Two genes control sperm formation and male fertility in mice.

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of PF16, a central axonemal protein essential for flagellar motility. In mice, two homologous genes have been identified: the ancestral on chromosome 2 and the evolutionary derived on chromosome 16. Although knockout mice ( ) are phenotypically normal, the surviving males are infertile. To further investigate the roles of SPAG6 and SPAG6L, we generated compound mutants by crossing the two knockout lines. Compound heterozygous ; mice are fertile, while all ; males are infertile despite grossly normal appearance. Histological and ultrastructural analyses revealed defective spermiogenesis, including abnormal chromatin condensation, malformed acrosome and manchette, and disorganized mitochondrial and fibrous sheath. Both SPAG6 and SPAG6L bind to SPINK2, a key regulator of acrosome function, but SPAG6 has an approximately 10-fold higher binding affinity than SPAG6L. Moreover, SPAG6 modulates testicular AKAP4 and SPAG16L levels, which are critical components of the fibrous sheath and central apparatus respectively. Notably, SPAG6 suppresses tubulin acetylation, whereas SPAG6L enhances this post-translational modification, suggesting antagonistic roles in microtubule assembly. Overall, our findings demonstrate that SPAG6 and SPAG6L coordinately regulate sperm formation and male fertility during evolution.