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. 2024 May 18;45(3):617–632. doi: 10.24272/j.issn.2095-8137.2023.280

Human-like adrenal features in Chinese tree shrews revealed by multi-omics analysis of adrenal cell populations and steroid synthesis

Jing-Hang Jiang 1,2,3,#, Yi-Fu Wang 1,2,#, Jie Zheng 1,2,#, Yi-Ming Lei 4,#, Zhong-Yuan Chen 1,5, Yi Guo 2, Ya-Jie Guo 1, Bing-Qian Guo 1, Yu-Fang Lv 1, Hong-Hong Wang 1, Juan-Juan Xie 1, Yi-Xuan Liu 1, Ting-Wei Jin 1, Bi-Qi Li 6, Xiao-Shu Zhu 4,*, Yong-Hua Jiang 1,5,7,*, Zeng-Nan Mo 1,2,*
PMCID: PMC11188597  PMID: 38766745

Abstract

The Chinese tree shrew (Tupaia belangeri chinensis) has emerged as a promising model for investigating adrenal steroid synthesis, but it is unclear whether the same cells produce steroid hormones and whether their production is regulated in the same way as in humans. Here, we comprehensively mapped the cell types and pathways of steroid metabolism in the adrenal gland of Chinese tree shrews using single-cell RNA sequencing, spatial transcriptome analysis, mass spectrometry, and immunohistochemistry. We compared the transcriptomes of various adrenal cell types across tree shrews, humans, macaques, and mice. Results showed that tree shrew adrenal glands expressed many of the same key enzymes for steroid synthesis as humans, including CYP11B2, CYP11B1, CYB5A, and CHGA. Biochemical analysis confirmed the production of aldosterone, cortisol, and dehydroepiandrosterone but not dehydroepiandrosterone sulfate in the tree shrew adrenal glands. Furthermore, genes in adrenal cell types in tree shrews were correlated with genetic risk factors for polycystic ovary syndrome, primary aldosteronism, hypertension, and related disorders in humans based on genome-wide association studies. Overall, this study suggests that the adrenal glands of Chinese tree shrews may consist of closely related cell populations with functional similarity to those of the human adrenal gland. Our comprehensive results (publicly available at http://gxmujyzmolab.cn:16245/scAGMap/) should facilitate the advancement of this animal model for the investigation of adrenal gland disorders.

Keywords: Tree shrew, Adrenal gland, Dehydroepiandrosterone, Genome-wide association studies, Disease model

INTRODUCTION

Androgen production by adrenal glands is important for reproductive and endocrine health across the human lifespan. The zona reticularis, the innermost region of the human adrenal cortex, is responsible for synthesizing the primary precursor androgen dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) (Lyraki & Schedl, 2021). DHEA promotes gonadal differentiation towards the male phenotype during embryonic development (Wang et al., 2022), and circulating levels of DHEA and DHEAS increase prior to puberty, reflecting down-regulation of the steroid enzyme HSD3B2 and up-regulation of the steroid enzymes CYB5A and SULT2A1 (Rosenfield, 2021; Witchel et al., 2020). After peaking with the maturation of reproductive and endocrine functions, the levels of DHEA and DHEAS and number of cells in the zona reticularis gradually begin to decline (Skiba et al., 2019). This decline is linked to a decrease in reproductive function and loss of ovarian follicles in women over the age of 37 (Ford, 2013). After menopause, the zona reticularis serves as the primary source of estrogen and precursor androgens such as DHEA, although in reduced quantities (Labrie, 2019). Dysregulation of the timing and production levels of DHEA and DHEAS can induce various disorders, including polycystic ovary syndrome (Ahn et al., 2021; De Medeiros et al., 2021; Goodarzi et al., 2015).

To elucidate the origins and treatment modalities of such disorders, suitable animal models that faithfully mimic clinical conditions while allowing extensive multi-omics analyses are imperative. This presents a challenge as the zona reticularis production and prepubertal increase in DHEA only occur in humans and a few other primates. Commonly used animal models for various disorders, such as mice, rats, and rabbits, lack the capability for hormone secretion in their adrenal cortex (Pihlajoki et al., 2015). Chimpanzees and certain nonhuman primates possess similar adrenal reticularis hormone-secreting functions (Abbott & Bird, 2009; Bernstein et al., 2012; Nguyen & Conley, 2008; Sabbi et al., 2020). Nevertheless, extensive research into primate adrenal function is limited by an array of challenges, including financial, logistical, ethical, and time constraints associated with large-animal research. Although the spiny mouse exhibits analogous web-like belt-shaped hormone secretion structures and functions, the state of spiny mouse animal husbandry remains underdeveloped (Pihlajoki et al., 2015; Quinn et al., 2013). Furthermore, ethical constraints limit the extent to which adrenal regulation can be experimentally manipulated in humans, necessary for understanding the pathogenesis and progression of adrenal disorders (Nonaka et al., 2019; Tezuka et al., 2021). Consequently, a more appropriate animal model of these disorders is urgently needed.

The Chinese tree shrew (Tupaia belangeri chinensis), a small-sized, rapidly reproducing species with large litters, represents promising prospects in this context, already gaining recognition as an effective model for studying infectious diseases, tumors, and immunological conditions (Li et al., 2023; Lu et al., 2021; Pan et al. 2022; Xiao et al., 2017; Zeng et al., 2023). Its genome has been fully sequenced and is available online (www.treeshrewdb.org) (Fan et al., 2013, 2019; Ye et al., 2021). Notably, the adrenal gland of the Chinese tree shrew features cortical and medullary structures similar to those found in humans, including an androgen-producing zona reticularis. While these characteristics underscore the potential of the tree shrew as a viable model for studying adrenal disorders, several critical questions must first be addressed: Does the zona reticularis synthesize DHEA and DHEAS? Are the range and arrangement of adrenal cell types similar to those in humans, and do they express the same key enzymes in steroid synthesis?

To examine these questions, we used single-cell RNA sequencing to map the range of cell types in the tree shrew adrenal gland and determined their distribution using spatial transcriptomics. We analyzed the expression of key steroid enzymes and the production of steroid hormones using immunohistochemistry and liquid chromatography-tandem mass spectrometry (Figure 1A). To assess the utility of the tree shrew as a model of human adrenal dysfunction, we compared adrenal cell types and their gene expression profiles across tree shrews, humans, macaques (Macaca fascicularis), and mice, and compared tree shrew expression profiles to genetic alterations associated with adrenal disorders. Our results identified extensive structural and functional similarities in the adrenal gland between tree shrews and humans. Our data (publicly available at http://gxmujyzmolab.cn:16245/scAGMap/) should substantially advance the development of tree shrews as a viable animal model of human adrenal disorders.

Figure 1.

