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 3

Intercellular interactions between Sertoli and other cell types in KS.

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Intercellular interactions between Sertoli and other cell types in KS. (...
(A) The circle diagrams depicting the relative number (left) or strength (right) of interactions in the cell-cell communication network between KS and control group. Red or blue edges represent an increase or decrease trend in signaling in KS group compared with control group, respectively. (B) Dot plot showing the relative strength of signaling pathways sent or received by each cell type (Endothelial, Sertoli, Peritubular myoid, Immune, Germ, Leydig). (C) Signaling pathways with the greatest variation between Sertoli cells and Leydig cells. PTN, pleiotrophin. (D) Heatmap shows the roles of different cell types in the MK signaling pathway. (E) Dot plot of ligand-receptor interactions in the MK pathway from different cell types to Sertoli in KS and control fetal testes. Commun. Prob., communication probability. (F) Expression of ligand MDK in testicular cell populations. (G) The expression of MDK in Leydig cells of KS and control. Statistical analysis was performed using 2-sided Wilcoxon’s rank-sum test; ****P < 0.0001. (H) The expression of MDK in control and KS primary Leydig cells using qRT-PCR. Data are presented as the mean ± SEM from 3 independent experiments. **P < 0.01. GAPDH was used as a loading control (n = 3). (I) The expression of LRP1 and NCL in Sertoli cells of KS and control. Statistical analysis was performed using 2-sided Wilcoxon’s rank-sum test; ****P < 0.0001. The triangle (▲) represents the arithmetic mean in each group. (J) The expression of LRP1 in control and KS primary Sertoli cells using qRT-PCR. Data are presented as the mean ± SEM from 3 independent experiments. ****P < 0.0001. GAPDH was used as a loading control (n = 3).