A homozygous nonsense variant in HENMT1 causes male infertility in humans and mice.

BACKGROUND: HENMT1 encodes a small RNA methyltransferase that plays a crucial role in mouse spermatogenesis through the methylation of the 3' end of PIWI-interacting RNAs. OBJECTIVES: Our study aims to elucidate the relationship between HENMT1 and male infertility in humans. MATERIALS AND METHODS: A consanguineous family, having a single non-obstructive azoospermia patient was recruited for pathogenic variants screening. The research includes genetic analysis and experimental validation using mouse models. The patient was diagnosed with non-obstructive azoospermia. Whole-exome sequencing and subsequent bioinformatic analyses were performed to screen for candidate pathogenic variants. The pathogenicity of the identified variant was assessed and studied in vivo using a mouse model that mimicked the patient's mutation. RESULTS: Through whole-exome sequencing, we identified a homozygous nonsense variant (c.555G > A, p.Trp185*) in HENMT1 in the patient. The presence of the mutant HENMT1 mRNA was detected in the patient's blood, and the truncated HENMT1 protein was observed in transfected HEK293T cells. The mutant mice modeling this HENMT1 variant displayed an infertile phenotype similar to that of the patient, characterized by spermiogenesis arrest. Further analysis revealed a significant derepression of retrotransposon LINE1 in the testes of the Henmt1 mutant mice, and increased apoptosis of spermatids. DISCUSSION AND CONCLUSION: Our findings provide the evidence of pathogenicity of the identified HENMT1 variant, thus shedding light on the indispensable role of HENMT1 in human spermatogenesis.

CCDC181 is required for sperm flagellum biogenesis and male fertility in mice.

The structural integrity of the sperm flagellum is essential for proper sperm function. Flagellar defects can result in male infertility, yet the precise mechanisms underlying this relationship are not fully understood. CCDC181, a coiled-coil domain-containing protein, is known to localize on sperm flagella and at the basal regions of motile cilia. Despite this knowledge, the specific functions of CCDC181 in flagellum biogenesis remain unclear. In this study, Ccdc181 knockout mice were generated. The absence of CCDC181 led to defective sperm head shaping and flagellum formation. Furthermore, the Ccdc181 knockout mice exhibited extremely low sperm counts, grossly aberrant sperm morphologies, markedly diminished sperm motility, and typical multiple morphological abnormalities of the flagella (MMAF). Additionally, an interaction between CCDC181 and the MMAF-related protein LRRC46 was identified, with CCDC181 regulating the localization of LRRC46 within sperm flagella. These findings suggest that CCDC181 plays a crucial role in both manchette formation and sperm flagellum biogenesis.

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

Three-dimensional reconstruction of rat sperm using volume electron microscopy.

Three-dimensional (3D) reconstruction serves as a crucial instrument for the analysis of biological structures. In particular, a comprehensive and accurate 3D ultrastructural examination of rat sperm is vital for understanding and diagnosing male fertility issues and the underlying causes of infertility. In this study, we utilize the automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) imaging technique, which is a highly effective method for 3D cellular ultrastructural analysis. Our findings reveal that during spermiogenesis, the volume of the nucleus significantly decreases, shrinking to just 10% of its original size. The acrosomal vesicles derived from the Golgi apparatus converge and elongate along the spermatid nucleus. These vesicles then attach to the nucleus via a cap-like structure, thereby defining the head side of the spermatozoa. In the initial stages of spermiogenesis, the mitochondria in spermatids are distributed beneath the cell membrane. As the process progresses, these mitochondria gradually migrate to the sperm tail, where they form the mitochondrial sheath. This sheath plays a crucial role in providing the energy required for the movement of the sperm. In addition, we reconstruct the mRNA-stroring structure-chromatoid body in sperm cells, which are cloud-like or net-like structures in the cytoplasm. The precise and comprehensive nature of 3D ultrastructural examination allows for a deeper understanding of the morphological process of spermiogenesis, thereby contributing to our knowledge of male fertility and the causes of infertility. Our research has significantly advanced the understanding of the 3D ultrastructure of sperm more comprehensively than ever before.

Loss of CCDC188 causes male infertility with defects in the sperm head-neck connection in mice.

