Decoding the pathogenesis of spermatogenic failure in cryptorchidism through single-cell transcriptomic profiling

Abstract

Cryptorchidism, commonly known as undescended testis, affects 1%-9% of male newborns, posing infertility and testis tumor risks. Despite its prevalence, the detailed pathophysiology underlying male infertility within cryptorchidism remains unclear. Here, we profile and analyze 46,644 single-cell transcriptomes from individual testicular cells obtained from adult males diagnosed with cryptorchidism and healthy controls. Spermatogenesis compromise in cryptorchidism links primarily to spermatogonium self-renewal and differentiation dysfunctions. We illuminate the involvement of testicular somatic cells, including immune cells, thereby unveiling the activation and degranulation of mast cells in cryptorchidism. Mast cells are identified as contributors to interstitial fibrosis via transforming growth factor β1 (TGF-β1) and cathepsin G secretion. Furthermore, significantly increased levels of secretory proteins indicate mast cell activation and testicular fibrosis in the seminal plasma of individuals with cryptorchidism compared to controls. These insights serve as valuable translational references, enriching our comprehension of testicular pathogenesis and informing more precise diagnosis and targeted therapeutic strategies for cryptorchidism.

Keywords: cryptorchidism; male infertility; spermatogonial stem cells; testicular interstitial fibrosis.

Graphical abstract

Figure 1

Single-cell transcriptome profiling and analysis of CR (A) Schematic of the experimental workflow. (B) Uniform manifold approximation and projection (UMAP) plot showing the major cell types (n = 38,587 cells) from CR samples (CR) (n = 3) and samples from healthy adult males (control) (n = 2). Dots represent individual cells, and colors represent different cell populations. (C) Separate UMAP plot from CR samples and samples from healthy adult males (control). (D) Cell ratio of cell types from (B). Colors represent different cell populations, consistent with (B). (E) Periodic acid-Schiff (PAS) staining of a testis section from a CR patient. Scale bar, 200 μm.

Figure 2

Impaired spermatogenesis in CR (A) Left: UMAP plot of all germ cells (n = 6,290 cells, 5 samples). The curve with the arrow represents the development trajectory of germ cells from SSCs to sperm. Right: separate UMAP plot of the CR group (n = 3) and control (n = 2). (B) Bar plot showing the cell ratio of each germ cell subset in (A). (C) IF staining of PRM1 in tissue sections of the CR group and control. Scale bars, 20 μm (PRM1) and 100 μm (merged). Control: testis samples from patients with obstructive azoospermia. (D) GO term enrichment of SSCs in downregulated genes. The size of the dot represents the gene count. The color of the dot represents the value of −log10 p value. (E) Left: IF staining of FGFR3 and ACTA2 in tissue sections of the CR group and control, showing the number of FGFR3+ cells significantly decreased in the CR group. Scale bar, 50 μm. Control: samples from adult males with obstructive azoospermia. Right: Quantification of the number of undifferentiated spermatogonia (FGFR3+) in a cross-section of each seminiferous tubule in different groups. Bars represent the mean with SD of 30 independent tubules in the CR group (n = 3) and controls (n = 3). The p value was calculated by Student’s t test. ∗∗∗∗p < 0.0001, ∗∗p < 0.01. (F) UMAP plot of 4 states for spermatogonia (merged SSCs and differentiating spermatogonia). Different colors represent different states. (G) Violin plots showing the expression of selected gene markers for different spermatogonium states. (H) Cell numbers of different spermatogonium states in the CR group and control. (I) The expression of classic cell-cycle-related genes in the CR group and control in different states. The color of the dot represents the average expression. (J) The expression of classic cell-cycle-related pathways in the CR group and control in different states.

