Chronic diabetic wounds are trapped in a persistent inflammatory state, largely due to macrophage failure to transition from pro-inflammatory (M1) to pro-reparative (M2) phenotypes. Here, we show that adipose-derived stem cell extracellular vesicles (ADSC-EVs) deliver functional mitochondria into diabetic wound macrophages, thereby restoring tricarboxylic acid (TCA) cycle-driven M2 polarization. Mechanistically, ADSC-EV-mediated mitochondrial transfer reactivates pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), increases TCA cycle flux, and suggests enhanced glutamine anaplerosis, as evidenced by ¹³C-glucose isotope tracing. This metabolic rewiring restores oxidative phosphorylation (OXPHOS), elevates oxygen consumption rate (OCR) and suppresses glycolysis. Consequently, ADSC-EV treatment reduces M1 macrophages and increases M2 macrophages, lowers pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, MCP1, p < 0.0001), and upregulates IL-10 in vitro, p < 0.0001). In a diabetic mouse wound model, a single course of ADSC-EVs accelerates wound closure at day 14 (p < 0.05), enhances re-epithelialization and collagen deposition, and reduces local oxidative stress and inflammation. Mitochondria‑depleted Rho-ADSC-EVs show markedly diminished effects, confirming that functional mitochondrial transfer is the primary driver. Our findings establish ADSC-EV-mediated mitochondrial transfer as a central metabolic reprogramming strategy that breaks the inflammatory lock in diabetic wounds and promotes healing.

Osteoarthritis (OA) is characterized by progressive cartilage degradation accompanied by limited intrinsic repair capacity. Stromal vascular fraction (SVF) transplantation has demonstrated potential for regenerating damaged joint tissue; however, the pathological microenvironment impairs SVF stemness, thereby limiting therapeutic efficacy. Herein, we developed a bioinspired hydrogel formed via the cross-linking of hyaluronic acid-grafted dopamine and a fibroblast growth factor 2-mimetic peptide-modified RADA16-I. This injectable hydrogel combined excellent gelation performance with multifunctionality, particularly in enhancing SVF proliferation and chondrogenic differentiation. The hydrogel activated forkhead box M1 (FOXM1) - mediated epigenetic reprogramming, enhancing DNA repair capacity and increasing chromatin accessibility at pluripotency loci. In a rat OA model, combined hydrogel and SVF transplantation significantly enhanced articular cartilage regeneration and ameliorated OA symptoms. This study provides preliminary evidence showing that a biomaterial-mediated epigenetic reprogramming strategy improves recovery in OA, highlighting the therapeutic potential of the novel hydrogel for stem cell-based regenerative medicine.

Bronchopulmonary dysplasia (BPD) is a major cause of chronic lung disease in both preterm infants and adults, but its etiology remains incompletely understood. In this study, F0 generation mice were exposed to polystyrene nanoplastics (PS-NPs), and F1 to F3 generations were obtained by breeding. Multi-omics sequencing including whole genome methylation sequencing, single cell transcriptome sequencing and transcriptome sequencing was performed on the lungs of offspring. The levels of Fe2+, lipid peroxidation products and key gene expression were determined. Male mice exposed to PS-NPs at environmentally relevant doses produced offspring (F1 and F2) that exhibited a typical BPD-like phenotype. Meanwhile, the F0 males showed diminished sperm motility, demonstrating that paternal PS-NPs exposure constituted an etiological factor for BPD in descendants. Mechanistic studies showed that PS-NPs exposure upregulated the expression of DNA methyltransferase Dnmt3a, leading to global hypermethylation of the sperm genome. Importantly, the hypermethylated promoter signature of the mitochondrial ferritin (FtMt) gene partially resisted epigenetic reprogramming and was transmitted to the lungs of offspring, resulting in persistently low FtMt expression in F1 and F2 lungs. This led to increased intracellular Fe2+ levels, subsequently triggered ferroptosis in alveolar epithelial cells, and ultimately impaired alveolarization. Knockdown of FtMt confirmed that FtMt deficiency was sufficient to induce ferroptosis and BPD-like lung injury both in vitro and in vivo. Furthermore, using in vitro fertilization of F0 sperm combined with Dnmt3a siRNA microinjection, we directly demonstrated that Dnmt3a is a key driver for FtMt to escape reprogramming and maintain its hypermethylation. In summary, this study reveals for the first time that paternal PS-NPs exposure causes BPD through a Dnmt3a-FtMt hypermethylation intergenerational and transgenerational axis, providing an epigenetic basis for understanding paternal derived chronic lung disease and potential targets for early intervention.

