Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) offer distinct advantages for clinical translation, including accessibility, scalability, and broad immunomodulatory capacity. However, the efficacy of systemically delivered UC-MSCs is constrained by suboptimal in vivo trafficking. Intravascular administration is limited by pulmonary first-pass sequestration, inefficient endothelial recruitment, blood-mediated inflammatory injury, and poor retention. While UC-MSCs homing is often conceptualized through a leukocyte adhesion paradigm, this model incompletely describes culture-expanded UC-MSCs, which exhibit heterogeneous expression of chemokine receptors, adhesion molecules, and selectin ligands. Furthermore, biodistribution and safety are critically determined by biophysical and hemocompatibility parameters, including cell size, deformability, cryopreservation status, and tissue factor (TF/CD142)-dependent procoagulant activity. This review synthesizes current understanding of UC-MSCs trafficking at the translational interface of biology and manufacturing. We examine canonical migratory mechanisms-chemokine signaling, integrin-mediated adhesion, extracellular matrix remodeling, and intracellular motility pathways-alongside underappreciated determinants of therapeutic performance: instant blood-mediated inflammatory reaction (IBMIR), complement-coagulation crosstalk, post-thaw functional impairment, donor variability, and route-dependent biodistribution. We also address the paradox wherein therapeutic benefit occurs despite minimal durable engraftment, implicating paracrine signaling, extracellular vesicles, and apoptosis-associated immune reprogramming as primary effectors. Finally, we evaluate strategies to enhance delivery and efficacy, including preconditioning, glycoengineering, receptor overexpression, route optimization, biomaterial-assisted retention, and migration-relevant potency assays under Good Manufacturing Practice (GMP). Advancing UC-MSCs therapy toward reproducible, mechanism-guided clinical application requires rigorous integration of hemocompatibility assessment, product characterization, and clinically informative cell tracking.

Background:Macrophages play a crucial role in the inflammatory response and fibrosis after myocardial infarction (MI). CMTM3 exerts important functions in the immune system and cardiovascular system. This study aims to explore the role and mechanism of CMTM3 in regulating macrophage-related inflammation after MI. Methods:The CMTM3-/- mouse MI model was established. The effects of CMTM3 on MI and macrophage-related inflammation in mice were evaluated by TTC, Masson, echocardiography, flow cytometry and Elisa. In vitro, the effects of CMTM3 on primary macrophages were assessed by flow cytometry, RT-qPCR and Elisa. The mechanism of CMTM3 regulating macrophages was explored by Western blot. Results:In the mouse MI model, CMTM3 expression was mainly increased in macrophages. CMTM3 deficiency resulted in an enlarged infarct size, increased collagen deposition, and deteriorated cardiac function. Further studies revealed that CMTM3 deficiency promoted macrophage polarization toward M1 types, and increase the production and secretion of inflammatory factors IL-1β, IL-6 and TNF-α. In vitro studies also confirmed CMTM3 deficiency promoted M1 macrophage differentiation and upregulated the expression of inflammatory factors. Mechanistically, CMTM3 can interact with PPARα, CMTM3 deficiency can inhibit PPARα activity, and increase the phosphorylation of NF-κB, thereby promoting macrophage inflammation. Conclusion:CMTM3 inhibits macrophage-related inflammation after MI by activating PPARα and inhibiting NF-κB phosphorylation. This study highlights the anti-inflammatory effect of CMTM3 in MI, and holds that CMTM3 can serve as a new target for improving cardiac remodeling after MI.

Pancreatic ductal adenocarcinoma (PDAC) carries a poor prognosis largely due to lack of efficient diagnostic means. We applied mass spectrometry-based high-coverage plasma proteome analysis accompanying with machine learning to develop a 5-protein diagnostic model: SNCA, GCLC, LBP, ALAD, and SORD. For differentiating PDAC from healthy controls (HCs), this model reached an area under the curve (AUC) of 0.973 with 100% sensitivity and 85% specificity in the discovery cohort, with nested cross-validation confirming robust performance (AUC = 0.958). Further validation centered on SNCA achieved an AUC of 0.835 in an independent validation cohort. SNCA also showed good diagnostic performance in PDAC patients with low CA19-9 level (AUC = 0.868), underscoring its potential value for this subgroup. Overall, these findings indicate SNCA as a promising candidate plasma diagnostic marker for PDAC.

