A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads

Abstract

Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 gestational weeks (GW) via 10× Genomics single-cell transcriptome techniques, reconstructed their location information by spatial transcriptomics. Relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple non-steroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the window of sexual differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

Keywords: Sc-RNA sequencing; adrenal glands; sexual differentiation; spatial transcriptomics; steroidogenic regulation network.

Figure 1

Global patterns of single-cell expression profiles of fetal adrenal glands. (A) Experimental schematic; 10 adrenal glands and 8 gonads in 10× Genomics; 2 adrenal glands in the Spatial transcriptome. (B) Uniform manifold approximation and projection analysis (UMAP) of the transcriptomes of fetal adrenal cells in 10× Genomics data (n = 75,482). The clusters were identified by marker genes, as shown in (C) C. Violin plot overview of the expression of selected marker genes by the fetal adrenal clusters. Detailed cell information and differentially expressed genes can be found in Table S2 .

Figure 2

Landscape and characteristics of fetal adrenal gland steroidogenic cells. (A) Uniform manifold approximation and projection analysis visualization of steroidogenic cells for 10× Genomics data (n = 10,651). (B) Heatmap of the top 10 differentially expressed genes (DEGs) between steroidogenic cell populations (n = 10,651). Color scale: yellow, high expression; purple, low expression. Detailed cell information and DEGs can be found in Table S3 . (C) Differentiation of the trajectory of steroidogenic cells using Dyno. The arrow direction indicates the trajectory of cell differentiation. (D) Dot plots of differential gene expression of three-stage steroidogenic cell groups (before, within, and after sexual differentiation). Detailed DEGs of steroidogenic cells between sexes before, within, and after the window of sexual differentiation can be found in Tables S7-9 . (E) Visualization of the spatial transcriptome shows the location of T1 and T3, T4 and T5 steroidogenic cells in 8GW fetal adrenals. (F) Immunohistochemical staining of CYP11B1 (up) and CYP21A2 (down) in the fetal adrenal glands spanning the window of sexual differentiation. Scale bar, 20 μm. n = 6.

Figure 3

Sexual dimorphic expression patterns of steroidogenic-related genes in steroidogenic cells. (A) Violin and dot plots of HSD3B2 gene expression patterns of female and male fetal adrenal steroidogenic cells spanning the window of sexual differentiation. (B) Immunofluorescence staining of StAR (green), HSD3B2 (red), and CYP17A1 (purple) in the female fetal adrenal glands spanning the window of sexual differentiation. Scale bar, 20 μm. n = 3.

Figure 4

Landscape and characteristics of fetal adrenal gland neurocytes. (A) Uniform manifold approximation and projection analysis visualization of adrenal neurocytes for 10× Genomics data (n = 10,812). (B) Heatmap of the top 10 differentially expressed genes (DEGs) between adrenal neurocyte populations. Detailed cell information and DEGs can be found in Table S4 . (C) Expression patterns of SRD5A1 and AKR1C2 exhibited by feature plot visualization, which are key enzymes of the DHT “backdoor pathway”. A gradient of gray, red, or green indicates low to high expression, and yellow indicates coexpression. (D) Visualization of the spatial transcriptome shows the locations with high expression of SRD5A1 and AKR1C2 in 8GW fetal adrenal tissues. (E) Immunofluorescence staining of SRD5A1 (purple), AKR1C2 (red), and CHGA (green) in fetal adrenal tissues. Scale bar, 20 μm. (F) Differentiation of the trajectory of fetal adrenal gland neurocytes using Dyno. The arrow direction indicates the trajectory of cell differentiation. (G) Feature plot visualization of SST in fetal adrenal neurocyte data, mainly expression in mature neurocytes (chromaffin cells and sympathoblasts). Violin plot of SST expression with sex differences.

Figure 5

Transcriptomic landscape of human fetal gonads. (A) Uniform manifold approximation and projection analysis of the transcriptomes of all-stage fetal gonadal cells split by sex (n = 53,508). (B) Violin plot overview of the expression of selected marker genes by gonad clusters. Detailed cell information and differentially expressed genes can be found in Table S6 . (C) Dot plot showing the expression of key enzymes for sex hormone biosynthesis (CYP11A1, CYP17A1, HSD3B2, HSD17B3, and CYP19A1) in different gonadal somatic cells. (D) CellChat analysis showing the cell–cell interaction of gonadal somatic cells in the testis (top). The signal interaction pathway networks are divided into two functions: niche-associated (activin, AMH, and opioid) and steroidogenic-associated (GH and melanocortin). The cell–cell interaction of gonadal somatic cells in the ovary (below). The signal interaction pathway networks are divided into two functions: niche-associated (BMP, NPY, and WNT) and steroidogenic-associated (FSH and KIT). .