A single-cell atlas of the testicular interstitium defines Leydig progenitor networks sustaining Leydig cell homeostasis across the lifespan
- Department of Sports Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, China
- Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, China
- Medical Research Institute, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, China
- Central Laboratory, The Second Affiliated Hospital of Fujian Medical University, China
- Department of Laboratory Medicine, The First Affiliated Hospital of Sun Yat-sen University, China
- Department of OB/GYN and Women’s Health, University of Louisville School of Medicine, United States
Figures
Figure 1 with 2 supplements
Characterization of cell populations in the mouse testicular interstitium.
(A) Schematic overview of the experimental design for single-cell RNA sequencing conducted on mouse testicular interstitium samples across multiple developmental stages (neonatal, adolescent, adult, middle-aged, and aged), with three replicates at each stage. (B) Uniform manifold approximation and projection (UMAP) visualization illustrating the heterogeneity and distribution of distinct cell types within the testicular interstitium across developmental stages. (C) UMAP visualization displays various developmental phases: neonatal (1 week), adolescent (1 month), adult (2 months), middle-aged (8 months), and aged (24 months). (D) Heatmap displaying the top 10 differentially expressed genes (DEGs) for each identified cell cluster. (E) Dot plot summarizing enriched Gene Ontology (GO) terms corresponding to each cell cluster, highlighting their distinct biological functions. (F) Violin plots demonstrating expression patterns of representative marker genes used to identify key interstitial cell populations. (G) Quantification of cell type proportions within the testicular interstitium across the different developmental stages. (H) Immunofluorescence staining revealing a subpopulation of ambiguous cells (ACs) characterized by co-expression of LC marker, Hsd3b1, and macrophage marker, F4-80, indicated by yellow fluorescence. Scale bar: 50 µm. (I) Immunofluorescence labeling for Hsd3b1 and Cd34 to distinguish LCs from MCs within the 2-week-old testicular interstitium. Scale bar: 50 µm. (J) Quantitative analysis of Cd34+ MCs within the testicular interstitium across different developmental stages.
Figure 1—figure supplement 1
Single-cell transcriptome profiling of the mouse testicular interstitium.
(A) Isolation of mouse testicular interstitial cells via fluorescence-activated cell sorting (FACS) for single-cell RNA sequencing (scRNA-seq). (B) A uniform manifold approximation and projection (UMAP) visualization showing all identified cell types from scRNA-seq, with analysis focused exclusively on cells from the testicular interstitium. (C) Dot plot displaying characteristic differentially expressed genes (DEGs) across identified cell types. (D) UMAP representation highlighting distinct cell populations within the mouse testicular interstitium across neonatal, prepubertal, adolescent, adult, and aged stages in mice. (E) UMAP plot showing expression patterns of selected marker genes. (F) Heatmap presenting the differentially DEGs between AC and LC. (G) Gene Ontology (GO) enrichment analysis of AC-upregulated and AC-downregulated DEGs (compared with LC). Gene expression levels are scaled and colored based on Z-scores, while enrichment significance is indicated by color and adjusted p-values. (H) Heatmap presenting the DEGs between AC and macrophages. (I) GO enrichment analysis of AC-upregulated and AC-downregulated DEGs (compared with macrophages). Gene expression levels are scaled and colored based on Z-scores, while enrichment significance is indicated by color and adjusted p-values.
Figure 1—figure supplement 2
Dynamic functional changes of testicular macrophages across the postnatal lifespan.
(A) Pseudotime trajectory analysis for macrophages across five postnatal developmental stages, depicted with distinct colors. (B) Heatmap illustrating temporal shifts in GO term enrichment for macrophages, mapped along the pseudotime continuum. (C) Expression trend plots for selected genes within macrophages across pseudotime, associated with processes such as proliferation, antigen processing and presentation, proteasomal protein degradation, and glutathione metabolism. (D) Violin plots reveal changes in functional scores for macrophages during development, assessing aspects such as migration, antigen processing and presentation, phagocytosis, cytokine production, and M1/M2 polarization. (E) Circle diagrams depict key signaling pathways initiated by macrophages, highlighting VCAM, IGF, VISFATIN, and GAS pathways. Edge colors match the signaling sources, and the width of each edge denotes the likelihood of communication, with thicker lines indicating more robust signaling.
Figure 2 with 2 supplements
Identification and characterization of dynamic Cd34-MC and LC subpopulations across developmental stages.