This study investigated the protective effects of pituitary adenylate cyclase-activating polypeptide (PACAP) against lipopolysaccharide (LPS)-induced acute epididymitis in mice, with a particular emphasis on its antioxidant and anti-inflammatory mechanisms within the epididymal microenvironment responsible for sperm maturation. Acute epididymitis was triggered by injecting LPS intraperitoneally, with concurrent PACAP administration. Epididymal tissues were collected for histological, immunofluorescence, RT-PCR, and in vitro fertilization analyses. The results demonstrated that PACAP markedly decreased the expression of Il-6 mRNA (2.87 fold decrease, p < 0.05) and Tnf-α mRNA (2.45 fold decrease, p < 0.05) in the cauda epididymis following LPS adminstration for 6 h, alleviated histopathological changes, and improved sperm motility (from 37.8 ± 4.1

Mesenchymal stromal cells (MSCs) are metabolically active and redox-sensitive therapeutic cells, with their therapeutic potency tightly linked to mitochondrial integrity and function. Beyond paracrine and immunomodulatory actions, MSCs can transfer functional mitochondria to damaged cells, restoring bioenergetics, maintaining redox homeostasis via ROS regulation, and facilitating tissue repair and regeneration. This review summarizes recent progress in MSC mitochondrial biology, highlighting how metabolic reprogramming, mitochondrial biogenesis, fusion-fission dynamics and mitophagy coordinately regulate MSC stemness, differentiation, senescence and therapeutic capacity. It outlines core redox regulatory networks covering mitochondrial ROS production (ETC Complexes I/III and reverse electron transport), non-mitochondrial oxidases (NADPH oxidases), and canonical antioxidant signaling (Nrf2/Keap1, thioredoxin/peroxiredoxin and glutathione/glutaredoxin). Redox-dependent post-translational modifications governing mitochondrial transfer machinery are emphasized, including cysteine oxidation of connexin 43, redox-regulated Drp1 phosphorylation, and oxidative modulation of Miro1-mediated mitochondrial trafficking. Major intercellular mitochondrial transfer routes, such as tunneling nanotubes, connexin 43-based intercellular communication and extracellular vesicles, are discussed under inflammatory, hypoxic and metabolic stress conditions. Preclinical studies across pulmonary, cardiovascular, neurological, renal, hepatic and immune-mediated diseases validate that MSC-derived mitochondrial transfer preserves ATP production, mitigates oxidative injury and remodels recipient cell immunometabolic phenotypes. Emerging engineering strategies to improve mitochondrial delivery and therapeutic outcomes are also reviewed, alongside translational bottlenecks including cell source heterogeneity, mitochondrial quality control, in vivo tracking, dosage optimization and long-term biosafety. Overall, MSC mitochondrial dynamics and intercellular transfer bridge redox biology, metabolism and regenerative medicine, offering mechanistic insights for next-generation precision regenerative therapies.

The testicular microenvironment, with Sertoli cells as a key component, plays a pivotal role in spermatogenesis. DHX37, a member of the DEAH-box family of RNA helicases, has been identified as a pathogenic gene in 46, XY disorders of sex development (DSD), underscoring its potential significance in testicular development. Here, we focus on elucidating the role of Dhx37 in maintaining Sertoli-cell survival. RIP-seq and RNAi-RNA-seq reveal that Dhx37 safeguards nucleolar integrity and PI3K–AKT signaling, suppresses p53-driven apoptosis, and its loss triggers pro-apoptotic splicing. Cell-specific Dhx37 knockout mice (Dhx37−/−) were subsequently generated to investigate the function of Dhx37 in testicular development. In the Dhx37−/− mice, we observed pronounced defects, including diminished testicular volume, lower testosterone levels, and marked vacuolization of the seminiferous tubules. Immunofluorescence staining revealed disruptions in both Sertoli and germ cell compartments, characterized by reduced cell proliferation and elevated apoptosis. The snRNA-seq disclosed marked changes in the expression of genes governing apoptosis and proliferation, findings that were further validated through qRT-PCR and Western blotting. In this study, we identified Dhx37 as a pivotal determinant of nucleolar architecture in murine testicular Sertoli cells. Preservation of the nucleolus safeguards supporting normal testicular morphogenesis.

