Search Results (58)

Primary ciliary dyskinesia (PCD) is a rare genetic disorder mainly characterized by impaired mucociliary clearance and chronic respiratory symptoms. From a consanguineous family, a male patient, although with respiratory complaints since birth, was diagnosed with PCD only in adulthood. Whole-exome sequencing disclosed a novel homozygous intronic single-nucleotide deletion, NM_001369.3(DNAH5):c.13723+4del, initially classified as of uncertain clinical significance. Digital highspeed videomicroscopy (HSVM) evidenced a null ciliary beating frequency; transmission electron microscopy showed absence of outer dynein arms (class-1); and immunofluorescence (IF) demonstrated markedly absent DNAH5 protein level in the apical cilia region with delocalization to the transition and basal-body regions. Bioinformatic analysis predicted altered splicing at the donor splice site of exon 78, whereas mRNA sequencing revealed two splicing defects: the mainly expressed transcript corresponding to exon 78 skipping and a minor transcript originated from a cryptic splice site in exon 78. The patient was infertile and showed severe oligoteratozoospermia. Sperm IF analysis revealed absence of DNAH5 from the flagellum with accumulation at the neck region. The family study confirmed homozygosity. The present results support a pathogenic role for the c.13723+4del variant and underscore the importance of integrating clinical, ultrastructural, DNA, mRNA and protein analyses to clarify and contribute to PCD diagnosis. Full article

The microstructure and ultrastructure of the sperm of Japanese eel, Anguilla japonica, artificially induced with weekly injections with carp pituitary (CP) and human chorionic gonadotropin (HCG), was studied, and milt from 10 out of 20 mature fish was collected. Two distinct morphological structures of A. japonica sperm had been observed with optical microscopy. The cell nucleus of one type of sperm was round or nearly round, the sperm was smaller in size, with 2.57 ± 0.62 μm of the long diameter of the cell nucleus, 2.11 ± 0.59 μm of the short diameter, and 37.35 ± 7.71 μm of the flagellum length. Another type was the eyebrow-shaped sperm, the sperm was relatively larger in size, with 7.66 ± 1.09 μm of the long axis, 2.54 ± 0.46 μm of the short axis, and 38.26 ± 9.02 μm of the flagellum length. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the anterior end of the round sperm lacked an acrosomal structure. The implantation fossa was located in the center of the base of the sperm head, and it was in the shape of a channel along the long axis of sperm. The centriolar complex was situated within the implantation fossa. There were 2–3 mitochondria distributed at the basal end of the nucleus. Sperm flagellum prolongated from the sleeve cavity, and the initial part of axoneme connected to the distal end of basal body. The axoneme displayed a typical “9 + 2” pattern. There was a spherical structure in the curving area for eyebrow-shaped sperm, and the axoneme formed a “9 + 0” pattern. The discovery of two different types of sperm in the Japanese eel has provided new perspectives for research on its artificial reproduction. Full article

