestion results,and the size of the target gene fragment was correct,and the expression plasmid was successfully constructed.The expression of SPAG6 was detected on postnatal day 12 and elevated between postnatal day 16 and 28.SPAG6 was localized in the acrosome of round spermatids.Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu /-Trp /-His.The transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei.Co-Immunoprecipitation assay showed Flag antibody could simultaneously precipitate SPINK2 and SPAG6 from cell lysate.The expression and acrosomal localization of SPINK2 were not detected in testicular germ cells of SPAG6 knockout mice by immunofluorescence analysis and Western blot.Conclusion: SPAG6 interacts with SPINK2 and probably regulates the formation of sperm acrosome through stabilizing SPINK2 during spermatogenesis. |
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