Mouse Testis Research Articles

Single-cell transcriptomics reveals the cellular dynamics of hexafluoropropylene oxide dimer acid in exerting mouse male reproductive toxicity

BackgroundHexafluoropropylene oxide dimer acid (GenX), a substitute for per- and polyfluoroalkyl substances, has been widely detected in various environmental matrices and foods recently, attracting great attention. However, a systematic characterization of its reproductive toxicity is still missing. This study aims to explore the male reproductive toxicity caused by GenX exposure and the potential cellular and molecular regulatory mechanisms behind it.ResultsNormally developing mice were exposed to GenX, and testicular tissue was subsequently analyzed and validated using single-cell RNA sequencing. Our results revealed that GenX induced severe testicular damage, disrupted the balance between undifferentiated and differentiated spermatogonial stem cells, and led to strong variation in the cellular dynamics of spermatogenesis. Furthermore, GenX exposure caused global upregulation of testicular somatic cellular inflammatory responses, increased abnormal macrophage differentiation, and attenuated fibroblast adhesion, disorganizing the somatic-germline interactions.ConclusionsIn conclusion, this study revealed complex cellular dynamics and transcriptome changes in mouse testis after GenX exposure, providing a valuable resource for understanding its reproductive toxicity.

A Single-Cell Transcriptome Atlas Characterizes the Immune Landscape of Human Testes During Aging.

Aging disrupts immune regulation, affecting tissue function and increasing vulnerability to various diseases. However, the effects of aging on immune cells within human testes are not well understood. In this study, we utilized single-cell RNA sequencing to profile immune cells from 33 human testis samples from individuals aged 21 to 69. Our analysis revealed key immune cell types, including CD8+ T cells, monocytes, cDC2 cells, and various macrophage subtypes within the testes. We observed an age-related change in monocytes and MRC1hi tissue-resident macrophage (TRM), a pattern consistent in both human and mouse testes. Individuals aged 40 and older showed notable shifts in pathways related to phagocytosis, cytokine signaling, and antigen presentation. Monocytes also exhibited pro-inflammatory characteristics, potentially contributing to the low-grade inflammation commonly associated with aging. Together, these findings provide insights into age-related immune cell alterations in human testes and uncover molecular mechanisms underlying these shifts, offering a valuable resource for understanding immune aging in the reproductive system.

Fine particulate matter (PM2.5) and the blood-testis-barrier: an in vivo and in vitro mechanistic study.

Fine particulate matter (PM2.5) is considered a major component of ambient particulate matter (PM). Exposure to PM2.5 was shown to be related to male reproductive system injury. Ferroptosis is regarded as an iron-dependent programmed cell death, that is associated with the pathological process. It has been reported that SIRT1 has protective effects on the male reproductive system. However, the underlying mechanisms of exposure to PM2.5 induced-testicular injury are still unexplored. In this study, we investigated the relationship between ferroptosis and male reproductive injury, after exposure to PM2.5 and the role of SIRT1/HIF-1α signaling pathway in this process. We established PM2.5 exposure model in vivo and in vitro using Sertoli cell Sirt1 conditional knockout C57BL/6 (cKO) mice testes and primary Sertoli cells. Hematoxylin and eosin (H&E) staining were conducted to examine the histology of the mice testes. Sperm parameters and biotin tracer assay were conducted to evaluate the effects of exposure to PM2.5 on the mice testes. Related markers and genes related to the blood-testis barrier (BTB) and ferroptosis were measured by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and immunofluorescence assay. siRNA transfection was used to evaluate the potential mechanism. Significant pathological damage and lower sperm quality were detected in mice testes exposed to PM2.5. We found that exposure to PM2.5 damaged the blood-testis barrier (BTB) and inhibited the expression level of the BTB-related proteins (including Connexin 43, Occludin, Claudin 11, N-Cadherin and ZO-1). According to the enrichment analysis results, ferroptosis and HIF-1α signaling pathway were significantly enriched in mice testes and primary Sertoli cells exposed to PM2.5. Subsequent experiments were conducted to verify the results of enrichment analysis and revealed differences in the expression levels of HIF-1α, ferroptosis-related genes (including GPX4, SLC7A11, ACSL4 and HO-1) and ferroptosis-related markers (including MDA, GSH and Fe2+), associated with lower expression of SIRT1 after exposure to PM2.5. These results suggest that PM2.5 exposure may be associated with ferroptosis and HIF-1α signaling pathway in male reproductive dysfunction. Taken together, in vivo and in vitro experiments verified that PM2.5 exposure in mice may lead to testicular dysfunction through new pathways. https://doi.org/10.1289/EHP14447.