The yak represents a distinct domestic animal species that predominantly inhabits the Qinghai–Tibet Plateau and adjacent areas, possessing considerable value in both scientific and economic contexts. Compared to animals that mainly dwell on plains, such as cattle, the sperm maturation process in yak exhibits a certain degree of species specificity to adapt to their unique reproductive needs in high-altitude environments. Serving as the main storage site for functionally competent sperm, the cauda epididymis plays an integral role in mediating their post-testicular maturation. MiRNAs are vital regulatory molecules in the epididymis, influencing sperm maturation by modulating gene expression after transcription. To investigate the unique regulatory mechanisms of sperm maturation in yak, this study compared the miRNA expression profiles in the cauda epididymis of yak and cattle using high-throughput small RNA (sRNA) sequencing. The comparative analysis identified and characterized sRNA populations in the cauda epididymis of yak and cattle, revealing a similar length distribution that peaked at 22 nt and a predominance of known miRNAs. Notably, eight miRNAs were found to be highly expressed in both species. Furthermore, the first-nucleotide bias differed significantly between known and novel miRNAs within each species. A total of 31 differentially expressed (DE) miRNAs were identified, with 11 upregulated and 20 downregulated in yak compared to cattle. Among these, bta-miR-1298 exhibited the most significant upregulation, while bta-miR-2344 displayed the most pronounced downregulation. Bioinformatic analysis linked the predicted target genes of these miRNAs to numerous critical signaling pathways, including calcium signaling, the mitogen-activated protein kinase (MAPK) signaling pathway, the Ras-associated protein 1 (Rap1) signaling pathway, and the cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway. Furthermore, eight significantly DE miRNAs, including bta-miR-2443, bta-miR-503-3p, bta-miR-6517, bta-miR-2440, bta-miR-2431-3p, bta-miR-2436-3p, bta-miR-6523a, and bta-miR-6775, were predicted to target genes involved in various aspects of sperm structural and functional maturation. These aspects include flagellum formation, sperm motility, chromatin remodeling, acrosome reaction, acrosome structure, sperm capacitation, chemotaxis, and nuclear chromatin condensation. Multiple miRNAs and their corresponding predicted target genes were analyzed by quantitative real-time PCR (qPCR), demonstrating an inverse correlation between miRNA expression and target gene levels. These findings reveal a distinct, species-specific miRNA signature in the yak cauda epididymis, which suggests a potential contribution to regulating the epididymal luminal environment and the process of sperm maturation. This study provides preliminary foundational data for elucidating the differences in sperm maturation mechanisms between yak and cattle, and offers potential novel targets for improving reproductive efficiency in plateau livestock. Full article

Parassi mullet (Mugil incilis) is an ecologically and economically important species that supports small-scale artisanal fisheries. However, scarce knowledge of its reproductive biology limits the development of management and conservation strategies. This research describes key milt and sperm characteristics, including milt volume, sperm concentration, motility, and ultrastructural features. Males produced an average of 40.0 ± 20 µL of milt, with sperm concentrations between 6.00 and 20.37 × 10⁹ spermatozoa mL⁻¹. Sperm motility varied between 10% and 80%, with a mean duration of 14.13 ± 4.49 min. Mature spermatozoa measured 33.79 ± 0.67 µm and exhibited a subspherical head without an acrosome, a short midpiece, and a cylindrical flagellum. The nucleus contains electron-dense heterogeneous chromatin. The centriolar complex was positioned outside the nuclear fossa consistent with Type II spermiogenesis. The flagellum comprises a main piece and tapering end piece. The axoneme had 9 + 0 arrangement at the basal body region and the typical 9 + 2 configuration along its length. These results provide the first detailed description of sperm morphology in parassi mullet and contribute to an understanding of its reproductive biology, supporting future applications in taxonomy, toxicology, conservation and aquaculture programs. Full article

Spermiogenesis requires extensive molecular and structural remodeling to produce motile sperm. Mutations in the testis-specific RNA methyltransferase NSUN7 are associated with defective fibrous sheath, impaired sperm motility, and male infertility. However, the underlying molecular mechanisms remain poorly understood. Here, we performed proteomic profiling of sorted, elongated, and round spermatids, as well as mature spermatozoa from Nsun7 knockout mice. We showed that NSUN7 is present at all stages of spermiogenesis and is most abundant in round spermatids, which corresponds to the formation of the flagellum and fibrous sheath assembly. Loss of NSUN7 altered the abundance of proteins essential for dynein arm assembly (PIH1D3, CCDC103, CCDC40), intraflagellar transport (IFT122), and fibrous sheath organization (AKAP3, AKAP4, ROPN1L). We also showed that the previously detected impaired retention of cytoplasm in elongated spermatids may be caused by plectin accumulation. Interestingly, no statistically significant changes were found in mature sperm proteomes upon Nsun7 inactivation. Our findings support a model in which NSUN7 primarily stabilizes protein complexes and coordinates flagellar assembly. This indicates that NSUN7 is a critical regulator of spermiogenesis, and its malfunction is a contributing factor to male infertility. Full article