Figure 1

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Comprehensive atlas of single-cell transcripts in Chinese tree shrew adrenal glands

A: Schematic of experimental procedure and bioinformatic analyses. LC, liquid chromatography; MS, mass spectrometry. B: Details of eight tree shrews used to generate single-cell RNA transcriptomes. F, female; M, male; A, mature (18 months); B, immature (3 months). C: Violin plot of marker gene expression (ordinate) in each adrenal cell cluster (abscissa). D: Visualization of 13 major adrenal cell types based on uniform manifold approximation and projection (UMAP). Cell types are annotated and color-coded. E: Distributions of adrenal cell types across individual tree shrews and groups of different sex. F: Relative proportions of major adrenal cell types across individual tree shrews.

MATERIALS AND METHODS

Animals

All animal procedures were approved by the Animal Experiment Review Board of Guangxi Medical University and conducted in accordance with the university and local regulations on animal handling and experimentation. The Chinese tree shrews were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences (China) and housed in a temperature-regulated room (25±2°C) under a regular 12 h light/dark cycle. Each animal received an intraperitoneal injection of pentobarbital (100 mg/kg) for anesthetization.

Single-cell RNA sequencing of tree shrew adrenal glands

The adrenal tissue of sexually immature (3 months old; two males and two females) and sexually mature tree shrews (18 months old; two males and two females) was carefully isolated using tweezers and scissors and transferred into a 1.5 mL microcentrifuge tube containing 1 mL of RPMI 1640 medium (catalog no. C11875500BT, Gibco, USA) supplemented with 10% fetal bovine serum (catalog no. SH30070.03, HyClone, USA). The adrenal tissue was cut into 1 mm3 pieces with scissors, washed twice with Dulbecco’s phosphate-buffered saline (D-PBS, catalog no. 311-425-CL, Wisent, Canada) to eliminate residual blood, and transferred to a 15 mL centrifuge tube, followed by the addition of 2 mL of digestive solution (RPMI 1640 containing 0.1 mg/mL Liberase TL enzyme (catalog no. 5401020001, Roche, Swiss) and 1 mg/mL DNase I (catalog no.10104159001, Roche, Swiss)) and digestion for 20 min in a 37°C water bath with gentle shaking. After digestion, fresh RPMI 1640 medium containing 10% fetal bovine serum was added to stop the reaction, and incompletely digested tissue was filtered using a 70 μm cell strainer (catalog no. 352350, Falcon, Japan). The cells in suspension were centrifuged at 350 ×g at 4°C for 6 min, with the pellet then washed with D-PBS, resuspended in 3 mL of RBC lysis buffer (catalog no. 420301, BioLegend, USA), incubated on ice for 5 min, and terminated with 10 mL of D-PBS. Subsequently, the cells were pelleted, washed with D-PBS, and resuspended.

The resulting single-cell suspension was combined with gel beads and partitioning oil before being loaded onto the 10× Genomics Chromium chip G from the 10× Genomics Single Cell kit (v.3.1). Using the Chromium Controller, the target cell count for each sample was set at 10 000. After generation of gel bead-in-emulsions (GEM), the samples were transferred to a polymerase chain reaction (PCR) tube for reverse transcription using a T100 Thermal Cycler (Bio-Rad, USA). The cDNA was purified and used to prepare libraries according to the 10× Genomics instructions. The libraries were sequenced by Annoroad Gene Technology (Beijing, China) using the NovaSeq 6000 platform (Illumina, USA).

Raw sequencing data were demultiplexed using the “mkfastq” application in 10× Genomics Cell Ranger (v.3.1.0) to generate files in Fastq format, which were processed using the “count” application with default settings. Experimental RNA sequences were annotated based on the tree shrew reference genome (www.treeshrewdb.org) using STAR aligner (Wang et al., 2022). Unique molecular identifiers (UMIs) were counted for further analysis.

The Read 10× function in the “Seurat” package in R (v.4.0.3) was used to generate count tables, which were filtered using the subset function to exclude cells with gene expression levels below 200 or above 4 500, as well as cells for which more than 20% of the transcriptome came from mitochondria. Single-cell data were normalized using the NormalizeData, FindVariableFeatures, and ScaleData functions. Principal component analysis (PCA) was performed using the RunPCA function, and the top 35 principal components were used to analyze uniform manifold approximation and projection (UMAP). Finally, cell cluster analysis was performed using the RunUMAP, FindNeighbors, and FindClusters functions.

Bulk RNA sequencing of tree shrew adrenal glands

Sexually immature (3 months old; four males and four females) and sexually mature tree shrews (18 months old; four males and four females) were euthanized via pentobarbital injection (100 mg/kg), and their adrenal glands were removed, immediately washed with cold phosphate-buffered saline (PBS), and flash-frozen in liquid nitrogen. RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent, USA), with the RNA then used to prepare mRNA libraries (Parkhomchuk et al., 2009), sequenced by Novogene (China) using the NovaSeq 6000 platform (Illumina, USA). Sequences were annotated using the tree shrew reference genome (www.treeshrewdb.org), and raw reads were filtered and trimmed before further analysis. Read counts for each gene were generated using featureCounts (v.1.5.0-p3), then used to calculate fragments per kilobase million (FPKM) for each gene.

Spatial transcriptomics of tree shrew adrenal glands

Sexually immature (3 months old; one male and one female) and sexually mature tree shrews (18 months old; one male and one female) were euthanized via an injection of pentobarbital (100 mg/kg), and their adrenal glands were removed, immediately washed with cold PBS, dried with absorbent paper, embedded in pre-cooled OCT-embedding medium (catalog no. 4583, Sakura, USA), frozen on dry ice, and stored at −80°C until sectioning (10 μm thickness) using a cryostat microtome (catalog no. CM3050S, Leica, Germany) at temperatures of −20°C for the blade and −10°C for the specimen head. These sections were placed onto the capture areas of Visium Spatial Tissue Optimization slides and Visium Spatial Gene Expression slides. The optimal permeabilization time was determined following the instructions in the “Tissue Optimization User Guide” (CG000238, revision D) in the Visium Spatial Gene Expression Reagent Kit. Spatial gene expression libraries were generated according to the “Spatial Gene Reagent Guidelines-Technical Note (CG000239)”. Libraries of adequate quality were sequenced by Annoroad Gene Technology using the NovaSeq 6000 platform (USA).

Sequences were mapped to the tree shrew reference genome (www.treeshrewdb.org). Spatial image information was checked and, if necessary, corrected by manually determining the location of the image and lattice location of the distribution of organizational and background information within the image using Loupe Browser 5 (10× Genomics). Based on these data, spatial location information was used to generate a spatial transcriptomic matrix using Space Ranger (v.1.0.0, 10× Genomics).

Spatial transcriptomic data were analyzed using the “Seurat” package following published guidelines (http://satijalab.org/seurat). The spatial transcriptomic matrix was imported using the Read10x_h5 and CreateSeuratObject functions to generate analysis objects. Spatial image information was integrated into the objects using the Read10x_Image function, and the quality of the results was evaluated using the VlnPlot and SpatialFeaturePlot functions, with the data then standardized using the SCTransform function. Genes expressed in specific locations were identified using the FindSpatiallyVariableFeatures function.