Single-cell multiomic comparison of mouse and rat spermatogenesis reveals gene regulatory networks conserved for over 20 million years.

Spermatogenesis is driven by dramatic changes in chromatin regulation, gene transcription, and protein expression. To assess the mechanistic bases for these developmental changes, we utilized multiomic single-cell/nucleus RNA sequencing (sc/snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to identify chromatin changes associated with transcription in adult mouse and rat testes. We characterized the relationships between the transcriptomes and chromatin of both species, including the divergent expression of Id4 in spermatogonial stem cells between species. Promoter accessibility and gene expression showed the greatest association during meiosis in both species. We mapped the cross-species conservation of putative regulatory regions for key spermatogenic genes, including Cd9 and Spam1, and investigated correlations and disconnects in chromatin accessibility, gene expression, and protein expression via antibody-derived tags. Using a gene regulatory network (GRN) model, we identified 40 core regulons conserved between mouse and rat germ cells, highlighting the relevance of chromatin-related factors in regulating the transcription of canonical genes across spermatogenesis.

TEX38 localizes ZDHHC19 to the plasma membrane and regulates sperm head morphogenesis in mice

Sperm morphogenesis is a tightly regulated differentiation process, disruption of which leads to sperm malfunction and male infertility. Here, we show that Tex38 knockout (KO) male mice are infertile. Tex38 KO spermatids exhibit excess retention of residual cytoplasm around the head, resulting in abnormal sperm morphology with backward head bending. TEX38 interacts and colocalizes with ZDHHC19, a testis-enriched acyltransferase catalyzing protein S-palmitoylation, at the plasma membrane of spermatids. ZDHHC19 and TEX38 are each downregulated in mouse testes lacking the other protein. TEX38 stabilizes and localizes ZDHHC19 to the plasma membrane of cultured cells and vice versa, consolidating their interdependence. Mice deficient in ZDHHC19 or harboring a C142S mutation that disables the palmitoyltransferase activity of ZDHHC19 display phenotypes resembling those of Tex38 KO mice. Strikingly, ZDHHC19 palmitoylates ARRDC5, an arrestin family protein regulating sperm differentiation. Overall, our findings indicate that TEX38 forms a stable complex with ZDHHC19 at the plasma membrane of spermatids, which governs downstream S-palmitoylation of proteins essential for morphological transformation of spermatids.

Mitochondrial ROS-associated integrated stress response is involved in arsenic-induced blood-testis barrier disruption and protective effect of melatonin.

Mitochondrial ROS-associated integrated stress response is involved in arsenic-induced blood-testis barrier disruption and protective effect of melatonin.

Study on the role of mitophagy and pyroptosis induced by nano-silver in testicular injury.

Study on the role of mitophagy and pyroptosis induced by nano-silver in testicular injury.

Diminazene aceturate, an ACE2 activator, ameliorates testicular injury in diet-induced obese mice.