Introduction Mitochondrial homeostasis is intimately associated with the toxic effects of exogenous chemicals, as well as the onset and progression of various diseases. Benzo[b]fluoranthene (BbF) is ubiquitously distributed across various environmental media. The association between BbF exposure and male reproductive damage has been recently discovered. However, the relevant mechanisms remain unexplored. Objectives To investigate the male reproductive toxicity and mechanisms of BbF. Methods We established BbF exposure models in both mice and GC-2 mouse spermatocyte cell lines to investigate BbF-associated reproductive toxicity. By integrating transcriptomic sequencing, bioinformatics analysis, and experimental validation, we demonstrated that disruption of mitochondrial homeostasis was the central mechanism. Furthermore, through N6-methyladenosine (m6A) sequencing combined with functional validation, we revealed the regulatory role of m6A modification in modulating mitochondrial homeostasis. Results BbF exposure triggered mitochondrial homeostasis disruption in spermatocytes and contributed to male reproductive toxicity. Specifically, BbF impaired mitochondrial biogenesis and oxidative phosphorylation via the p53/PGC-1α/TFAM signaling pathway. As a compensatory response, PINK1/Parkin-associated mitophagy was activated, thereby partially alleviating cellular damage. To further investigate the regulatory mechanisms of m6A modification in the aforementioned mitochondrial dysfunction process, we observed that BbF exposure resulted in reduced expression of YTHDF2 in spermatocytes. The downregulation of YTHDF2 slowed the degradation of Trp53 mRNA, leading to elevated levels of both Trp53 mRNA and p53 protein, thereby inducing the p53/PGC-1α/TFAM-mediated mitochondrial damage. On the other hand, we demonstrated that METTL3 exerted a crucial protective effect by regulating mitophagy. Particularly, METTL3 enhanced the m6A modification of Mark4 mRNA, thereby stabilizing the transcript, increasing both mRNA and protein levels of MARK4. Furthermore, the METTL3/MARK4 signaling axis promoted PINK1/Parkin-associated mitophagy, which served as a protective mechanism. Conclusion As a result of the confrontation between YTHDF2/p53/PGC-1α/TFAM-mediated mitochondrial damage and PINK1/Parkin-associated mitophagy regulated by METTL3/MARK4, BbF disrupted mitochondrial homeostasis, ultimately leading to mitochondrial dysfunction and spermatocyte apoptosis.

Meiosis, a specialised form of cell division, is essential for sexual reproduction, which requires the proper formation of synaptonemal complex (SC) and homologous recombination (HR). However, the regulatory mechanisms underlying these processes remain incompletely understood. Here, we demonstrate that SOX30 is a key transcriptional regulator of male meiotic synapsis and recombination. In Sox30 -knockout mice, zygotene spermatocytes accumulate with synapsis defects. SOX30 deficiency disrupts the SC central element components SYCE1, SYCE2, and TEX12 distribution. Furthermore, disrupted γ-H2AX distribution reveals impaired DNA double-strand break repair and the persistence of recombination proteins RAD51 and RPA2 in late spermatocytes confirms defective homologous recombination repair (HRR) which results in reduced crossover formation in Sox30 -knockout mice spermatocytes. Mechanistically, SOX30 directly binds to SYCE1/SYCE2 promoters to modulate their transcription, thereby regulating SC assembly and HRR. Restoring SOX30 expression effectively rescues meiotic defects. Importantly, transcriptome co-expression analysis in non-obstructive azoospermia (NOA) testes identifies SOX30 as a central regulator of NOA transcriptional networks. Collectively, these findings underscore SOX30's crucial role in meiotic synapsis and recombination, highlighting its therapeutic potential for NOA.

