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 2

Defect and heterogeneity of fetal Sertoli cells in KS.

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Defect and heterogeneity of fetal Sertoli cells in KS. (A) UMAP plot of ...
(A) UMAP plot of all Sertoli cells (4 samples). Each dot corresponds to an individual cell, and colors represent different clusters and samples. (B) XIST and FOSB expression patterns drawn in UMAP. (C) Immunofluorescence costaining of SOX9 (green) and FOSB (red). Scale bars, 50 μm. White arrow indicates FOSB– Sertoli cell, and red arrow indicates FOSB+ Sertoli cell. (D) Ratios of cells in G0, S, and G2/M phases. (E) The expression levels of SOX9 and AMH were significantly different among control, XIST-positive, and XIST-negative KS samples. Statistical analysis was performed using 2-sided Wilcoxon’s rank-sum test; ****P < 0.0001. (F) GO terms enriched in fNorm Sertoli cells. The x axis is –log10(P value), and the size of the dots represents count. (G) GO terms enriched in fKS XIST+ Sertoli cells. The x axis is –log10(P value), and the size of the dots represents count. (H) GO terms enriched in fKS XIST– Sertoli cells. The x axis is –log10(P value), and the size of the dots represents count. (I) Immunofluorescence staining of ZO-1, β-catenin, and occludin. Scale bars, 50 μm. Insets original magnification, ×600. Fluorescence signal intensities of 3 proteins are shown as mean ± SEM. Statistical analysis was performed using unpaired 2-sided t tests (n = 4).