Integration of single-cell RNA sequencing and spatial transcriptomics

Within the “Seurat” package, single-cell RNA sequencing data were standardized using the SCTransform function and subjected to PCA using the RunPCA function. “Anchors” facilitating the integration of single-cell sequencing and spatial transcriptomics were identified using the FindTransferAnchors function, then used to map the first type of data onto the second type using the TransferData function based on a “predictions.assay” matrix. During this process, a gene expression matrix based on single-cell RNA sequencing and cell classification information, as described above, served as a reference. This matrix was incorporated into spatial transcriptomic datasets, with the resulting images visualized using the SpatialFeaturePlot function.

Definition of cell types in tree shrew adrenal gland

The FindAllMarkers function in the “Seurat” package was used to analyze the unique gene expression in each adrenal cell cluster, with differentially expressed genes (DEGs) defined through comparison with the CellMarker database (http://bio-bigdata.hrbmu.edu.cn/CellMarker/).

Interactions between adrenal cell types in tree shrews

Input data and cell group labels were generated using the data.input function in “Seurat”, with the resulting identification data incorporated into the “CellChat” package in R (v.1.1.1) (Jin et al., 2021). Interactions between cell types, specifically those involved in secretion signaling or cell-cell interactions, were extracted using the CellChatDB.use function.

Developmental pseudotime analysis of adrenal cell types in tree shrews

Input data, with S4 and S3 steroidogenic cells and Schwann cell precursors (SCPs) defined as “root” cells (see Results), were filtered and normalized using the “Seurat” package. Pseudotime analysis was conducted for various cell types using the recommended settings in the “monocle3” package (v.2.10.1) in R, with trajectories visualized using the plot_dimred function.

Enrichment of tree shrew gene functions based on single-cell RNA sequencing

Gene sets were obtained from single-cell objects in the “Seurat” package. Gene names were converted from ‘symbol’ to Entrez ID using the “org.Hs.eg.db” package (v.3.8.2) (Wang et al., 2022), and Gene Ontology enrichment in genes was analyzed using the “clusterProfiler” package (v.3.12.0) (Yu et al., 2012). Terms associated with P<0.05 were considered enriched. Dot plots and gene-concept networks were created using the “enrichplot” package (v.1.4.0) (Wang et al., 2022).

Cell cycling by individual adrenal cells in tree shrews

Each cell type was examined for its progression through the cell cycle using a set of cell cycle genes (Wang et al., 2022) with the “Seurat” package. Gene expression levels were used to compute a “cycle score”. Cell types with scores <2 were classified as aperiodic, while those with scores of at least 2 were classified as proliferating.

Comparisons of new adrenal transcriptomic data from tree shrews with previously published data and other species

Gene expression levels were compared across four species: tree shrews, macaques, mice, and humans. Single-cell RNA sequencing data of the adrenal glands from three adult humans were taken from our previous study (Huang et al., 2021). Eleven single-cell RNA sequencing datasets from M. fascicularis were downloaded from a publicly available database (https://db.cngb.org/nhpca/download) and integrated with the previously reported adrenal cell types in macaques (Han et al., 2022). Three single-cell RNA sequencing datasets of the adrenal glands from mice were obtained from the Gene Expression Omnibus (Lai et al., 2020) (accession code GSE134355). Cell populations were identified based on UMAP analysis of the annotated information. Data on bulk RNA in adrenal glands from mice and humans were taken from the Gene Expression Omnibus (Chen et al., 2019) (accession code GSE121051) and Genotype-Tissue Expression Project (https://gtexportal.org/), respectively. Genes in different species were homology-transformed using the “biomaRt” package (v.2.56.0).

Orthologous genes across the four species were identified and analyzed. Correlations in gene expression were then examined using Spearman rank correlation and log2(TPM+1). Correlation analyses of single-cell data and cross-species gene comparisons were integrated with the normalized data. Average gene expression of each cell type was calculated using the AverageExpression function in the “Seurat” package. The “KEGGREST” package (v.1.22.0) was used to sort gene sets characteristic of each cell type related to genetic information processing, human diseases, metabolism, cellular processes, environmental information processing, and organismal systems. To clarify functional differences among adrenal cell types in different species, cell types showing enrichment in the abovementioned KEGGREST pathways were determined using the “GSVA” package (v.1.42.0). Gene expression in tree shrew adrenal cell types was correlated with the results of genome-wide association studies in healthy humans and patients with polycystic ovary syndrome, hypercortisolism, primary adrenal insufficiency, primary aldosteronism, hypertension, and pheochromocytoma in the GWAS Catalog (www.ebi.ac.uk/gwas/) and literature (Supplementary Table S1).

Primary adrenal cell culture

Primary adrenal cell cultures from six sexually mature tree shrews were established and cultured in 6-well plates (catalog no. 3516, Corning, USA) in RPMI-1640 supplemented with 10% fetal bovine serum (catalog no. SH30070.03, HyClone, USA), 1% penicillin/streptomycin (catalog no. 15140-122, Gibco, USA), and 2 mmol/L L-glutamine (catalog no. 25030081, Gibco, USA), as described previously (Wang et al., 2022). After 4–6 h of culture at 37°C in a humidified environment with 5% CO2, the medium in some plates, but not the control plates, was supplemented with 0.1 mmol/L cholesterol (catalog no. C3045, Sigma, USA), then cultured for another 24 h at 37°C in a humidified environment with 5% CO2, collected, and assayed for DHEA, as described below. Plates treated with cholesterol were paired with control plates from the same animal.

Liquid chromatography-tandem mass spectrometry of steroid hormones in tree shrew adrenal glands

Sexually immature (3 months old; four males and four females) and sexually mature tree shrews (18 months old; four males and four females) were euthanized via a pentobarbital injection (100 mg/kg). Their adrenal glands were then removed, washed thoroughly with cold PBS, homogenized, added to 400 μL of methanol, shaken for 5 min, cooled on ice for 5 min, and centrifuged for 10 min at 4°C and 12 000 r/min. Alternatively, for the culture supernatants (see above), 400 μL of methanol was added to the medium, shaken for 10 min, cooled on ice for 10 min, and centrifuged for 10 min at 4°C and 12 000 r/min. The supernatant was then removed, concentrated, redissolved in 100 μL of pure methanol, shaken for 5 min, and centrifuged for 3 min at 4°C and 12 000 r/min.

For both sample types, 80 μL of supernatant was analyzed by liquid chromatography-mass spectrometry on the UPLC-ExionLC AD system coupled to a QTRAP® 6500+ System (UPLC, ExionLC AD, https://sciex.com.cn/; MS, QTRAP® 6500+ System, https://sciex.com/). Steroid hormone levels were detected using MetWare (http://www.metware.cn/).

