The Ancient and Evolved Mouse Sperm-Associated Antigen 6 Genes Have Different Biologic Functions In Vivo

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, an axonemal central pair protein involved in flagellar motility. In mice, two Spag6 genes have been identified. The ancestral gene, on mouse chromosome 2, is named Spag6. A related gene originally called Spag6, localized on mouse chromosome 16, evolved from the ancient Spag6 gene. It has been renamed Spag6-like (Spag6l). Spag6 encodes a 1.6 kb transcript consisting of 11 exons, while Spag6l encodes a 2.4 kb transcript which contains an additional non-coding exon in the 3'-end as well as the 11 exons found in Spag6. The two Spag6 genes share high similarities in their nucleotide and amino acid sequences. Unlike Spag6l mRNA, which is widely expressed, Spag6 mRNA expression is limited to a smaller number of tissues, including the testis and brain. In transfected mammalian cells, SPAG6/GFP is localized on microtubules, a similar localization as SPAG6L. A global Spag6l knockout mouse model was generated previously. In addition to a role in modulating the ciliary beat, SPAG6L has many unexpected functions, including roles in the regulation of ciliogenesis/spermatogenesis, hearing, and the immunological synapse, among others. To investigate the role of the ancient Spag6 gene, we phenotyped global Spag6 knockout mice. All homozygous mutant mice were grossly normal, and fertility was not affected in both males and females. The homozygous males had normal sperm parameters, including sperm number, motility, and morphology. Examination of testis histology revealed normal spermatogenesis. Testicular protein expression levels of selected SPAG6L binding partners, including SPAG16L, were not changed in the Spag6 knockout mice, even though the SPAG16L level was significantly reduced in the Spag6l knockout mice. Structural analysis of the two SPAG6 proteins shows that both adopt very similar folds, with differences in a few amino acids, many of which are solvent-exposed. These differences endow the two proteins with different functional characteristics, even though both have eight armadillo repeats that mediate protein-protein interaction. Our studies suggest that SPAG6 and SPAG6L have different functions in vivo, with the evolved SPAG6L protein being more important. Since the two proteins have some overlapping binding partners, SPAG6 could have functions that are yet to be identified.