Rapid spermatogenesis and structural analysis of the manchette in Chinese soft-shelled turtle, Pelodiscus sinensis

Male infertility, often caused by structural and functional sperm defects, remains genetically unexplained in a substantial proportion of cases. ATP1A4 encodes a testis-specific isoform of the Na+, K+-ATPase, a membrane enzyme crucial for maintaining cellular ionic homeostasis. Previous studies on Atp1a4 knockout mice have demonstrated severe defects in sperm motility and flagellar architecture; however, the contribution of ATP1A4 variants to human male reproduction remains to be elucidated. In this study, we identified compound biallelic variants in ATP1A4, a missense variant (c.2578 T>A, p.Tyr860Asn) and a frameshift variant (c.2582del, p.Gly861Aspfs*5), in a patient presenting with severe oligoasthenoteratozoospermia. Both variants markedly affected ATP1A4 protein expression. Morphological analyses revealed coiled and folded flagella, disrupted mitochondrial sheaths, and irregular head morphology in the patient's spermatozoa. Expression profiling revealed that ATP1A4 was highly enriched in post-meiotic spermatids and localized along the entire flagellum of mature sperm in both humans and mice, indicating a critical role in flagellar assembly and structural integrity. Notably, intracytoplasmic sperm injection (ICSI) in this patient resulted in low fertilization efficiency and failed implantation, suggesting a potential adverse impact of ATP1A4 deficiency on sperm functional competence beyond motility. These findings broaden the genetic spectrum of oligoasthenoteratozoospermia and highlight ATP1A4 as a potential gene associated with human male infertility.

Integrating transcriptomics and metabolomics reveals the molecular landscape of sperm maturation driven by regional differentiation in the epididymis of Guizhou-Guiqian semi-fine wool sheep.

Nuclear pore complexes, composed of nucleoporins (Nups), are critical for bidirectional nucleocytoplasmic transport and regulate various cellular processes, thus contributing significantly to organismal development. Intriguingly, the integral Nup107 complex member, Nup43, is linked with premature ovarian insufficiency. Here, we report that Nup43 plays essential roles in Drosophila fertility, and Nup43 null mutant females and males are sterile. In mutant females, after fertilization with wildtype, mitotic progression during embryonic development is severely disrupted at early divisions, leading to irregular mitotic divisions. However, male sterility results from sperm development arrest at the canoe stage. In Nup43 mutants, sperm maturation is suspended, and nuclear elongation, nuclear shaping, and actin cone assembly aspects of individualization complex formation are adversely affected. Further, the Nup43 transgene completely rescues spermiogenesis defects in mutants. Myosin VI (jar), an actin-based motor, interacts with Nup43 and rescues the actin cone assembly but not the sterility defects seen in Nup43 null mutants. In essence, we report a novel non-canonical function for Nup43 in Drosophila fertility and spermiogenesis and propose that Nup43 facilitates sperm individualization by promoting jar-dependent actin cone assembly.

A stable connection between the sperm head (containing the nucleus and acrosome) and tail (containing the axoneme, mitochondrial sheath, and periaxonemal structures) is critical for fertility. This connection is mediated by a series of nuclear, cytoplasmic, and centriole components that make up the head-tail coupling apparatus (HTCA). Impairment of the HTCA leads to a severe type of infertility called acephalic spermatozoa syndrome, where sperm heads become detached from their tails. Acephalic spermatozoa syndrome can be found in species across the animal kingdom from flies to humans. Here, we review the literature on HTCA establishment and final HTCA architecture in mature sperm in different species, focusing on common themes throughout. Despite a large diversity of sperm structures and HTCA linkages in different species, we propose that there are significant commonalities and underlying themes that shape the HTCA throughout the animal kingdom. In particular, we emphasize the role of centrioles in mediating linkage between the head and tail. Given that the molecular architecture of the HTCA is poorly understood, focusing on commonalities of HTCA structure and development is important for extrapolating data from model organisms to combat human infertility.

Metabotropic glutamate receptors in the testis: An integrative bioinformatic review of neuroendocrine, reproductive, and Neuropsychopharmacological aspects.

