Identification of TSSK1 and TSSK2 as Novel Targets for Male Contraception.

The testis-specific serine kinases (TSSKs) are post-meiotically expressed in testicular germ cells. Their testis-specific expression, together with their putative role in phosphorylation pathways, suggests that TSSKs have relevant roles in spermiogenesis, sperm function, or both. Independent and knockout mice, as well as the double / KO males, are sterile. However, the double KO results are silent regarding the individual roles of TSSK1 and TSSK2. The aim of this study was to develop independent mutant mouse models of and , using CRISPR/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one and two independent mutant lines. Natural mating mutant and homozygous males but not females were found to be sterile. Additionally, homozygous males have lower sperm numbers and decreased motility, and were infertile in vitro. Anti-TSSK2 antibodies were validated against mutants and used in Western blot and immunofluorescence experiments. TSSK2 is localized to the sperm head; importantly, it is present in the testes and sperm from mutant mice, confirming individual mutation. Our results indicate that both TSSK1 and TSSK2 are individually essential for male reproduction and support both kinases as suitable nonhormonal male contraceptive targets.