IntroductionEnvironmental factors mediated testicular vacuolization injury is a prevalent occurrence, and its etiology and mitigation strategies have long remained inadequately elucidated.MethodsA testicular injury model was established using Triptolide (TP), with Icariin (ICA) employed as a rescue compound. Function and mechanism investigations primarily employed testicular coefficient and sperm concentration, H&E staining, immunofluorescence staining, and western blot analysis. Single-cell RNA-sequencing (scRNA-seq) predominantly examined the impacts of TP and ICA on various cell populations within the testis at a single-cell resolution.ResultsWe successfully established a TP-induced model of testicular injury and identified ICA as a protective agent in alleviating testicular vacuolization. Moreover, we delineated a comprehensive single-cell transcriptome profile of ICA in the repair of testicular injury, revealing the pivotal role of Sertoli-germline communications in the genesis of testicular vacuolization and the reparative process mediated by ICA. Furthermore, our investigation unveiled that ICA mitigated TP-induced damage to niche integrity through signatures associated with the blood-testis barrier (BTB), thereby averting substantial germ cell loss via the germline associated-ferroptosis signatures, potentially a key factor in the occurrence of testicular vacuolization injury. Additionally, we also identified ferroptosis-related molecules for testicular vacuolization injury.DiscussionWe suggest that TP-induced testicular injury disrupts the spermatogenic microenvironment mediated by the BTB, leading to the formation of testicular vacuoles through the ferroptosis pathway in germ cells. Our findings offer fresh perspectives for ICA on mitigating this process.

The ubiquitin-proteasome system (UPS) represents an evolutionarily conserved machinery governing proteostasis through spatiotemporal regulation of protein degradation. While spermatogenesis involves multilayered regulatory mechanisms spanning translation to dynamic post-translational modifications (PTMs), the identity of UPS-associated E3 ligases orchestrating germ cell-specific protein turnover remains elusive. Here, we identify a testis-specific E3 ubiquitin ligase complex comprising elongin B/C, Cullin-2 (CUL2), RING-box protein-1 (RBX1), and SOCS box protein ASB9, designated ECSASB9. Genetic ablation of ECSASB9 in mice via ubiquitous Asb9 knockout (KO) or spermatid-specific elongin B/C conditional KO disrupts spermiogenesis and compromises fertility. Mechanistic studies reveal that ECSASB9 engages tubulin beta 4 A (TUBB4A) through substrate recognition, catalyzing K48-linked polyubiquitination at lysine 379 (K379) to promote proteasomal degradation. Notably, Tubb4aK379R knock-in (KI) mice phenocopy the spermiogenesis defects observed upon ECSASB9 deficiency. Clinically, we identify three hemizygous missense variants in X-linked ASB9 among Chinese males with idiopathic infertility. Male mice bearing orthologous ASB9 variant exhibit oligoasthenoteratozoospermia (OAT) and subfertility, mirroring human phenotypes. Taken together, our findings establish ECSASB9 as an important regulator of spermatogenic proteostasis and provide mechanistic insights into UPS-mediated tissue-specific degradation, while implicating ASB9 variants in male infertility pathogenesis.

AbstractThe Drosophila stem cell niche harbours two principal stem cell populations: germline stem cells (GSCs) and cyst stem cells (CySCs), whose self-renewal and differentiation are stringently governed by niche-derived regulatory factors. Nevertheless, the mechanistic role of the 20S core particle (CP)—the catalytic core of the 26S proteasome—within this niche remains poorly elucidated. In this study, we reveal that three 20S CP subunits, Prosα5, Prosβ2 and Prosβ5, mediate non-cell autonomous effects in the niche. Loss of function of Prosα5, Prosβ2 or Prosβ5 in cyst cells disrupts CySC differentiation, impairs early-stage germline differentiation and culminates in testicular dysgenesis, aberrant GSC-like cluster formation and male sterility. Moreover, we establish that diminished levels of these proteasome subunits trigger the accumulation of cell adhesion molecules and Cyclin proteins. Collectively, our findings offer novel insights into the regulatory functions of the 20S CP within the Drosophila testicular stem cell niche.

