FBXO24 ensures male fertility by preventing abnormal accumulation of membraneless granules in sperm flagella.

Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 plays a critical role in preventing the accumulation of importins and RNP granules in sperm flagella.

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†.

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.

CIB4 is essential for the haploid phase of spermatogenesis in mice†.

Spermatogenesis is a complex developmental process that involves the proliferation of diploid cells, meiotic division, and haploid differentiation. Many genes are shown to be essential for male fertility using knockout (KO) mice; however, there still remain genes to be analyzed to elucidate their molecular mechanism and their roles in spermatogenesis. Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitously expressed protein that possesses three paralogs: CIB2, CIB3, and CIB4. It is reported that Cib1 KO male mice are sterile due to impaired haploid differentiation. In this study, we discovered that Cib4 is expressed strongly in mouse and human testis and begins expression during the haploid phase of spermatogenesis in mice. To analyze the function of CIB4 in vivo, we generated Cib4 KO mice using the CRISPR/Cas9 system. Cib4 KO male mice are sterile due to impaired haploid differentiation, phenocopying Cib1 KO male mice. Spermatogenic cells isolated from seminiferous tubules demonstrate an essential function of CIB4 in the formation of the apical region of the sperm head. Further analysis of CIB4 function may shed light on the etiology of male infertility caused by spermatogenesis defects, and CIB4 could be a target for male contraceptives because of its dominant expression in the testis.

Testis-enriched kinesin KIF9 is important for progressive motility in mouse spermatozoa.

Kinesin is a molecular motor that moves along microtubules. Kinesin family member 9 (KIF9) is evolutionarily conserved and expressed strongly in mouse testis. In the unicellular flagellate Chlamydomonas, KLP1 (ortholog of KIF9) is localized to the central pair microtubules of the axoneme and regulates flagellar motility. In contrast, the function of KIF9 remains unclear in mammals. Here, we mutated KIF9 in mice using the CRISPR/Cas9 system. Kif9 mutated mice exhibit impaired sperm motility and subfertility. Further analysis reveals that the flagella lacking KIF9 showed an asymmetric waveform pattern, which leads to a circular motion of spermatozoa. In spermatozoa that lack the central pair protein HYDIN, KIF9 was not detected by immunofluorescence and immunoblot analysis. These results suggest that KIF9 is associated with the central pair microtubules and regulates flagellar motility in mice.

RSPH6A is required for sperm flagellum formation and male fertility in mice.

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice.This article has an associated First Person interview with the first author of the paper.