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.

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.

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.

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.

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.

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.

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.

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.

Expression dynamics indicate the involvement of SPG7 in the reproduction and spermiogenesis of Phascolosoma esculenta.

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis.

Peroxisomes are organelles enclosed by a single membrane and are present in various species. The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs. However, little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs. Through transcriptomic and proteomic analyses, we observed heterogenous peroxisomal components among different organs, as well as between testicular somatic cells and different developmental stages of germ cells. As Pex3 is expressed in both germ cells and Sertoli cells, we generated Pex3 germ cell- and Sertoli cell-specific knockout mice. While Pex3 deletion in Sertoli cells did not affect spermatogenesis, the deletion in germ cells resulted in male sterility, manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells. Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins. These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis, but not for Sertoli cells.

CCDC146 is required for sperm flagellum biogenesis and male fertility in mice.

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.

An Improved Method for Purification of the Residual Bodies from the Seminiferous Tubules of Mice.

Human fertility is declining in Western countries, and it is becoming increasingly clear that male infertility plays a pivotal role in the overall fertility decline. To understand the process that drives successful male germ cell maturation, the study of spermatogenesis of model organisms, such as mice, is essential. Residual bodies (RBs) play an important role in the last stages of spermatogenesis. They are formed at the time when post-meiotic spermatids undergo sequential differentiation steps so that the acrosome and flagellum are developed, the nucleus is markedly condensed, and the cytoplasm is lost. The masses of lost cytoplasm become RBs. Our recent work has shown that RB dynamics are highly sensitive to even small fertility defects. It was also noted that the transcriptome and proteome of RBs changes in response to spermatogenic defects. Thus, RBs represent an excellent and highly sensitive entity for studying male fertility. Previously published protocols for RB purification had some major limitations: they produced an RB fraction that was heavily contaminated with spermatozoa and erythrocytes or required tens of grams of starting material. In addition, most of the available protocols were developed for purification of RBs from rat testes. Here, we present a protocol that allows the isolation of 2.5-3 × 106 RBs from mouse testes with a purity of 98% from only 1 g of starting material. The purified material can be used for various downstream applications to study male fertility, such as transcriptome and proteome analyses, super-resolution microscopy, and electron and cryo-electron microscopy, amongst many others. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: An improved method for purification of the residual bodies from the seminiferous tubules of mice.

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.

Integrated single cell transcriptome sequencing analysis reveals species-specific genes and molecular pathways for pig spermiogenesis.

Mammalian spermatogenesis is a highly complicated and intricately organized process involving spermatogonia propagation (mitosis) and meiotic differentiation into mature sperm cells (spermiogenesis). In pigs, spermatogonia development and the role of somatic cells in spermatogenesis were previously investigated in detail. However, the characterization of key molecules fundamental to pig spermiogenesis remains less explored. Here we compared spermatogenesis between humans and pigs, focusing on spermiogenesis, by integrative testicular single-cell RNA sequencing (scRNA-seq) analysis. Human and pig testicular cells were clustered into 26 different groups, with cell-type-specific markers and signalling pathways. For spermiogenesis, pseudo-time analysis classified the lineage differentiation routes for round, elongated spermatids and spermatozoa. Moreover, markers and molecular pathways specific to each type of spermatids were examined for humans and pigs, respectively. Furthermore, high-dimensional weighted gene co-expression network analysis (hdWGCNA) identified gene modules specific for each type of human and pig spermatids. Hub genes (pig: SNRPD2.1 related to alternative splicing; human: CATSPERZ, Ca[2+] ion channel) potentially involved in spermiogenesis were also revealed. Taken together, our integrative analysis found that human and pig spermiogeneses involve specific genes and molecular pathways and provided resources and insights for further functional investigation on spermatid maturation and male reproductive ability.

CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility.

Sperm flagellum plays a crucial role in male fertility. Here, we generated Ccdc183 knockout mice using the CRISPR/Cas9 system to reveal the protein function of the testis-specific protein CCDC183 in spermiogenesis. We demonstrated that the absence of CCDC183 causes male infertility with morphological and motility defects in spermatozoa. Owing to the lack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface and nucleus during spermiogenesis, which causes subsequent loss of cytoplasmic invagination around the flagellum. As a result, the flagellar compartment does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In addition, ectopic localization of accessory structures, such as the fibrous sheath and outer dense fibers, and abnormal head shape as a result of abnormal sculpting by the manchette are observed in Ccdc183 knockout spermatids. Our results indicate that CCDC183 plays an essential role in cytoplasmic invagination around the flagellum to form functional spermatozoa during spermiogenesis.