To determine the gamete-specific etiology of severe or total fertilization failure after intracytoplasmic sperm injection (ICSI) and to evaluate the efficacy of individualized assisted oocyte activation (AOA) strategies guided by sperm phospholipase C-zeta (PLCζ) content. A pilot topographic analysis on PLCζ content in ejaculated, epididymal, and testicular spermatozoa was performed to better understand the fertilization capability of spermatozoa at different maturational stages. The prospective case series included 135 couples with complete or severe fertilization failure following ICSI, who were screened for PLCζ content and allocated as having oocyte- or sperm-related deficiency based on PLCζ assay results. According to gamete-specific deficits, cycles were treated with AOA, that is, ionophore-only, rhPLCζ-only, or combined treatment. Embryological and clinical outcomes of AOA cycles were compared with those of historical non-AOA ICSI cycles. PLCζ content varied according to the maturity and origin of spermatozoa in the pilot study. Compared with historical cycles, AOA using ionophore and rhPLCζ each rescued fertilization and improved pregnancy outcomes with comparable efficiency. Notably, dual AOA (ionophore + rhPLCζ) yielded the greatest improvements in fertilization (24.1% vs. 73.2%, P < 0.0001), implantation (6.5% vs. 31.7%, P < 0.01), and ongoing pregnancy rates (0% vs. 63.3%, P < 0.0001), even in cycles using testicular spermatozoa. The introduction of recombinant PLCζ enabled specific correction of gamete-specific fertilization failure. Dual AOA (ionophore + rhPLCζ) was particularly effective in cases of compounded etiology and with immature spermatozoa, supporting a shift from empiric to individualized AOA strategies.

ABSTRACT Omphisa fuscidentalis is a univoltine moth whose larvae undergo an unusually prolonged diapause (~9 months) inside bamboo internodes – a unique life‑history trait that may shape its reproductive biology. Despite its ecological and economic importance as an edible insect, little is known about its reproductive system. This study presents the first detailed analysis of testis development and sperm ultrastructure across larval, prepupal, and adult stages using light, scanning electron, and transmission electron microscopy. We describe a novel transformation from paired testes with four follicles in larvae to a fused single testis with eight follicles in the prepupal stage. Furthermore, we document sperm dimorphism in detail, highlighting structural specializations in both eupyrene and apyrene spermatozoa. Noteworthy features include micronuclei formation during apyrene spermiogenesis, a dense cap in mature apyrene sperm, a shoe‑sole-shaped mitochondrial derivative, and a developmental shift in eupyrene flagella from a hand-like to a sunshine-like configuration. These ultrastructural changes likely reflect adaptive strategies associated with diapause. By integrating multi-scale imaging, this work offers new insights into the reproductive development of O. fuscidentalis and provides a valuable reference for future comparative studies on sperm evolution and reproductive adaptations in insects with complex life cycles.

Spermatogenesis is a highly energy-dependent and tightly regulated differentiation process in the male reproductive system, characterized by dynamic, stage-specific metabolic adaptations during spermatogonial proliferation, meiosis and sperm maturation. Accumulating evidence suggests that successful spermatogenesis is associated with a coordinated program of metabolic reprogramming, which involves the sequential and context-dependent utilization of distinct bioenergetic pathways rather than dependence on a single energy source. Importantly, metabolic reprogramming represents a physiological and tightly controlled process, whereas metabolic dysfunction arises from its disruption or dysregulation. Studies using mouse models, supported by single-cell omics and metabolic analyses, indicate stage-associated transitions from glycolysis to oxidative phosphorylation (OXPHOS), followed by increased utilization of alternative substrates such as fatty acids and amino acids during germ cell development. These transitions are orchestrated by interconnected networks involving energy-sensing pathways, endocrine regulation and metabolite-driven epigenetic modifications. Disruption of this finely tuned metabolic reprogramming leads to metabolic dysfunction, characterized by oxidative stress, mitochondrial impairment, meiotic defects and chromatin instability, ultimately compromising spermatogenic homeostasis and contributing to idiopathic male infertility. Consistent evidence from experimental and clinical studies further suggests that systemic metabolic disorders, including obesity, diabetes, and dyslipidaemia, as well as exposure to endocrine-disrupting chemicals, may impair metabolic coupling between Sertoli cells and germ cells, thereby exacerbating testicular metabolic imbalance. These disturbances exacerbate mitochondrial dysfunction, one-carbon metabolic imbalance, and blood-testis barrier (BTB) disruption, ultimately leading to reduced sperm quality and fertility. Building on these insights, we propose a metabolism-centred classification framework that stratifies idiopathic male infertility into glycolysis-impaired, OXPHOS-deficient, lipid-overload, and one-carbon metabolism-dysregulated subtypes. We further discuss the translational potential of targeting metabolic pathways through mitochondrial-directed antioxidants, modulation of energy-sensing signalling, and nutritional interventions. Finally, we highlight the need for future studies integrating in vivo metabolic imaging, testicular organoid models and non-invasive seminal metabolomic biomarkers, alongside well-designed clinical trials, to advance metabolism-based precision diagnostics and therapeutics for male infertility.