N6-methyladenosine (m6A) is extensively involved in complex spermatogenesis while being extremely sensitive to environmental exposure. Numerous studies have revealed the toxicity of fine particulate matter (PM2.5) to the male reproductive system, but the specific epigenetic mechanisms involved have been underexplored. Here, we investigated the effect of m6A modification on PM2.5-induced male reproductive impairment by establishing a real-time PM2.5-exposed mouse model and a GC-2spd cell model. PM2.5 exposure resulted in damage to the spermatogenic epithelium and mitochondrial abnormalities in spermatocytes and significantly reduced sperm motility in mice. Gene enrichment analyses of testicular tissue differential m6A modified genes were significantly enriched to spermatogenesis in the PM2.5-treated mice compared with the control group, and the expression of the methylase WTAP was markedly decreased after PM2.5 exposure. Moreover, PM2.5 exposure resulted in a significant reduction in the expression of the spermatogenesis-related gene Hmgb2, as well as in the level of the Hmgb2 m6A modification. Transcriptome sequencing and verification experiments suggested that Hmgb2 may regulate spermatocyte ATP levels. In addition, we demonstrated that the m6A methylase WTAP affects Hmgb2 mRNA stability via m6A modification. Our study provides new insights into PM2.5-induced damage to spermatogenesis and reduced sperm motility.

Sox30 has recently been demonstrated to be a key regulator of spermatogenesis. However, the precise roles of Sox30 in the testis remain largely unclear. Here, the specific functions of Sox30 in testicular cells were determined by single-cell sequencing and confirmed via pathological analyses. Sox30 loss appears to damage all testicular cells to different extents. Sox30 chiefly drives the differentiation of primary spermatocytes. Sox30 deficiency causes spermatocyte arrest at the early phase of meiosis I, with nearly no normally developing second spermatocytes and three new spermatocyte -subclusters emerging. In addition, Sox30 seems to play important roles in the mature phenotypes of Sertoli and Leydig cells, and the proliferation and differentiation of spermatogonia. The developmental trajectory of germ cells begins with spermatogonia and splits into two different spermatocyte branches, with Sox30-null spermatocytes and wild-type spermatocytes placed at divergent ends. An opposite developmental trajectory of spermatocyte subclusters is observed, followed by incomplete development of spermatid subclusters in Sox30-null mice. Sox30 deficiency clearly alters the intercellular cross-talk of major testicular cells and dysregulates the transcription factor networks primarily involved in cell proliferation and differentiation. Mechanistically, Sox30 appears to have similar terminal functions that are involved mainly in spermatogenic development and differentiation among major testicular cells, and Sox30 performs these similar crucial roles through preferential regulation of different signalling pathways. Our study describes the exact functions of Sox30 in testicular cell development and differentiation and highlights the primary roles of Sox30 in the early meiotic phase of germ cells.

Chchd10 protein is crucial for sustaining mitochondrial dynamics, physiology and functions, and has been reported to be most abundantly in myocardial cells and skeletal muscle. However, nothing is known for the expression pattern of Chchd10 in gonadal development. Here, we characterized the expression patterns of Chchd10 gene during embryonic gonad development and postnatal testis development in mice, as well as the expression pattern of Chchd10 gene in human puberty testis and young adult testis using publicly available datasets. Besides, we investigated the expression and distribution of Chchd10 in mice testis by RT-qPCR and immunofluorescence and analyzed the possible role and mechanism of Chchd10 in the testis. We noticed that Chchd10 showed abundant expression in embryonic testis compared to ovaries and dynamically expressed during embryonic and postnatal testis development in mice. In addition, Chchd10 was highly abundant within testicular Sertoli cells populations both in embryonic and postnatal mice and mainly located in the mitochondria of Sertoli cells in mice. Furthermore, CHCHD10 was not only enriched in Sertoli cells, but also highly expressed in tMΦ of human puberty testis and adult testis. CHCHD10 may participate in testicular development by regulating multiple biological processes of Sertoli cells. Taken together, our data indicated that Chchd10 appears to be important during testicular development, particularly in the functional modulation of Sertoli cells. Our study revealed the expression profile of Chchd10 gene during testicular development for the first time and will provide new ideas for further studying the function and molecular mechanism of Chchd10.