Sperm proteases are involved in several gamete interaction events leading to fertilization. This report presents a detailed analysis of the expression and localization of serine protease PRSS38 in human and in mouse spermatozoa and its involvement in fertilization-related events, using bioinformatics, cellular, biochemical, molecular, and functional approaches. Bioinformatics analyses included genomics and data analysis, prediction of protein subcellular localization and post-translational modifications, Self-Organizing Maps (SOMs) unsupervised training with other serine proteases, protein modeling (AlphaFold), and genetic variant analysis. For cellular, biochemical, and functional studies, human semen samples were obtained from healthy normozoospermic volunteers, and cauda epididymal sperm were collected from adult Balb-c/C57 mice. PRSS38 presence was detected in human and mouse sperm protein extracts by Western immunoblotting. Sperm PRSS38 subcellular localization was determined by fluorescence immunocytochemistry. Human sperm–oocyte interaction events were assessed by means of the mouse Cumulus Penetration Assay (CPA) using mouse COCs, the Human Hemizona Assay (HZA), and the ZP-free hamster egg Sperm Penetration Assay (SPA). Mouse sperm–oocyte interactions were evaluated by means of in vitro fertilization (IVF) with COCs and denuded oocytes. PRSS38 is proposed to be a GPI-anchored serine protease (active site: His-100, Asp-150, and Ser-245) based on bioinformatics analyses. Using commercial antibodies, protein forms of the expected Mr (human: 31 kDa; mouse: 32 and 24 kDa) were specifically immunodetected in protein sperm extracts. Immunocytochemical analysis revealed a specific PRSS38 signal in the human sperm acrosomal region, equatorial segment, and flagellum. Mouse sperm PRSS38 was immunolocalized in the equatorial segment and hook. Human sperm preincubation with specific antibodies resulted in inhibition (p < 0.05) of CPA, HZA, and SPA. Mouse sperm preincubation with PRSS38 antibodies impaired (p < 0.05) homologous IVF using COCs and denuded oocytes. Genetic variants affecting residues involved in the GPI anchor and the catalytic triad were found in individuals from the general population whose PRSS38 protease function could be altered. This study provides, for the first time, an integrated analysis of PRSS38 in human and mouse sperm, contributing to our understanding of mammalian fertilization and male infertility. Full article

Sperm flagellum defects are tightly associated with male infertility. Centriolar satellites are small multiprotein complexes that recruit satellite proteins to the centrosome and play an essential role in sperm flagellum biogenesis, but the precise mechanisms underlying this role remain unclear. Serologically defined colon cancer autoantigen protein 8 (SDCCAG8), which encodes a protein containing eight coiled-coil (CC) domains, has been associated with syndromic ciliopathies and male infertility. However, its exact role in male infertility remains undefined. Here, we used an Sdccag8 mutant mouse carrying a CC domains 5–8 truncated mutation (c.1351–1352insG p.E451GfsX467) that models the mutation causing Senior–Løken syndrome (c.1339–1340insG p.E447GfsX463) in humans. The homozygous Sdccag8 mutant mice exhibit male infertility characterized by multiple morphological abnormalities of the flagella (MMAF) and dysmorphic structures in the sperm manchette. A mechanistic study revealed that the SDCCAG8 protein is localized to the manchette and centrosomal region and interacts with PCM1, the scaffold protein of centriolar satellites, through its CC domains 5–7. The absence of the CC domains 5–7 in mutant spermatids destabilizes PCM1, which fails to recruit satellite components such as Bardet–Biedl syndrome 4 (BBS4) and centrosomal protein of 131 kDa (CEP131) to satellites, resulting in defective sperm flagellum biogenesis, as BBS4 and CEP131 are essential to flagellum biogenesis. In conclusion, this study reveals the central role of SDCCAG8 in maintaining centriolar satellite integrity during sperm flagellum biogenesis. Full article

