Abstract
Sex differences in mammalian complex traits are prevalent and are intimately associated with androgens1,2,3,4,5,6,7. However, a molecular and cellular profile of sex differences and their modulation by androgens is still lacking. Here we constructed a high-dimensional single-cell transcriptomic atlas comprising over 2.3 million cells from 17 tissues in Mus musculus and explored the effects of sex and androgens on the molecular programs and cellular populations. In particular, we found that sex-biased immune gene expression and immune cell populations, such as group 2 innate lymphoid cells, were modulated by androgens. Integration with the UK Biobank dataset revealed potential cellular targets and risk gene enrichment in antigen presentation for sex-biased diseases. This study lays the groundwork for understanding the sex differences orchestrated by androgens and provides important evidence for targeting the androgen pathway as a broad therapeutic strategy for sex-biased diseases.
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Data availability
Customizable visualizations and analyses can be executed in our web tool (https://casadbtools.com). The raw data for scRNA-seq have been deposited in the GSA (https://ngdc.cncb.ac.cn/gsa/) under CRA006610. The processed data for scRNA-seq have been deposited in the OMIX under OMIX001083. The mm10 genome reference was obtained from: https://ftp.ensembl.org/pub/release-93/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz. Source data are provided with this paper.
Code availability
The codes used for analysing and visualizing the data in this study are available in the GitHub repository (https://github.com/lifei176/Single-cell-atlas-of-sex-differences-and-androgen-effects)45 and in Zenodo (https://doi.org/10.5281/zenodo.10784644)46.
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Acknowledgements
We thank B. Wu, G. Chen and W. Bian for providing technical help at the CEMCS (SIBCB) Core Facility, the Genome Tagging Project Center at the CEMCS for technical support, and H. Dong for the schematic drawing. This study was supported by the National Key Research and Development Program of China (no. 2023YFC2506401, 2020YFA0509002, 2022YFC2504602 and 2022YFA1004800), the National Science Fund for Distinguished Young Scholars (32125013 and T2125002), the National Natural Science Foundation of China (92253304, 82241230, 82341007, T2341007, 12131020, 31930022 and T2350003), the Basic Frontier Science Research Program of Chinese Academy of Sciences (ZDBS-LY-SM015), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020905, XDB38040400), the Shanghai Science and Technology Committee (21XD1424200, 21ZR1470100 and 23JS1401300), the Shanghai Municipal Science and Technology Major Project, the China National Postdoctoral Program for Innovative Talents (BX2021306), the China Postdoctoral Science Foundation (2023M743484 and 2023000162), the Innovative Research Team of High-level Local Universities in Shanghai (SHSMUZDCX20211800), the Science and Technology Commission of Shanghai Municipality (23JS1401300), JST Moonshot R&D (JPMJMS2021), the New Cornerstone Science Foundation through the XPLORER PRIZE, the Guangdong Basic and Applied Basic Research Foundation (2023A1515011075), and the Shenzhen Bay Laboratory.
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Contributions
D.G., F.B., L.C. and C.Y. conceived and designed the project. F.L., X.X., Q.J., X.-M.W., C.Y., L.C., F.B. and D.G. wrote the manuscript. F.L., X.X., Q.J., X.-M.W., Q.W., Y.C., C.Y., L.C., F.B. and D.G. provided edits to the manuscript. F.L., P.D. and M.H. performed the experiments. H.S., X.S., Y.P., J.X., D.L., W.W. and Y.Z. provided experimental support. F.L., X.X., Q.J. and X.-M.W. performed the computational and statistical analyses. Z.Z. constructed the web tool.
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Extended data figures and tables
Extended Data Fig. 1 AR-positive cells are prevalent across tissues.
a, b, Immunochemical staining for AR across tissues in male mice (a) and female mice (b). c, Schematic for the generation of the systemic AR knockout in male mice. d, Immunochemistry staining on AR across multiple tissues of Rosa26CreERT2/+; Arflox/Y male mice 2 weeks post tamoxifen or corn oil treatment. Scale bar represents 50 μm. In a, b and d, experiments were repeated independently with similar results for three times.