BACKGROUND & AIMS:Inflammatory bowel disease leads to increased risk of developing colitis-associated colon cancer (CAC). CMTM3 has a higher methylation level in colon cancer, and accumulating evidence suggests that chemokine-like factor-like MARVEL transmembrane domain-containing member 3 (CMTM3) participates in inflammation and cancer development. METHODS:We explored the signs of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced CAC in wild-type (WT) and Cmtm3 deficiency (Cmtm3-/-) mice. Experimental colitis was induced in Cmtm3-/- mice as well as mice with endothelial cell-specific deletion of Cmtm3. Disease phenotypes were investigated by body weight, disease activity index (DAI), colon length, histology, immune cell infiltration, and intestinal permeability. The mechanism was analyzed using bone marrow reconstitution, immunofluorescent staining, Western blot, immunoprecipitation, and pull-down experiments. RESULTS:We found CMTM3 promoted CAC by aggravating colitis. Further, we revealed endothelial cell-specific deletion of Cmtm3 inhibited the colitis development. In vitro and in vivo mechanistic studies revealed that CMTM3 drove colitis by increasing clathrin-dependent downregulation of vascular endothelial-cadherin, thus causing vascular permeability. We further identified that CMTM3 interacted with clathrin heavy chain and inhibited clathrin heavy chain ubiquitination and proteasome-dependent degradation. Interestingly, Cmtm3 knockout and imatinib mesylate both targeted vascular permeability and had comparable efficacy. CONCLUSIONS:Our study indicates that CMTM3 promotes CAC by aggravating colitis through causing vascular permeability, providing insights into targets for development of future therapies.

Zhushao Granules (ZSG) had exhibited beneficial effects in the treatment of ulcerative colitis (UC) as an effective herbal prescription in Traditional Chinese Medicine. However, the underlying anti-inflammatory mechanism of ZSG remains unclear. This study aimed to decipher the mechanism of ZSG against UC combining network pharmacology and animal-based experiments. Network pharmacology was employed to identify active components and therapeutic targets of ZSG against UC. The protein–protein interaction (PPI) network was constructed among the therapeutic targets using the STRING database, and GO and pathway analyses were carried out using DAVID. Then, the “herb-component-target-pathway” network based on therapeutic targets was established and the topological parameters were subsequently calculated to identify hub active components, targets and pathways by Cytoscape. Finally, the therapeutic function and the special pathway of ZSG against UC were validated using a TNBS-induced UC model in BABL/c mice. Ninety-four active components of ZSG and 460 potential targets were acquired from the Encyclopedia of Traditional Chinese Medicine and Tradition Chinese Medicine Systems Pharmacology Database and Analysis Platform. 884 potential targets of UC were obtained from OMIM and HINT. Sixty-two overlapping potential targets were identified as therapeutic targets of ZSG against UC. PPI network filtered out 61 therapeutic targets. GO and pathway analyses extracted 48, 25, and 98 terms corresponding to biological processes, molecular functions and Reactome pathways, respectively. Enrichment analysis suggested that the therapeutic targets were mainly involved in immune regulation, especially RIP-mediated NF-κB activation via ZBP1. Topological analysis of the “herb-component-target-pathway” network recognized 9 hub components, 20 hub targets and 18 hub pathways. The animal-based experiments revealed that ZSG ameliorated symptoms and histological changes in TNBS-induced colitis by significantly inhibiting the ZBP1/RIP/NF-κB pathway. ZSG might alleviate the mucosal damage and ameliorate colitis via targeting ZBP1/RIP/NF-κB pathway, which laid the theoretical foundation for the clinical application and further study of ZSG and provided new insights into UC treatment.

