Single-Cell RNA Sequencing Defines the Regulation of Spermatogenesis by Sertoli-Cell Androgen Signaling

Abstract

Androgen receptor (AR) signaling is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which AR acts between male germ cells and somatic cells during spermatogenesis have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking AR in Sertoli cells (SCARKO) and single-cell transcriptomic sequencing (scRNA-seq), the cell specific targets of AR action as well as the genes and signaling pathways that are regulated by AR are being identified. In this study, we collected scRNA-seq data from wild-type (WT) and SCARKO mice testes at p20 and identified four somatic cell populations and two male germ cell populations. Further analysis identified that the distribution of Sertoli cells was completely different and uncovered the cellular heterogeneity and transcriptional changes between WT and SCARKO Sertoli cells. In addition, several differentially expressed genes (DEGs) in SCARKO Sertoli cells, many of which have been previously implicated in cell cycle, apoptosis and male infertility, have also been identified. Together, our research explores a novel perspective on the changes in the transcription level of various cell types between WT and SCARKO mice testes, providing new insights for the investigations of the molecular and cellular processes regulated by AR signaling in Sertoli cells.

Keywords: AR; knockout; male infertility; scRNA-seq; spermatogenesis.

FIGURE 1

Schematic overview of scRNA-seq using WT and SCARKO testes at P20. (A) The sequencing analysis process of scRNA-seq. (B) Sequencing metrics after quality control of scRNA-seq datasets. (C) HE stanning of the testis from WT and SCARKO mice at P20. Asterisks indicate spermatocyte. Scale bar, 20 μm. (D) AR protein expression in WT and SCARKO testes at P20 analyzed by immunofluorescent staining. AR and nuclei were labeled by anti-AR antibody (green) and DAPI (blue), respectively. Scale bar, 20 μm.

FIGURE 2

ScRNA-seq profiles of P20 WT and SCARKO testicular cells and cell type identification. (A) Heatmap of differentially expressed genes (DEGs) from the five major cell types. Top: cell cluster; right: major cell types and representative enriched marker genes for each cell type. (B) Representative markers for each cell type. (C,D) tSNE and clustering analysis of combined single-cell transcriptome data from P20 WT and SCARKO testicular cells. Each dot represents a single-cell and cell clusters are distinguished by colors. (E) The percentage of cell numbers in each cluster in P20 WT and SCARKO testicular cells. (F) The number of DEGs (up and down-regulated) in each cluster in SCARKO testicular cells.

FIGURE 3

Identification of Sertoli cell clusters in WT and SCARKO mice. (A,B) UMAP plot of SCs from P20 WT and SCARKO mice. (A) SC clusters; (B) sample source. (C) Expression patterns of marker genes for SCs visualized in tSNE plots. (D) The percentage of cell numbers in each cluster in P20 WT and SCARKO testicular SCs. (E,F) Monocle pseudotime trajectory analysis of the SC clusters. (E) SC clusters; (F) segregated by sample source. Arrow indicates the inferred developmental direction from the analysis shown in (A). (G) Heatmap of DEGs from different SC subsets following the trajectory timeline shown in (E). Top: pseudotime directions; right: the number of DEGs and the representative biological processes.

FIGURE 4

Loss of functional Sertoli AR affects cellular activity and alters the transcriptome of Sertoli cells. (A) UMAP plot inferring the cell-cycle phase based on expression of a large set of G2/M- and S-phase genes (47). (B) Percentages of SCs in different cell-cycle phases from different WT and SCARKO mice. (C) Heatmap of apoptosis-associated genes in SCs of WT and SCARKO mice. (D) Heatmap of DEGs between WT and SCARKO Sertoli cells. (E,F) GO term analysis of up-regulated (E) and down-regulated (F) genes in SCs of SCARKO mice. (G) Gene regulatory networks of DEGs shown in (D). (H) Violin plot showing the expression levels of DEGs in WT and SCARKO SCs. (I) qPCR results showing the expression fold change of DEGs showed in (H).

FIGURE 5

Identification of spermatogonia clusters in WT and SCARKO mice. (A,B) UMAP plot of spermatogonia from P20 WT and SCARKO mice. (A) spermatogonia clusters; (B) sample source. (C) Gene expression patterns of selected marker genes corresponding to each cellular state on the UMAP plots. (D) The percentage of cell numbers in each state in P20 WT and SCARKO spermatogonia. (E,F) Monocle pseudotime trajectory analysis of the spermatogonia clusters. spermatogonia clusters; (F) segregated by sample source. Arrow indicates the inferred developmental direction from the analysis shown in (A). (G) Heatmap of DEGs from different spermatogonia subsets following the trajectory timeline shown in (E). Top: pseudotime directions; right: the number of DEGs and the representative biological processes.

FIGURE 6

Identification of DEGs in spermatogonia from WT and SCARKO mice. (A) Heatmap of DEGs between WT and SCARKO Sertoli cells. (B,C) GO term analysis of up-regulated (B) and down-regulated (C) genes in SCs of SCARKO mice. (D) Gene regulatory networks of DEGs shown in (A). (E) Violin plot showing the expression levels of DEGs in WT and SCARKO spermatogonia.