(A) Uniform manifold approximation and projection (UMAP) visualization illustrating distinct cell populations within mouse testicular interstitium at various developmental stages (neonatal, adolescent, adult, middle-aged, and aged). (B) Dot plot depicting key differentially expressed genes (DEGs) characterizing each Leydig cell subtype. (C) Monocle-generated pseudotime trajectory tracing developmental transitions and differentiation paths among Leydig cell subtypes. (D) Pseudotime analysis revealing dynamic developmental transitions specifically enriched in progenitor Cd34-MC1 cells toward mature Leydig cell lineages. (E) Heatmap of top DEGs and their corresponding Gene Ontology (GO) terms enriched within the Cd34-MC subsets. (F) Pseudotime plot highlighting gene expression patterns unique to Cd34-MC1 cells. (G) Representative immunofluorescence images demonstrating co-localization of Cd34 and Sox4 in interstitial cells at 3 weeks of age. Scale bar: 10 µm. (H) UMAP visualization showing the distribution of human interstitial cell populations across infant and adult testicular samples (data sourced from GSE124263). (I) Pseudotime trajectory analysis elucidating developmental trajectories of human testicular interstitial cell populations. (J) UMAP plots highlighting the distribution of CD34-MC subpopulations in human testes across developmental stages. (K) Violin plots illustrating relative gene expression (z-score normalized log-transformed values) of indicated genes in Cd34-MC subpopulations.
Figure 2—figure supplement 1
Subpopulation classification within Leydig cell (LC) and mesenchymal cell (MC) across developmental stages.
(A) UMAP visualization showing LC and MC subtypes across various developmental phases: neonatal (1 week), adolescent (1 month), adult (2 months), middle-aged (8 months), and aged (24 months). (B) Heatmap of the top ten differentially expressed genes for each LC and MC subtype, with expression levels normalized and visualized through a Z-score-based color gradient. (C) GO enrichment analysis for each LC subtype, with significance indicated by color corresponding to adjusted p-values. (D) Schematic representation of testosterone biosynthesis, emphasizing genes implicated in the process. (E) Dot plot detailing gene expression relevant to testosterone synthesis across stages of LC lineage differentiation. (F) Immunofluorescence staining of testis sections showing the expression patterns of representative markers distinguishing Mki67⁺ Leydig cells (Mki67-LC), ambiguous cells (ACs), progenitor Leydig cells (PLCs), and mature Leydig cells (MLCs). (G) Immunofluorescence staining of in vitro–cultured cells demonstrating the expression of representative markers: Col1a1, Plin1, Cyp51, and Hsd3b1. (H) Pseudotime trajectory analysis delineates differentiation pathways from undifferentiated MCs towards mature LCs, showcasing expression evolution within the LC lineage from neonatal to aged stages.
Figure 2—figure supplement 2
Molecular characterization of Cd34+ mesenchymal cell (MC) subpopulations.
(A) Dot plot illustrating expression profiles of previously reported stem Leydig cell (SLC) markers across distinct Leydig cell subtypes. (B) Dot plot illustrating expression profiles of previously reported SLC markers in Cd34⁺ MC subpopulations across defined age cohorts. (C) Violin plots depicting the expression patterns of previously reported SLC markers across distinct Cd34⁺ MC subpopulations. (D) Violin plots showing the expression patterns of novel SLC markers across distinct Cd34⁺ MC subpopulations. (E) UMAP analysis reveals cell type distribution within the testicular interstitium of infant (n=2) and adult (n=2) human testes, based on the public dataset GSE124263. (F) Dot plot illustrates marker gene expression profiles, identifying key cell types within the human testicular interstitium. (G) Pseudotime trajectory analysis delineates differentiation pathways from infancy (dark blue) to adulthood (light blue) stages. (H) Pseudotime analysis reveals dynamic gene expression profiles and developmental transitions towards LC lineages within the testicular interstitium of human testes.
Figure 3 with 1 supplement
Age-associated alterations in glutathione metabolism within testicular interstitial cells.