Male contraception remains an unmet need. Na,K-ATPase α4 (NKA α4), a specific Na⁺/K⁺ transporter of the sperm flagellum, is an attractive target for male contraception. NKA α4 is critical for sperm motility and fertility, and its deletion in male mice causes complete infertility. Our previous structure–activity relationship (SAR) studies on a cardenolide scaffold identified a highly selective, safe NKAα4 inhibitor, but its complex, heavily hydroxylated structure posed challenges for modification and optimization. To address this, we employed a structural simplification strategy to synthesize novel steroidal and non-steroidal analogs and examined their effects on NKAα4 inhibition and sperm motility. Both series reduced sperm motility (up to ~50%), with IC₅₀ values in the picomolar range. Compounds 13 and 45 displayed specificities for NKAα4 over NKAα1, did not affect sperm viability, and showed no reversibility in vitro. Notably, 45, featuring a hexahydronaphthalene core and a benzyltriazole moiety at C5, exhibited potent, highly selective NKAα4 inhibition, reduced sperm motility in vitro and in vivo, and blocked fertilization in vitro. This highlights 45 as a promising lead for non-hormonal male contraception and indicates that the newly generated series of compounds possess the key characteristics needed for further development as potential non-hormonal male contraceptive agents. Full article

Type 1 diabetes (T1D) is a chronic metabolic disease defined by sustained hyperglycemia, leading to oxidative stress (OS) and systemic complications, including male subfertility. This study investigates the potential impact of T1D-induced OS on microtubule (MTs) dynamics and microtubule-associated proteins (MAPs) in the testis and spermatozoa (SPZ). Using a streptozotocin-induced T1D rat model, we examined the expression and localization of key MAPs, including Microtubule Affinity-Regulating Kinase 4 (MARK4), Microtubule-Associated Protein 1A (MAP1A), Dynein Light Chain LC8-Type 1 (DYNLL1), Prolyl Endopeptidase (PREP), and Radial Spoke Head 6 Homolog A (RSPH6A), alongside sperm functional parameters. Our findings showed that T1D significantly impaired the expression and distribution of these proteins, which may affect MTs organization and be associated with cytoskeletal disorganization, and impaired germ cell differentiation. Moreover, T1D rats exhibited reduced sperm count, viability, and motility, accompanied by increased DNA fragmentation and chromatin defects. Elevated levels of 4-hydroxy-2-nonenal (4-HNE), a marker of OS, were detected in SPZ, particularly in the acrosome and flagellum, correlating with mitochondrial dysfunction and ATP depletion. Additionally, decreased intracellular Ca²⁺ levels, downregulation of Cation Channel of Sperm (CATSPER) and Voltage-Dependent Anion Channel 3 (VDAC3), and altered tubulin acetylation, possibly due to imbalanced Alpha-Tubulin N-Acetyltransferase 1 (ATAT1) and Histone Deacetylase 6 (HDAC6) expression, were also associated with impaired sperm motility. The combined data suggest that T1D-induced OS is linked to disrupted MTs dynamics, which may contribute to testicular dysfunction and reduced sperm quality, potentially affecting male fertility. A better understanding of these associations may support the development of therapeutic strategies to mitigate the reproductive consequences of T1D and improve male fertility outcomes. Full article

Utilizing high-resolution microscopy in conjunction with a new antibody highly specific for rat alpha4 Na,K-ATPase, we describe changes in alpha4 expression during spermatogenesis and in sperm incubated under capacitating and noncapacitating conditions. Immunohistochemical analyses showed alpha4 expression at low levels in spermatogonia and in pachytene spermatocytes. Alpha4 then becomes highly expressed on round spermatids and the midpiece of elongated spermatozoa within the seminiferous tubules. In noncapacitating conditions, alpha4 was confined mainly to the flagellum of mature sperm; however, under capacitating conditions, sperm acquired intense alpha4 staining along the acrosomal region of the sperm head. To visualize the precise localization of alpha4 in the sperm head, we performed an ultrastructural analysis using immuno-scanning electron microscopy. Under capacitating conditions, sperm exhibited alpha4 staining along the dorsal surface of the sperm head associated with the acrosome. In addition, after 4 h of incubation in motility buffer, we observed an increase in alpha4 protein in sperm that could be blocked with chloramphenicol, a mitochondrial-type ribosome inhibitor. These findings demonstrate that both the localization and expression level of alpha4 Na,K-ATPase are dynamic aspects of sperm maturation and suggest that sperm motility and capacitation may be supported by these changes to the location and amount of this protein. Full article