Obesity is a prevalent health concern globally, often associated with testicular damage that can lead to male infertility. This study investigates the improvement of obesity-induced testicular damage by ACE2 agonist diminazene aceturate (DIZE) and explores the role of the ACE2/Ang 1-7/MasR axis in this process. DIZE improved obesity and metabolic disturbances in diet-induced obesity (DIO) mice. An increase in sperm count and motility, along with improved morphology and increased male fertility, was observed after DIZE treatment. Both serum and intratesticular testosterone levels showed an increase. Moreover, ACE2/Ang 1-7/MasR protein levels in the testes were restored, which inhibited germ cells apoptosis, lipid accumulation and oxidative stress, and elevated testosterone synthesis-related proteins. However, the MasR antagonist A779 partially counteracted the improving effects of DIZE on metabolism and the testes of DIO mice. This study shows that DIZE treatment has protective effects against obesity-induced testicular injury by activating the ACE2/Ang 1-7/MasR axis.

Investigation of the transcriptome and metabolome of the cerebral cortex and testes in Cntnap4-deficient mice

Investigation of the transcriptome and metabolome of the cerebral cortex and testes in Cntnap4-deficient mice

TMC7 is required for spermiogenesis and male fertility by regulating TGN-derived vesicles.

TMC7 is required for spermiogenesis and male fertility by regulating TGN-derived vesicles.

Lead Causes Lipid Droplet Accumulation by Impairing Lysosomal Function and Autophagic Flux in Testicular Sertoli Cells

Lead (Pb) is one of the most common environmental pollutants that negatively impacts male reproductive health. Thus far, the underlying molecular mechanisms of Pb-induced reproductive toxicity are still not well understood. In this study, 64 male ICR mice were given drinking water with Pb (0, 100, 200, and 300 mg/L) for 90 days. We found that exposure to 300 mg/L Pb resulted in reduced sperm quality and elevated autophagy-related protein levels in the mouse testes. Our findings indicate that the Pb hindered the autophagic clearance by impairing the lysosomes’ function and then obstructing the fusion of lysosomes and autophagosomes. The autophagy cycle obstruction prevented the lipid droplets from breakdown and led to their accumulation in the Sertoli cells. In turn, the ccytotoxic effects that resulted from the interruption of the autophagy maturation stage, instead of the elongation phase, could be alleviated by either Chloroquine or Bafilomycin A1. Furthermore, exposure to 400 μM Pb initiated the TFE3 nuclear translocation and caused the increased expression of its target genes. Then, the knockdown of TFE3 reduced the formation of the autophagosome. In addition, the use of the antioxidant NAC notably enhanced the autophagic activity and reduced the occurrence of lipid droplets in the Sertoli cells. This study demonstrated that Pb disrupted the autophagic flow, which caused lipid droplet accumulation in the TM4 cells. Consequently, focusing on the maturation stage of autophagy might offer a potential therapeutic approach to alleviate male reproductive toxicity caused by Pb exposure.

GSK1016790A, a TRPV4 Agonist, Repairs Spermatogenic Dysfunction Caused By Diabetes.

As a devastating metabolic disease, diabetic testicular damage can lead to impaired spermatogenesis and sexual dysfunction. Transient receptor potential cation channel subfamily vanilloid 4 (TRPV4) plays complex roles in the pathogenesis of diabetes and its complications and plays a major role in the development of apoptosis and related mechanisms. This study aimed to investigate the effects of the TRPV4 agonist GSK1016790A on sperm parameters, spermatogenic cell apoptosis and testicular histopathology in STZ-induced diabetic mice. A model of diabetes was induced in 8-week-old male ICR mice by an intraperitoneal injection of STZ for five consecutive days. At week 7, the mice were intraperitoneally injected with the TRPV4 agonist GSK1016790A or normal saline (NS) for 7days. At week 8, the animals were sacrificed. The results revealed that the expression of TRPV4 in the testes of STZ-induced diabetic mice decreased, and the testes exhibited the destruction of tissue structure, decreases in the total sperm count and viability, an increase in the sperm deformity rate, and an increase in apoptosis. These symptoms were reversed by increased TRPV4 expression following the injection of the TRPV4 agonist GSK1016790A. Increased TRPV4 expression can ameliorate cell apoptosis in the STZ-induced testes of diabetic mice and alleviate spermatogenic dysfunction in the testes.

