Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and β-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.

BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases.
METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development.
RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated.
CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

Iodoacetic acid (IAA) is an emerging unregulated iodinated disinfection byproduct with high toxicity and widespread exposure. IAA has potential reproductive toxicity and could damage male reproduction. However, the underlying mechanisms and toxicological targets of IAA on male reproductive impairment are still unclear, and thus Sprague-Dawley rats and Leydig cells were used in this work to decode these pending concerns. Results showed that after IAA exposure, the histomorphology and ultrastructure of rat testes were abnormally changed, numbers of Leydig cells were reduced, the hypothalamic-pituitary-testis (HPT) axis was disordered, and testosterone biosynthesis was inhibited. Proteomics analyses displayed that oxidative stress, endoplasmic reticulum stress, and steroid hormone biosynthesis were involved in IAA-caused reproductive injury. Antioxidant enzymes were depleted, while levels of ROS, MDA, 8-OHdG, and γ-H2A.X were increased by IAA. IAA triggered oxidative stress and DNA damage, and then activated the GRP78/IRE1/XBP1s and cGAS/STING/NF-κB pathways in Leydig cells. The two signaling pathways constructed an interactive network by synergistically regulating the downstream transcription factor CHOP, which in turn directly bound to and negatively modulated steroidogenic StAR, finally refraining testosterone biosynthesis in Leydig cells. Collectively, IAA as a reproductive toxicant has anti-androgenic effects, and the GRP78/IRE1 and cGAS/STING pathway crosstalk through CHOP facilitates IAA-mediated testosterone decline.

As an emerging environmental endocrine disruptor, polystyrene microplastics (PS-MPs) are considered to have the anti-androgenic feature and impair male reproductive function. To explore the adverse effects of PS-MPs on testosterone synthesis and male reproduction and further elucidate underlying mechanisms, BALB/c mice and Leydig cells were employed in the present work. The results indicated that 50 μm PS-MPs accumulated in mouse testes and were internalized into the cytoplasm. This not only damaged the testicular histomorphology and ultrastructure, but also reduced the viability of Leydig cells and the serum level of GnRH, FSH, LH, and testosterone. After PS-MPs exposure, the ubiquitination degradation and miR-425-3p-targeted modulation synergistically contributed to the suppression of GPX1, which induced oxidative stress and subsequently activated the PERK-EIF2α-ATF4-CHOP pathway of endoplasmic reticulum (ER) stress. The transcription factor CHOP positively regulated the expression of SRD5A2 by directly binding to its promoter region, thereby accelerating testosterone metabolism and ultimately lowing testosterone levels. Besides, PS-MPs compromised testosterone homeostasis via interfering with the hypothalamic-pituitary-testis (HPT) axis. Taken together, PS-MPs possess an anti-androgenic characteristic and exert male reproductive damage effects. The antioxidant enzyme GPX1 plays a crucial role in the PS-MPs-mediated testosterone decline.

Obesity is a growing epidemic among reproductive-age men, which can cause and exacerbate male infertility by means of associated comorbidities, endocrine abnormalities, and direct effects on the fidelity and throughput of spermatogenesis. A prominent consequence of male obesity is a reduction in testosterone levels. Natural products have shown tremendous potential anti-obesity effects in metabolic diseases. This study aimed to investigate the potential of apigenin (AP) to alleviate testicular dysfunction induced by a high-fat diet (HFD) and to investigate the underlying mechanisms, focusing on endoplasmic reticulum stress (ERS) and testosterone synthesis. A murine model of obesity was established using HFD-fed mice. The effects of AP on obesity, lipid metabolism, testicular dysfunction, and ERS were assessed through various physiological, histological, and molecular techniques. Administration of AP (10 mg/kg) ameliorated HFD-induced obesity and testicular dysfunction in a mouse model, as evidenced by decreased body weight, improved lipid profiles and testicular pathology, and restored protein levels related to testosterone. Furthermore, in vitro studies demonstrated that AP relieved ERS and recovered testosterone synthesis in murine Leydig cells (TM3) treated with free fatty acids (FFAs). It was also observed that AP rescued testosterone synthesis enzymes in TM3 cells, similar to that observed with the inhibitor of the PERK pathway (GSK2606414). In addition, ChIP, qPCR, and gene silencing showed that the C/EBP homologous protein (CHOP) bound directly to the promoter region of steroidogenic STAR and negatively modulated its expression. Collectively, AP has remarkable potential to alleviate HFD-induced obesity and testicular dysfunction. Its protective effects are attributable partly to mitigating ERS and restoring testosterone synthesis in Leydig cells.

Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323 testicular single cells from young and old mice, shedding light on 1032 telocytes-an underexplored testicular cell type in previous research. Our study unveiled 916 age-related differentially expressed genes (age-DEGs), with telocytes emerging as the cell type harboring the highest count of age-DEGs. Of particular interest, four genes (Klk1b21, Klk1b22, Klk1b24, Klk1b27) from the Kallikrein family, specifically expressed in Leydig cells, displayed down-regulation in aged testes. Moreover, cell-type-level splicing analyses unveiled 1838 age-related alternative splicing (AS) events. While we confirmed the presence of more age-DEGs in somatic cells compared to germ cells, unexpectedly, more age-related AS events were identified in germ cells. Further experimental validation highlighted 4930555F03Rik, a non-coding RNA gene exhibiting significant age-related AS changes. Our study represents the first age-related single-cell transcriptomic investigation of testicular telocytes and Kallikrein genes in Leydig cells, as well as the first delineation of cell-type-level AS dynamics during testicular aging in mice.

Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1β and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.

Late-onset hypogonadism (LOH) is an age-related syndrome characterized by deficiency of serum testosterone produced by Leydig cells. Previous evidence suggested that microRNA (miR)-361-3p can serve as a promising biomarker for LOH. Nonetheless, its detailed function and molecular mechanism in LOH remain unclarified. The 24-month-old male mice were selected as an animal LOH model, and mouse Leydig cell line TM3 was stimulated with H2O2. ELISA was employed for testosterone level evaluation. Hematoxylin-eosin staining was implemented for histologic analysis of mouse testicular tissues. Western blotting and RT-qPCR were utilized for evaluating molecular protein and RNA expression, respectively. Functional experiments were conducted to test miR-361-5p roles. Luciferase reporter assay was for verifying the interaction between miR-361-5p and protein inhibitor of activated STAT 1 (PIAS1). miR-361-5p displayed a decreased level in the testes of LOH mice. Overexpressing miR-361-5p attenuated Leydig cell loss in the testis and elevated serum and intratesticular testosterone levels in LOH mice. H2O2 stimulation impaired TM3 cell viability, proliferation and intracellular testosterone production and enhanced cell apoptosis. miR-361-5p targeted PIAS1 in TM3 cell. PIAS1 upregulation counteracted miR-361-5p overexpression-mediated alleviation of cell apoptosis and elevation of testosterone synthesis in H2O2-stimualetd TM3 cells. miR-361-5p ameliorates LOH progression by increasing testosterone production and alleviate Leydig cell apoptosis via downregulation of PIAS1.

Tris (2-ethylhexyl) phosphate (TEHP) is a frequently used organophosphorus flame retardant with significant ecotoxicity and widespread human exposure. Recent research indicates that TEHP has reproductive toxicity. However, the precise cell mechanism is not enough understood. Here, by using testicular mesenchymal stromal TM3 cells as a model, we reveal that TEHP induces apoptosis. Then RNA sequencing analysis, immunofluorescence, and western blotting results show that THEP inhibits autophagy flux and enhances endoplasmic reticulum (ER) stress. Moreover, the activation of the ER stress is critical for TEHP-induced cell injury. Interestingly, TEHP-induced ER stress is contributed to autophagic flux inhibition. Furthermore, pharmacological inhibition of autophagy aggravates, and activation of autophagy attenuates TEHP-induced apoptosis. In summary, these findings indicate that TEHP triggers apoptosis in mouse TM3 cells through ER stress activation and autophagy flux inhibition, offering a new perspective on the mechanisms underlying TEHP-induced interstitial cytotoxicity in the mouse testis.

Fine particulate matter (PM2.5), a significant component of air pollution particulate matter, is inevitable and closely associated with increasing male reproductive disorder. However, the testicular targets of PM2.5 and its toxicity related molecular mechanisms are still not fully understood. In this study, the conditional knockout (cKO) mice and primary Leydig cells were used to explore the testicular targets of PM2.5 and the related underlying mechanisms. First, apparent the structure impairment of seminiferous tubules, Leydig cells vacuolization, decline of serum testosterone and sperm quality reduction were found in male wild-type (WT) and Sirt1 knockout mice after exposure to PM2.5. Enrichment analyses revealed that differentially expressed genes (DEGs) were enriched in steroid hormone biosynthesis, ferroptosis, and HIF-1 signaling pathway in the mice testes after exposure to PM2.5, which were subsequently verified by the molecular biological analyses. Notably, similar enrichment analyses results were also observed in primary Leydig cells after treatment with PM2.5. In addition, Knockdown of Sirt1 significantly increased PM2.5-induced expression and activation of HIF-1α, which was in parallel to the changes of cellular iron levels, oxidative stress indicators and the ferroptosis markers. In conclusion, this highlights that PM2.5 triggers ferroptosis via SIRT1/HIF-1α signaling pathway to inhibit testosterone synthesis in males. These findings provide a novel research support for the study that PM2.5 causes male reproductive injury.