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. 2025 May 15:13:RP97958.
doi: 10.7554/eLife.97958.

scRNA-seq and scATAC-seq reveal that Sertoli cell mediates spermatogenesis disorders through stage-specific communications in non-obstructive azoospermia

Shimin Wang #  1   2 , Hongxian Wang #  3 , Bicheng Jin  4 , Hongli Yan  5 , Qingliang Zheng  1 , Dong Zhao  2
Affiliations

scRNA-seq and scATAC-seq reveal that Sertoli cell mediates spermatogenesis disorders through stage-specific communications in non-obstructive azoospermia

Shimin Wang et al. Elife. .

Abstract

Non-obstructive azoospermia (NOA) belongs to male infertility due to spermatogenesis failure. However, evidence for cell type-specific abnormalities of spermatogenesis disorders in NOA remains lacking. We performed single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on testicular tissues from patients with obstructive azoospermia (OA) and NOA. HE staining confirmed the structural abnormalities of the seminiferous tubules in NOA patients. We identified 12 germ cell subtypes (spermatogonial stem cell-0 [SSC0], SSC1, SSC2, diffing-spermatogonia [Diffing-SPG], diffed-spermatogonia [Diffed-SPG], pre-leptotene [Pre-Lep], leptotene-zygotene [L-Z], pachytene [Pa], diplotene [Di], spermatids-1 [SPT1], SPT2, and SPT3) and 8 Sertoli cell subtypes (SC1-SC8). Among them, three novel Sertoli cell subtype phenotypes were identified, namely SC4/immature, SC7/mature, and SC8/further mature Sertoli cells. For each germ or Sertoli cell subtype, we identified unique new markers, among which immunofluorescence confirmed co-localization of ST3GAL4, A2M, ASB9, and TEX19 and DDX4 (classical marker of germ cell). PRAP1, BST2, and CCDC62 were co-expressed with SOX9 (classical marker of Sertoli cell) in testes tissues also confirmed by immunofluorescence. The interaction between germ cell subtypes and Sertoli cell subtypes exhibits stage-specific-matching pattern, as evidenced by SC1/2/5/7 involving in SSC0-2 development, SC3 participating in the whole process of spermiogenesis, SC4/6 participating in Diffing and Diffed-SPG development, and SC8 involving in the final stage of SPT3. This pattern of specific interactions between subtypes of germ cell and Sertoli cell was confirmed by immunofluorescence of novel markers in testes tissues. The interaction was mainly regulated by the Notch1/2/3 signaling. Our study profiled the single-cell transcriptome of human spermatogenesis and provided many potential molecular markers for developing testicular puncture-specific marker kits for NOA patients.

Keywords: Sertoli cell; cell biology; germ cell; human; non-obstructive azoospermia; single cell ATAC-sequencing; single-cell RNA-sequencing.

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Conflict of interest statement

SW, HW, BJ, HY, QZ, DZ No competing interests declared

Figures

Figure 1.
Figure 1.. Overview of major cell types and cellular attributes of testes of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) patients.
(A) Representative images of HE staining of testicular tissues of OA and NOA patients. (B) Schematic of the experimental design for scRNA-seq and scATAC-seq. (C) UMAP analysis of human testicular cells in scRNA-seq results. (D) UMAP analysis of human testicular cells in scATAC-seq results. (E) Heatmap of expression of markers for the 12 cell types. (F) UMAP analysis of testicular cell population.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. scATAC-seq analysis of the entire cell subpopulation.
(A) UMAP analysis of 10 cell subsets in scATAC-seq results. (B) Accessibility peak plots of markers for cell subpopulations.
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Integrating scRNA-seq and scATAC-seq analysis of all cell types.
Figure 2.
Figure 2.. Identification of germ cell subtypes.
(A) UMAP analysis of 12 germ cell subsets in scRNA-seq results. (B) Pseudotime analysis of 12 germ cell subpopulations. (C) Gene expression patterns of marker genes on UMAP plots. (D) State of germ cell subsets using pseudotime analysis. (E) Immunofluorescence double staining of markers of newly identified spermatocyte subpopulations with the classical marker DDX4. (F) Accessibility peak plots of markers for newly identified germ cell subpopulations.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Integrating scRNA-seq and scATAC-seq analysis of germ cell subtypes.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Analysis of germ cell markers in spermatogenesis.
(A) Heatmap shows marker gene expression of germ cell subtypes. (B) Gene expression patterns of germ cell markers on violin plots. (C) Distribution diagram of germ cell subsets using pseudotime analysis.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. scATAC-seq clustering.
(A) Heatmap of germ cell markers in scATAC-seq results. (B) Heatmap of motifs in scATAC-seq results.
Figure 2—figure supplement 4.
Figure 2—figure supplement 4.. Distribution characteristics of differentially expressed genes (DEGs) in germ cells from the obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups.
(A) Volcano plot of DEGs in germ cells for the OA group vs. the NOA group. (B) The expression levels of the top 5 DEGs in the OA and NOA groups. (C) GO and (D) KEGG enrichment analysis of DEGs. (E) The circos plot shows the genomic location information of differential peaks in germ cells between the OA and NOA groups.
Figure 2—figure supplement 5.
Figure 2—figure supplement 5.. Normalized chromatin accessibility of genes involved in cell cycle in germ cells between the obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups.
Accessibility peak plots of CDC6 (A), ESCO2 (B), FBXO5 (C), MTBP (D), and SMC1B (E).
Figure 3.
Figure 3.. Analysis of germ cells at different stages.
(A) Germ cell subsets at different stages of the cell cycle. (B) Heatmap of cell cycle in germ cells. (C) Transcriptional dynamics of driver genes of germ cell subtypes. (D) The main stage of spermatogenesis. (E) Beam analysis of differentially expressed genes at different stages of spermatogonial stem cell (SSC).
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Germ cell function analysis and epigenetic analysis.
(A–C) Analysis of beam cluster transcription factors and its GO terms. (D) Heatmap of transcription factors in germ cells.
Figure 4.
Figure 4.. Identification of Sertoli cell subtypes and epigenetic analysis.
(A) UMAP analysis showed eight Sertoli cell subsets in scRNA-seq results. (B) Heatmap of Sertoli cell subtype marker genes in eight Sertoli cell subsets. (C) Gene expression patterns of Sertoli cell subtype marker genes on violin plots. (D) Accessibility peak plots of markers for newly identified Sertoli cell subpopulations.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Integrating scRNA-seq and scATAC-seq analysis of Sertoli cell subtypes.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Distribution characteristics of differentially expressed genes (DEGs) in Sertoli cells from the obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups.
(A) Volcano plot of DEGs in Sertoli cells for the OA group vs. the NOA group. (B) The expression levels of the top 5 DEGs in the OA and NOA groups. (C) GO and (D) KEGG enrichment analysis of DEGs. (E) scATAC-seq analysis of differential peaks between the two groups.
Figure 4—figure supplement 3.
Figure 4—figure supplement 3.. Normalized chromatin accessibility of genes involved in cell cycle in Sertoli cells between obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups.
Accessibility peak plots of BUB18 (A), ESCO2 (B), KNL1 (C), SMC1B (D), and SMC3 (E).
Figure 5.
Figure 5.. The development of Sertoli cells.
(A) State of Sertoli cells using pseudotime analysis. (B) Distribution of Sertoli cell subtypes in different states. (C) Potential temporal differentiation of SC subgroups using RNA velocity. (D) Sertoli cells division diagram and representative markers. (E) Immunofluorescence of Sertoli cell subtype markers in testicular tissues from obstructive azoospermia (OA) patients. The scale bar represents 20 μm.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. GO and Qusage analysis of different states of Sertoli cells.
(A) GO analysis of different states of Sertoli cells. (B) Qusage analysis of different states of Sertoli cells.
Figure 6.
Figure 6.. Correlation analysis of germ and Sertoli cell expression in different groups.
(A) Cell proportion of germ cell subtypes in different groups. (B) Cell proportion of Sertoli cell subtypes in different groups. (C) Pseudotime analysis of germ cell subtypes in different groups. (D) Pseudotime analysis of Sertoli cell subtypes in different groups. (E) Distribution map of germ cell subtypes in different samples. (F) Distribution map of Sertoli cell subtypes in different samples. (G) Spearman correlation between germ and Sertoli cells. (H) GSE149512 was used to verify the correlation between SC3 and germ cells.
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. Distribution characteristics of differentially expressed genes (DEGs) in Sertoli cell subtype SC3 from the obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) groups.
(A) Volcano plot of DEGs in SC3 for the OA group vs. the NOA group. (B) The expression levels of the top 5 DEGs in the OA and NOA groups. (C) GO and (D) KEGG enrichment analysis of DEGs. (E) scATAC-seq analysis of differential peaks between the two groups. (F) Accessibility peak plots of VTN.
Figure 6—figure supplement 2.
Figure 6—figure supplement 2.. The interaction between germ cells and other cells.
Figure 6—figure supplement 3.
Figure 6—figure supplement 3.. GO analysis of germ cells.
Figure 6—figure supplement 4.
Figure 6—figure supplement 4.. GO analysis of Sertoli cells.
Figure 6—figure supplement 5.
Figure 6—figure supplement 5.. Correlation analysis of germ and Sertoli cell expression.
(A) The interaction between germ cells and Sertoli cells through the Notch signaling pathway. (B) Accessibility peak plots of NOTCH1/2/3.
Figure 7.
Figure 7.. Co-localization of subpopulations of Sertoli cells and germ cells.
(A) Representative images of HE staining of testicular tissues of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) patients. (B) Immunofluorescence analysis of germ cell and Sertoli cell subtype markers in testicular tissues. Blue DAPI indicates nuclei. The colors of the markers and cells indicate the fluorescent color of the corresponding antibody. The scale bar represents 20 μm.
Figure 8.
Figure 8.. Schematic diagram of spermatogenesis and stage specificity of Sertoli cell subtypes.
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Update of

  • doi: 10.1101/2024.04.09.588807
  • doi: 10.7554/eLife.97958.1
  • doi: 10.7554/eLife.97958.2

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