Small RNA data sets of mouse testes and ovaries before and after sexual maturity

For a considerable period, reproductive health, fertility, and reproductive-related diseases have posed challenges to human well-being, as well as to the conservation of endangered species and the advancement of animal husbandry. PANDORA-seq, a recently introduced sequencing technique, demonstrates heightened sensitivity towards highly modified small RNAs like tsRNA and rsRNA. In this research endeavor, we leveraged PANDORA-seq to capture the small RNA expression profiles of mouse testes and ovaries pre- and post-sexual maturation. Our investigation successfully pinpointed an array of abundantly expressed small RNAs across various tissues, encompassing tsRNA, rsRNA, piRNA, miRNA, snoRNA, and ysRNA. Next, we conducted an expression profile analysis of these small RNAs to assist researchers in screening and validating them for various areas of interest. This dataset is poised to become an invaluable resource for exploring the postnatal development of testes and ovaries, offering new insights into the epigenetic mechanisms underlying germ cell production and differentiation.

Male germ cells with Bag5 deficiency show reduced spermiogenesis and exchange of basic nuclear proteins

Bcl-2 associated athanogene-5 (BAG5) represents a unique BAG cochaperone family member, regulating chaperone activity. We first demonstrated significant differences in Bag5 expression by RNA seq analysis of teratozoospermia and healthy male sperm samples, but the genetic and molecular mechanisms governing this process remain elusive. We further found that BAG5 has highest expression in human and mouse testes. BAG5 expression is elevated in late stage pachytene spermatocytes and spermatids. Targeted Bag5 inactivation in mice induces massive apoptosis in male germ cells and abrogates male infertility. The ordered loading of sperm basic nuclear proteins on chromatin is altered, with lost TNPs and PRMs, resulting in severe sperm head deformity and partial 9 + 2 microtubule structure disorder. In terms of mechanism, immunoprecipitation (IP)-mass spectroscopy (MS) revealed BAG5 interacts with HSPA2, a testis-specific HSP70 family member regulating the transcription of the transition protein TNPs as well as spermatogenesis. RNA-sequencing assessment of Bag5 deficient testis confirmed Bag5 participation in transcriptional repression and revealed significant changes in Hspa2 expression. Bag5 deficiency resulted in decreased levels of HSPA2, germ cell apoptosis and subsequent inappropriate nuclear protein deposition and chromatin condensation. Decreased BAG5 expression levels in patients with non-obstructive azoospermia and oligoasthenospermia were also detected. These results uncovered an intriguing HSPA2-mediated key function of BAG5, which may constitute a potential prognostic biomarker of male infertility.

Age-Dependent Clonal Expansion of Non-Sperm-Forming Spermatogonial Stem Cells in Mouse Testes.

In male mammals, spermatogonial stem cells (SSCs) are essential for sustaining lifelong spermatogenesis within the testicular open niche, a unique environment that allows SSC migration over an extended niche area. As SSCs undergo continuous mitotic division, mutations accumulate and are transmitted to the descendant SSC clones. Therefore, SSC clonal fate behaviors, in terms of their efficiencies in completing spermatogenesis and undergoing expansion within the niche, influence sperm genomic diversity. We aimed to elucidate the effects of physiological aging on SSC clonal fate behavior within the testicular open niche. We used single-cell RNA sequencing, lineage tracing, and intravital live imaging to investigate SSC behavior in aged mouse testes, where spermatogenesis, although reduced, persists. We found that undifferentiated spermatogonia maintained gene expression heterogeneity during aging. Among these, GFRα1+ cells, which exhibited state heterogeneity, showed accelerated proliferation and persistent motility, continuing to function as SSCs in older mice. In contrast, a subset of SSCs characterized by low Egr4 and Cops5 expression did not contribute to spermatid formation. These non-sperm-forming SSC clones increased in proportion among the total SSC clones and expanded spatially within the testicular open niche in old mice, a phenomenon not observed in young mice. The expansion of non-sperm-forming SSC clones in aged testes suggests that they occupy a niche space, limiting the availability of functional SSCs and potentially reducing sperm production and genetic diversity. These findings highlight age-specific clonal characteristics as hallmarks of stem cell aging within the testicular open niche and provide novel insights into the mechanisms governing reproductive aging.