Caseinolytic peptidase P (ClpP) plays a key role in maintaining cellular homeostasis for mitochondrial quality control. However, the specific function of ClpP during meiosis and its subcellular localization in spermatocytes remain poorly understood. To investigate the function of ClpP in spermatocyte meiosis. ClpP expression was examined in mouse spermatocytes, and tamoxifen was utilized to achieve spatiotemporal-specific deletion of Clpp mediated by Ddx4-CreERT2 in spermatocytes. We analyzed the meiotic progression of Clpp conditional KO (ClppcKO) using spermatocyte chromosome spreading, combined with immunofluorescence and transmission electron microscopy, to determine the morphology and number of spermatocyte mitochondria in ClppcKO mice. A progressive increase in ClpP expression levels was evident from the leptotene stage to the pachytene stage in mouse spermatocytes, and a decrease in ClpP expression was observed from the diplotene stage to the metaphase I (MI) stage. Compared with wild-type male mice, adult ClppcKO male mice had reduced testis size and no mature spermatozoa in their epididymides. A large proportion of pachytene and diplotene spermatocytes, as well as round or elongated spermatids, were eliminated from the seminiferous tubules of the ClppcKO mice. The mitochondria of ClppcKO spermatocytes appeared as "giant mitochondria." However, ClppcKO spermatocytes exhibited normal meiotic synapsis and impaired recombination, with reduced RAD51 foci and abnormal MLH1 localization. ClpP is critical for spermatocyte survival and mitochondrial integrity during meiosis. As a consequence of its deficiency, meiotic progression and spermatogenesis are disrupted, highlighting its essential role in the meiosis of spermatocytes.

The evolutionary timing of the origin of secretory proteins underlying post-mating reproductive processes remains uncharacterized in vertebrates. We dated the origin of 2520 human tissue-specific genes encoding secretory proteins across vertebrate evolution, finding that the male reproductive (MR) secretome underwent a dramatic expansion during the eutherian (placental mammal) diversification, experiencing a 6.8-fold gene gain-the largest increase compared to any other secretome or transition in vertebrate evolution. These genes are predominantly expressed in the epididymis, where they protect sperm and drive sperm maturation, influencing essential post-mating reproductive processes and male reproductive outcome. In contrast, MR secretome genes that originated along other evolutionary branches are primarily associated with sperm structure, motility, egg binding, and fusion. These findings provide molecular evidence for a major reconfiguration of male reproductive biology during placental mammal diversification through the MR secretome, affecting primarily the epididymis.

In brief: As a major family of RNA binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in the entire RNA metabolism process and have been increasingly recognized as essential regulators of spermatogenesis. This review systematically summarizes the latest advances in the functions, molecular mechanisms, and clinical applications of hnRNPs in mammalian spermatogenesis. Abstract: Spermatogenesis in mammals is a complex and highly orchestrated process of cellular development that necessitates precise regulation at the transcriptional, post-transcriptional, and translational levels. RNA binding proteins (RBPs) are integral to this process, particularly due to the occurrence of two transcriptional arrest phases in spermatogenic cells. Among these RBPs, heterogeneous nuclear ribonucleoproteins (hnRNPs) constitute a significant family that engages in the entire RNA metabolism process through various RNA-binding domains. Recent research has underscored their essential role in mammalian spermatogenesis. This article provides a comprehensive review of the expression patterns of hnRNP proteins and elucidates their mechanisms of action during critical stages of spermatogenesis, including the maintenance and differentiation of spermatogonial stem cells, meiosis in spermatocytes, and the formation and maturation of sperm. It further examines their functions in supporting cellular development, proliferation, and the maintenance of the blood-testis barrier. Additionally, the article explores the association between aberrant hnRNP proteins and male infertility, highlighting their potential clinical applications, and summarizes recent advancements in the field. This review provides theoretical references for in-depth analysis of the molecular regulatory network of spermatogenesis, and also opens up new directions for clinical diagnosis and treatment of male infertility.

Comprehensive S-acylation profiling of the porcine epididymis and exosomes reveals a role in cargo sorting and long-distance trafficking.

Decreased autophagic activity in spermatozoa from infertile men adversely affects early embryonic development.

Fertility Considerations for Gender-Diverse People.

