Sperm morphogenesis is a tightly regulated differentiation process, disruption of which leads to sperm malfunction and male infertility. Here, we show that Tex38 knockout (KO) male mice are infertile. Tex38 KO spermatids exhibit excess retention of residual cytoplasm around the head, resulting in abnormal sperm morphology with backward head bending. TEX38 interacts and colocalizes with ZDHHC19, a testis-enriched acyltransferase catalyzing protein S-palmitoylation, at the plasma membrane of spermatids. ZDHHC19 and TEX38 are each downregulated in mouse testes lacking the other protein. TEX38 stabilizes and localizes ZDHHC19 to the plasma membrane of cultured cells and vice versa, consolidating their interdependence. Mice deficient in ZDHHC19 or harboring a C142S mutation that disables the palmitoyltransferase activity of ZDHHC19 display phenotypes resembling those of Tex38 KO mice. Strikingly, ZDHHC19 palmitoylates ARRDC5, an arrestin family protein regulating sperm differentiation. Overall, our findings indicate that TEX38 forms a stable complex with ZDHHC19 at the plasma membrane of spermatids, which governs downstream S-palmitoylation of proteins essential for morphological transformation of spermatids.

BackgroundMale infertility is an intricate multifactorial disease involving the interplay between genetic and environmental factors. Genetic anomalies account for more than 15% of all male infertility cases; however, diagnosing them exhibits enormous challenges due to variable symptomatic presentations and limited knowledge of gene functions. Therefore, a thorough investigation into gene regulatory networks underlying male reproduction is demanded to improve patient counseling and infertility treatment.ObjectiveIn this study, we aimed to identify testis-expressed genes essential for male fertility.MethodsWe searched public databases, such as the National Center for Biotechnology Information (NCBI), Ensembl genome browser, the Human Protein Atlas (HPA), and the Mammalian Reproductive Genetics Database V2 (MRGDv2), to identify genes predominantly expressed in male reproductive tissues. Genetically engineered mouse lines lacking individual genes of interest were generated using either targeted gene replacement or the CRISPR/Cas9 system. To determine the gene functions, we analyzed fertility, testis weight, testis and epididymis histology, and sperm motility and morphology in adult knockout (KO) male mice.ResultsThrough the in silico screen, we identified 18 testis-expressed genes, including coiled-coil domain containing 182 (Ccdc182), EF-hand calcium-binding domain 15 (Efcab15), family with sequence similarity 187, member B (Fam187b), family with sequence similarity 24, member A (Fam24a), family with sequence similarity 24, member B (Fam24b), glial cell line derived neurotrophic factor family receptor alpha 2 (Gfra2), GLI pathogenesis-related 1 like 1, 2, and 3 (Glipr1l1-3), interleukin 3 (Il3), IZUMO family member 4 (Izumo4), peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1, retrogene 1 (Pin1rt1), solute carrier family 22 (organic cation transporter), member 16 (Slc22a16), sperm microtubule inner protein 2 (Spmip2), testis expressed 51 (Tex51), transmembrane and coiled-coil domains 2 (Tmco2), and tripartite motif family-like 1 and 2 (Triml1/2). The KO males displayed no obvious health problems, and normal mating behavior, fecundity, testis and epididymis histology, and sperm morphology and motility.Discussion and ConclusionOur findings indicate that these 18 testis-expressed genes are individually dispensable for male reproduction in mice. Disseminating such genes would promote our understanding of male reproduction and expedite the discovery of novel key male factors. Although we anticipate that mutations in these genes may not impair fertility in men, their enrichment in male germ cells makes them potential biomarkers for sperm count, quality, and morphological anomalies.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. Author summary

Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components. Male fecundity was determined by consecutively pairing knockout males with wild-type females and comparing the fecundity of wild-type controls. Phenotypic analyses of testis appearance and weight, testis and epididymis histology, and sperm movement were further carried out to examine any potential spermatogenic or sperm maturation defect in mutant males. In this study, we uncovered 13 testis- or epididymis-enriched evolutionarily conserved genes that are individually dispensable for male fertility in mice. Owing to their dispensable nature, it is not feasible to use these targets for the development of a male contraceptive.