Sertoli cell-only syndrome (SCOS) represents the most severe form of non-obstructive azoospermia and is closely associated with developmental abnormalities in prospermatogonia (ProSG). However, the mechanisms governing ProSG development remain incompletely understood. Here, we demonstrate that WSB2 plays a critical regulatory role in ProSG development through a non-canonical mechanism independent of its established function as a substrate-recognition receptor for the Elongin B/C-Cullin-SOCS (ECS) E3 ubiquitin ligase complex. Wsb2 knockout (Wsb2 KO) mice exhibited rapid depletion of ProSG and SCOS-like phenotypes. Mechanistically, loss of WSB2 led to significantly decreased expression of the O-GlcNAcase MGEA5 in ProSG, resulting in elevated O-GlcNAcylation levels and subsequent DNA double-strand break (DSB) formation. We further found that WSB2 stabilizes MGEA5 by competitively binding to it and thereby preventing its ubiquitination by the E3 ligase XIAP. Importantly, treatment with the O-GlcNAc transferase inhibitor OSMI-1 effectively restored the ProSG population in Wsb2 KO mice. Collectively, this study uncovers a previously uncharacterized non-canonical function of WSB2, a conventional ECS complex subunit, in regulating ProSG development and establishes a critical link between O-GlcNAcylation homeostasis and germ cell maintenance.

Background: Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are systemic, autoimmune inflammatory diseases. Although overlapping clinical and genetic features have been reported, the presence of a direct causal relationship between them remains uncertain. Methods: A bidirectional two-sample Mendelian randomization (MR) analysis was performed using genetic variants as instrumental variables to evaluate potential causal associations between RA and SLE. Sensitivity analyses were performed to assess heterogeneity, horizontal pleiotropy and the robustness of the findings. Results: Genetically predicted RA did not have a significant causal effect on susceptibilityto SLE. Conversely, genetically predicted SLE did not demonstrate a significant causal effect on RA risk. Sensitivity analyses supported the stability and reliability of these findings, with no evidence of substantial heterogeneity or directional pleiotropy. Conclusion: We concluded that RA and SLE may share genetic susceptibility and environmental triggers without a unidirectional causal relationship.

The decline in testicular function with age has raised significant concerns. Long non-coding RNA (lncRNA) influences a wide array of physiological processes, including spermatogenesis. Nevertheless, the precise roles and regulatory mechanisms of lncRNA in testicular aging remain elusive. This investigation delves into the function of lncRNA:CR43306 in governing spermatogenesis during testicular aging. Depletion of lncRNA:CR43306 in 40-day-old Drosophila testes resulted in the hindrance of spermatogenesis, particularly affecting elongated spermatids, leading to functional senescence. Additionally, differentially expressed gene (DEG) expression analysis through bulk RNA-seq unveiled genes linked to elongated spermatids and cell adhesion. Our findings underscore the pivotal role of lncRNA:CR43306 in testicular aging by influencing cell adhesion. These discoveries illuminate the regulatory pathways of lncRNA in testicular aging and offer valuable insights for potential studies and therapeutic interventions.

The biochemical composition underlying cytoplasmic residual body (RB) and arrested spermatogonia remains unresolved in spermatogenesis, in part due to limited approaches for intact-tissue metabolic interrogation. Here we develop Super-multiplexed Label-free Raman Imaging (SLRI) to generate 2D/3D biochemical maps of intact Drosophila testes. SLRI delineates major testicular cell types and provides the first in situ, stage-resolved mapping of cytoplasmic RBs at two discrete phases. We further identify a differentiation-arrest-associated metabolic trajectory in spermatogonial clusters, characterized by coordinated spatiotemporal decay of specific biochemical species. Across 42 spatiotemporally variant metabolites and metabolic pathways, SLRI reveals RB- and arrest-enriched signatures. Notably, RBs and arrested spermatogonia display significant biochemical overlap, alongside unique spectral signatures. Furthermore, we uncovered a ring-like, punctate arrangement of lipids, amino acids, and vitamins encircling RBs, suggesting structured metabolite partitioning during cytoplasmic remodeling. Collectively, SLRI establishes a general framework for high-dimensional metabolic mapping and remodeling analysis in intact tissues.

