TSSK3, a novel target for male contraception, is required for spermiogenesis

Abstract

We have previously shown that members of the family of testis-specific serine/ threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

Testis‐specific serine kinase 3 is required for sperm morphogenesis and male fertility

“…For example, quite commonly, elongated spermatids that should have undergone spermiation at stage VIII were observed in later stages IX–XI; early elongating spermatids that are characteristic of stage IX could be observed at XII; and, elongating spermatids that should have been fully developed at stage I were instead underdeveloped (Figure 3). In agreement with the report by Nayyab et al., 24 we found that elongated spermatids did not line the lumen at stage VII (Figure 3), so technically stage VII does not exist in these mice; however, it is obvious that the KO tubules progress to stage IX since irregularly shaped round spermatids can be found, despite unreleased, fully elongated spermatids still embedded in the epithelium (Figure 3). We also observed significant vacuolization of germ cells in histological sections of Tssk3 KO testes.…”

“…Our data that show localization of TSSK3 to the tail suggests that it interacts with and/or results in the phosphorylation of the cytoskeleton, 59 as we observed significant changes in the phosphorylation state of ACTL7A and ACTL9, which are “actin‐like” constituents of the cytoskeleton (Figure 5D). The interaction of TSSK3 and cytoskeletal proteins was also suggested in the previous report 24 . However, because Tssk3 KO males do not phenocopy Actl7a and Actl9 KO males that only show a reduction in sperm counts, and had more severe phenotypes, 47,48 other proteins (presented above) must also function downstream of TSSK3.…”

“…While Nayyab et al 24 . generated Tssk3 KO mice with local gene indels (−19 bp, −6+19 bp, and 47 bp) and reported similar phenotype results as our TSSK3 KO, the functional pathways and substrates of TSSK3 remain unknown.…”

“…While Nayyab et al 24 generated Tssk3 KO mice with local gene indels (−19 bp, −6+19 bp, and 47 bp) and reported similar phenotype results as our TSSK3 KO, the functional pathways and substrates of TSSK3 remain unknown. In the present report, we created Tssk3 KO mice with a deletion of 1551 bp, including most of the open reading frame, using the CRISPR/Cas9 system and revealed that TSSK3 plays an essential role in formation of sperm and male fertility.…”

Mutation of Serine protease 1 Induces Male Sterility in Bombyx mori

“…The testis-specific serine kinase family (TSSK) has six members, which are all expressed post-meiotically during spermiogenesis. The members of the TSSK family have high homologies in their kinase domains, and their defects lead to sterility without exhibiting somatic abnormalities ( Nayyab et al, 2021 ). In Drosophila melanogaster , the serine protease Seminase acts as seminal fluid protein component and initiates protease cascade signaling pathway through hydrolysis, thus participating in early post-copulation reproductive regulation.…”