Androgen receptor ubiquitination links KCTD13 to genitourinary tract defects.

The potassium channel tetramerization domain containing 13 (KCTD13) protein is a substrate-specific adapter for cullin3-based E3 ubiquitin ligase. Patients with copy number variants at this locus exhibit genitourinary tract anomalies. In this study, we show that decreased androgen receptor (AR) protein level correlated with increased AR ubiquitination in the testis of Kctd13-deficient mice, suggesting that KCTD13 inhibits AR ubiquitination. KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR, confirming the role of KCTD13 as an adaptor of CUL3 ligase. Recombinant KCTD13 directly binds to recombinant AR, and the BTB domain of KCTD13 is critical for binding both the N-terminal domain of AR and STUB1. Moreover, KCTD13 dose-dependently decreased STUB1 binding to AR resulting in decreased AR ubiquitination. KCTD13 ΔBTB was unable to bind to AR and subsequently failed to block STUB1-mediated AR ubiquitination, strongly suggesting that reduced AR ubiquitination is dependent on KCTD13 ability to dissociate AR/STUB1 complex. Furthermore, KCTD13 increased the expression of AR target gene, FOXJ1, whereas KCTD13 ΔBTB had no effect. Our data reveal a distinctive mode of action of KCTD13 on AR ubiquitination depending on the E3 ubiquitin ligase involved: (1) KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR; and (2) KCTD13 decreased STUB1-mediated AR ubiquitination by decreasing STUB1 binding to AR thus preventing AR ubiquitination. We hypothesize that in the testes of Kctd13-deficient mice, the absence of KCTD13 results in increased binding of STUB1 to AR leading to increased AR ubiquitination and degradation.

SATINN v2: automated image analysis for mouse testis histology with multi-laboratory data integration.

Analysis of testis histology is fundamental to the study of male fertility, but it is a slow task with a high skill threshold. Here, we describe new neural network models for the automated classification of cell types and tubule stages from whole-slide brightfield images of mouse testis. The cell type classifier recognizes 14 cell types, including multiple steps of meiosis I prophase, with an external validation accuracy of 96%. The tubule stage classifier distinguishes all 12 canonical tubule stages with external validation accuracy of 63%, which increases to 96% when allowing for ±1 stage tolerance. We addressed generalizability of SATINN, through extensive training diversification and testing on external (non-training population) wildtype and mutant datasets. This allowed us to use SATINN to successfully process data generated in multiple laboratories. We used SATINN to analyze testis images from 8 different mutant lines, generated from 3 different labs with a range of tissue processing protocols. Finally, we show that it is possible to use SATINN output to cluster histology images in latent space, which, when applied to the 8 mutant lines, reveals known relationships in their pathology. This work represents significant progress towards a tool for robust, automated testis histopathology that can be used by multiple labs.

Loss of lncRNAs 1700101O22Rik and 1700027A15Rik causes sperm malformation and subfertility.