Non-obstructive azoospermia represents one of the most intractable forms of male infertility, with its onset frequently associated with meiotic abnormalities. To date, the pivotal molecular mechanisms governing meiosis remain incompletely understood. This study demonstrates that CMTR2 (Cap methyltransferase 2) exerts a critical regulatory function in male germ cell meiosis through a mechanism independent of its methyltransferase activity. Germ cell-specific Cmtr2 knockout mice (Ddx4-GcKO) exhibited meiotic prophase I zygotene arrest and male sterility. Ddx4-GcKO spermatocytes displayed phenotypic abnormalities, including defective DNA double-strand break repair and compromised crossover formation and recombination. Subsequent investigations revealed a marked reduction in the messenger RNA (mRNA) stability of key meiotic genes, Speedy/RINGO cell cycle regulator family member A (Spdya) and moloney leukemia virus 10 Like 1 (Mov10l1), in Ddx4-GcKO testes. Mechanistic studies indicated that CMTR2 directly interacts with the 5 ' untranslated regions of these genes. Importantly, CMTR2 selectively mediates 2 '-O-methylation of Spdya mRNA, whereas its regulation of Mov10l1 mRNA stability is independent of its enzymatic activity. This study is the first to uncover the non-canonical role of CMTR2 in meiotic regulation, enhancing our comprehension of meiotic molecular mechanisms and offering novel insights into the etiology of male sterility.

Recent research has underscored the widespread presence of plastic in diverse ecosystems, prompting concerns about its potential long-term impacts on organisms. However, the reproductive toxicity of polystyrene nanoparticles (PSNPs) is contingent on factors, such as drug concentrations, exposure duration, delivery method, and PSNP characteristics. Consequently, the evaluation of testicular toxicity stemming from chronic PSNP exposure warrants a systematic investigation. This study extensively probed the potential toxicity of PSNPs on testes using multiexposure models. Three distinct long-term exposure paradigms (adult, gestational, and cross-generational exposure) were executed to assess PSNP-induced testicular toxicity (80 nm) in Drosophila. Nevertheless, spermatogenesis exhibited no discernible alterations following exposure to 75, 150, and 300 mu g/mL of PSNPs in the adult and gestational exposure models. Furthermore, cross-generational exposure to PSNPs did not perturb testicular phenotype or offspring fertility from the F1 to F4 generations. These findings imply that chronic consumption of low-dose PSNPs may not suffice to induce testicular dysfunction, providing fresh insights into the dosage and duration of PSNP exposure.

Endometriosis involves ectopic growth of endometrial-like tissue, yet the spatial transcriptomic and metabolic landscape of ovarian endometriomas remains poorly understood. This investigation presents a comprehensive multi-omics analysis of ovarian endometriomas incorporating single-cell RNA sequencing in conjunction with Digital Spatial Profiler-Whole Transcriptome Atlas for spatial transcriptomics, and non-targeted Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging for spatially resolved metabolomics. Our integrated analysis confirms the importance of cell adhesion, ECM-receptor interaction, and focal adhesion pathways in disease context. We identify XBP1, VCAN, and CLDN7 as key markers in epithelial cells, and THBS1 in perivascular cells. Spatially resolved metabolomics further reveals altered activity of cytochrome P450 enzymes, lipoprotein particles, and cholesterol metabolism in mesenchymal regions, along with several undefined metabolites enriched in epithelial areas of endometriomas compared to ovarian cortex controls. These findings reveal potential markers and metabolic pathways linked to ovarian endometriomas, offering a foundation for future diagnostic and therapeutic strategies.