Anatomical structure of tree shrew adrenal glands

Adrenal glands from sexually immature (3 months old; three males and three females) and sexually mature tree shrews (18 months old; three males and three females) were sectioned (4 μm thickness), dewaxed with xylene, rehydrated through a decreasing ethanol gradient (95%, 80%, then 70%, 1 min at each concentration), stained with hematoxylin-eosin, and analyzed using a NanoZoomer S60 digital slide scanner (catalog no. C13210-01, Hamamatsu, Japan).

The adrenal glands from one sexually mature male tree shrew were extracted following pentobarbital overdose, immediately washed with cold PBS, fixed for 2–4 h at 4°C in Gluta fixation buffer (catalog no. P1126, Solarbio, China), rinsed three times (15 min each) with 0.1 mol/L phosphate buffer (pH 7.4), treated for 2 h at room temperature with 1% osmium acid in 0.1 mol/L phosphate buffer, dehydrated through an increasing gradient of alcohol (50%, 70%, 80%, 90%, 95%, twice in 100%, 15 min at each concentration), and washed twice in 100% anhydrous acetone for 15 min each. Glands were treated with a 1:1 mixture of acetone and EMBed 812 (catalog no. 90529-77-4, SPI, Singapore) at 37°C for 2–4 h, followed by a 1:2 mixture at 37°C overnight and pure EMBed 812 at 37°C for 5–8 h. The embedded glands were polymerized in a 60°C oven for 48 h, with ultrathin sections then prepared on copper grids. The grids were stained in the dark for 15 min with a 2% solution of uranium acetate in alcohol, followed by 15 min in 2.6% lead citrate, then dried at room temperature overnight. Samples were imaged using an HT7700 transmission electron microscope (Hitachi, Japan).

Expression of key steroid enzymes and other proteins in tree shrew adrenal glands

Paraffin-embedded adrenal glands from sexually immature (3 months old; three males and three females) and sexually mature tree shrews (18 months old; three males and three females) were sliced to a thickness of 4 μm, dewaxed, hydrated, and subjected to antigen retrieval by boiling for 5–10 min in EDTA (C1034, Solarbio, China) at pH 8.5. Endogenous peroxidases were then inactivated by incubating the sections at room temperature with 3% H2O2, followed by one of the following primary antibodies overnight at 4°C: rabbit anti-human STAR (1:500; catalog no. NBP1-33485, Novus, USA), rabbit anti-human CYP11B1 (1:250; catalog no. bs-3898R, Bioss, China), rabbit anti-human CYP11B2 (1:250; catalog no. bs-10161R, Bioss, China), rabbit anti-human SUTL2A1 (1:300; catalog no. PA5-83561, Invitrogen, USA), rabbit anti-human CYP21A2 (1:75; catalog no. ab230327, Abcam, UK), rabbit anti-human CYB5 (1 μg/mL; catalog no. ab69801, Abcam, UK), rabbit anti-human CHGA (1:2500; catalog no. ab283265, Abcam, UK), and rabbit anti-human MPZ (1:200; catalog no. bs-0337R, Bioss, China). The sections were then treated with biotin-tagged goat anti-rabbit or anti-mouse secondary antibodies (catalog no, SAP-9100, ZSGB-BIO, China), followed by alkaline phosphatase-labeled streptavidin (catalog no, SAP-9100, ZSGB-BIO, China) at 37°C for 10–15 min. The sections were counterstained with hematoxylin, then colored with 3,30-diaminobenzidine tetrahydrochloride solution (catalog no. ZLI9018, ZSGB-BIO, China), dehydrated through an increasing alcohol gradient (70%, 80%, 90%, then 100% alcohol, 2 min at each concentration), and analyzed using a NanoZoomer S60 digital slide scanner (Hamamatsu, Japan).

Statistical analysis

As specified above, statistical analyses were carried out using R v.4.0.3. Unpaired two-tailed Student’s t-test was used to evaluate differences in parametric data. A difference with a P-value less than 0.05 was considered statistically significant.

RESULTS

Human-like diversity of cell types and anatomical structure in tree shrew adrenal glands

Cell type diversity in the adrenal glands of sexually immature and mature Chinese tree shrews was mapped using single-cell RNA sequencing (Figure 1A). After filtering out damaged cells, dead cells, and putative cell doublets, a dataset containing 47 096 cell transcriptomes from eight animals was obtained (Figure 1B; Supplementary Figure S1A). We identified 13 cell types based on the expression of canonical marker genes (in parentheses): endothelial cells (EGFL7), stromal cells (CDH3), T cells (CD3D), macrophages (C1QA), fibroblast cells (DCN), steroidogenic cells (CYP17A1), erythroid cells (HBG), neutrophil cells (S100A12), sympathoblast cells (PENK), dendritic cells (LY6H), SCPs (MPZ), angiotensinogen-expressing cells (AGT), and apoptotic cells (CIDEA) (Figure 1C, D). All cell types closely resembled those previously identified in the human adrenal gland cell (Huang et al., 2021). The relative proportions of these 13 cell types were similar between male and female tree shrews, and between sexually mature and immature animals (Figure 1E, F).

Hematoxylin-eosin staining of adrenal gland sections from tree shrews, regardless of age or sexual maturity, showed a medulla and cortical structure comprising a zona glomerulosa, zona fasciculata, and zona reticularis (Supplementary Figure S1B), resembling the structure of the human adrenal gland (Rosenfield, 2021). Transmission electron microscopy revealed an abundance of lipid droplets and mitochondria in the tree shrew adrenal cortex (Supplementary Figure S1C), implying the ability to perform steroid metabolism. Norepinephrine-producing cells in the medulla showed abundant dense granules but fewer mitochondria, whereas adrenaline-producing cells showed abundant mitochondria but fewer dense granules.

Interactions among adrenal cell types in tree shrews

To explore the regulatory mechanisms of steroid production, CellChat was utilized to examine the interactions and intercellular communication across adrenal cell types. Four incoming cell patterns were identified, with pattern 3 primarily associated with steroid hormone metabolism (Supplementary Figure S2A, B). Three outgoing cell patterns were identified, with pattern 2 predominantly associated with steroid hormone metabolism. Cortical steroidogenic cells and stromal cells showed similar incoming and outgoing patterns, consistent with several studies suggesting complex interplay and signaling pathways between steroidogenic and supportive cells, including stromal, myocyte, endothelial, and immune cells (Huang et al., 2021 ; Kater et al., 2022). The connections between steroidogenic and stromal cells in tree shrews were stronger in the cortex than in other parts of the adrenal gland (Supplementary Figure S2C).

A signaling pathway was identified between myocytes and steroidogenic cells that appeared to be mediated by the angiotensin II precursor AGT (Supplementary Figure S2D). AGT is known to promote adrenal gland growth and development as well as maintain normal adrenal function and blood pressure homeostasis (Matsuyama et al., 2021). Thus, our results suggest that steroidogenic cells may use AGT to regulate adrenal function.