The testis is a key reservoir of long non-coding RNAs, yet their physiological roles in male reproduction remain debated. Notably, long non-coding RNAs 1700101O22Rik (22Rik) and 1700027A15Rik (15Rik) are uniquely expressed in the mouse testis. Previous research indicates that both 22Rik and 15Rik play roles in male reproductive processes; however, it is still unclear whether their effects on fertility are cumulative or compensatory. To investigate the influence of simultaneous deletion of 22Rik and 15Rik on male reproduction and whether there are additive effects. 22Rik and 15Rik knockout mice were generated using CRISPR-Cas9, and double knockout mice were obtained through co-caging. To investigate reproductive phenotypes, we utilized computer-aided sperm analysis, acrosome reaction assessments, in vitro fertilization techniques, and sperm morphology analysis. Additionally, RNA sequencing and RNA binding protein immunoprecipitation were employed to explore the regulatory mechanisms of 22Rik and 15Rik. The simultaneous deletion of 22Rik and 15Rik led to abnormal sperm morphology, impaired acrosome reaction, and reduced in vitro fertilization. Sperm count and fertility were also decreased in double knockout male mice. Compared to the knockout of long non-coding RNA 22Rik, reproductive abnormalities were somewhat exacerbated but largely similar to those observed with 15Rik knockout alone because of shared targeted genes, particularly Y chromosome-linked genes. Additionally, these abnormal phenotypes may be linked to reduced expression of transition protein 1 and dysfunction of the HSF2‒Rik22‒Rik15 complex in double knockout mice. Our study demonstrates for the first time that simultaneous knockout of these two long non-coding RNAs adversely affects sperm morphology and function by disrupting the HSF2‒Rik22‒Rik15 complex. Moreover, many overlapping regulated genes suggest that 22Rik and 15Rik may share similar regulatory mechanisms at the molecular level. This research sheds light on the causes and mechanisms behind sperm malformation and impaired male fertility.

Characterization of PRDM9 Multifunctionality in Yak Testes Through Protein Interaction Mapping.

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks during spermatogenesis. PRDM9 determines the localization of recombination hotspots by interacting with several protein complexes in mammals. The function of PRDM9 is not well understood during spermatogenesis in mice or yaks. In this study, we applied yeast two-hybrid assays combined with next-generation sequencing techniques to screen the complete set of PRDM9-interacting proteins and explore its novel functions in yak spermatogenesis. Our results showed that 267 PRDM9-interacting proteins were identified. The gene ontology (GO) analysis of the interacting proteins revealed that the GO terms were primarily associated with spermatogenesis, positive regulation of double-strand break repair via homologous recombination, RNA splicing, the ubiquitin-dependent ERAD pathway, and other biological processes. MKX and PDCD5 were verified to be strongly interacting with PRDM9 and expressed in prophase I of meiosis in both mouse and yak testes. The localizations of RNA splicing genes including THOC5, DDX5, and XRCC6 were expressed in spermatocytes. Cattleyak is the hybrid offspring of a yak and a domestic cow, and the male offspring are sterile. The gene expression of the interacting proteins was also examined in the sterile male hybrid of yak and cattle. Among the 58 detected genes, 55 were downregulated in cattleyak. In conclusion, we established a complete PRDM9 interaction network, and a novel function of PRDM9 was identified, which will further promote our understanding of spermatogenesis. It also provides new insights for the study of hybrid male sterility.

Tributyl Phosphate Induced Male Reproductive Toxicity in Mice.

Tributyl phosphate (TBP) is an emerging hazardous material that is ubiquitous in the environment. However, whether TBP affects the male reproductive system remains unknown. In this study, we exposed male ICR mice and mouse spermatocyte GC-2 cells to TBP to explore the male reproductive toxicity of TBP. The results revealed that TBP (50 and 100 mg·kg-1·day-1) decreased sperm count and motility, damaged testicular structure, and induced apoptosis in mice after 35 days of oral gavage. TBP (10, 50, and 200 μM) inhibited cell viability, induced apoptosis, and reduced the ratio of BCL2/BAX in GC-2 cells after 24 h of exposure. TBP (50 and 200 μM) triggered the buildup of reactive oxygen species and induced a reduction in the mitochondrial membrane potential within GC-2 cells. In addition, TBP led to gut microbiota dysbiosis and metabolic profile alterations and increased the abundance of Prevotellaceae, which are negatively associated with sperm motility. Furthermore, TBP caused inflammation, induced the infiltration of Th17 cells into the mouse small intestine, and stimulated IL-17A signaling in mouse testes. In conclusion, TBP induced male reproductive toxicity via the gut-testis axis, oxidative stress, and apoptosis, offering fresh perspectives on TBP's reproductive hazards in male mammals.