Gamete Biology

A Heterozygous Mutation Disrupting the SPAG16 Gene Results in Biochemical Instability of Central Apparatus Components of the Human Sperm Axoneme

Abstract
The SPAG16 gene encodes two major transcripts, one for 71 kDa SPAG16L, the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for 35 kDa SPAG16S, a nuclear protein representing the C-terminus (exons 11-16) of SPAG16L. We previously reported a targeted mutation in exon 11 of the Spag16 gene which impaired spermatogenesis and prevented transmission of the mutant allele in chimeric mice. Here we describe a heterozygous mutation in exon 13 of the SPAG16 gene, causing a frame shift and premature stop codon, affording the opportunity to compare mutations with a similar impact on SPAG16L and SPAG16S on male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of whom were fertile. Freeze-boiling of isolated sperm from both affected males resulted in the loss of SPAG16L protein, SPAG6, another central apparatus protein which interacts with SPAG16L, and the 28 kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freeze-boiling cycles of sperm extracts from mice heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation affecting both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by sperm from mice heterozygous for a mutation affecting SPAG16L.

Key words: Spag16L • axoneme • central apparatus • mutation • sperm motility