Fu Huang ¹ Jiexia Wang ² Hu Wang ³ Jingfeng Xu ³ Mengjie Qin ⁴ Xin Wen ² Shuyan Cao ⁵ Xiaoju Guan ¹ Ping Duan ² Haolin Chen ^1* Congde Chen ^1*

• ¹ Department of Pediatrics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China
• ² Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China
• ³ Department of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China
• ⁴ Department of Pharmacology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China
• ⁵ Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China

Abstract Background: The mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques. Methods: Interstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks. Results: Seven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week. Conclusion: Seven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis.