基于单细胞RNA测序解析人与小鼠睾丸中SARS-CoV-2相关受体的时空表达特征

doi:10.3969/j.issn.1674-8115.2021.04.002

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (4): 421-426.doi: 10.3969/j.issn.1674-8115.2021.04.002

基于单细胞RNA测序解析人与小鼠睾丸中SARS-CoV-2相关受体的时空表达特征

罗嘉强¹^,²(), 赵亮宇¹^,², 姚晨成¹^,², 朱子珏¹^,², 邢晓宇³, 李朋¹^,², 田汝辉¹^,², 陈慧兴¹^,², 孙杰³, 李铮¹^,²()

^1.上海交通大学附属第一人民医院泌尿外科中心男科，辅助生殖医学科，上海 200080
^2.上海交通大学泌尿外科研究所男性健康评估中心，上海市生殖医学重点实验室，上海 200080
^3.上海交通大学医学院附属上海儿童医学中心泌尿外科，上海 200127

收稿日期:2020-07-07 出版日期:2021-04-28 发布日期:2021-05-14
通讯作者: 李铮 E-mail:luojiaq@sjtu.edu.cn;lizhengboshi@sjtu.edu.cn
作者简介:罗嘉强（1994—），男，博士生；电子信箱：luojiaq@sjtu.edu.cn。
基金资助:
国家重点研发计划(2017YFC1002003);上海市科学技术委员会项目(17JC1420103)

Single-cell RNA sequencing reveals the spatio-temporal expression profile of SARS-CoV-2 related receptor in human and mouse testes

Jia-qiang LUO¹^,²(), Liang-yu ZHAO¹^,², Chen-cheng YAO¹^,², Zi-jue ZHU¹^,², Xiao-yu XING³, Peng LI¹^,², Ru-hui TIAN¹^,², Hui-xing CHEN¹^,², Jie SUN³, Zheng LI¹^,²()

^1.Urologic Medical Center, Department of Andrology, Department of Assisted Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
^2.Center for Men's Health, Shanghai Key Lab of Reproductive Medicine, Institute of Urology, Shanghai Jiao Tong University, Shanghai 200080, China
^3.Department of Urology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2020-07-07 Online:2021-04-28 Published:2021-05-14
Contact: Zheng LI E-mail:luojiaq@sjtu.edu.cn;lizhengboshi@sjtu.edu.cn
Supported by:
National Key Research and Development Program of China(2017YFC1002003);Program of Science and Technology Commission of Shanghai Municipality(17JC1420103)

摘要/Abstract

摘要：

目的·基于单细胞RNA测序（single-cell RNA sequencing，scRNA-Seq），分析严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）受体血管紧张素转化酶2（angiotensin-converting enzyme 2，ACE2）在人和小鼠睾丸中的时空表达特征。方法·收集生理性各发育阶段的人睾丸组织10例和C57BL/6小鼠睾丸组织9例，用酶消化成单细胞悬液后经scRNA-Seq标准处理程序得到细胞-基因表达矩阵。经过质量控制、数据标准化、批次效应处理和聚类降维后，利用已知的睾丸细胞标志物注释各群细胞，以明确在发育的不同阶段中人和小鼠睾丸中ACE2表达模式和差异。结果·鉴定出人和小鼠均有的睾丸细胞亚群9个，包括3群生殖细胞（精原细胞、精母细胞和精子细胞/精子）和6群体细胞（Sertoli细胞、巨噬细胞、血管平滑肌细胞、内皮细胞、Leydig细胞和管周肌样细胞）。从空间分布来看，在成人睾丸中，ACE2主要表达于Sertoli细胞，在Leydig细胞、管周肌样细胞和生殖细胞也有表达。从时间尺度上看，人Sertoli细胞的ACE2转录丰度随着睾丸的发育而增加，青春期后Sertoli细胞中ACE2表达显著高于幼儿期和儿童期（P=0.000）。从小鼠睾丸发育各阶段来看，Ace2表达模式与人体均有显著不同。在5周龄成年C57BL/6小鼠睾丸中，Ace2的转录水平低且主要表达于血管平滑肌细胞（P=0.000），而Sertoli细胞中Ace2阳性细胞数则极少。结论·SARS-CoV-2可能主要通过Sertoli细胞感染人类睾丸，常规C57BL/6小鼠模型不适用于模拟SARS-CoV-2感染对人类睾丸的功能影响。

关键词: 严重急性呼吸综合征冠状病毒2, 单细胞RNA测序, 血管紧张素转化酶2, 病毒性睾丸炎, sertoli细胞

Abstract:

Objective·To analyze the spatio-temporal expression profile of angiotensin-converting enzyme 2 (ACE2), the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in human and mouse testes based on single-cell RNA sequencing (scRNA-Seq).