BACKGROUND:Human spermatogonial stem cells (SSCs) exhibit a remarkable capacity for proliferation, crucial for sustaining spermatogenesis throughout life. While the Cullin-RING E3 ubiquitin ligase 2 (CRL2) complex is known to regulate various cellular functions, its precise role in human SSCs has not been fully elucidated. This study aimed to investigate a novel variant of the CRL2 complex, termed CRL2LRRC41, and its role in SSC function. METHODS:We utilized molecular biology techniques, including gene knockdown and functional assays, to assess the effects of CRL2LRRC41 on the proliferative and migratory abilities of human spermatogonial stem cell-like cell (SSCLC) line. Additionally, we employed proteomics and biochemical approaches to identify potential substrates of CRL2LRRC41. We specifically focused on ATP-dependent RNA helicase DDX5, a known regulator of spermatogenesis, to explore its interaction with CRL2LRRC41 and the downstream molecular mechanisms involved. RESULTS:Our findings revealed that the disruption or dysfunction of CRL2LRRC41 led to reduced proliferative and migratory abilities in human SSCLCs. Through our investigation, we identified DDX5 as a ubiquitination substrate of CRL2LRRC41. Notably, the ubiquitination of DDX5 fosters its interaction with the RNA-binding protein ELAVL1, without directing DDX5 towards degradation via the ubiquitin-proteasome system (UPS). This interaction enhances the stability of the downstream transcript, Noggin (NOG), thereby supporting human SSCLC proliferation and migration. CONCLUSIONS:This study provides the first identification of the CRL2LRRC41 complex in human SSCLCs and elucidates the molecular mechanisms by which CRL2LRRC41 facilitates SSCLC function via ubiquitination-mediated protein interactions. These findings offer novel insights into the molecular underpinnings of male infertility.

Recent studies have highlighted RNA modifications as integral regulators of gene expression during spermatogenesis. Ribosomal RNAs (rRNAs) are the most abundant RNA in cells, while the function and clinical relevance of rRNA modifications in spermatogenesis remain poorly understood. Here, we identified 4 pathogenic heterozygous variants of METTL5 in 1,427 patients with male infertility characterized as oligoasthenoteratozoospermia (OAT). The pathogenic variants of METTL5 led to the significantly decreased expression level of METTL5. Null mutation of 18S rRNA methyltransferase Mettl5 led to male infertility attributed to OAT during spermiogenesis, presenting defects in both the sperm head and tail. Notwithstanding the absence of notable changes in global translation after Mettl5 loss, we observed a compromised translational efficiency of mRNAs encoding proteins crucial for spermiogenesis, including Gk2, Akap4, Fsip2, Odf2, and Pgk2. Intriguingly, therapeutic interventions via intracytoplasmic sperm injection in OAT couples with these variants resulted in successful pregnancies. These insights not only identify METTL5-mediated 18S rRNA m6A modification as a novel genetic determinant for OAT but also offer a new target to the genetic counseling, clinical diagnosis, and potential treatment strategies for male infertility.

Recent evidence consolidates the deleterious impact of environmental exposure on testicular damage. Environmental exposures can instigate testicular toxicity, causing damage to the Sertoli-Sertoli cell-mediated blood-testis barrier (BTB) integrity, alterations in hormone levels orchestrated by aberrant Leydig cells, and disruption of spermatogenesis. Despite diverse study designs and methodologies, a consensus is emerging on how environmental factors induce oxidative stress by elevating ROS levels, affecting autophagy through pathways such as the ROS-mediated mTOR signaling pathway, ultimately culminating in testicular damage. This review synthesizes existing literature on how environmental exposures, including metals, air pollutants, industrial contaminants, and pesticides, disturb testicular homeostasis via autophagy-mediated oxidative stress, highlighting recent significant advancements. It also explores interventions like antioxidant support and autophagy regulation to alleviate testicular damage. These findings underscore the importance of elucidating the mechanisms of autophagy influenced by environmental exposures in disrupting the equilibrium of oxidative stress, identifying potential drug targets, and establishing a groundwork for enhancing future treatments and clinical management of testicular injuries.