(A) K-means clustering analysis of genes exhibiting differential expression patterns in testicular interstitial cell populations during aging. (B) Gene set enrichment analysis (GSEA) illustrating significantly downregulated metabolic pathways in aged interstitial cells (p < 0.05). (C) GSEA showing enrichment of immune-related pathways among genes upregulated in older interstitial cells. (D) Targeted steroid hormone metabolomics analysis comparing the testicular lipid profile between young (3-month-old) and aged (24-month-old) mice (n = 5). (E) Dot plot illustrating correlation between upregulated metabolites and differentially expressed genes (DEGs) in aged interstitial cells. (F) Expression trends of essential glutathione metabolism pathway genes across developmental stages, highlighting decreased activity in older interstitial cells. (G) Relative abundance of reduced glutathione (GSH) quantified in testes from young (3-month-old) and aged (24-month-old) mice (n = 5, p < 0.01), presented as mean ± SEM. (H) A ranked plot of super-enhancers (SEs) showcases those with the highest median H3K27ac scores across Cd34-MC1, Cd34-MC2, and Cd34-MC3 cells. (I) Representative ChIP-seq tracks displaying H3K27ac enrichment at the Nrf2 genomic locus in interstitial cells at various ages. (J) Violin plots illustrating age-associated changes in Nrf2 gene expression within the Cd34-MC1 cell population. (K) GSEA showing enrichment of reactive oxygen species (ROS)-related signaling pathways influenced by Nrf2 activity specifically in aging Cd34-MC1 cells.
Figure 3—figure supplement 1
Transcriptional changes and metabolic alterations in the testicular interstitium during aging.
(A) Principal component analysis (PCA) score plot demonstrating distinct metabolite profiles between the young and aged groups (n=5). (B) Volcano plot displaying differentially abundant metabolites comparing testes from 3-month-old and 24-month-old mice (n=5). (C) Heatmap showing key metabolites that differ significantly between young and aged groups. (D) Violin plot illustrates Gpx4 and Gsta4 expression across specific subpopulations. (E) Heatmap shows that aging-related genes in the GenAge database are expressed in testicular subpopulations of aged mice. (F) The intensity of H3K27ac at the transcription start site (TSS) in Cd34-MC1, Cd34-MC2, and Cd34-MC3 cells. (G) Protein-protein interaction (PPI) analysis indicates the involvement of Nrf2 in both glutathione and reactive oxygen species (ROS) metabolism pathways.
Figure 4 with 1 supplement
Cd34-MC1 cell transplantation enhances testosterone production and Leydig cell regeneration in ethylene dimethanesulfonate (EDS)-treated and aged mice.
(A) Schematic illustrating the experimental design for Cd34-MC1 cell transplantation in EDS-treated mice. (B) Representative histological analysis by hematoxylin and eosin staining of testis sections showing structural restoration post-transplantation of Cd34-MC1 cells. Scale bar: 100 µm. (C) Immunofluorescence images confirming an increased presence of Leydig cells marked by Hsd3b1 expression in the testicular interstitium following Cd34-MC1 cell transplantation. Scale bar: 50 µm. (D) Serum testosterone levels measured at indicated time points post-EDS treatment demonstrating significant restoration following Cd34-MC1 transplantation. Data are presented as mean ± SEM. *p < 0.05 compared to EDS-treated controls, n = 6. (E) Improved sperm progressive motility (PR) observed at day 20 post-transplantation compared to controls. Data presented as mean ± SEM. *p < 0.05; **p < 0.01 compared to EDS-treated group, n = 6. (F) Experimental schematic depicting Cd34-MC1-tdTomato cell transplantation into testes of Lhcgr−/− mice. (G) Immunofluorescence images illustrating successful engraftment and differentiation of Cd34-MC1-tdTomato cells into Lhcgr+ and Hsd3b1+ Leydig cells within the interstitium of Lhcgr−/− mouse testes. Data are presented as mean ± SEM. *p < 0.05, n = 3, Scale bar: 50 µm. (H) Immunofluorescence comparison of Leydig cell regeneration capability between Cd34-MC1 (2-week-old) and Cd34-MC3 (24-month-old) transplanted cells, indicating a superior regenerative potential of younger progenitor cells. Scale bar: 50 µm. (I) Quantitative analysis revealing significantly fewer Hsd3b1-positive Leydig cells derived from transplanted Cd34-MC3 compared to Cd34-MC1 cells in Lhcgr−/− mouse testes. Data are presented as mean ± SEM. *p < 0.05, n = 3. (J) Statistical analysis comparing serum testosterone levels in Cd34-MC1-transplanted Lhcgr−/− mice with or without human chorionic gonadotropin (hCG) treatment. Data are presented as mean ± SEM. *p < 0.05, n = 3.
Figure 4—figure supplement 1
Restoration of testicular function by transplantation of Cd34-MC1 in Leydig cell (LC)-disrupted mouse models.