Graphical We examined SARS-CoV-2 infection in the epididymis of K18-hACE2 mice, focusing on the cauda region. Thirty mice were divided into control (CG), 2-day infection (2D), and 5-day infection (5D) groups. The animals were intranasally infected with SARS-CoV-2. The 5D group showed a significant increase in IL-6, TNF-a, and IL-1b, high mRNA levels of Ifna, Ifnb, and Ifng, and infiltration of CD8+ T cells. SARS-CoV-2 directly infects smooth muscle cells, leading to atrophy of the smooth muscle layer. The infection impaired the basement membrane and downregulated blood–epididymis barrier-related genes Ocln and Jam-a. Clear cells (CCs) exhibited increased apical V-ATPase and immunoexpression of hACE2, spike, and nucleocapsid. Interestingly, the infected groups showed a greater number of juxtaposed CCs with PCNA- and Ki67-positive nuclei. The findings also revealed a newly identified NF-kB-driven inflammatory-proliferative response in CCs. By combining in vivo and in silico analysis of a parallel LPS-stimulation RNA-seq dataset, we uncovered significant co-enrichment of the TNF-a–NF-kB pathway alongside cell cycle pathways (G2/M, E2F). The protein–protein interaction network identified key nodes linking the inflammatory NF-kB pathway to the cell cycle machinery. This in silico model was confirmed in vivo, demonstrating colocalization of phosphorylated NF-kB and PCNA in the CC rows. Alterations in spermatozoa, including decreased PNA labeling, ultrastructural changes, and reduced Crisp1 mRNA, were also observed. Therefore, SARS-CoV-2 infection causes structural and functional changes in the epididymal cauda, leading to smooth muscle cell death and basement membrane disruption. Overall, the results shows that SARS-CoV-2 infection induces dysfunction in the cauda, and points CCs as players in the epididymal immune response.Lay summarySARS-CoV-2, the virus that causes COVID-19, can reach many organs beyond the lungs, including the male reproductive tract. We studied how this virus affects epididymis, a sperm-carrying tube where sperm mature and are stored, in a genetically modified mouse model that expresses the human ACE2 receptor, which allows SARS-CoV-2 infection. We demonstrated that infection triggers a strong local inflammatory response and impairs the muscular wall and spermatozoa. The virus also infects specialized clear cells that control the epididymal fluid pH where sperm are stored. Following infection, clear cell groups showed proliferation and possible acidification of the sperm storage microenvironment. All these combined changes suggest that the normal environment required for sperm maturation is disrupted. Our findings raise concern that severe COVID-19 may have lasting effects on male fertility and highlight the epididymis as an important but overlooked target organ of this infection.

Spermatogenesis is precisely regulated by an intricate genetic network, but the biological roles of numerous testis-enriched genes remain unelucidated. This study aimed to systematically investigate the expression pattern, subcellular localization and functional significance of Testis-Expressed Gene 29 (Tex29) in mice and male fertility. Integrated molecular, cellular, and animal model approaches were employed. Tex29 mRNA and protein expression were analyzed by molecular and immunofluorescence staining techniques. CRISPR/Cas9-mediated genome editing was used to generate Tex29-knockout (KO) mice. Fertility assessment, histological examination of testes, sperm quality analysis, and transmission/scanning electron microscopy were performed on Tex29-KO mice. Additionally, whole-exome sequencing was conducted in 165 infertile men to identify Tex29 variants. Tex29 mRNA was specifically expressed in testes, first detectable on postnatal day 18 and gradually upregulated during testicular maturation. TEX29 protein was specifically localized to the acrosome of spermatids and mature sperm throughout spermiogenesis. Tex29-KO males exhibited normal fertility with litter sizes comparable to wild-type (WT) controls, and their seminiferous tubules retained intact structure with all spermatogenic stages. No significant differences in sperm concentration, viability, or motility were observed between Tex29-KO and WT mice. Although Tex29-KO sperm maintained normal overall morphology and canonical "9 + 2" axonemal structure in the flagellum, a subset showed acrosomal membrane abnormalities in the apical region. In vitro fertilization (IVF) rates and blastocyst development were uncompromised in Tex29-KO mice. Two synonymous TEX29 variants (c.66C > T, p.Asp22Asp; c.207C > A, p.Ile69Ile) were identified in 7 of 165 infertile men, and four couples carrying these variants achieved live births. TEX29 is a novel testis-specific acrosomal marker protein essential for maintaining normal acrosomal membrane integrity during murine spermiogenesis. Notably, Tex29 is dispensable for spermatogenesis and male fertility in mice. The functional role of TEX29 in human spermatogenesis and fertility remains to be fully determined due to limited clinical evidence. These findings provide valuable insights for basic research on acrosome biogenesis and male infertility associated with acrosomal abnormalities.