IntroductionChronic epididymitis threatens male fertility, yet the role of LYG1 in this disease is unclear. LYG1, an immunomodulatory protein, is highly expressed in the mouse epididymis, especially the cauda region prone to fibrosis. This study aimed to explore whether Lyg1 deficiency exacerbates LPS-induced epididymal inflammation, fibrosis and sperm dysfunction.MethodsSixty-four wild-type (WT) and Lyg1 knockout (KO) mice were divided into four groups. LPS was used to induce chronic epididymitis, with PBS as control. After 42 days, histological staining, flow cytometry, oxidative stress detection, transcriptomic profiling, sperm function analysis and In Vitro Fertilization (IVF) were performed to evaluate related indexes.ResultsUnder physiological conditions, Lyg1 KO mice had normal reproductive phenotypes. However, LPS-induced inflammation led to more severe epididymal damage, enhanced oxidative stress, abnormal ECM pathways and worse sperm dysfunction (reduced acrosomal integrity and functional proteins) in KO mice. IVF showed a more significant decrease in fertilization rate in KO mice.DiscussionLYG1 regulates the balance between epididymal inflammation and tissue repair. Its deficiency aggravates LPS-induced epididymal fibrosis and sperm dysfunction. This study identifies LYG1-dependent pathways as potential therapeutic targets for chronic epididymitis-related infertility.

The CMTM family plays pivotal roles in various physiological and pathological processes, including those associated with the male reproductive system. Among them, CMTM2 exhibits specific expression in testicular tissue; however, its relationship with spermatogenesis, sperm function, and the underlying molecular mechanisms remains unclear. This study systematically investigated the localization of CMTM2 in human testis and sperm, as well as its correlation with spermatogenesis and sperm quality. The results revealed that CMTM2 expression was specifically localized in human testis, predominantly in germ cells, with significantly lower levels observed in sperm from patients with asthenozoospermia and teratozoospermia compared to normospermic individuals. Immunofluorescence analysis revealed that CMTM2 was predominantly localized in the middle piece of sperm, with significant reductions in infertile samples. Western blot analysis confirmed decreased CMTM2 levels in sperm from asthenozoospermia, oligozoospermia, and teratozoospermia patients, indicating a strong correlation between CMTM2 expression and sperm quality. Furthermore, CMTM2 expression positively correlated with sperm progressive motility, but not with sperm concentration, suggesting its potential role in regulating sperm motility in both normal and asthenozoospermia patients. The findings suggested a significant correlation between the CMTM2 expression and spermatogenesis as well as sperm quality, thereby providing valuable insights for further investigations into the molecular mechanisms involved. These results lay a foundational basis for exploring the potential roles of other members within the CMTM family in male reproduction.

Background:To investigate the molecular mechanisms by which PACAP alleviates LPS-induced epididymitis, focusing on its anti-inflammatory and antioxidant pathways in the epididymal microenvironment. Methods: A mouse model of acute epididymitis was induced by LPS injection. The study assessed the levels of inflammatory markers (IL-6 and TNF-α mRNA) in the cauda epididymis, sperm motility and morphology, morphological alterations in the epididymis, and the expression of inflammation and antioxidant-related genes. PACAP treatment was administered to evaluate its effects on these parameters. Results: Results showed elevated levels of IL-6 and TNF-α mRNA in the cauda epididymis due to LPS injection. PACAP treatment effectively reduced these inflammatory markers and improved compromised sperm motility and morphology. PACAP also ameliorated morphological changes in the epididymis and mitigated the LPS-induced increase in leukocyte and macrophage markers. Gene expression analysis revealed that PACAP co-treatment suppressed LPS-induced upregulation of Il-6 and Tnf-ɑ in caput tissues. In cauda tissues, PACAP significantly reduced the expression of LPS-elevated Tnf-ɑ and IL-1β. PACAP up-regulated the antioxidant genes Cat and Sod1, which were down-regulated by LPS. IVF experiments demonstrated that PACAP restored the effects of LPS on sperm-egg fusion and embryo development. In conclusion: PACAP played a crucial role in maintaining epididymal function and sperm quality in the presence of inflammation. ### Competing Interest Statement The authors have declared no competing interest.