The most prominent communication pathway among adrenal cell types, particularly steroidogenic, fibroblast, and stromal cells, appeared to be mediated by fibroblast growth factor (FGF), which also may play a central role in steroidogenesis (Supplementary Figure S2D). These results are consistent with the fact that disruption of a certain FGF receptor 2 isoform impairs adrenal gland growth and development and alters steroidogenic enzyme expression in the gland (Guasti et al., 2013). FGF is also known to determine the fate of neuroendocrine progenitor cells and regulate their proliferation (Chung et al., 2016).

Another important signaling molecule among the adrenal cell types was hepatocyte growth factor (HGF) (Supplementary Figure S2D), which is known to regulate interactions between granulosa and theca cells (Zachow et al., 2000) and stimulate the proliferation of granulosa cells by activating the MAPK pathway, while simultaneously inhibiting the synthesis of E2, which is dependent on progesterone and follicle-stimulating hormone (FSH) (Taniguchi et al., 2004).

The insulin-like growth factor (IGF) (Supplementary Figure S2D) signaling pathway was also identified, which plays a crucial role in the growth and differentiation of the adrenal cortex, facilitating cell growth and survival through the MAPK and PI3K-AKT pathways. In the prepubertal stage, cells expressing IGF or its receptor are located, together with adrenal progenitor cells, beneath the capsule in the peripheral cell layer (Belgorosky et al., 2009). Activation of the IGF signaling pathway positively influences the proliferation of adrenal precursor cells and their migration towards the inner region, thereby promoting the development of specific adrenal zones, including the zona reticularis. Additionally, IGF signaling is known to participate in adrenal zone development, differentiation, and steroidogenesis (Dumontet & Martinez, 2021).

Results also showed that the Wnt signaling factor mediated some communication between steroidogenic and stromal cells (Supplementary Figure S2D). Consistently, Wnt signaling is thought to participate in adrenal cortex development, stem cell functions, and cortical homeostasis (Little III et al., 2021). Furthermore, the WNT and PKA signaling pathways mutually regulate the differentiation of cortical cells, leading to the synthesis of steroid hormones and enabling responses to stressors from both internal and external environments, as well as endocrine stimulation (Drelon et al., 2016; Pignatti et al., 2017).

The communication pathway network identified in the tree shrew adrenal gland overlapped extensively with that reported in humans (Huang et al., 2021). Notably, our data suggested that, as in the human adrenal gland, steroidogenic and supportive cells interact closely in the tree shrew gland through a regulatory network that controls steroid hormone biosynthesis.

Various tree shrew steroidogenic adrenal cells show characteristics of stem cells

We identified seven steroidogenic adrenal cell clusters (Figure 2A), with pseudotime developmental analysis identifying clusters S3 and S4 as the initial populations, and clusters S1, S2, and S5 are relatively mature populations, expressing CYP17A1 and STAR (Figure 2B; Supplementary Figure S3A). S1 and S2 expressed higher levels of cell maturation markers GJA1 and DAPL1 compared to other clusters (Figure 2C; Supplementary Figure S3A, B). The transcription factors NR5A1 (Urs et al., 2007) and FOXO1 were highly expressed in S1 and S2 (Supplementary Figure S3C). FOXO1 up-regulates genes involved in steroid hormone biosynthesis and metabolism and inhibits NR5A1 to down-regulate adrenocorticosteroid biosynthesis (Kinyua et al., 2018). NR5A1 and FOXO1 were also highly expressed in S7 and S5, respectively. The transcriptome of S1 was enriched in genes involved in cellular energy metabolism (Figure 2D), likely ensuring adequate energy for the synthesis and metabolism of adrenal steroid hormones. The transcriptome of S2 was enriched in genes associated with steroid biosynthesis and lipid, adenosine triphosphate (ATP), and cholesterol metabolism (Supplementary Figure S3D), suggesting active endocrine functions in cluster S2.

Figure 2.

Figure 2

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Comprehensive characterization of steroidogenic cells in tree shrew adrenal glands

A: Distribution of steroidogenic cell subpopulations S1–S7. B: Pseudotime analysis of adrenal steroidogenic cells. C: Expression of stem cell marker VSNL1 and mature cell marker GJA1 in steroidogenic cells. D: Gene Ontology analysis of subpopulations S1 and S4. E: Spatial distribution of subpopulations S1 and S4 in adrenal gland, based on spatial transcriptomics. F, female; M, male; A, mature (18 months); B, immature (3 months).

In contrast, clusters S3 and S4 expressed stem cell marker VSNL1 (Zubkova et al., 2021) (Figure 2C; Supplementary Figure S3A), which helps maintain adrenal precursor cell proliferation and differentiation, ultimately giving rise to functional steroidogenic cells in sexual maturity (Trejter et al., 2015). These two clusters also showed high expression of AGTR1, a regulator of vasopressin and aldosterone secretion (Supplementary Figure S3B). Cluster S3 also expressed three genes associated with stem cells, PLCL1, CACNB2, and ANGPTL1 (Cheng et al., 2021; Ye et al., 2020) (Supplementary Figure S3A), while cluster S4 also expressed GADD45A, whose protein product is proposed to drive “stem cell stemness” by lengthening telomeres or enhancing telomere methylation (Diao et al., 2018). Both S3 and S4 expressed genes encoding several transcription factors linked to stem cell stemness, including TCF7L2, POU4F2, SIX2, and MAFB (Karner et al., 2011; Park et al., 2016; Wenzel et al., 2020; Zhang et al., 2013) (Supplementary Figure S3C). The transcriptome of cluster S4 showed enrichment in genes related to the regulation of translation, mRNA catabolism, and translational initiation (Figure 2D), while the transcriptome of S3 showed enrichment in muscle cell contraction and action potential processes (Supplementary Figure S3D). Collectively, these findings suggest that clusters S3 and S4 give rise to steroidogenic cells.

Spatial transcriptomics identified clusters S3 and S4 as progenitor cells in the subcapsular region (Figure 2E; Supplementary Figure S3E). S4 also showed a punctate distribution in the internal adrenal zones. These data suggest that steroid stem cell-like progenitor cells are potentially scattered throughout the adrenal zones (Figure 2E), where they can be induced to self-renew and differentiate.

The mature S1 and S2 clusters were predominantly localized in the inner region (Figure 2E; Supplementary Figure S3E). Our findings indicated that adrenal steroidogenic cells arose through “centripetal” migration and differentiation following a subcapsular-zona glomerulosa-zona fasciculata-zona reticularis pattern, confirming the mechanism by which zonation is formed in the human adrenal gland (Drelon et al, 2016; Freedman et al., 2013; Xing et al., 2015).

