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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 suggests 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 work 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 supports this kinase as a suitable candidate for the development of novel non-hormonal male contraceptives.

Mammalian sperm have to undergo capacitation to fertilize the egg. At the molecular level, capacitation involves cAMP synthesis, protein kinase A activation, and downstream increase in tyrosine phosphorylation. In addition, during capacitation, mammalian sperm actively generate reactive oxygen species (ROS). It has been proposed that ROS modulate phosphorylation pathways; however, the crosstalk between these signaling processes is not well-understood. In the present study, we used loss- and gain-of-function approaches to evaluate the interconnection between ROS and phosphorylation. We showed that BSA and HCO3 - , but not Ca2+ , in the capacitation media are required for ROS production. The synergic effect of these compounds was neither mediated by HCO3 - stimulation of cAMP synthesis nor by BSA-induced cholesterol efflux. The capacitation-induced ROS generation was blocked in the presence of superoxide dismutase (SOD), catalase, and apocynin. However, none of these compounds affected cAMP-dependent or tyrosine phosphorylation. On the other hand, the addition of NADPH to the media induced ROS generation in sperm incubated in the absence of BSA and HCO3 - without upregulating cAMP-dependent or tyrosine phosphorylation signaling. Most interestingly, catalase, but not SOD, blocked in vitro fertilization suggesting a role for H2 O2 in this process.

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests 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 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model., The established role of TSSKs in spermiogenesis and in male fertility, together with their unique kinase features, strongly supports this family of protein kinases as a very promising drug discovery target for development of a non-hormonal male contraceptive.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes.

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

SummaryThe CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper ion channel complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 interacts with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. Interestingly, transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm in vitro. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 may regulate CatSper assembly, target the CatSper complex to flagellar plasma membrane, and function as a calcium sensor. The identification of C2CD6 as an essential subunit may facilitate the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

The X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile. Here, we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

SummaryThe X-linked geneRlimplays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemicRlimknockout (KO) die around implantation, maleRlimKO mice appear healthy and are fertile, raising questions as to the pressures drivingRlimgene selection during evolution. Here we report an important role forRlimin testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of theRlimgene leads to lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility andin vitrofertilization rates. Targeting the conditionalRlimcKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions ofRlimin male reproduction specifically in round spermatids during spermiogenesis with likely evolutionary implications.

Mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation in order to acquire the ability to fertilize. Although the increase in phosphorylation associated with mouse sperm capacitation is well established, the identity of the proteins involved in this signaling cascade remains largely unknown. Tandem mass spectrometry (MS/MS) has been used to identify the exact sites of phosphorylation and to compare the relative extent of phosphorylation at these sites. In the present work, we find that a novel site of phosphorylation on a peptide derived from the radial spoke protein Rsph6a is more phosphorylated in capacitated mouse sperm. The Rsph6a gene has six exons, five of which are conserved during evolution in flagellated cells. The exon containing the capacitation-induced phosphorylation site was found exclusively in eutherian mammals. Transcript analyses revealed at least two different testis-specific splicing variants for Rsph6a.Rsph6a mRNA expression was restricted to spermatocytes. Using antibodies generated against the Rsph6a N-terminal domain, western blotting and immunofluorescence analyses indicated that the protein remains in mature sperm and localizes to the sperm flagellum. Consistent with its role in the axoneme, solubility analyses revealed that Rsph6 is attached to cytoskeletal structures. Based on previous studies in Chlamydomonas reinhardtii, we predict that Rsph6 participates in the interaction between the central pair of microtubules and the surrounding pairs. The findings that Rsph6a is more phosphorylated during capacitation and is predicted to function in axonemal localization make Rsph6a a candidate protein mediating signaling processes in the sperm flagellum.