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 <i>Tssk3</i> and <i>Tssk6</i> knockout mice, as well as the double <i>Tssk1</i>/<i>Tssk2</i> 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 <i>Tssk1</i> and <i>Tssk2</i>, using CRISPR/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one <i>Tssk1</i> and two independent <i>Tssk2</i> mutant lines. Natural mating mutant <i>Tssk1</i> and <i>Tssk2</i> 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 <i>Tssk2</i> 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 <i>Tssk1</i> 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.