The mechanism underlying metabolic dysfunction-associated steatohepatitis (MASH) to hepatocellular carcinoma (HCC) is elusive, and whether circRNA can serve as biomarker or therapeutic target for MASH/HCC needs to be systematically explored. Integrative transcriptomic analysis of circRNA from MASH and HCC were performed. Multi-cohort analyses of serum and tissues from MASH and HCC patients (n = 206) were conducted. Mechanisms are explored via RNA-protein interaction assays, CRISPR-mediated knockdown, and xenograft/PiggyBac-mediated mice models. circSMEK1 is significantly decreased in MASH/HCC tissues and serum, correlating with tumor size, vascular invasion, and overall survival. Mechanistically, nuclear circSMEK1 binds hnRNPK, promoting its ubiquitin-mediated degradation, suppressing IGF2 transcription and PI3K/AKT signaling. Loss of circSMEK1 elevated autocrine IGF2 in HCC promoting tumor growth, also activated AKT in cancer-associated fibroblasts through paracrine, fostering an immunosuppressive microenvironment. SF3B4 overexpression drove circSMEK1 depletion in HCC. In murine models, circSMEK1 restoration inhibited tumor growth and metastasis. circSMEK1 is a tumor-suppressor in MASH/HCC through the hnRNPK-IGF2-AKT axis. The serum level of circSMEK1 has non-invasive diagnostic value for HCC (AUC = 0.790), as well as potential diagnostic utility for early HCC or high-risk MASH, owing to its key role in bridging MASH to HCC progression. Restoring of circSMEK1, alone or combined with IGF2 inhibitors, proposing a novel therapeutic strategy for HCC.

BACKGROUND:Cisplatin (CDDP), a widely used chemotherapeutic agent, induces reproductive toxicity primarily by damaging the blood-testis barrier (BTB) and triggering oxidative stress. This study aimed to investigate whether human umbilical cord mesenchymal stem cells (hUC-MSCs) protect against CDDP-induced BTB dysfunction and ferroptosis in mice, with implications for preserving fertility in chemotherapy patients. METHODS:Male C57 mice were randomized into four groups: control, CDDP-treated, hUC-MSCs-treated, and CDDP+hUC-MSCs-treated. An additional CDDP+Ferrostatin-1 (Fer-1, a ferroptosis inhibitor) group was included to validate ferroptosis involvement. Testicular histology, sperm quality, BTB integrity (via Evans blue permeability assay), and oxidative stress markers were evaluated. Ferroptosis-related (GPX4, NRF2, COX2, TFR1) and BTB-related (N-cadherin, ZO-1, Connexin 43) proteins were assessed by immunofluorescence and Western blotting. In vitro fertilization (IVF) was used to evaluate fertility. RESULTS:CDDP induced significant testicular damage, reduced sperm quality, increased BTB permeability, and disrupted BTB proteins. It also triggered ferroptosis, as evidenced by decreased GSH, elevated MDA, downregulated GPX4/NRF2, and upregulated COX2/TFR1. hUC-MSCs reversed these changes: restoring GSH levels, reducing MDA, normalizing ferroptosis-related proteins, and repairing BTB integrity. Fer-1 mimicked these effects, confirming ferroptosis as a key mechanism. IVF showed hUC-MSCs restored embryonic development (two-cell and blastocyst rates) to normal. CONCLUSIONS:hUC-MSCs protect against CDDP-induced reproductive injury by inhibiting ferroptosis (especially in Sertoli cells), repairing BTB, and restoring fertility. Their transient retention and low immunogenicity support their potential as a safe therapeutic strategy for preserving fertility in chemotherapy patients.