Extended Data Fig. 2 Effects of androgens on body weight and tissue weight.
a, Quantification of the changes in body weight of MS (n = 15), MC (n = 16), FS (n = 27) and FD (n = 29) mice. n denotes the number of mice. Data are mean ± s.e.m. b, Volcano plot showing the weight changes in each tissue induced by androgen deprivation in male mice. The tissues with decreased weight in response to androgen deprivation are marked in blue, while the tissues with increased weight in response to androgen deprivation are marked in red. c, Heatmap showing the weight changes in each tissue induced by androgen deprivation in male mice. d, Volcano plot showing the changes in the weight of each tissue induced by androgen supplementation in female mice. The tissues with decreased weight in response to androgen supplementation are marked in blue, while the tissues with increased weight in response to androgen supplementation are marked in red. e, Heatmap showing the weight changes in each tissue induced by androgen supplementation in female mice. f, Summary of the effects of androgens on the weight of each tissue. g, Representative tissues, including the salivary gland (androgen-promoting, left), thymus (androgen-suppressing, middle) and liver (nonsignificant changes, right) tissue of MS, MC, FS and FD mice. In b, d, statistical comparisons were performed using two-tailed Student’s t test.
Extended Data Fig. 3 Expression levels of representative markers.
a, Feature plot showing the RNA expression levels of representative markers for tissue-common cell lineages, including Ptprc for immune cells, Pecam1 for endothelial cells, Col1a1 for fibroblasts and Tagln for smooth muscle cells. b, Feature plot showing the RNA expression levels of representative markers for tissue-specific cell lineages, including Alb for hepatocytes in the liver, Lrp2 for proximal tubule cells in the kidney, Sftpc for AT2 cells in the lung and Krt20 for enterocytes in the colon and intestine.
Extended Data Fig. 4 Androgens modulate sex-biased cellular populations.
a, Scatter plot showing the correlation between sex differences (log2(MS vs. FS)) and the differences in the proportions of all the cell types across tissues between FD and FS (log2(FD vs. FS)). b, Similar to (a), but for the correlation between log2(MS vs. FS) and log2(MC vs. MS). c, Heatmap denoting the impacts of sex and androgens on the proportions of all cell types across tissues. The color key represents the log2-transformed fold change in the cell proportions. d, Dot plot showing the Pearson correlation efficiency calculated based on sex differences (log2(MS vs. FS)) and the differences in cell composition between MC and MS (log2(MC vs. MS)) (x-axis), as well as sex differences (log2(MS vs. FS)) and the differences in cell composition between FD and FS (log2(FD vs. FS)) (y-axis). The dot color represents the tissue type. e, Scatter plot showing the correlation between sex differences (log2(MS vs. FS)) and the differences in salivary cell composition between FD and FS (log2(FD vs. FS), left) or the differences in salivary cell composition between MC and MS (log2(MC vs. MS), right). f, Bar plot denoting the cellular composition of salivary gland across four conditions. g, UMAP plot showing the cell composition of salivary gland based on four conditions involving sex and androgens. Two thousand cells for each condition were randomly extracted for visualization. h, Bubble plot (left) and heatmap (right) denoting the impacts of sex and androgens on the proportions of all salivary cell types. The size indicates the proportion. The color key represents the log2-transformed fold change in cell proportions. In a, b, and e, P values are shown; the test statistic is based on Pearson’s product moment correlation coefficient and follows a t distribution; level of confidence interval is 0.95.
