The Role Of Sperm-associated Antigen 6(SPAG6)and Its Binding Protein In Regulating Spermatogenesis

Objective: To study the interaction between sperm-associated antigen 6(SPAG6)and various proteins,and to explore the role of SPAG6 in the regulation of spermatogenesis in mice.Methods: SPAG6 localization in male germ cells was examined by immunofluorescence staining(IF).A yeast two-hybrid assay was conducted on a mouse testicular cDNA library using a full length SPAG6 as bait,and selected potential binding partners were valiadated by direct yeast two-hybrid assay.The expression levels of SPAG6,Snapin and COPS5 during the first wave of spermatogenesis(postnatal day 6,8,12,16,20,24,28,30,35 and 42)were examined by Western blotting.Interaction between SPAG6 and these proteins were further examined by co-localization in CHO cells and Co-Immunoprecipitation(Co-IP)in COS-1 cells.Testis histology of Spag6 knockout mice and the wild-type mice was examined by H&E staining;Localization of Snapin,COPS5,and SPAG16 L in testicular germ cells were compared between the control mice and Spag6 knockout mice by IF.Results: In wild-type mice,SPAG6 was present in cytoplasmic vesicles in spermatocytes,the acrosome of round and elongating spermatids,and the manchette of elongating spermatids.Comparing with wild-type mice,Spag6 knockout mice showed abnormal morphology in the late stages of spermatogenesis.The armadillo repeat domain of mouse SPAG6 was used as a bait for a yeast two-hybrid screen,and several proteins with diverse functions appeared multiple times,including Snapin,COPS5 and TCTE3.Snapin has a similar localization to SPAG6 in male germ cells.It is localized in the cytoplasm of CHO cells and co-localized with SPAG6 when the two proteins were co-expressed.COPS5 was present throughout the cytoplasm in CHO cells,it clustered near the nuclear membrane when it was co-expressed with SPAG6.Co-IP assay showed that Flag antibody simultaneously precipitated flag-tagged Snapin,COPS5,TCTE3 and SPAG6 from the lysates prepared from the cells expressing these proteins,further confirming that SPAG6 and Snapin,COPS5,TCTE3 interact,respectively.SPAG16 L was found in the cytoplasm of round spermatids and the manchette of elongating spermatids.Although SPAG16 L was still existed in cytoplasm of round spermatids,while it lost its localization to the manchette in Spag6-deficient mice.Conclusion: SPAG6 interacts with multiple proteins,including Snapin,COPS5,etc.,and modulates germ cell development in different developmental stages during spermatogenesis.