CMTM6, a regulator of PD -L1 stability, has been implicated in the development of various cancers. However, the expression and role of CMTM6 in hepatocellular carcinoma (HCC) remains controversial. Our study revealed a negative correlation between CMTM6 expression and HCC prognosis through bioinformatics analysis and immunofluorescence staining. CMTM6 expression was also positively associated with alpha-fetoprotein (AFP) levels, supporting its potential as a prognostic marker for HCC. Using Cmtm6 knockout mice, we found that Cmtm6 deficiency inhibited HCC formation and cell proliferation in primary liver cancer models induced by DEN and DEN/CCl4. In HCC cell lines, CMTM6 promoted cell proliferation and interacted with 8-catenin, stabilizing it by preventing ubiquitination. In conclusion, our study suggested that CMTM6 upregulation promotes HCC cell proliferation through the 8-catenin pathway, making it a potential therapeutic target for HCC treatment.

CMTM6, a regulator of PD-L1 stability, has been implicated in the development of various cancers. However, the expression and role of CMTM6 in hepatocellular carcinoma (HCC) remains controversial. Our study revealed a negative correlation between CMTM6 expression and HCC prognosis through bioinformatics analysis and immunofluorescence staining. CMTM6 expression was also positively associated with alpha-fetoprotein (AFP) levels, supporting its potential as a prognostic marker for HCC. Using Cmtm6 knockout mice, we found that Cmtm6 deficiency inhibited HCC formation and cell proliferation in primary liver cancer models induced by DEN and DEN/CCl4. In HCC cell lines, CMTM6 promoted cell proliferation and interacted with β-catenin, stabilizing it by preventing ubiquitination. In conclusion, our study suggested that CMTM6 upregulation promotes HCC cell proliferation through the β-catenin pathway, making it a potential therapeutic target for HCC treatment.

Janus two-dimensional (2D) polymeric materials present asymmetric dual surfaces that enable a variety of applications of biosensors, catalysts, drug delivery, etc. This work reports the evaporation-induced interfacial self-assembly of amphiphilic block copolymer poly(ethylene glycol)-b-poly(N-(2-phenylethyl) glycine) (PEG-b-PNPE) at the air-water interface. The PEG-b-PNPE was initially assembled into a monolayer with a uniform thickness of similar to 2.5 +/- 0.1 nm, which was mechanically compressed into a bilayer structure by Langmuir-Blodgett (LB) technology as surface pressure is exceeding the critical collapse pressure. Both monolayer and bilayer nanostructure span over hundreds of microns in both dimensions. The surface contact angle of the sample was measured by dynamic/static optical contact angle/interface tensiometer, which showed asymmetric wettability on the air side and the water side. The evolution from a monolayer to a bilayer was further tracked by an atomic force microscope. By the evaporation-induced interfacial self-assembly at the air-water interface, we also prepared macroscopic Janus films with a diameter of similar to 3.5 mm. The obtained micro/macro-scale 2D materials have potential applications in nanoscience and biomedicine.

Chemotherapeutic drugs can cause reproductive damage by affecting sperm quality and other aspects of male fertility. Stem cells are thought to alleviate the damage caused by chemotherapy drugs and to play roles in reproductive protection and treatment. This study aimed to explore the effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on alleviating paclitaxel (PTX)-induced spermatogenesis and male fertility defects. An in vivo PTX-induced mice model was constructed to evaluate the reproductive toxicity and protective roles of hUC-MSCs in male fertility improvement. A 14 day PTX treatment regimen significantly attenuated mice spermatogenesis and sperm quality, including affecting spermatogenesis, reducing sperm counts, and decreasing sperm motility. hUC-MSCs treatment could significantly improve sperm functional indicators. Mating experiments with normal female mice and examination of embryo development at 7.5 days post-coitum (dpc) showed that hUC-MSCs restored male mouse fertility that was reduced by PTX. In IVF experiments, PTX impaired sperm fertility and blastocyst development, but hUC-MSCs treatment rescued these indicators. hUC-MSCs’ protective role was also displayed through the increased expression of the fertility-related proteins HSPA2 and HSPA4L in testes with decreased expression in the PTX-treated group. These changes might be related to the PTX-induced decreases in expression of the germ cell proliferation protein PCNA and the meiosis proteins SYCP3, MLH1, and STRA8, which were restored after hUC-MSCs treatment. In the PTX-treated group, the expression of testicular antioxidant proteins SIRT1, NRF2, CAT, SOD1, and PRDX6 was significantly decreased, but hUC-MSCs could maintain these expressions and reverse PTX-related increases in BAX/BCL2 ratios. hUC-MSCs may be a promising agent with antioxidant and anti-apoptosis characteristics that can maintain sperm quality following chemotherapy treatment.