Extended Data Fig. 5 Cross-cell type effects of sex and androgens on biological pathways.
a-f, Dot plot showing the top 50 cross-cell type biological pathways based on the upregulated DEGs (a) and downregulated DEGs (b) between MS and FS, the upregulated DEGs (c) and downregulated DEGs (d) between MC and MS, and the upregulated DEGs (e) and downregulated DEGs (f) between FD and FS. The x-axis represents the number of cell types sharing the target biological pathway. Gradient color represents the number of tissues sharing the target biological pathway. g, h, Lollipop plot showing the remarkable androgen-associated genes in the “neutrophil chemotaxis” pathway (g) and the “T-cell activation” pathway (h). The x-axis represents the number of cell types sharing the target gene. i, Scatter plot showing the correlation of log2FC in the DEGs which locates on autosomes between sex differences and MC vs. MS based on all of the cell types from 17 tissues. j, Scatter plot showing the correlation of log2FC in the DEGs which locates on autosomes between sex differences and FD vs. FS based on all of the cell types from 17 tissues. In i, j, P values are shown; the test statistic is based on Pearson’s product moment correlation coefficient and follows a t distribution.
Extended Data Fig. 6 The effects of androgen supplementation on ovariectomized female mice and castrated male mice.
a, Schematic diagram of the experimental design to investigate the effects of androgens on the tissue weight, gene expression and cell composition in ovariectomized female mice (FOD vs. FO). b, Heatmap showing the tissue weight changes induced by androgen supplementation in ovariectomized female mice. c, Representative tissues, including the kidney and salivary gland (androgen-promoting), thymus and spleen (androgen-suppressing), and brain and lung (nonsignificant changes) of FO and FOD mice. d, Scatter plot showing the correlation of androgen supplementation-induced cell proportional differences between intact (FD vs. FS) and ovariectomized (FOD vs. FO) female mice across tissues. Level of confidence interval is 0.95. e, Scatter plot showing the correlation of DEG expression differences between FD vs. FS and FOD vs. FO mice (left). Heatmap denoting the log2FC in the expression of the DEGs from a single cell type across the four comparisons, where DEGs are colored according to their tissue of origin (right). f, Similar to (a) but for castrated male mice (MCD vs. MC). g, Similar to (b) but for androgen supplementation in castrated male mice. h, Similar to (c) but for MC and MCD mice. i, Scatter plot showing the correlation of cell proportional differences between the comparisons of MC vs MCD and MC vs. MS across tissues. Level of confidence interval is 0.95. j, Similar to (e) but for comparison between MC vs. MCD and MC vs. MS mice. In d, e, i, and j, P values are shown; the test statistic is based on Pearson’s product moment correlation coefficient and follows a t distribution. In each condition (FOD, FO and MCD), each tissue contained at least three biological replicates (three mice).
Extended Data Fig. 7 AASB-DEGs and their functionally enriched pathways across tissues.
a, Network of all AASB-DEGs in each cell type across tissues. Red edges and blue edges represent the positive AASB-DEGs and the negative AASB-DEGs, respectively. The gray node represents the AASB-DEG, and the colored node represents the cell type. b, Scatter plot showing the correlation between the numbers of AASB-DEGs and Ar RNA expression. c, Scatter plot showing the correlation between the numbers of AASB-DEGs and Gapdh RNA expression. d, e, Representative cross-tissue AASB-DEGs and their relevant cell types, including Ace (positive AASB-DEG, d) and Hsd11b1 (positive AASB-DEG, e). f, Lollipop plot denoting the shared biological pathways across tissues based on positive (left top) or negative (left bottom) AASB-DEGs. Network of significantly enriched biological pathways based on AASB-DEGs in each cell type across tissues (right). In b, c, P values are shown; the test statistic is based on Pearson’s product moment correlation coefficient and follows a t distribution; level of confidence interval is 0.95.
