Single-Cell RNA Sequencing Reveals Dynamic Transcriptional Landscape of Testicular Maturation in Dezhou Donkeys

Testicular development and spermatogenesis are critical for male reproduction, but their molecular mechanisms in Dezhou donkeys remain understudied. This study used single-cell RNA sequencing (scRNA-seq) to analyze testicular tissues from Dezhou donkeys at juvenile (2 months), pre-pubertal (12 months), and mature (24 months) stages. A total of 24,606 high-quality cells were profiled, constructing a comprehensive single-cell transcriptional atlas. Unsupervised clustering identified nine major cell types: three germ cell subtypes (spermatogonia, spermatocytes, spermatids) and six somatic cell subtypes (Leydig cells, Sertoli cells, peritubular muscle cells, macrophages, endothelial cells, T cells). Key marker genes (AMH, TNP1, UTF1, ZMYND10) were validated by immunofluorescence. Pseudotemporal trajectory analysis revealed sequential germ cell differentiation (spermatogonia → spermatocytes → spermatids) and Sertoli cell maturation (immature → mature), while Leydig cells and peritubular muscle cells shared common progenitors. CellChat analysis identified critical ligand–receptor pairs in BMP, IGF, WNT, and FSH pathways, which regulate testicular development. This study provides the comprehensive single-cell transcriptional map of Dezhou donkey testicular development, elucidating key molecular mechanisms of germ and somatic cell maturation. The findings offer valuable insights into donkey reproductive biology, supporting breeding improvement and male infertility research.