Polystyrene nanoplastics (PS-NPs) have been reported to accumulate in the testes and constitute a new threat to reproductive health. However, the exact effects of PS-NPs exposure on testicular cells and the underlying mechanisms remain largely unknown. The C57BL/6 male mice were orally administered with PS-NPs (80 nm) at different dosages (0, 10, and 40 mg/kg/day) for 60 days, and GC-1 cells were treated with PS-NPs in this study. Enlarged seminiferous tubule lumens and a loose and vacuolated layer of spermatogenic cells were observed in PS-NPs-exposed mice. Spermatogenic cells which may be one of the target cells for this reproductive damage, were decreased in the mice from PS-NPs group. PS-NPs caused spermatogenic cells to undergo senescence, manifested as elevated SA-β-galactosidase activity and activated senescence-related signaling p53-p21/Rb-p16 pathways, and induced cell cycle arrest. Mechanistically, Gene Ontology (GO) enrichment suggested the key role of reactive oxygen species (ROS) in PS-NPs-induced spermatogenic cell senescence, and this result was confirmed by measuring ROS levels. Moreover, ROS inhibition partially attenuated the senescence phenotype of spermatogenic cells and DNA damage. Using the male health atlas (MHA) database, Sirt1 was filtrated as the critical molecule in the regulation of testicular senescence. PS-NPs induced overexpression of the main ROS generator Nox2, downregulated Sirt1, increased p53 and acetylated p53 in vivo and in vitro, whereas these disturbances were partially restored by pterostilbene. In addition, pterostilbene intervention significantly alleviated the PS-NPs-induced spermatogenic cell senescence and attenuated ROS burst. Collectively, our study reveals that PS-NPs exposure can trigger spermatogenic cell senescence mediated by p53-p21/Rb-p16 signaling by regulating the Sirt1/ROS axis. Importantly, pterostilbene intervention may be a promising strategy to alleviate this damage.

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancer worldwide, has an exceedingly poor prognosis. Tandem C2 domain nuclear protein (TC2N) has been implicated in tumorigenesis and serves as an oncogene or tumor suppressor in different types of cancer. Here, we explore the possible regulatory activities and molecular mechanisms of TC2N in HCC progression. However, TC2N expression was significantly upregulated in HCC tissues and hepatoma cell lines, and this upregulation was positively correlated with tumor progression in HCC patients. The ectopic overexpression of TC2N accelerated the proliferation, migration, and invasion of HCC cells, whereas its knockdown showed the opposite effects. Bioinformatics analysis showed that TC2N participates in the regulation of the Wnt/β-catenin signaling pathway. Mechanistically, TC2N activated the Wnt/β-catenin signaling pathway by regulating the expression levels of β-catenin and its downstream targets CyclinD1, MMP7, c-Myc, c-Jun, AXIN2, and glutamine synthase. Furthermore, the deletion of β-catenin effectively neutralized the regulation of TC2N in HCC proliferation and metastasis. Overall, this study showed that TC2N promotes HCC proliferation and metastasis by activating the Wnt/β-catenin signaling pathway, indicating that TC2N might be a potential molecular target for the treatment of HCC.

