Perfluorohexane sulfonic acid (PFHxS) is extensively used in waterproof coatings and fire-fighting foams, and several studies have found it to be a potential health hazard, but there is still unknown about its effects on spermatogenesis. Our results showed that PFHxS-treated mice have significant reproductive toxicity, including a decrease in sperm count and motility, and the levels of sex hormones (P < 0.05). Concurrently, structural abnormalities are observed in sperm, affecting ≈60-75% of those in the PFHxS-treated group. Additionally, it is found that the structure of the blood-testis barrier (BTB) is damaged after PFHxS treatment, leading to higher expression levels of inflammatory cytokines in the microenvironment for spermatogenesis. Moreover, the expression of proteins associated with mitochondrial biogenesis, including PTEN-induced kinase 1 (PINK1) and NADPH oxidase 4 (NOX4), is dysregulated in the testes after PFHxS treatment. Based on metabolome data, the differential metabolite 3-hydroxybutanoic acid is identified in the PFHxS-treated group, which can regulate the histone Kac levels, especially H3K4ac and H3K9ac. In summary, the results of this study suggest that in the testes of PFHxS-treated mice, inflammatory factors disrupt the mitochondrial function and metabolic profiles and hinder the progress of gene transcription through histone Kac, ultimately causing sperm dysfunction.

Getah virus (GETV) is a zoonotic virus transmitted via a mosquito-vertebrate cycle. While previous studies have explored the epidemiology and pathogenicity of GETV in various species, its molecular mechanisms remain largely unexplored. This study investigated the impact of GETV infection and associated molecular mechanisms on reactive oxygen species (ROS) and autophagy levels in mouse Leydig cells both in vivo and in vitro. The male mice and TM3 cells were treatment with N-acetylcysteine (NAC) to reduce cellular ROS levels. Rapamycin (Rapa) and 3-Methyladenine (3- MA) were used to change autophagy in both infected and uninfected TM3 cells. The findings revealed that GETV infection in mouse testes speciffcally targeted Leydig cells and induced oxidative stress while enhancing autophagy in testicular tissue. Using TM3 cells as an in vitro model, the study confirmed GETV replication in this cell line, triggering increased ROS and autophagy levels. Treatment with N-acetylcysteine (NAC) to reduce cellular ROS levels markedly reduced autophagy in testicular tissue and TM3 cells infected with GETV. Interestingly, the use of rapamycin (Rapa) and 3-Methyladenine (3- MA) led to autophagy change in both infected and uninfected TM3 cells, with no signiffcant alterations in cellular ROS levels. These results indicate that GETV infection elevates ROS levels, subsequently inducing autophagy in mouse Leydig cells. We also found that autophagy plays an important role in GETV replication. When autophagy levels were reduced using NAC and 3-MA, a corresponding decrease in TCID50 was observed. Conversely, upregulation of autophagy using Rapa resulted in an increase in TCID50 of GETV. Therefore, we speculate that GETV may exploit the autophagy pathway to facilitate its replication. These ffndings illuminate the interplay between GETV and host cells, providing valuable insights for therapeutic strategies targeting autophagy in GETV infections.

Gametogenesis is a process in which dysfunctions lead to infertility, a growing health and social problem worldwide. In both spermatogenesis and oogenesis, post-transcriptional gene expression regulation is crucial. Essentially, all mRNAs possess non-templated poly(A) tails, whose composition and dynamics (elongation, shortening, and modifications) determine the fate of mRNA. Moreover, gametogenesis, especially oogenesis, represents a unique instance of the complexity of poly(A) tails metabolism, with oocyte-specific waves of cytoplasmic polyadenylation. In this context, we provide a comprehensive transcriptomic dataset focusing on mRNA poly(A) tail composition and dynamics in murine testes and ovaries. It consists of RNA samples isolated from wild-type and transgenic mice lacking TENT5 polymerases, which can extend poly(A) tails in the cytoplasm. TENT5 deficiencies have serious consequences. For instance, the defect of TENT5D causes infertility in humans. The data described here are generated mainly using the Oxford Nanopore Direct RNA Sequencing (DRS) method, which provides ground-truth information about mRNA molecules, including poly(A) tail length and nucleotide content. For instance, we show the prevalence of uridilated tails in testicular mRNAs.