Tree shrew steroidogenic adrenal cells produce DHEA similar to human zona reticularis cells

Results showed that all seven subpopulations of steroidogenic adrenal cells in tree shrews expressed STAR, CYP11A1, and CYP11B2 (Figure 3A). Clusters S1, S2, S4, S6, and S7 also expressed HSD3Bli1, HSD11B2, and CYP17A1. Due to incomplete identification and annotation of tree shrew transcriptome data, along with the utilization of a specialized mapping approach for the gene HSD3Bli1, this gene may exhibit a functional state similar to that of HSD3B1 (Ye et al., 2021). Clusters S1, S2, S5 and S7 also showed high CYB5A expression, while stem cell-like clusters S3 and S4 showed high HSD3B expression.

Figure 3.

Figure 3

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Analysis of key enzyme genes involved in steroid synthesis in tree shrew adrenal gland

A: Expression of STAR, CYP11A1, HSD3B, HSD3Bli1, CYP11B2, HSD11B1, HSD11B2, CYB5A, and CYP17A1 in adrenal steroidogenic cells. B: Spatial distribution and expression of steroidogenic cell markers STAR, CYP11B2, CYP17A1, and CYB5A, based on spatial transcriptomics. F, female; M, male; A, mature (18 months); B, immature (3 months). C: Immunohistochemical analysis of adrenal tissue sections against proteins encoded by STAR, CYP11B1, CYP11B2, CYP21A2, CYB5, and SULT2A1.

Results further showed that the zona reticularis-specific gene CYB5A (Rosenfield, 2021) was predominantly expressed in the adrenal corticomedullary junction, while CYP17A1-positive (Dumontet & Martinez, 2021) cells were observed in the cortex, implying DHEA steroidogenesis in the adrenal gland (Figure 3B, C). STAR and CYP11B2 were also expressed in the adrenocortex (Figure 3B), while immunohistochemical analysis identified positive expression of metabolic enzymes such as STAR, CYP11B1, CYP11B2, and CYP21A2 (Figure 3C), and liquid chromatography-mass spectrometry confirmed the production not only of DHEA (60.59±27.82 ng/g adrenal tissue) but also of mineralocorticoids and glucocorticoids (Figure 4A). Stimulating primary adrenal cell cultures with cholesterol significantly boosted DHEA synthesis (Figure 4B, P=0.001<0.05).

Figure 4.

Figure 4

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Establishing production of DHEA in tree shrew adrenal gland

A: Quantification of steroid hormones in tree shrew adrenal tissue using liquid chromatography-mass spectrometry. B: Production of DHEA by primary cultures of adrenal cells after stimulation with cholesterol (Chol) or not (Control). DHEA was assayed using liquid chromatography-mass spectrometry.

The expression of SULT2A1, the gene responsible for the sulfation of DHEA in humans and other primate species (Dumontet & Martinez, 2021; Rosenfield, 2021), was not detected based on single-cell RNA sequencing, spatial transcriptomics, or immunohistochemical analysis of the tree shrew adrenal gland. Similarly, DHEAS was not detected through liquid chromatography-mass spectrometry (Figure 4A). These findings suggest that the tree shrew adrenal gland produces DHEA, similar to the human adrenal gland, whereas, in contrast to humans, the bioactive form of DHEA acting on downstream target organs is not DHEAS but some other derivative(s).

Analysis of adrenal medullary neurocytes reveals functional similarities and differences between tree shrews and humans

Clustering analysis of adrenal medullary cells led to the identification of five subpopulations of neuroendocrine cells (N1–N5), which expressed CHGA, DBH, and TH; one subpopulation of SCPs, which expressed MPZ, PLP1, and GLDN; and one subpopulation of fibroblasts, which expressed CDH19, HTRA1, and COL4A1 (Supplementary Figure S4A–C). These seven subpopulations displayed distinct sex and age variations, particularly in the N5 subpopulation, which primarily originated from the adrenal medulla of sexually mature male tree shrews (Supplementary Figure S5A). These findings suggest that the medulla transcriptome may depend on sex and sexual maturation to a greater extent than the adrenal cortex. CHGA but not MPZ was expressed in the adrenal medulla based on spatial transcriptomics (Supplementary Figure S4D) and immunohistochemical analysis (Supplementary Figure S4E, F). Thus, the observed differences in medullary cell subpopulations may be an artifact of single-cell digestion and handling, consistent with our previous research (Huang et al., 2021).

Pseudotime analysis of medullary cells suggested that the neuroendocrine cells originated from SCPs (Supplementary Figure S5B). The N2 neuroendocrine subpopulation strongly expressed genes associated with “bridge cell” functions, such as PHOX2B, ASCL1, and ID2 (Chen et al., 2012; Dong et al., 2020; Furlan et al., 2017) (Supplementary Figure S5C). Thus, we propose that N2 cells may serve as “bridge cells” between SCPs and mature neuroendocrine cells, consistent with our previous findings in human embryonic adrenal glands (Wang et al., 2022) and other work on the origin and development of adrenal medullary cells in humans (Dong et al., 2020; Furlan et al., 2017), indicating conservation across species.

Consistent with the identification of communication between medullary neuroendocrine cells and steroidogenic cells (Supplementary Figure S2), our study demonstrated that STAR, CYP11A1, CYP17A1, and CYP11B2 were expressed in the medulla (Figure 3B, C; Supplementary Figure S5D). These observations suggest a role of the medulla in steroid metabolism, in contrast to the situation in humans, where the medulla participates in catecholamine but not steroid metabolism (Streit et al., 2022).

Human-like steroid hormone synthesis pathways may be the same in tree shrew adrenal glands

Our comparison of adrenal cell populations across species suggested considerable conservation from mice to humans (Figure 5A). However, comparative analysis also indicated that the M. fascicularis and mouse adrenal glands lacked a zona reticularis, consistent with our integrated data, which also supported the absence of a zona reticularis in the mouse adrenal.

Figure 5.

Figure 5

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Transcriptomic comparison of adrenal tissues from humans, tree shrews, macaques, and mice

A: Atlas of single-cell RNA sequencing in adrenal gland. From left to right: human, tree shrew, macaque, and mouse. B: Expression of genes related to DHEA synthesis: STAR, CYP11A1, CYP17A1, CYB5A, and DHEA metabolism: SULT2A1 and HSD3B2. From top to bottom: human, tree shrew, macaque, and mouse. C: Correlations of cell subpopulations among adrenal cell types, with darker red indicating stronger correlation. D: Correlations of expression of genes related to DHEA synthesis across species, based on bulk adrenal RNA sequencing: STAR, CYP11A1, CYP17A1, and CYB5A.

Further analysis revealed that a variety of adrenal cell types in both human and tree shrew glands expressed enzymes involved in the synthesis and metabolism of DHEA (Figure 5B). In contrast, SULT2A1, essential for DHEAS production, was absent in tree shrews or mice, unlike in humans. This gene was minimally expressed in macaques, which seemingly lacked adrenal gland expression of CYP11A1 and CYB5A. Additionally, the mouse adrenal gland showed no Cyp17a1 or Hsd3b2 expression.