Extended Data Fig. 8 Dissection of the effects of androgens on the immune compartment.
a, Dot plot showing the expression patterns of representative markers for nine major immune cell types. The dot size represents the percentage of cells with detectable gene expression. The color key represents the expression levels. b-d, Heatmap denoting sex differences (b) and the effects of androgen deprivation (c) and androgen supplementation (d) on immune cell proportions across tissues. The color key represents the log2-transformed fold change in the cell proportion. Light gray indicates that the comparison was performed on two cell types with cell numbers less than 10.
Extended Data Fig. 9 Enriched biological pathways based on disease-shared risk genes.
a, Pie chart showing the categorization of sex-biased diseases in the 17 tissues in our atlas. b, Heatmap denoting the differences in the incidence rates of sex-biased diseases between males and females. Representative male-biased and female-biased diseases are marked in red and blue, respectively. c, Network denoting the top 30 significantly enriched biological pathways based on the 662 risk genes that were shared by multiple sex-biased diseases. Node size represents gene number. Distinct biological pathway modules are marked in different colors. d, Dot plot denoting the significantly enriched biological pathways based on the 662 disease-shared risk genes of sex-biased diseases. Significantly enriched biological pathways were defined by the hypergeometric distribution and P values were adjusted by the Benjamini-Hochberg method (BH) by using EnrichGO function implanted in the clusterProfiler.
Extended Data Fig. 10 Sex and androgens influence MHC gene expressions.
a, b, Volcano plot denoting the MHC genes that showed significant differential expression (a) and their tissue of origin (b) based on the comparison of MS vs. FS. DEGs were defined by Wilcoxon Rank Sum test and P values were adjusted by the Bonferroni correction using the FindMarkers function implanted in Seurat. c, d, Volcano plot denoting the MHC genes that showed significant differential expression (c) and their tissue of origin (d) based on the comparison of MC vs. MS. DEGs were defined by Wilcoxon Rank Sum test and P values were adjusted by the Bonferroni correction using the FindMarkers function implanted in Seurat. e, f, Volcano plot denoting the MHC genes that showed significant differential expression (e) and their tissue of origin (f) based on the comparison of FD vs. FS. DEGs were defined by Wilcoxon Rank Sum test and P values were adjusted by the Bonferroni correction using the FindMarkers function implanted in Seurat. g, Gating strategy for bone marrow immature B cells (CD19+/B220+/AA4.1+/IgM+) and relative H2-Eb1 mRNA expression level quantified by qRT–PCR in the FACS-sorted bone marrow immature B cells of FD and FS mice. n = 6 mice examined per group. h, Gating strategy for spleen macrophages (CD45+/Ly6G+/F4/80+) and relative H2-Ab1 mRNA expression level quantified by qRT–PCR in the FACS-sorted spleen macrophages of FD and FS mice. n = 5 mice examined per group. i, Schematic to explain how we categorized human sex-biased diseases into five major groups using our scRNA-seq data. In g, h, Data are mean ± s.e.m. P values are shown; statistical comparisons were performed using two-tailed Student’s t test.
Extended Data Fig. 11 Serum levels of corticosterone.
Serum levels of corticosterone in FD, FS, MS and MC mice (FD: n = 5 mice; FS: n = 13 mice; MS: n = 5 mice; MC: n = 6 mice). n denotes the number of mice. Data are mean ± s.e.m.
Supplementary information
Supplementary Table 1
General information for atlas.
Supplementary Table 2
Cell proportion.
Supplementary Table 4
Significantly enriched biological pathways.
Supplementary Table 5
Information for scRNA-seq data of FOD, FO and MCD conditions.
Supplementary Table 6
AASB-DEGs and pathways.
Supplementary Table 7
Sex-biased diseases and their risk genes.
Supplementary Table 8
Cell-type enrichment for sex-biased diseases.
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Li, F., Xing, X., Jin, Q. et al. Sex differences orchestrated by androgens at single-cell resolution. Nature (2024). https://doi.org/10.1038/s41586-024-07291-6
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DOI: https://doi.org/10.1038/s41586-024-07291-6
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