(A) Bright-field microscopy images showing clonal sphere formation capacity of Cd34-MC1 and Cd34-MC2 cells derived from single cells. Scale bar: 10 µm. (B) Immunofluorescence staining showing the differentiation of Cd34-MC1 and Cd34-MC2 into LCs, marked by LC-specific marker expression. (C) Flow cytometry results showing the percentage of Cd34+/Cd36- (CD34-MC1) cells in the testes of 3-week-old mice. (D) Quantification of seminiferous tubule diameter in the two groups. Three sections per slide and three slides per mouse testis were counted. Data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (compared to the control group, n = 6). (E) Quantification of the number of Hsd3b1+ cells in the three groups. Data are presented as the mean ± SEM. ***, p < 0.001 (compared to the EDS-treated group, n = 6). (F) Immunofluorescence imaging with anti-Sycp3 antibody to identify meiotic spermatocytes in the testes. (G) Quantification showing a marked increase in Sycp3+ seminiferous tubules per section in Cd34-MC1-transplanted mice versus those receiving EDS treatment only. (H) Evaluation of sperm motility differences between Cd34-MC1-transplanted mice and their non-transplanted counterparts. (I) Comparative imagery of tdTomato-leaky mice against standard inducible tdTomato mice (uninduced). (J) FACS-based isolation of Cd34-MC1 cells from 2-week-old tdTomato-leaky mice for subsequent transplantation. (K) Flow cytometry assessment revealing the presence of tdTomato+ cells in the testes of Lhcgr knockout mice. (L) Flow cytometry showing the proportion of Cd34+ MCs, and the immunofluorescence staining was performed to assess expression of Sox4 in sorted Cd34+ cells in 3-week-old and 24-week-old groups.
Figure 5 with 1 supplement
SE landscape reveals Sox4 as a crucial transcription factor in maintaining stemness in Cd34-MC1.
(A) Venn diagram illustrating Cd34-MC1-specific SEs compared with other Cd34-MC subtypes. (B) Gene Ontology (GO) enrichment analysis of SE-associated genes in three distinct Cd34-MC subpopulations. (C) Gene set enrichment analysis (GSEA) demonstrating significant enrichment of Sox4-regulated genes in pathways essential for maintaining stem cell populations. (D) Violin plots showing Sox4 expression levels across distinct Cd34-MC subsets. (E) ChIP-seq profiles highlight H3K27ac mark enrichment at the Sox4 locus within Cd34-MC1, Cd34-MC2, and Cd34-MC3. (F) Immunofluorescence images confirming successful Sox4 knockdown (green, Sox4 shRNA) in Cd34-MC1 cells, co-labeled with Sox4 (purple) and Leydig cell marker Hsd3b1 (red), after 48 hr of shRNA-mediated Sox4 suppression. Scale bar: 50 µm. Statistical significance indicated as *p < 0.05; **p < 0.01; ***p < 0.001, n = 3. (G) qPCR analysis evaluating LC gene expression changes following 48 hr of Sox4 knockdown in Cd34-MC1 cells. *p < 0.05; **p < 0.01; ***p < 0.001, n = 3. (H) Schematic depicting transplantation of Cd34-MC1 cells with or without Sox4 knockdown into Lhcgr−/− mice. (I) Immunofluorescence staining demonstrating reduced numbers of Hsd3b1-positive Leydig cells derived from Sox4-knockdown Cd34-MC1 cells compared to controls in Lhcgr−/− mouse testes. Scale bar: 50 µm, n = 3. (J) Summary diagram illustrating the key findings of this research.
Figure 5—figure supplement 1
Construction of Sox4 and Cd34 knockdown Cd34-MC1.
(a) qPCR analysis showing the knockdown efficiency of Sox4 shRNAs in Cd34-MC1 cells. Statistical significance indicated as *p < 0.05; **p < 0.01; ***p < 0.001, n = 3. (b) Fluorescence showing the infection efficiency of Sox4 shRNA lentiviruses in Cd34-MC1 cells. (c) qPCR analysis demonstrating the knockdown efficiency of Cd34 shRNAs in Cd34-MC1 cells. Statistical significance indicated as *p < 0.05; **p < 0.01; ***p < 0.001, n = 3. (d) Fluorescence showing the infection efficiency of Cd34 shRNA lentiviruses in Cd34-MC1 cells. (e) qPCR analysis revealing altered Leydig cell gene expression 48 hours after Cd34 knockdown in Cd34-MC1 cells. Statistical significance indicated as *p < 0.05; **p < 0.01; ***p < 0.001, n = 3.
Additional files
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Supplementary file 1
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Chemicals, Peptides, and Recombinant Proteins.
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A single-cell atlas of the testicular interstitium defines Leydig progenitor networks sustaining Leydig cell homeostasis across the lifespan
eLife 14:e100396.
https://doi.org/10.7554/eLife.100396