In terms of the transcriptome, several mouse adrenal cell types were more similar than tree shrew or macaque cell types to the human counterparts, including for natural killer (NK) cells, fibroblasts, endothelial cells, myeloid cells, and zona fasciculata cells (Figure 5C). Nevertheless, the transcriptomes of adrenal stromal and steroidogenic cells in tree shrews were strongly correlated with counterpart cells in the human zona fasciculata, zona glomerulosa, and zona reticularis. Furthermore, adrenal tissue from both tree shrews and humans expressed STAR, CYP11A1, CYP17A1, and CYB5A, while CYP17A1 was expressed to a negligible extent in the mouse adrenal tissue, consistent with the lack of a zona reticularis (Figure 5D).

Encouraged by these similarities, we integrated the known metabolic pathways of human adrenal steroid hormones (Dumontet & Martinez, 2021; Rosenfield, 2021) with our bulk adrenal RNA sequencing and liquid chromatography-mass spectrometry analyses to propose a comprehensive steroid hormone metabolism map for tree shrew adrenal glands (Figure 1A). Enzymes involved in human pathways included STAR, CYP11A1, CYP17A1, and HSD3B2/1, with key enzymes CYP11B2, CYP11B1, and CYB5A responsible for aldosterone, cortisol, and DHEA synthesis, respectively. The map suggested striking similarities in steroid metabolism between tree shrews and humans, particularly in the biosynthesis of androgens in the zona reticularis. Indeed, profiles of enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were similar between tree shrews and humans for immune-related and steroidogenic adrenal cell types (Figure 6A). These observations create a compelling case for the development of the tree shrew as a model for human adrenal disorders.

Figure 6.

Figure 6

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Correlations between transcriptomic profiles in different species and enrichment in KEGG pathways related to adrenal disorders

A: Enrichment of transcriptomes from different adrenal cell types in KEGG signaling pathways. Darker red indicates stronger correlation between KEGG signaling pathways and adrenal cell types. B: Scores describing associations of transcriptomes of adrenal cell types with signaling pathways related to human disease (HD), organismal systems (OS), metabolism (MB), environmental information processing (EIP), cellular processes (CP), and genetic information processing (GIP). Darker red indicates higher score.

Correlations between tree shrew transcriptomics and adrenal disorders in humans

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is a significant biological event that links stress, depression, and the onset of diabetes (Joseph & Golden, 2017). DHEA has shown therapeutic benefits in animal models of diabetes and obesity, by increasing insulin secretion from the pancreas and enhancing liver, adipose tissue, and muscle sensitivity to insulin (Aoki & Terauchi, 2018). Here, we found that the transcriptome of tree shrew steroidogenic cells was enriched in gene pathways associated with type I or II diabetes mellitus, insulin resistance, and AGE-RAGE signaling in diabetes-related complications (Figure 6B), as well as Cushing’s syndrome, a typical adrenal disease, and pathways related to cortisol synthesis and secretion.

Adrenopause (Dumontet & Martinez, 2021; Nunes-Souza et al., 2020), a unique physiological process that occurs in the human adrenal gland, is characterized by a decrease in the concentration of DHEA synthesized by the zona reticularis with age. Interestingly, the transcriptomes of the tree shrew adrenal cell types, including cortical stromal cells, fibroblasts, endothelial cells, and SCPs, were highly enriched in cellular processes related to aging (Figure 6B). These findings highlight the potential for the tree shrew to serve as a model of adrenal aging.

Adrenal transcriptomes from tree shrews and humans also overlapped regarding enrichment in pathways related to steroid biosynthesis, steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion as well as signaling pathways involving gonadotropin-releasing hormone (GnRH), Notch, Hippo, Wnt, and AMPK (Figure 6B), which regulate adrenal zonation formation and adrenal endocrine activity.

Nearly all adrenal cell types in tree shrews, but none in mice, expressed RAB5B, PROX1, and PLGRKT, whose mutations are linked to polycystic ovary syndrome risk (Figure 7). Similarly, these genes are expressed in various human adrenal cell types in the zona glomerulosa, zona fasciculata, and zona reticularis (Shi et al., 2011). Mutations in other genes were also detected in the adrenal steroidogenic cells of tree shrews, including ARMC5 (Gagliardi et al., 2014) and FH (Stratakis & Boikos, 2007), which are implicated in the risk of hypercortisolism in humans. Corresponding mutations in mice and macaques showed a weak correlation with steroidogenic cells. Certain mutations were also detected in HSD11B2 and ARMC5 (Scholl, 2022), which are closely linked to primary aldosteronism risk in humans. The expression levels of SH2B3, GATA3, and SPACA6, closely related to the risk of Addison’s disease (primary adrenal insufficiency) in humans (Eriksson et al., 2021; Mitchell et al., 2015), were strongly correlated with steroidogenic cells in the cortical cell subpopulations in tree shrews. The tree shrew medullary cell subpopulations also showed strong expression of SDHAF2 and SDHD, polymorphisms of which are associated with the risk of pheochromocytoma in humans (Welander et al., 2012). Furthermore, certain adrenal cell types in tree shrews expressed genes whose polymorphism has been linked to hypertension (Padmanabhan & Dominiczak, 2021).

Figure 7.

Figure 7

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Bioinformatic correlations between gene expression profiles of adrenal cell subtypes in different species and genes linked to adrenal disorders in humans

Heatmap showing correlations between genetic risk factors of adrenal dysfunction and transcriptomes of adrenal cell types from different species. Darker red indicates stronger correlation. PA, primary aldosteronism; PAI, primary adrenal insufficiency; PCOS, polycystic ovary syndrome.

Overall, these observations strongly suggest that tree shrews can provide a rich resource for understanding the genetic and molecular basis of numerous disorders involving dysregulation or dysfunction of the adrenal gland in humans.

DISCUSSION

The present study provides robust anatomical, transcriptional, and biochemical evidence that Chinese tree shrew adrenal glands mimic those of humans, offering a viable model for investigating the mechanisms and treatments adrenal disorders. Notably, we identified 13 major adrenal cell types and used bioinformatic analysis to reveal similarities in their interactions and steroid synthesis pathways with those of the human adrenal gland. Of note, our results showed that unlike the macaque and mouse glands, the tree shrew gland produced substantial levels of DHEA, akin to humans, but, similar to macaques and mice, did not produce DHEAS, which exerts important functions in downstream organs in humans. Thus, further research is needed to identify the DHEA metabolite(s) in tree shrews that act on downstream organs. We also successfully mapped specific adrenal cell types and pathways in tree shrews onto genetic pathways known to be dysregulated in human adrenal disorders, including Cushing’s disease, primary aldosteronism, polycystic ovary syndrome, and hypertension. Our findings strongly suggest that the tree shrew may be the most appropriate small-animal model so far explored for human adrenal disorders. To accelerate the continuing exploration and development of the Chinese tree shrew as a disease model, our data have been deposited on a publicly accessible website (http://gxmujyzmolab.cn:16245/scAGMap/).