This study aims to explore the male reproductive toxicity of Benzo[b]fluoranthene (BbF) and related mechanisms. The results of computational toxicology analysis indicated male reproductive toxicity of BbF was related to apoptosis of Leydig cells and that Akt/p53 pathway might play a key role. In experiments, BbF induced testosterone decline, decreased concentration and motility of sperm and aggravated testicular pathological injury in mice. Besides, BbF led to apoptosis in Leydig cells, and decreased expressions of p-Akt and Bcl2, while improving the expressions of p53, Bax and Cleaved Caspase-3 in vivo and in vitro. Further, compared with BbF group, Akt activator SC79 significantly reduced cell apoptosis rate, improved cell viability, promoted the expressions of p-Akt and p-Mdm2, and reversed the above molecular expressions. Similarly, p53 inhibitor Pifithrin-α also significantly enhanced the cell vitality, alleviated the apoptosis of TM3 cells induced by BbF, and decreased the expressions of Bax and Cleaved Caspase-3, with the up-regulation of Bcl2. To sum up, by inhibiting Akt-Mdm2 signaling, BbF activated the p53-mediated mitochondrial apoptosis pathway, further inducing the apoptosis of Leydig cells, therefore resulting in testosterone decline and male reproductive damage. Besides, this study provided a valid mode integrating computational toxicology and experimental approaches in toxicity testing.

Environmental hazards are an increasing concern due to the rapid pace of industrialization. Among these hazards, noise and carbon monoxide (CO) are common risk factors and have been shown to cause serious health problems. However, existing studies focused on the individual effects of noise and CO exposure and the combined effects of these two factors remain poorly understood. Our study aimed to examine the combined effects of noise and CO exposure on testicular function by constructing individual and combined exposure models. Our findings indicated that combined exposure to noise and CO was associated with a higher risk of testicular damage and male reproductive damage when compared to exposure alone. This was evidenced by poorer semen quality and more severe pathological damage to the testis. This combined exposure led to higher levels of oxidative stress and apoptosis in the testes, with bioinformatics analyses suggesting the signaling pathways involved in these responses. Specifically, activation of the P53 signaling pathway was found to contribute to the testicular damage caused by the combined exposure. Encouragingly, pterostilbene (PTE), a novel phytochemical, alleviated combined exposure-induced testicular damage by reducing oxidative stress and germ cell apoptosis. Overall, we identified joint reproductive toxicity resulting from the exposure to noise and CO, and found that PTE is a promising potential treatment for injuries caused by these factors. The cover image is based on the Research Article Effects and possible mechanisms of combined exposure to noise and carbon monoxide on male reproductive system in rats by Yingqing Li et al., https://doi.org/10.1002/tox.23927.

Chronic diabetic wounds remain a globally recognized clinical challenge. They occur due to high concentrations of reactive oxygen species and vascular function disorders. A promising strategy for diabetic wound healing is the delivery of exosomes, comprising bioactive dressings. Metformin activates the vascular endothelial growth factor pathway, thereby improving angiogenesis in hyperglycemic states. However, multifunctional hydrogels loaded with drugs and bioactive substances synergistically promote wound repair has been rarely reported, and the mechanism of their combinatorial effect of exosome and metformin in wound healing remains unclear. Here, we engineered dual-loaded hydrogels possessing tissue adhesive, antioxidant, self-healing and electrical conductivity properties, wherein 4-armed SH-PEG cross-links with Ag+, which minimizes damage to the loaded goods and investigated their mechanism of promotion effect for wound repair. Multiwalled carbon nanotubes exhibiting good conductivity were also incorporated into the hydrogels to generate hydrogen bonds with the thiol group, creating a stable three-dimensional structure for exosome and metformin loading. The diabetic wound model of the present study suggests that the PEG/Ag/CNT-M + E hydrogel promotes wound healing by triggering cell proliferation and angiogenesis and relieving peritraumatic inflammation and vascular injury. The mechanism of the dual-loaded hydrogel involves reducing the level of reactive oxygen species by interfering with mitochondrial fission, thereby protecting F-actin homeostasis and alleviating microvascular dysfunction. Hence, we propose a drug-bioactive substance combination therapy and provide a potential mechanism for developing vascular function-associated strategies for treating chronic diabetic wounds.

