Objective: To explore the expression and regulatory function of sperm-associated antigen 6 (SPAG6) in the formation of the sperm acrosome in mice.
Methods: The expression of SPAG6 during the first wave of spermatogenesis on postnatal days (PN) 8, 12, 16, 20, 24, 28, 30 and 35 was examined by Western blot and the localization of SPAG6 in the testicular germ cells was determined by immunofluorescence. The expression plasmids of SPAG6 and serine protease inhibitor Kazal-type 2 (SPINK2) were constructed, the interaction between SPAG6 and SPINK2 in the AH109 and CHO cells examined by yeast two-hybrid and co-localization assays, and the expression and localization of SPINK2 in the testicular germ cells of the SPAG6-knockout (SPAG6 KO) mice detected by immunofluorescence.
Results: SPAG6 was highly expressed between PN 16 and 28 and localized in the acrosome of the round spermatids. Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu/-Trp/-His, and the transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei. The expression and acrosomal localization of SPINK2 were not found in the testicular germ cells of the SPAG6-KO mice.
Conclusions: SPAG6 interacts with SPINK2 and probably participates in the formation of the sperm acrosome by stabilizing the expression of SPINK2 during spermatogenesis.
Keywords: male infertility; mouse; serine protease inhibitor Kazal-type 2; sperm acrosome; sperm-associated antigen 6.