Mesenchymal cells constitute the primary structural support elements within endometriotic lesions, yet their pivotal roles in endometriotic pathogenesis remain largely uncharted. This study aimed to construct a single-cell atlas of endometriosis using samples from three ovarian tissues affected by endometriosis and three normal ovarian tissues. Through the utilization of scRNA-seq, we have unveiled six distinct mesenchymal subclusters in normal and endometriosis-afflicted ovaries, elucidating the diverse functions of mesenchymal populations in endometriosis. Our comprehensive analysis has revealed that mesenchymal cells predominantly engage in three key functions: ribosome-mediated protein synthesis and processing, cell adhesion facilitating intercellular support and communication, and a range of metabolic processes. Furthermore, our findings have identified several pivotal differentially expressed genes (e.g. C3, FN1, COL3A1, COL1A1, NRXN3), primarily associated with the complement and coagulation cascades, extracellular matrix (ECM) regulation, ECM receptor interactions, and cell adhesion molecules. In essence, our study provides a comprehensive transcriptomic dataset and novel insights into adhesive molecule and integrin networks within mesenchymal subclusters in endometriosis. This, in effect, has deepened the understanding of the pathomechanisms governing this condition.

Testicular aging has profound effects on spermatogenesis, sperm function, and the spermatogenic microenvironment, contributing to reduced male fertility. However, the precise molecular mechanisms by which mitochondria influence spermiogenesis during aging still remain largely unclear. Vha68-3 KO flies were generated using the CRISPR/Cas9 technique. Testicular phenotypes and functions were mainly observed through immunofluorescence staining and transmission electron microscopy. Multi-omics study was mainly conducted through single-cell RNA sequencing and transcriptome–metabolomics association analysis. Vha68-3 binding proteins were identified via liquid chromatography–tandem mass spectrometry. The therapeutic potential of modulating mitochondrial metabolism for testicular aging mainly relied on the dietary intake of related compounds in fruit flies. In this study, we identified Vha68-3, a testis-specific subunit of the V-type adenosine triphosphate (ATP) synthase, predominantly localized in the tails of elongated spermatids, as a key age-related regulator of male fertility and spermatid elongation in Drosophila testes. Crucially, Vha68-3 deficiency impaired mitochondrial homeostasis in elongated spermatids during testicular aging. Through a multi-omics approach, including single-cell transcriptomics, protein interaction mapping of Vha68-3, and transcriptome–metabolome integration, we identified pyruvate metabolism as a critical pathway disrupted by Vha68-3 deficiency. Moreover, dietary supplementation with pyruvate (PA), S-lactoylglutathione (SLG), and phosphoenolpyruvate (PEP) effectively alleviated mitochondrial dysfunction and testicular aging linked to Vha68-3 deficiency. Our findings uncover novel mechanisms by which mitochondrial metabolism regulates spermatid elongation and propose potential therapeutic strategies to combat mitochondrial metabolic disorders in aging testes.

The E3 ubiquitin ligase RNF187, also known as RING domain AP1 coactivator-1, is a member of the RING finger family. RNF187 is indispensable for the proliferation and migration of GC-1 cells derived from mouse spermatogonia and GC-2 cells derived from spermatocytes. However, it remains unclear whether RNF187 plays a crucial role in the self-renewal and migration of human spermatogonial stem cells (SSCs). In this study, we observed a positive correlation between RNF187 expression and the proliferation and migration of human SSCs. Through co-immunoprecipitation and mass spectrometry analyses, we identified WD repeat-containing protein 77 (WDR77) as an interacting partner of RNF187. Specifically, RNF187 recognises the K118 site of WDR77 through lysine 48-linked polyubiquitination, subsequently mediating its degradation via the ubiquitin-proteasome system (UPS). Further studies have revealed that decreased expression of WDR77 diminishes the symmetric dimethylation at H4R3 (H4R3me2s) catalysed by its interacting protein, the arginine methyltransferase PRMT5. This, in turn, relieves the transcriptional repression of early growth response protein 1 (EGR1), a positive regulator for human SSC maintenance. In conclusion, this study has unveiled a pivotal role for RNF187 in the proliferation and migration of human SSCs. This may provide a promising strategy for addressing non-obstructive azoospermia (NOA) caused by SSC dysfunction.