Na⁺,K⁺-ATPase is the active ion transport system that maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane of most animal cells. Na⁺,K⁺-ATPase is constituted by the association of two major subunits, a catalytic α and a glycosylated β subunit, both of which exist as different isoforms (in mammals known as α1, α2, α3, α4, β1, β2 and β3). Na⁺,K⁺-ATPase α and β isoforms assemble in different combinations to produce various isozymes with tissue specific expression and distinct biochemical properties. Na⁺,K⁺-ATPase α4β1 is only found in male germ cells of the testis and is mainly expressed in the sperm flagellum, where it plays a critical role in sperm motility and male fertility. Here, we report the molecular structure of Na⁺,K⁺-ATPase α4β1 at 2.37 Å resolution in the ouabain-bound state and in the presence of beryllium fluoride. Overall, Na⁺,K⁺-ATPase α4 structure exhibits the basic major domains of a P-Type ATPase, resembling Na⁺,K⁺-ATPase α1, but has differences specific to its distinct sequence. Dissimilarities include the site where the inhibitor ouabain binds. Molecular simulations indicate that glycosphingolipids can bind to a putative glycosphingolipid binding site, which could potentially modulate Na⁺,K⁺-ATPase α4 activity. This is the first experimental evidence for the structure of Na⁺,K⁺-ATPase α4β1. These data provide a template that will aid in better understanding the function Na⁺,K⁺-ATPase α4β1 and will be important for the design and development of compounds that can modulate Na⁺,K⁺-ATPase α4 activity for the purpose of improving male fertility or to achieve male contraception. Full article

Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO⁺) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO⁻). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO⁻, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols. Full article

Spermatogenesis is a complex biological process crucial for male reproduction and is characterized by intricate interactions between testicular somatic cells and germ cells. Due to the cellular heterogeneity of the testes, investigating different cell types across developmental stages has been challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable approach for addressing this limitation. Here, we conducted an unbiased transcriptomic study of spermatogenesis in sexually mature 4-month-old Hezuo pigs using 10× Genomics-based scRNA-seq. A total of 16,082 cells were collected from Hezuo pig testes, including germ cells (spermatogonia (SPG), spermatocytes (SPCs), spermatids (SPTs), and sperm (SP)) and somatic cells (Sertoli cells (SCs), Leydig cells (LCs), myoid cells (MCs), endothelial cells (ECs), and natural killer (NK) cells/macrophages). Pseudo-time analysis revealed that LCs and MCs originated from common progenitors in the Hezuo pig. Functional enrichment analysis indicated that the differentially expressed genes (DEGs) in the different types of testicular germ cells were enriched in the PI3K–AKT, Wnt, HIF-1, and adherens junction signaling pathways, while the DEGs in testicular somatic cells were enriched in ECM–receptor interaction and antigen processing and presentation. Moreover, genes related to spermatogenesis, male gamete generation, sperm part, sperm flagellum, and peptide biosynthesis were expressed throughout spermatogenesis. Using immunohistochemistry, we verified several stage-specific marker genes (such as UCHL1, WT1, SOX9, and ACTA2) for SPG, SCs, and MCs. By exploring the changes in the transcription patterns of various cell types during spermatogenesis, our study provided novel insights into spermatogenesis and testicular cells in the Hezuo pig, thereby laying the foundation for the breeding and preservation of this breed. Full article

The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins. Full article

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. Full article