Background: Studies on male reproductive toxicity of microplastics are still scarce and the precise mechanism is not distinct.Methods: C57BL/6 male mice were given oral gavage treatments treated with 5 & mu;m (MPs) and 80 nm (NPs) polystyrene microplastics every day for 60 consecutive days in a row at dosages of 0, 10 and 40 mg/kg/d. The major damage of MPs and NPs were assessed by the assays in vivo and in vitro. Transcriptome sequencing was applied to screen the key involved pathways.Results: In the 10 mg/kg/d NPs group, there was an increase in testicular organ coefficient, and in the 40 mg/kg/ d MPs group, an increase in epididymal weight was observed. Vacuolization of spermatogenic cell layer, interstitial congestion, and germ cell apoptosis were found in the testes of MPs and NPs treatment mice at different dose groups. Higher apoptosis rate was observed in GC-2 cells after MPs and NPs treatment at different concentrations. Transcriptome analysis suggested that p53 pathway might be the key signal pathway of the cell apoptosis, and the expressions of p53 and other markers of cell apoptosis were indeed altered after exposure to MPs and NPs.Conclusions: MPs and NPs can cause reproductive toxicity in male mice through inducing apoptosis of spermatogenic cells via p53 signaling pathway, indicating MPs and NPs exposure be an unnegligible risk factor for reproductive health in male mice.

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancer worldwide, has an exceedingly poor prognosis. Tandem C2 domain nuclear protein (TC2N) has been implicated in tumorigenesis and serves as an oncogene or tumor suppressor in different types of cancer. Here, we explore the possible regulatory activities and molecular mechanisms of TC2N in HCC progression. However, TC2N expression was significantly upregulated in HCC tissues and hepatoma cell lines, and this upregulation was positively correlated with tumor progression in HCC patients. The ectopic overexpression of TC2N accelerated the proliferation, migration, and invasion of HCC cells, whereas its knockdown showed the opposite effects. Bioinformatics analysis showed that TC2N participates in the regulation of the Wnt/I3-catenin signaling pathway. Mechanistically, TC2N activated the Wnt/I3-catenin signaling pathway by regulating the expression levels of I3-catenin and its downstream targets CyclinD1, MMP7, c-Myc, c-Jun, AXIN2, and glutamine synthase. Furthermore, the deletion of I3-catenin effectively neutralized the regulation of TC2N in HCC proliferation and metastasis. Overall, this study showed that TC2N promotes HCC proliferation and metastasis by activating the Wnt/I3-catenin signaling pathway, indicating that TC2N might be a potential molecular target for the treatment of HCC.(c) 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.

Fine particulate matter (PM2.5) has been reported to cause various types of damage to male reproductive system, but the research on the underlying mechanisms is still insufficient. This study attempted to explore the underlying mechanisms of this widely concerning environmental health problem through in vivo and in vitro exposure models. Significant pathological damage and abnormal mitochondria in spermatocytes were observed in the real-time PM2.5 exposure animal model. In addition, significant alterations in key biomarkers of iron metabolism and ferroptosis were found in testis tissues. Notably decreased cell viability was found in vitro. Moreover, the ferroptosis pathway was significantly enriched in the transcriptome enrichment analysis. Subsequent experiments showed that the two core events of ferroptosis, iron overload and lipid peroxidation, occurred in spermatocytes after PM2.5 treatment. Moreover, lipid metabolic genes (Acsl4 and Aloxe3) and the antioxidant gene Gpx4 were found to be key target genes of ferroptosis caused by PM2.5 in spermatocytes. Importantly, further studies showed that the damaging effect could be reversed by the iron chelator deferoxamine mesylate (DFOM) and the lipid peroxidation inhibitor ferrostatin-1 (Fer-1), which further confirmed the role of ferroptosis in PM2.5 toxicity. Our study revealed the vital role of ferroptosis in PM2.5-induced male reproductive damage, providing novel insights into the air pollution-induced decrease in male fertility.