Spermatogenesis is one of the most complex processes of cell differentiation and its failure is a major cause of male infertility. Therefore, a proper model that recapitulates spermatogenesis in vitro has been long sought out for basic and clinical research. Testis organ culture using the gas-liquid interphase method has been shown to support spermatogenesis in mice and rats. However, the conventional method using agarose gel has limitations including medium replacement efficiency and live imaging because agarose absorbs medium and is not transparent. To overcome this issue, we developed a new device using microporous membranes and oxygen-permeable materials. Mouse testes sandwiched between a microporous polyethylene terephthalate (PET) membrane on top and an oxygen-permeable 4-polymethyl-1-pentene polymer (PMP) membrane base maintained spermatogenesis over months. The chamber volume was minimized to 0.1% of the culture medium. Weekly time-lapse live imaging enabled us to observe transgenically fluorescent acrosome and nuclear shape formation throughout spermatogenesis. Finally, we obtained healthy fertile offspring from spermatozoa generated in our system. The device could be used not only for basic research to understand spermatogenesis but also for applied research, such as diagnosing and treating male infertility.

BP-3 is the most widely used ultraviolet absorber, but its toxic effects and mechanisms far from being elucidated. This study evaluated the male developmental reproductive toxicities and mechanism of low-doses of BP-3. The results indicated that BP-3 (2.28 and 228 μg/L) led to a decrease in sperm quantity, quality and testosterone level, impaired blood-testis barrier (BTB) integrity and cytoskeleton, accompanied by aggravated oxidative stress in testes of mice on postnatal day 56 (PND 56). Notably, chemokine CCL27, a driver of oxidative stress, was significantly upregulated induced by BP-3. Similar disrupted effects were detected in testes of mice on PND14, which could be antagonized by ICI 182780 (estrogen receptor antagonist). Mechanistically, BP-3 directly interacted with ER, which boosted CCL27 expression, reactive oxygen species (ROS) accumulation, and BTB and cytoskeleton impairment. In vitro, si-CCL27 and/or ROS scavenger treatment significantly antagonized BP-3-induced oxidative stress and the decrease of BTB and cytoskeleton related genes in TM4 cells. These findings demonstrate that prolonged exposure to low-doses of BP-3 resulted in detrimental effects on testicular development through activation of the ER/CCL27/ROS axis. This study provides a novel perspective understanding the male reproductive toxicity risk caused by BPs exposure at low-doses.

[This retracts the article DOI: 10.1155/2021/6644093.].

Shenling Baizhu San improves spermatogenic dysfunction in hyperuricemia mice by regulating Sirt3/Nrf2 to inhibit testicular ferroptosis.

The molecular basis of spermatogenesis, which is a tightly regulated spatiotemporal process in testicular seminiferous tubules where germ cell differentiation and somatic-germ cell interactions drive sperm production, remains incompletely understood. Histological staining techniques lack molecular resolution, while scRNA-seq loses spatial context. Conventional spatial transcriptomics (approx. 100 μm resolution) is too coarse-grained for testicular cells (10–20 μm in diameter), leading to mixed-cell signals. In this study, we used Salus-STS high-resolution spatial transcriptomics (∼1 μm resolution) and Salus Cellbins Algorithm to characterize the spatial transcriptomic profile of mouse testes at single-cell level. Integrating the spatial data with scRNA-seq via RCTD annotated major cell subtypes, whose distributions aligned with histology. Pseudotime and spatial gradient analyses revealed a basement membrane-to-lumen developmental axis, with luminal genes (e.g., Prm2) enriched in sperm maturation and basal genes (e.g., mt-Nd4) linked to mitochondrial metabolism—validated by PPI and GO analyses. The biological relevance of these marker genes is underscored by the fact that mutations in Prm2 and mt-Nd4 are known to be associated with human male infertility, highlighting their potential diagnostic value. This work enables high-resolution dissection of spermatogenesis’ spatiotemporal dynamics, providing insights into male reproductive biology.