Ovarian clear cell carcinoma (OCCC) represents a rare and aggressive subtype of epithelial ovarian cancer with distinctive clinical and molecular characteristics. However, the identification, origin, and molecular features of the malignant epithelial cells in OCCC remain poorly studied. We establish an OCCC-associated transcriptional landscape using single-cell RNA sequencing and investigated the properties of epithelial cells in tissues from normal ovaries, ovarian endometriosis, primary OCCC and recurrent OCCC to assess the status of malignant epithelial cells. We identify a specific subcluster of malignant epithelial cells and further analyze them to discover 173 candidate factors associated with OCCC. Regulon and pseudotime trajectory analyses reveal six transcription factors (TFs) and their corresponding targets among these candidate factors, highlighting their roles in OCCC onset and reoccurrence. Through experimental validation, we confirm the crucial involvement of STAT3, KLF5, and TRIM28 in the proliferation and migration of OVISE cells. Silencing these three TFs also results in the down-regulation of their associated TF targets linked to OCCC. Overall, we characterize complex malignant-like cell populations at single-cell resolution and highlighted several TFs and their targets, providing essential resources for understanding the regulatory mechanisms underlying OCCC initiation and recurrence.

Transgender women (transfemales) often undergo gender-affirming hormone therapy (GAHT). However, the testicular impacts of feminising hormones present heterogeneity due to the complexity of testicular regulatory mechanisms. In this study, we analyzed approximately 49,385 single-cell transcriptomes from transfemale human testicular tissue, comparing cellular composition with that of cisgender male individuals across a range of ages. Our approach included clustering of cell types, identification of marker genes, pseudotime analysis of germ cells, and comprehensive cell-cell interaction analyses. We employed immunohistochemistry, quantitative real-time PCR, and immunostaining to validate the key molecular signatures identified in the pathways of interest. GAHT led to a significant reduction in spermatogenic cells, accompanied by an unexpected increase in Sertoli cell numbers per seminiferous tubule, suggesting disrupted germ cell-Sertoli cell interactions. Molecular analyses revealed upregulation of genes such as Decorin (DCN), Myoglobin (MB), and Beta-2-Microglobulin (B2M) in Sertoli cells, with enrichment in pathways related to cell adhesion, cytokine response, and wnt signaling. Notably, β-catenin was significantly elevated and translocated into the nucleus of Sertoli cells determined by immunostaining analysis. Additionally, collagen fiber infiltration disrupted the testicular microenvironment, further impairing germline-Sertoli cell communication. This study provides novel insights into the testicular alterations associated with GAHT in transfemales, particularly highlighting the role of germline-Sertoli cell interactions in testicular injury. Our findings contribute to a deeper understanding of the testicular response to feminizing hormones, offering a foundation for future therapeutic strategies.

Polycystic ovary syndrome (PCOS), a prevalent multisystem endocrine disorder affecting women of reproductive age, is characterized by intertwined reproductive-metabolic axis dysfunction encompassing ovarian impairment, insulin resistance, and systemic metabolic dysregulation. Nevertheless, the intricate pathophysiology, especially regarding the coordinated RNA/protein targets among distinct ovarian cellular populations, remains inadequately elucidated at the single-cell level. Herein, we have developed a single-cell transcriptomic atlas of human ovaries, highlighting the dysregulation of LUM and SOD3 in mesenchymal and epithelial compartments in PCOS. By utilizing Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) technology, we have concurrently characterized transcriptomes and 163 antibody-derived tags (ADTs) in human ovarian cells, establishing the inaugural multi-omic atlas of PCOS ovaries derived from single-cell transcriptomes and cell-surface proteomes. Our analysis has revealed a strong correlation between RNA and protein expression levels across major ovarian cellular populations. Particularly, CD86 and CD14 displayed significant upregulation in PCOS epithelial cells, with functional enrichment suggesting the regulation of PCOS through cytokine-cytokine receptor interactions and cell adhesion signatures. Through a methodical analysis of concordant differentially expressed protein-RNA pairs, we have ascertained that HLA-DRA was upregulated in PCOS. These discoveries lay the groundwork for integrating single-cell transcriptomics with protein profiling to elucidate previously unexplored facets of coordinated protein-RNA pairs for PCOS.