Figure 3

Emergence and degranulation of mast cells in CR (A) Left: UMAP plot of immune cells and non-immune cells. Right: separate UMAP plot of the CR group and control and the corresponding cell ratio. Different colors represent different cell types. (B) Left: tSNE (t-Distributed Stochastic Neighbor Embedding) plot of mast cells (n = 1,057 cells; gray, control; blue, CR). Right: tSNE plot of mast cells for each individual. (C) Heatmap showing the expression of exocytosis-related genes in immune cells between the CR group and control. The scaled gene expression levels are colored according to Z score. (D) GO term enrichment of CR mast cells in upregulated genes compared with control mast cells. The size of the dot represents the gene count. The color of the dot represents the value of −log10 p value. (E) Violin plots showing the score of enriched functions (positive regulation of cell activation, mast cell degranulation, and regulation of immune effector process) between the CR group and control. (F) Heatmap showing the expression of genes associated with mast cell activation (top) and degranulation (bottom) between the CR group and control. The scaled gene expression levels are colored according to Z score. (G) IF staining of CC1 (chymase) and CTSG in tissue sections of the CR (n = 3) and control group (n = 3), showing the number of CC1+ cells significantly increased in the CR group. Scale bars, 100 μm (left) and 5 μm (right). Control: samples from adult males with obstructive azoospermia. Dotted lines represent cell boundaries. A white arrow indicates scattered granules out of cells. (H) Quantification of mast cells in the CR group and control. Statistics of cell numbers were compared between two groups. The p value was calculated by Student’s t test. ∗∗∗∗p < 0.0001.

Figure 4

Intercellular interactions between mast cells and other cell types in CR (A) Heatmap illustrating the cell-cell interactions between the CR group and the control group. The color gradient indicates the degree of difference, with red hues signifying stronger communication between these two cell types in the CR group. On the heatmap, the y axis signifies the sender, while the x axis represents the receiver. (B) Barplot showing the global cell communications number and strength in the CR group and control. (C) The incoming signal pathway patterns of the CR group. Left: cell types involved in different patterns. Right: signal pathways involved in different patterns. (D) Dot plot showing different signal pathways from different cell types (outgoing) in the CR group. Dot size represents the contribution of different cell types. Color represents different cell types. (E) Comparison of differential signal ligand-receptor pairs in the CR group and control (signals sent from mast cells to various germ cells and somatic cells). Dot color represents the contribution of differential signal ligand-receptor pairs in different cell types. (F) Distribution and expression of the PAR signal pathway in the CR group. (G) IF staining of PARD3 in tissue sections of the CR group and control, showing the expression of PARD3 significantly increased in the CR group under the same experimental condition. Scale bar, 50 μm. Control: samples from adult males with obstructive azoospermia.

Figure 5

Fibrosis of testicular interstitial cells in CR (A) Separate UMAP plot showing the distribution of LICs (Leydig-like cells) and myoid cells. Different colors represent different samples. (B) The numbers of LICs and myoid cells in the CR groups and control. (C) Bar plot showing the upregulated GO terms enriched in Leydig/interstitial cells in the CR group compared with control. The length of the bar represents the gene count. The color represents the different functions (red, biological process; blue, molecular function). (D) IF staining of collagen I in tissue sections of the CR group and control. Scale bar, 100 μm. Control: samples from adult males with obstructive azoospermia. (E) IF staining of collagen III in tissue sections of the CR group and control. Scale bar, 100 μm. Control: samples from adult males with obstructive azoospermia. (F) Heatmap showing the score of functional gene sets between the CR group and control. Color represents the score. (G) Western blot showing that the protein content of TGF-β1 is increased in the CR group. Control: samples from adult males with obstructive azoospermia.Protein quantification was analyzed using ImageJ software (right). ∗∗∗p < 0.001. (H) The protein concentration in seminal plasma between the CR group (n = 25) and control (n = 16). The p value was calculated by Mann-Whitney test. ∗∗p < 0.01, ∗p < 0.05. (I) The correlation of chymase and PINP (left)/CXCL8 (right) between the CR group (n = 25) and control (n = 16). CC, Spearman correlation coefficient.

Figure 6

Oxidative stress-related alterations of Sertoli cells in CR (A) UMAP plot showing Sertoli cells in the CR group and control. Different colors represent different groups. The donut chart represents the cell ratio between the CR group and control. (B) Left: IF staining of SOX9 and ACTA2 in tissue sections of the CR group (n = 3) and control (n = 3). Scale bar, 100 μm. Control: samples from adult males with obstructive azoospermia. Right: quantification of the number of Sertoli cells (SOX9+) in a cross-section of each seminiferous tubule in different groups. Bars represent the mean with SD of 20 independent tubules per group. n = 6 human samples. The p value was calculated by Student’s t test. (C) Heatmap showing the DEGs enriched in the CR group and control. DEGs are calculated by Seurat::FindMarkers (p_ < 0.05, |avg_log2FC| ≥ 0.5). (D) GO term enrichment of Sertoli cells in the CR group in upregulated genes compared with control. (E) The expression of Sertoli cell functional genes. The scaled gene expression levels are colored according to Z score. (F) GSEA score (CR vs. control) of the ROS metabolic process. (G) Schematic model of this study.