Cisplatin (CDDP) is a chemotherapeutic drug that is used to treat many different types of tumors. However, it also has significant adverse effects on male reproduction, which are partially mediated oxidative damage. Melatonin (MLT) is a promising antioxidant that can be used for reproductive protection. In this paper, we investigated the effect of CDDP on spermatogenesis, as well as MLT's potential role in reproductive protection. CDDP (5 mg/kg BW) significantly reduced male mice testosterone levels and decreased sperm vitality and progressive motility. Additionally, a lower percentage of stage VII and VIII seminiferous tubules were observed in CDDP-treated mice. MLT administration significantly alleviated CDDP-induced testicular damages, CDDP-induced lowered male fertility in vivo, and enhanced in vitro embryonic development of two cells and blastocysts. These changes may be due to CDDP-mediated spermatogenesis defects in germ cell and Leydig cell proliferation, which are reflected in abnormal PCNA, SYCP3, and CYP11A1 expression levels and can be improved by MLT. CDDP treatment significantly decreased the total antioxidant capacity (TAC), as well as SOD and GSH levels, and increased MDA levels in mice testis, leading to increased apoptosis of germ cells and increased BAX/BCL2 ratios in mice testis. MLT treatment may reduce germ cell apoptosis by reducing oxidative damage in mice testis. This study demonstrated that CDDP affects sperm fertility by altering germ cell and Leydig cell proliferation via increased oxidative damage and that MLT can attenuate these damages. Our work provides potential information for further research on the toxic effects of CDDP and the protective effects of MLT on male reproduction.

As a chemotherapeutic drug, cyclophosphamide (CP) has a negative impact on male fertility due to its reproductive toxicity. Melatonin (Mel) promotes the male reproductive system and increases testosterone synthesis. This study is aimed at exploring the molecular mechanism of Mel as a protector of male fertility against CP-induced cytotoxicity. A CP toxicity model was established in adult ICR male mice by intraperitoneal injection of 100 mg/kg CP every other day for a week. Protective effects of Mel on the testis from CP-induced damage were evaluated using four groups of ICR male mice that received intraperitoneal injections of normal saline, 100 mg/kg CP, 10 mg/kg Mel, or the same dosage of CP and Mel, respectively. Testis morphology was observed by hematoxylin and eosin (HE) staining. Sperm quality parameters were evaluated, and sperm function was studied by in vitro fertilization (IVF). Proliferation, meiosis, and pyroptosis markers were examined by western blot. Results showed that CP treatment induced testis toxicity in a time-dependent manner with the most severe damage to the testis at two weeks post CP treatment. CP-treated mice showed reduced testicular weight and impaired spermatogenesis by downregulating PCNA and SYCP3, reduced serum testosterone levels, decreased sperm counts and motility, increased seminiferous tubule vacuolization, and oxidative damage to spermatogenic cells. All these effects, apart from testicular weight, could be ameliorated by Mel administration. The IVF experiment revealed that CP treatment reduced the rates of sperm fertilization and blastocyst development, which were also enhanced by Mel. Mel-treated mice also showed increased expression of proliferation-associated protein PCNA and meiosis-associated proteins REC8, STRA8, and SYCP3, which were all reduced by CP. Furthermore, Mel inhibited the pyroptosis of spermatogenic cells by reducing GSDMD and IL18 expression. In conclusion, this study indicated that Mel might protect the testis from CP-induced DNA damage to germ cells through the alleviation of pyroptosis.