Capacitation is a key maturation process that enables spermatozoa to acquire fertilizing ability and can be induced in vitro using capacitation media. Because capacitation protocols differ markedly among laboratories, we compared three compositionally distinct Hepes-, Tris-, and TALP-based media. This study was performed in boar spermatozoa using 3–6 biological replicates of pooled ejaculates depending on the assay, with 46 ejaculate samples from 12 boars in total. The aim was to determine whether such non-standardized conditions differentially affect signaling pathways leading to capacitation and thereby influence the detection of commonly used capacitation markers. We found clear differences among the tested media. All three induced capacitation-associated events, but their functional and molecular effects were not equivalent. The Hepes-based medium supported sperm motility most effectively, increasing total and progressive motility to 60.0% and 48.7%, respectively, after 1 h of incubation and maintaining the highest motility throughout the incubation period. In contrast, the Tris-based medium maintained lower but relatively stable motility, whereas the TALP-based medium showed a rapid decline in total motility from 53.1% to 15.2% during the first hour. The TALP-based medium induced the highest and most sustained protein kinase A (PKA) activity, reaching 0.047 U/mL at 0 h and 0.040 U/mL after 3 h, whereas the Hepes- and Tris-based media showed lower and less sustained activity ranging from 0.003 to 0.030 U/mL during incubation. In addition, distinct patterns of protein tyrosine phosphorylation were observed depending on the medium used. In particular, the TALP-based medium containing bicarbonate and bovine serum albumin (BSA) and the Hepes-based medium with the highest BSA concentration were associated with the highest levels of total protein tyrosine phosphorylation. Phosphoproteomic analysis further revealed condition-specific phosphorylation events, indicating that sperm maturation is dynamically regulated by the surrounding molecular environment. In contrast, no significant differences were detected in oxidative phosphorylation or in electron transport system complexes among the tested media. These findings show that differences in capacitation media composition, particularly in bicarbonate and BSA content, can markedly alter signaling outcomes and the interpretation of capacitation markers, with important implications for reproductive technologies and experimental standardization.

With the advancement of next-generation sequencing technologies, transfer RNA (tRNA)-derived small RNAs (tsRNAs) have been progressively elucidated in their biogenesis and classification. tsRNAs are derived from precursor tRNAs (pre-tRNAs) or mature tRNAs and are classified into tRNA-derived fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs) based on their specific endonuclease cleavage sites and are functionally involved in diverse biological processes. tsRNAs are widely distributed within cells and are also present in the male reproductive system. Specifically, tRFs are the predominant tsRNA subspecies with established regulatory functions, whereas tiRNAs are less abundant and their roles remain less characterized. Notably, tsRNAs are particularly abundant in mature sperm, serving as vectors of paternal epigenetic inheritance. Multiple studies have shown that environmental factors and dietary modifications can significantly alter tsRNA profiles in the male reproductive system. Moreover, sperm tsRNAs regulate embryonic quality and are closely associated with male fertility. Collectively, these findings reveal the diverse roles of tsRNAs in male reproductive health and their potential as clinical biomarkers.

To characterise histological patterns and heterogeneity in testicular biopsies from azoospermic men, assess bilateral concordance, evaluate morphometric parameters for a structured reporting template and place findings in context with the existing literature on testicular biopsy evaluation. Retrospective cross-sectional study of 112 men with non-obstructive azoospermia who underwent open testicular biopsy at a single centre between 2006 and 2019. Archived H&E-stained slides were digitised and re-evaluated. Morphological changes were classified into predefined categories at whole-biopsy and individual tubule level, enabling detailed characterisation of hypospermatogenesis and mixed atrophy. Mean seminiferous tubule diameter, lamina propria thickness and interstitial Leydig cell quantity were measured and compared between histological groups. Bilateral biopsies were obtained in 104/112 patients (92.9%), and 26.9% showed discordant histological patterns. Mature spermatozoa were present bilaterally in 43.8% and unilaterally in 7.7% of bilaterally biopsied patients. Hypospermatogenesis was the predominant pattern (42.9%), followed by normal spermatogenesis (19.6%), Sertoli cell-only syndrome (23.2%), maturation arrest (12.5%) and tubular hyalinisation (1.8%). Two-thirds of hypospermatogenesis biopsies had heterogeneous mixed patterns. Mixed atrophy, a non-spermatozoa-producing heterogeneous pattern, was subdivided into 10 clusters based on the relative proportions of tubular types. Greater spermatogenic impairment was associated with reduced tubule diameter, increased lamina propria thickness and Leydig cell hyperplasia. Testicular biopsies in azoospermic men show marked intratesticular and intertesticular heterogeneity, and bilateral sampling reveals clinically relevant discordance in many patients. Simple quantitative morphometry complements qualitative assessment. A structured reporting template may standardise terminology, improve interobserver agreement and support evidence-based decisions in daily male infertility care.