Single-cell analysis of fetal testis reveals dysfunction of human Leydig cells in Klinefelter syndrome
Tong Yan, Guancheng Chen, Jie Zhang, Wenjing Jia, Nan Lu, Shuping Jin, Haotian Zhang, Yichen Zhao, Lu Jiang, Jing Wu, Qing Liu, Chenghao Situ, Hui Zhu, Yan Li, Quan Wang, Xiaoyu Yang, Chao Qin, Xiaofeng Song, Qing Cheng, Xuejiang Guo
Tong Yan, Guancheng Chen, Jie Zhang, Wenjing Jia, Nan Lu, Shuping Jin, Haotian Zhang, Yichen Zhao, Lu Jiang, Jing Wu, Qing Liu, Chenghao Situ, Hui Zhu, Yan Li, Quan Wang, Xiaoyu Yang, Chao Qin, Xiaofeng Song, Qing Cheng, Xuejiang Guo
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Research Article Cell biology Reproductive biology

Single-cell analysis of fetal testis reveals dysfunction of human Leydig cells in Klinefelter syndrome

Abstract

Klinefelter syndrome (KS), the most common sex chromosome aneuploidy (affecting approximately 1 in 650 live male births), causes severe infertility. The extra X chromosome can impair the development of fetal germ cells, but its effects on somatic cells, especially Leydig cells, are still not well known. We performed single-cell RNA-sequencing analysis of fetal KS and control testicular cells and found 2 clusters of KS Sertoli cells, with the XIST-negative cluster showing distinct gene expression pattern and abnormally increased G2/M ratio. Fetal KS Leydig cells showed increased proliferation and immature differentiation with high level of MAPK signaling pathway and X-linked EIF1AX. Inhibition of MAPK signaling partially rescued overproliferation and defective differentiation and androgen secretion in KS Leydig cells, while overexpression of EIF1AX recapitulated the phenotypes of increased proliferation and decline in testosterone synthesis capacity in the Leydig cell line. These findings reveal the early pathological mechanisms of KS somatic cells and lay the groundwork for developing early intervention strategies.

Authors

Tong Yan, Guancheng Chen, Jie Zhang, Wenjing Jia, Nan Lu, Shuping Jin, Haotian Zhang, Yichen Zhao, Lu Jiang, Jing Wu, Qing Liu, Chenghao Situ, Hui Zhu, Yan Li, Quan Wang, Xiaoyu Yang, Chao Qin, Xiaofeng Song, Qing Cheng, Xuejiang Guo

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Figure 1

Single-cell transcriptome profiling and analysis of KS and loss of prespermatogonia in KS testis.

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Single-cell transcriptome profiling and analysis of KS and loss of presp...
(A) Schematic diagram of experimental workflow. Fetal samples (KS n = 2, control n = 2) and adult samples (KS n = 2, control n = 1). (B) Uniform manifold approximation and projection (UMAP) plot illustrating the major cell types (n = 38,952 cells) from KS samples (n = 2) and samples from aborted male fetuses (n = 2). Each dot corresponds to an individual cell, and the colors denote different cell types. (C) Expression patterns of marker genes displayed on the UMAP plot. (D) Heatmap of marker genes in each cluster and GO term enrichment. (E) Fraction of each cell types isolated from KS and normal samples. (F) UMAP plot of all germ cells from the embryonic sample (n = 1,866 cells, 4 samples). Each dot corresponds to an individual cell, and the colors denote different cell types. (G) Immunofluorescence costaining of DDX4 (green) and MAGEA4 (red). Scale bars, 50 μm. (H) The number of DDX4+MAGEA4– cells and MAGEA4+ cells in the fetal gonads of male control fetuses and KS fetuses. Data are mean ± SEM. Statistical analysis was performed using unpaired 2-sided t tests. n = 15 regions from 3 independent samples or 20 regions from 3 independent samples in each group. (I) Dot plot showing the expression of selected gene markers for different stages of germ cells. (J) The expression level of 4 selected genes located on the X chromosome with significant difference between control and KS samples. Statistical analysis was performed using 2-sided Wilcoxon’s rank-sum test; ****P < 0.0001.