Single-cell RNA sequencing reveals a cellular atlas of the sheep testis.

Spermatogenesis is a highly regulated biological process involving various cell types, making the accurate identification and classification of different cell populations challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving cellular heterogeneity at high resolution. In this study, 10× Genomics Chromium™ scRNA-seq was employed to analyze 15,826 testicular cells from three 6-month-old sexually mature Hu sheep, and a high-resolution single-cell transcriptomic atlas was generated. Specifically, six somatic cell subtypes and five germ cell subtypes were identified, which revealed the differentiation trajectory of spermatogenesis. Notably, pseudo-time analysis suggested that Leydig cells and peritubular myoid cells may originate from a common progenitor lineage in sheep. Highly expressed genes were enriched in pathways such as cAMP signaling, PI3K-Akt, and ECM-receptor interaction. Furthermore, immunohistochemical analysis validated specific marker proteins for certain cell types. Collectively, these findings provide novel insights into spermatogenesis and serve as a valuable reference for the in vitro culture of male germ cells.