As a crucial member of the Suppressor of Cytokine Signaling (SOCS) family, SOCS7 regulates various physiological processes, including insulin resistance, inflammation, and tumor suppression. However, its role in male germ cells remains poorly understood. This study aims to investigate the function of SOCS7 in spermatogenesis and uncover its potential regulatory mechanisms. We conducted bioinformatics analyses to examine the expression profile of Socs7 in the testes, generated Socs7-knockout (KO) mice using CRISPR/Cas9 genome editing, and assessed testicular morphology through histological and immunohistochemical staining. Semen quality was evaluated using computer-assisted sperm analysis (CASA), and testicular apoptosis was examined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Bioinformatics analysis revealed high expression of Socs7 in both human and mouse testes. However, Socs7-KO mice exhibited normal fertility, with no significant differences in testicular morphology, sperm quality, or spermatogenesis compared to wild-type (WT) mice. Additionally, testicular apoptosis in Socs7-KO mice was not significantly altered. Our study demonstrates that although Socs7 is highly expressed in the testes, its deletion does not impair male fertility or spermatogenesis in mice. These findings provide valuable insights into the role of SOCS7 in male reproduction and help prevent unnecessary duplication of research efforts.

Bioactive molecules are mostly chiral, and their enantiomers often exhibit different biological properties, including pharmacological effects and toxicity. Recent biochemical studies have revealed that certain trace-level chiral metabolites are associated with specific pathological conditions, including chronic kidney disease and metabolic disorders. This finding highlights the importance of enantioselective imaging techniques that can help visualize the spatial distribution and dynamic behavior of individual enantiomers. Although enantioselective biochemical analyses, such as those based on chromatography or electrophoresis, have proven effective in separating enantiomers, progress in enantioselective imaging methods has been limited. Ion mobility spectrometry/mass spectrometry imaging (IMS/MSI) has emerged as a powerful tool with the potential to enable the enantioselective imaging of minor chiral metabolites. However, suitable chemical structures that can achieve both sufficient resolving power for the target enantiomer in IMS and high ionization efficiency in MSI remain unclear. This review highlights the development of enantioselective imaging methods based on on-tissue chiral derivatization and IMS/MSI. After exploring suitable chiral derivatization reagents, we designed a new charged chiral tag that enabled complete separation of a pair of enantiomers by IMS, sensitive detection of D,L-2-hydroxyglutaric acid by mass spectrometry, and the visualization of their distribution in the mouse testis by IMS/MSI. This approach can be further expanded to analyze other chiral molecules and has great potential for unveiling the enantioselective distribution and dynamics of minor chiral metabolites in biological tissues.

With the increasing importance of posttranscriptional regulation, in particular translational regulation in development and disease, researchers are developing a methodology to dissect the interactions between RNA binding proteins and their RNA targets. One key aspect of this investigation is identifying RNA targets of those RNA-binding proteins using the UV-cross-linking immunoprecipitation approach. Such a crosslinking immunoprecipitation (CLIP) methodology is potent in identifying targets of RNA-binding proteins in many cell lines. An extension of such a technology is to apply the CLIP approach to tissue and organs to reveal RNA targets of physiological relevance. Here, we summarize our methods in identifying RNA targets from mouse testes and fly testes by enhanced CLIP (eCLIP) method and propose this eCLIP method could be applied to testes of most animal species if an effective antibody is available to an RNA binding protein of interest.