Acephalic spermatozoa syndrome (ASS) represents a rare genetic and reproductive disease, which is defined as semen composed of mostly headless spermatozoa. The connecting piece in the neck region, also known as the head-to-tail coupling apparatus (HTCA), plays a crucial role in the tight linkage between the sperm head and tail. Dysfunction of this structure can lead to separation of sperm heads and tails, and male infertility. Using the mouse as an experimental model, several proteins have been identified as associated with the HTCA and disruption of these proteins causes acephalic spermatozoa. However, the molecular mechanism underlying this morphologic anomaly and HTCA remains elusive. In this study, we focused on coiled-coil domain containing 188 (Ccdc188), which shows testis-enriched expression. To elucidate the physiological role of CCDC188, we generated a knockout (KO) mouse line using the CRISPR/Cas9 system. Ccdc188 KO male mice were sterile, indicating that CCDC188 is indispensable for male fertility. Most Ccdc188-null spermatozoa were acephalic. Transmission electron microscopy revealed that while the sperm HTCA could assemble properly without CCDC188, the HTCA failed to attach to the nucleus during spermiogenesis, leading to sperm head and neck separation. In addition, we found almost all of the spermatozoa in the cauda epididymis lacked a mitochondrial sheath. Taken together, we demonstrated that CCDC188 plays a crucial role in forming a tight sperm head-neck junction.

Erratum: Karyotype depends on sperm head morphology in some amniote groups.

[This corrects the article DOI: 10.3389/fgene.2024.1396530.].

FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility.

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 and that FBXO24 could ubiquitinate IPO5. 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 is involved in the degradation of IPO5, disruption of which may lead to the accumulation of abnormal RNP granules in sperm flagella.

CCDC189 depletion leads to oligo-astheno-teratozoospermia and male infertility in mice.

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the poly(A)-binding protein (PABP) family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

Motor proteins, spermatogenesis and testis function.

The role of motor proteins in supporting intracellular transports of vesicles and organelles in mammalian cells has been known for decades. On the other hand, the function of motor proteins that support spermatogenesis is also well established since the deletion of motor protein genes leads to subfertility and/or infertility. Furthermore, mutations and genetic variations of motor protein genes affect fertility in men, but also a wide range of developmental defects in humans including multiple organs besides the testis. In this review, we seek to provide a summary of microtubule and actin-dependent motor proteins based on earlier and recent findings in the field. Since these two cytoskeletons are polarized structures, different motor proteins are being used to transport cargoes to different ends of these cytoskeletons. However, their involvement in germ cell transport across the blood-testis barrier (BTB) and the epithelium of the seminiferous tubules remains relatively unknown. It is based on recent findings in the field, we have provided a hypothetical model by which motor proteins are being used to support germ cell transport across the BTB and the seminiferous epithelium during the epithelial cycle of spermatogenesis. In our discussion, we have highlighted the areas of research that deserve attention to bridge the gap of research in relating the function of motor proteins to spermatogenesis.

The Role of Tubulin Polymerization-Promoting Protein2 (TPPP2) in Spermatogenesis: A Narrative Review.

Tubulin polymerization-promoting protein2 (TPPP2) is one of the three paralogs of mammalian TPPP proteins. Its possible role in spermatogenesis is described in this narrative review. TPPP2 is expressed specifically in the male reproductive system, mainly in testes and sperm, and also in the epididymis. In testes, TPPP2 is exclusively expressed in elongating spermatids; in the epididymis, it is located in the middle piece of the sperm tail. TPPP2 is involved in spermiogenesis, in steps which are determinative for the formation and morphology of spermatids. The inhibition of TPPP2 decreases sperm motility (the curvilinear velocity of sperms), probably due to influencing mitochondrial energy production since TPPP2 knockout mice possess an impaired mitochondrial structure. There are data on the role of TPPP2 in various mammalian species: human, mouse, swine, and various ruminants; there is a significant homology among TPPP2s from different species. Experiments with Tppp2-/--mice show that the absence of TPPP2 results in decreased sperm count and serious dysfunction of sperm, including decreased motility; however, the in vitro capacitation and acrosome reaction are not influenced. The symptoms show that Tppp2-/--mice may be considered as a model for oligoasthenozoospermia.

Axonemal tubules in the distal sperm tail of Wolbachia-infected Drosophila simulans males contain ring-like intraluminal structures that persist after axoneme fragmentation.

Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the reproductive alterations, Wolbachia also causes cytoplasmic incompatibility that leads to embryo death when infected males are crossed with uninfected females, thus selecting infected females. However, the presence of Wolbachia has important fitness costs and infected Drosophila simulans males produce less sperm than their uninfected counterparts. Such sperm suffer, indeed, of some structural alterations that hinder their proper function. We took advantage of the fact that several sperm have abnormal distal regions of the tail, in which the plasma membrane is broken and the axonemal components splayed, making the ultrastructural aspects clearly observable. We found that axoneme reduction in the distal region of the sperm does not follow a unique pattern as observed in other insects, but occurs by losing accessory tubules or peripheral doublets. The axonemal tubules contain distinct coaxial ring-like structures that are still observed after axoneme fragmentation and form large clusters of several units.

Karyotype depends on sperm head morphology in some amniote groups.

The karyotype of an organism is the set of gross features that characterize the way the genome is packaged into separate chromosomes. It has been known for decades that different taxonomic groups often have distinct karyotypic features, but whether selective forces act to maintain these differences over evolutionary timescales is an open question. In this paper we analyze a database of karyotype features and sperm head morphology in 103 mammal species with spatulate sperm heads and 90 sauropsid species (birds and non-avian reptiles) with vermiform heads. We find that mammal species with a larger head area have more chromosomes, while sauropsid species with longer heads have a wider range of chromosome lengths. These results remain significant after controlling for genome size, so sperm head morphology is the relevant variable. This suggest that post-copulatory sexual selection, by acting on sperm head shape, can influence genome architecture.

Homozygous deleterious variants in the C-terminal of TDRD5 impair spermiogenesis, causing severe oligoasthenoteratozoospermia in humans.

BACKGROUND: PiRNA pathway factors, including evolutionarily conserved Tudor domain-containing proteins, play crucial roles in suppressing transposons and regulating post-meiotic gene expression. TDRD5 is essential for retrotransposon silencing and pachytene piRNA biogenesis; however, a causal link between TDRD5 variants and human infertility has not yet been established. OBJECTIVE: To identify the likely pathogenic variants of TDRD5 in infertile men, characterised by azoospermia or severe oligozoospermia. MATERIAL AND METHODS: Potential candidate variants were identified and confirmed using whole-exome and Sanger sequencing. Haematoxylin and eosin staining, immunofluorescence, and ultrastructural analyses were performed to investigate the structural and functional abnormalities of spermatozoa. The pathogenicity of the identified TDRD5 variants was verified using in vitro experiments. Functional effects of the C-terminal nonsense variant were assessed via histology, immunofluorescence staining, and small-RNA sequencing. Intracytoplasmic sperm injection (ICSI) was also performed to evaluate the efficacy of the clinical treatment. RESULTS: We identified a homozygous missense variant (c.3043G > A, p.A1015T) and a homozygous nonsense variant (c.2293G > T, p.E765*) of TDRD5 in two unrelated infertile men. Both patients exhibited severe oligoasthenoteratozoospermia, characterised by the presence of spermatozoa with multiple heads and/or flagella, as well as acrosomal hypoplasia. In vitro experiments revealed that the p.A1015T variant caused a diffuse distribution of TDRD5 granules, whereas the p.E765* variant led to the production of a C-terminal truncated protein with nuclear localisation, instead of the typical cytoplasmic localisation observed for the wild-type protein. Functional investigations also revealed that truncation of the C-terminal region of TDRD5 could potentially lead to a decline in the expression levels of intermitochondrial cement and chromatoid body components, such as MIWI (PIWIL1) and UPF1, and a slight decrease in the abundance of pachytene piRNA, ultimately resulting in compromised spermiogenesis. ICSI may be an effective treatment for these deficiencies. DISCUSSION AND CONCLUSION: This study implicates TDRD5 as a novel candidate gene in the pathogenesis of human male infertility, emphasising the contribution of piRNA pathway genes to male infertility. In addition, our data suggest that ICSI could be a promising treatment for infertile men harbouring TDRD5 variants.

Gene expression programs in mammalian spermatogenesis.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies.

Running the gauntlet: challenges to genome integrity in spermiogenesis.

Species' continuity depends on gametogenesis to produce the only cell types that can transmit genetic information across generations. Spermiogenesis, which encompasses post-meiotic, haploid stages of male gametogenesis, is a process that leads to the formation of sperm cells well-known for their motility. Spermiogenesis faces three major challenges. First, after two rounds of meiotic divisions, the genome lacks repair templates (no sister chromatids, no homologous chromosomes), making it incredibly vulnerable to any genomic insults over an extended time (typically days-weeks). Second, the sperm genome becomes transcriptionally silent, making it difficult to respond to new perturbations as spermiogenesis progresses. Third, the histone-to-protamine transition, which is essential to package the sperm genome, counterintuitively involves DNA break formation. How spermiogenesis handles these challenges remains poorly understood. In this review, we discuss each challenge and their intersection with the biology of protamines. Finally, we discuss the implication of protamines in the process of evolution.