The effects of ambient fine particles on male reproductive health have raised widespread concern. Although various studies have made progress in understanding the causes of PM2.5 impairment to male reproductive health, the particular underlying mechanisms of the damage remain largely unclear, demanding more research in new directions. Previous research has revealed that DNA methylation is vital in the male reproductive system and that it is sensitive to environmental changes. However, there hasn't been enough investigation into the involvement of DNA methylation in PM2.5-induced male reproductive toxicity. This research found that real-world PM2.5 exposure, which mimicked everyday human exposure, may lead to testicular dysfunction including spermatogenesis and steroidogenesis impairment. Genome-wide DNA methylation analysis suggested that PM2.5 significantly affected the testicular DNA methylome. Candidate genes and networks related to spermatogenesis and steroidogenesis were discovered using integration analysis of methylome and transcriptome. After PM2.5 exposure, the testicular methylation levels of key genes such as Cyp11a1 and Pax8 were increased in mice, and their consequent reduced expression may impair the testosterone and sperm production process. This study might give basic information and new insights into the regulatory mechanisms of DNA methylation in PM2.5-induced male reproductive damage.

Sertoli cells play a pivotal role in the complex spermatogenesis process. This study aimed to investigate the effects of PM2.5 on Sertoli cells using the TM4 cell line and a real time whole-body PM2.5 exposure mouse model, and further explore the underlying mechanisms through the application of metabolomics and transcriptomics. The results in vivo and in vitro showed that PM2.5 reduced Sertoli cells number in seminiferous tubules and inhibited cell proliferation. PM2.5 exposure also induced Sertoli cell dysfunction by increasing androgen binding protein (ABP) concentration, reducing the blood-testis barrier (BTB)-related protein expression, and decreasing glycolysis capacity and lactate production. The results of transcriptomics, metabolomics, and integrative analysis of multi-omics in the TM4 Sertoli cells revealed the activation of xenobiotic metabolism, and the disturbance of glutathione and purine metabolism after PM2.5 exposure. Further tests verified the reduced GSH/GSSG ratio and the elevation of xanthine oxidase (XO) activity in the PM2.5-exposed TM4 cells, indicating that excessive reactive oxygen species (ROS) was generated via metabolic disorder caused by PM2.5. Moreover, the redox imbalance was proved by the increase in the mitochondrial ROS level, superoxide dismutase (SOD) and catalase (CAT) activity, as well as the activation of the Nrf2 antioxidative pathway. Further study found that the redox imbalance caused by PM2.5 induced DNA damage response and cell cycle arrest. Additionally, PM2.5 induced ferroptosis through iron overload and lipid peroxidation. Taken all together, our study provided new insights for understanding proliferation inhibition and dysfunction of TM4 Sertoli cells exposed to PM2.5 via metabolic disorder and redox imbalance-mediated DNA damage response and ferroptosis.

The effects of ambient fine particles on male reproductive health have raised widespread concern. The particular underlying mechanisms of the damage remain largely unclear and demand more research in new directions. Previous research has revealed that DNA methylation plays an important role in male reproductive development and is also vulnerable to environmental influences. However, there hasn't been enough investigation into the involvement of DNA methylation in PM2.5-induced male reproductive toxicity. Here, we establish a real-time PM2.5 exposure model and revealed that PM2.5 exposure could lead to testicular dysfunction including spermatogenesis impairment and steroid hormone dysfunction. In particular, the decrease in the testicular global level of 5-methylcytosine (5mC) indicated a possible association of DNA methylation with testicular injury induced by PM2.5 exposure. Further genome-wide methylation analysis revealed genomic hypomethylation of testicular DNA and identified more than 1000 differentially methylated regions in both CAP and UA versus FA, indicating that PM2.5 exposure, even low-dose, could modulate the testicular methylome. Furthermore, integrated analysis of methylome and transcriptome identified some key methylated genes and networks, which may be involved in spermatogenesis and synthesis of steroid hormone. The testicular methylation levels of key genes especially Cyp11a1 and Pax8 raised, and their consequent reduced expression may impair the testosterone and sperm production process. Our research provides fundamental knowledge as well as novel insights into the possible involvement of DNA methylation in PM2.5-induced male reproductive harm.