Transmembrane proteins are known to play pivotal roles in spermatogenesis and male fertility. Recent transcriptomic analyses have suggested that the serine incorporator 4 (SERINC4), a multi-pass transmembrane protein, is highly expressed in testicular germ cells, yet its physiologic function in male reproduction remains unclear. This study aimed to investigate the in vivo role of SERINC4 in spermatogenesis and male fertility using a Serinc4-knockout (KO) mouse model. Single-cell RNA sequencing (scRNA-seq) data were analyzed to determine the expression pattern of SERINC4 in human and Serinc4 in mouse testis. Serinc4-KO mice were generated by CRISPR/Cas9-mediated deletion of exon 5. Phenotypic analyses included fertility testing, testis histology, sperm evaluation using computer-assisted sperm analysis (CASA), immunofluorescence for germ and somatic cell markers, and TUNEL assay for germ cell apoptosis. Although scRNA-seq analyses revealed high SERINC4 expression in spermatids of human testes and high Serinc4 expression in spermatocytes of mouse testes, its deficiency did not impair male fertility, testicular morphology, or spermatogenesis in mice. No significant differences were observed between wild-type (WT) and Serinc4-KO mice in terms of sperm count, motility, or testis to body weight ratio. In addition, TUNEL assay revealed no significant change in germ cell apoptosis. Under physiologic conditions, Serinc4 appears to be dispensable for normal spermatogenesis and male fertility in mice. Future studies are needed to investigate its role under pathologic or aging-related conditions.

Systemic lipopolysaccharide (LPS)-induced inflammation has a significant impact on various organs, including the male reproductive system. In this study, we have demonstrated that LPS-induced inflammation causes oxidative stress in mouse testes, reduces expression of genes encoding the catalytic subunit of glutamate-cysteine ligase (Gclc) and superoxide dismutase 2 (Sod2). Inflammation suppressed transcription of genes involved in differentiation and metabolic regulation of testicular cells and sperm maturation: in the LPS group, the expression of the Amh, Lepr, Eif2b4 genes was approximately 3 times lower compared to the control group. The intake of probiotic microorganisms caused a decrease in the intensity of lipid peroxidation, which was manifested in a decrease in the level of conjugated dienes (CD) compared to the LPS group, contributed to maintaining the level of expression of genes supporting the antioxidant status, as well as genes supporting the functionality of the mouse testes. The data obtained suggest that probiotics may be considered as potential tools for maintaining male reproductive function under conditions of inflammatory processes.

Measuring mRNA abundance at the level of the whole transcriptome has become an indispensable tool for the investigation of fundamental biological processes in higher eukaryotes. However, traditional transcriptomic methods are destructive and ignore the three-dimensional organization of cells within tissues and organs. Spatial transcriptomics is a cutting-edge technology that begins to address this limitation and facilitates transcriptomic analyses within the normal spatial context of two-dimensional tissue sections. This method permits a comprehensive appreciation of cellular heterogeneity according to spatial organization and overcomes the critical limitation of traditional bulk methods by preserving tissue architecture. Spermatogenesis is an exemplary cell lineage that varies in a spatial manner. Specifically, the stages of the cycle of the seminiferous epithelium elaborate the cell type variation along the length of seminiferous tubules, which are revealed in two-dimensional space as distinct cellular associations in seminiferous tubule cross-sections. Interpreting the wealth of available single-cell RNA-seq data requires appreciation of cycle stage, which can only be accomplished by understanding each germ cell's spatial context. In this chapter, we provide an overview of spatial transcriptome technologies and step-by-step guidelines for application of one such method from Curio Biosciences, Seeker (SlideSeq V2) to the testis. The text elaborates on testicular tissue preparation and quality assessment, section and spatial transcriptome library preparation, and bioinformatic methods for data analysis using a simple exemplary analysis of round spermatids in the mouse testis.

Mammalian spermatogenesis is an intricate process resulting in the formation of male haploid gametes. In order to attain a better understanding of the complexity of this process, the development of a reproducible cytometry method to isolate germ cells is much needed. Here, we describe a reproducible cytometry protocol to isolate germ cells from adult mouse testis. We paid special attention to the optimization of what are considered the most critical steps, including testis disaggregation, antibody titration, cell staining, and germ cell discrimination by FACS.

Cross-species comparative single-cell transcriptomics highlights the molecular evolution and genetic basis of male infertility.