The sperm ultrastructure of Spermophagus kuesteri Schilsky, 1905 (Chrysomelidae, Bruchinae) and considerations on the relationships of Cucujiformia superfamilies.

The sperm ultrastructure of the bean-weevil Spermophagus kuesteri (Bruchinae) was studied to verify the congruence of the new position of the subfamily within Chrysomelidae. The results indicated a positive answer to the question supporting a close relationship between Chrysomelidae and Curculionidae, a finding confirmed also by molecular data. Moreover, the sperm morphology of Divales cinctus, a member of Melyridae (Cleroidea) allowed to confirm the different sperm organization between members of this superfamily and Phytophaga (Chrysomeloidea + Curculionoidea). While studying the spermiogenesis of S. kuesteri, some sperm cysts showed aberrant cells provided with two flagella in the same plasma membrane. These aberrant sperm could be the result, during early spermiogenesis, of irregular processes involving the canal rings between spermatids.

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis.

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and male fertility.

Spermiogenesis is a critical, post-meiotic phase of male gametogenesis, in which the proper gene expression is essential for sperm maturation. However, the underFlying molecular mechanism that controls mRNA expression in the round spermatids remains elusive. Here, we identify that FBXO24, an orphan F-box protein, is highly expressed in the testis of humans and mice and interacts with the splicing factors (SRSF2, SRSF3, and SRSF9) to modulate the gene alternative splicing in the round spermatids. Genetic mutation of FBXO24 in mice causes many abnormal splicing events in round spermatids, thus affecting a large number of critical genes related to sperm formation that were dysregulated. Further molecular and phenotypical analyses revealed that FBXO24 deficiency results in aberrant histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling along sperm flagella, ultimately leading to male sterility. In addition, we discovered that FBXO24 interacts with MIWI and SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination. Furthermore, we show that FBXO24 depletion could lead to aberrant piRNA production in testes, which suggests FBXO24 is required for normal piRNA counts. Collectively, these data demonstrate that FBXO24 is essential for sperm formation by regulating mRNA alternative splicing and MIWI degradation during spermiogenesis.

Deficiency of nucleosome-destabilizing factor GLYR1 dampens spermatogenesis in mice.

Aberrant sperm morphology hinders sperm motility and causes male subfertility. Spermatogenesis, a complex process in male germ cell development, necessitates precise regulation of numerous developmental genes. However, the regulatory pathways involved in this process remain partially understood. We have observed the widespread expression of Glyr1, the gene encoding a nucleosome-destabilizing factor, in mouse testicular cells. Our study demonstrates that mice experiencing Glyr1 depletion in spermatogenic cells exhibit subfertility characterized by a diminished count and motility of spermatozoa. Furthermore, the rate of sperm malformation significantly increases in the absence of Glyr1, with a predominant occurrence of head and neck malformation in spermatozoa within the cauda epididymis. Additionally, a reduction in spermatocyte numbers across different meiotic stages is observed, accompanied by diminished histone acetylation in spermatogenic cells upon Glyr1 depletion. Our findings underscore the crucial roles of Glyr1 in mouse spermiogenesis and unveil novel insights into the etiology of male reproductive diseases.

Autophagy core protein BECN1 is vital for spermatogenesis and male fertility in mice†.

Mammalian spermatogenesis is a highly complex multi-step biological process, and autophagy has been demonstrated to be involved in the process of spermatogenesis. Beclin-1/BECN1, a core autophagy factor, plays a critical role in many biological processes and diseases. However, its function in spermatogenesis remains largely unclear. In the present study, germ cell-specific Beclin 1 (Becn1) knockout mice were generated and were conducted to determine the role of Becn1 in spermatogenesis and fertility of mice. Results indicate that Becn1 deficiency leads to reduced sperm motility and quantity, partial failure of spermiation, actin network disruption, excessive residual cytoplasm, acrosome malformation, and aberrant mitochondrial accumulation of sperm, ultimately resulting in reduced fertility in male mice. Furthermore, inhibition of autophagy was observed in the testes of germ cell-specific Becn1 knockout mice, which may contribute to impaired spermiogenesis and reduced fertility. Collectively, our results reveal that Becn1 is essential for fertility and spermiogenesis in mice.