Our results further revealed that the tree shrew adrenal gland contained steroidogenic cells in the cortex, beneath the zona fasciculata and adjacent to the medulla. The identification of steroidogenic cells, their expression of CYB5A, and the biochemical detection of DHEA in the tree shrew adrenal gland point to a zona reticularis similar to that of humans, indicating parallel steroid-metabolizing pathways in both species (Figure 1A) (Dumontet & Martinez, 2021; Little III et al., 2021; Rosenfield, 2021), with aldosterone, cortisol, DHEA, and other androgens as end products. Surprisingly, our experimental methods failed to detect the presence of SULT2A1, the enzyme responsible for sulfating DHEA (Dumontet & Martinez, 2021; Rosenfield, 2021; Turcu et al., 2020), indicating a gap in our understanding of how DHEA is transported to target organs for metabolism, which warrants further research.

Our findings should be interpreted with some caution. The presence of zona glomerulosa, zona fasciculata, and zona reticularis in our 3-month-old tree shrews suggests a level of adrenal gland maturation. As such, future studies should examine younger and older tree shrews to map the structure and function of the adrenal gland over the lifespan, facilitating a detailed comparison with human adrenal physiology. We also failed to identify certain key human steroid-metabolizing enzymes in the tree shrew sequences, potentially due to the incomplete annotation of several sequences. Subsequent sequencing efforts should endeavor to resolve these knowledge gaps. Moreover, our analysis of the regulatory networks affecting steroid metabolism did not encompass the roles of the ovaries, testes, or adipocytes, all of which regulate adrenal gland activity in humans (Ford, 2013; Labrie, 2019; Paulukinas et al., 2022). An exhaustive examination of the HPA axis in tree shrews is essential for a comprehensive comparison with its human counterpart, enhancing the utility of tree shrews as a model for studying human adrenal gland physiology and pathology.

CONCLUSIONS

The comprehensive transcriptomic map obtained at single-cell resolution offers valuable insights into the regulation of steroid hormone metabolism, specifically adrenal androgens like DHEA, in the tree shrew adrenal glands. This knowledge lays the foundation for further investigation into the complex mechanisms governing human adrenal function and the development of disease models. Furthermore, this study contributes to our understanding of adrenal physiology, with important implications for future research in this field.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

zr-45-617-S1.pdf (5.9MB, pdf)

Acknowledgments

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

Z.N.M., Y.H.J., and X.S.Z. contributed to the concept and design of the original research. J.H.J., Z.Y.C., and Y.G. collected the animal samples. Y.J.G., B.Q.G., and Y.F.L. participated the single-cell RNA, Bulk RNA, and spatial transcriptomics experiments. J.H.J., Z.Y.C., and G.Y. prepared the primary adrenal cells and performed liquid chromatography-tandem mass spectrometry. H.H.W. and J.J.X contributed to data acquisition. Y.F.W., J.Z., and Y.M.L. analyzed the data. Y.X.L., T.W.J., and B.Q.L performed tissue sectioning and immunohistochemistry. Z.N.M., Y.H.J., and X.S.Z. contributed to supervision and project administration. J.H.J., Y.F.W., J.Z., and Y.M.L. performed the original draft preparation. J.H.J. and Y.F.W. wrote the manuscript. All authors read and approved the final version of the manuscript.

ACKNOWLEDGMENTS

Gratitude is extended to the Kunming Institute of Zoology, Chinese Academy of Sciences, for the provision of Chinese tree shrews. We would like to express our appreciation to Dr. Armando Chapin Rodriguez for his assistance with language editing. We also wish to thank the reviewers for their dedicated efforts in enhancing the quality of this article.

Funding Statement

This work was supported by the Key Research and Development Program of Guangxi (2021AB13014), Major Project of Guangxi Innovation Driven (AA18118016), National Key Research and Development Program of China (2017YFC0908000), Natural Key Research and Development Project (2020YFA0113200), National Natural Science Foundation of China (81770759, 82060145, 31970814), Natural Science Foundation of Guangxi Zhuang Autonomous Region (2021JJA140912), Advanced Innovation Teams and Xinghu Scholars Program of Guangxi Medical University, Guangxi Key Laboratory for Genomic and Personalized Medicine (19-050-22, 19-185-33, 20-065-33, 22-35-17), Major Project of Scientific Research and Technology Development Plan of Nanning (20221023), Guangxi Natural Science Foundation (2022GXNSFAA035641), and Self-funded Project of Health Commission of Guangxi Zhuang Autonomous Region (Z-A20230620)

Contributor Information

Xiao-Shu Zhu, Email: xszhu@csu.edu.cn.

Yong-Hua Jiang, Email: jiangyonghua@stu.gxmu.edu.cn.

Zeng-Nan Mo, Email: mozengnan@gxmu.edu.cn.

DATA AVAILABILITY

The adrenal gland data are available at http://gxmujyzmolab.cn:16245/scAGMap. The single-cell RNA sequencing data can be accessed via the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/gsa/) under accession number CRA011453 and Science Data Bank (https://www.scidb.cn/c/zoores) using the data private access link https://www.scidb.cn/en/s/QZzE7f (DOI:10.57760/sciencedb.j00139.00111). The mouse adrenal gland sequencing data used in this study can be accessed from the GEO database using accession numbers GSE134355 and GSE121051. The human adrenal gland sequencing data were sourced from the Genotype-Tissue Expression Project (https://gtexportal.org/). The macaque (Macaca fascicularis) adrenal gland sequencing data were obtained from a publicly accessible database (https://db.cngb.org/nhpca/download).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data to this article can be found online.

zr-45-617-S1.pdf (5.9MB, pdf)

Data Availability Statement

The adrenal gland data are available at http://gxmujyzmolab.cn:16245/scAGMap. The single-cell RNA sequencing data can be accessed via the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/gsa/) under accession number CRA011453 and Science Data Bank (https://www.scidb.cn/c/zoores) using the data private access link https://www.scidb.cn/en/s/QZzE7f (DOI:10.57760/sciencedb.j00139.00111). The mouse adrenal gland sequencing data used in this study can be accessed from the GEO database using accession numbers GSE134355 and GSE121051. The human adrenal gland sequencing data were sourced from the Genotype-Tissue Expression Project (https://gtexportal.org/). The macaque (Macaca fascicularis) adrenal gland sequencing data were obtained from a publicly accessible database (https://db.cngb.org/nhpca/download).

Articles from Zoological Research are provided here courtesy of Editorial Office of Zoological Research, Kunming Institute of Zoology, The Chinese Academy of Sciences

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