Exploring transcriptomic databases to identify and experimentally validate tissue-specific consensus reference gene for gene expression normalization in BALB/c mice acutely exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Diabetes is a chronic metabolic condition that causes testicular damage by high oxidative stress, rendering 35% of afflicted people infertile. Shilajit is a traditional Indian medicine known for its antioxidant, antidiabetic, and aphrodisiac properties. However, its effectiveness on diabetes‐induced testicular dysfunction remains unclear. Therefore, the current investigation aimed to determine whether Shilajit could restore testicular functions in diabetic mice. Two days postpartum male Parkes mice received a single intraperitoneal injection of streptozotocin (STZ) (90 mg/kg BW) to induce diabetes. Three months postinjection, the effects of daily Shilajit (100 and 200 mg/kg BW) treatment were evaluated for one spermatogenic cycle in adult diabetic mice, using Empagliflozin (10 mg/kg BW) as a positive control. In STZ‐induced diabetic mice, testicular functions were compromised due to disruptions in testosterone biosynthesis, changes in germ‐cell ratios, and increased oxidative stress and apoptosis. Shilajit restored glycemic status in diabetic mice by significantly decreasing serum glucose, insulin level, and homeostasis model assessment of insulin resistance (HOMA‐IR) value while increasing insulin sensitivity. These effects were comparable to those observed with conventional antidiabetic medication Empagliflozin. Further, Shilajit stimulates steroidogenesis and germ cell dynamics of diabetic mice by increasing the activity of StAR, 3 β ‐HSD, and 17 β ‐HSD enzymes and 1C:2C, 4C:S‐Ph, and 1C:4C germ cell ratios, respectively. Shilajit also improves blood–testis barrier (BTB) functioning by increasing expression of ZO‐1, Connexin‐43, N‐Cadherin, and β ‐catenin as well as oxidative status and apoptosis by modulating NF‐E2–related factor 2 (Nrf‐2)/heme oxygenase 1 (HO‐1) signaling and Bax/Bcl‐2 ratio. Subsequently, Shilajit improved the histoarchitecture of testis and epididymis in diabetic mice and recovered both qualitative and quantitative sperm parameters, as seen by higher percentages of sperm motility, viability, and normal sperm morphology as well as increased sperm numbers in cauda epididymis. In summary, Shilajit restores glycemic status, increases insulin sensitivity, stimulates steroidogenesis, and improves testicular functions through Sertoli cell and Nrf‐2/HO‐1 signaling in STZ‐induced diabetic mice.

Phthalates (PHA) are common environmental pollutants used extensively in the plastics sector. Exposure to PHA negatively impacts both human and animal health. Thymoquinone (TQ), an active ingredient of black cumin seed, exhibits potential pharmacological effects against many illnesses. The study aimed to investigate how TQ affected the reproductive parameters and histo-morphology of the testes in male Swiss albino mice that has been given PHA. Twenty-four male mice, aged 28 to 30 days, were utilized and allocated into three groups, each including eight animals. Group A (the control) received standard mice pellets combined with olive oil; Group B was supplied PHA; and Group C was given both TQ and PHA mixed with mice pellets. All mice were reared at 26–30°C for 60 days. Blood, serum, and organs were obtained and processed using established protocols. PHA induced an elevation in body weight in male mice. The administration of TQ normalized body weight in PHA-treated mice. Administration of PHA to male mice resulted in a significant decrease in blood levels of thyroxine (T4) and testosterone (p &lt;0.01), whereas the administration of TQ led to an increase in these two hormones. PHA resulted in a substantial (p&lt;0.01) decrease in sperm count and motility, accompanied by an increase in abnormalities, whereas TQ mitigated these sperm characteristics. Degenerative and necrotic alterations were observed in the seminiferous tubules of the testis in PHA-treated male mice which was altered by TQ. In conclusion, the integration of TQ may alleviate the adverse effects generated by PHA.

A long-term mouse testis organ culture system to identify germ cell damage induced by chemotherapy.