Methods·Ten testicular tissues from humans and nine testicular tissues from C57BL/6 mice with normal developmental stages were collected and digested into single cell suspensions by enzyme, and then the cell-gene expression matrixes were obtained by scRNA-Seq standard processing procedures. After quality control, data standardization, batch effect processing, clustering, and dimensionality reduction, each subgroup of cells was annotated based on known testicular cell bio-markers to clarify the expression patterns and differences of ACE2 in human and mouse testes with normal developmental stages.

Results·In this study, nine testicular cell subgroups found in human and mice were identified, including three subgroups of germ cells (spermatogonia, spermatocytes, and spermatids/sperm) and six subgroups of somatic cells (Sertoli cells, macrophages, vascular smooth muscle cells, endothelial cells, Leydig cells, and peritubular myoid cells). In terms of spatial distribution, ACE2 was predominantly expressed in human Sertoli cells in adult testes, and also expressed in Leydig cells, peritubular myoid cells, and germ cells. In terms of time scale, the transcription abundance of ACE2 in human Sertoli cells increased with the development of testis, and the expression level of ACE2 in Sertoli cells after puberty was significantly higher than that in infancy and childhood (P=0.000). Judging from the stages of mouse testicular development, the expression patterns of Ace2 were both significantly different from those in humans. In testis of the 5-week-old adult C57BL/6 mouse, the transcription level of Ace2 was low and it was mainly expressed in vascular smooth muscle cells (P=0.000), while the number of Ace2 positive cell in Sertoli cells was extremely low.

Conclusion·SARS-CoV-2 may mainly infects human testis through Sertoli cells, and the conventional C57BL/6 mouse model is not suitable to simulate the effect of SARS-CoV-2 infection on human testicular function.

Key words: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), single-cell RNA sequencing (scRNA-Seq), angiotensin-converting enzyme 2 (ACE2), viral orchitis, Sertoli cell

中图分类号:

R691.3

罗嘉强, 赵亮宇, 姚晨成, 朱子珏, 邢晓宇, 李朋, 田汝辉, 陈慧兴, 孙杰, 李铮. 基于单细胞RNA测序解析人与小鼠睾丸中SARS-CoV-2相关受体的时空表达特征[J]. 上海交通大学学报(医学版), 2021, 41(4): 421-426.

Jia-qiang LUO, Liang-yu ZHAO, Chen-cheng YAO, Zi-jue ZHU, Xiao-yu XING, Peng LI, Ru-hui TIAN, Hui-xing CHEN, Jie SUN, Zheng LI. Single-cell RNA sequencing reveals the spatio-temporal expression profile of SARS-CoV-2 related receptor in human and mouse testes[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 421-426.

图/表 3

图1 人和小鼠睾丸细胞的scRNA-Seq分析Note:A. t-SNE plot of testicular cells from 5 adult OA patients. B. t-SNE plot of 5-week-old mouse testicular cells. C. Expression patterns of markers on t-SNE plots of human testicular cells. D. Expression patterns of markers on t-SNE plots of mouse testicular cells. SPGs—spermatogonia; SPCs—spermatocytes; SPTs—spermatids/sperm; SCs—Sertoli cells; MACs—macrophages; VSMs—vascular smooth muscle cells; ENCs—endothelial cells; LCs—Leydig cells; PTMs—peritubular myoid cells. FGFR3—fibroblast growth factor receptor 3; SYCP3—synaptonemal complex protein 3; TNP1—transition protein 1; VWF—von willebrand factor; DLK1—delta like non-canonical notch ligand 1; MYH11—myosin heavy chain 11; MUSTN1—musculoskeletal embryonic nuclear protein 1; SOX9—(sex-determining of Y)-Box transcription factor 9; Gfra1—glial cell derived neurotrophic factor family receptor α 1.

Fig 1 scRNA-Seq analysis of human and mouse testicular cells

图2 ACE2和FURIN在人睾丸中的表达模式Note:A. Feature plot for ACE2 expression across all cell populations of adult testicles. B. Feature plot for FURIN expression across all cell populations of adult testicles. C. Violin plot for ACE2 and FURIN expression across all cell populations of adult testicles. D. Changes of expression levels of ACE2 in human testicular Sertoli cells at different developmental stages from birth to adult. E. Immunohistochemical staining of ACE2 in the testicle of adult male (×400). F. Immunohistochemical staining of FURIN in the testicle of adult male (×400).

Fig 2 Expression patterns of ACE2 and FURIN in human testicles

图3 Ace2在小鼠睾丸中的表达模式Note:A. Feature plot for Ace2 expression across all cell populations of 5-week-old mouse testicle. B.Violin plot for Ace2 expression across all cell populations of 5-week-old mouse testicle. C. Changes of expression levels of Ace2 in mouse testicular Sertoli cells. D. Changes of expression levels of Ace2 in mouse testicular vascular smooth muscle cells from 3 days and 5 weeks after birth.

Fig 3 Expression patterns of Ace2 in mouse testicles

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基于单细胞RNA测序解析人与小鼠睾丸中SARS-CoV-2相关受体的时空表达特征

Single-cell RNA sequencing reveals the spatio-temporal expression profile of SARS-CoV-2 related receptor in human and mouse testes

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