Epididymis development is the basis of male reproduction and is a crucial site where sperm maturation occurs. In order to further understand the epididymal development of yak and how to regulate sperm maturation, we conducted a multi-omics analysis. We detected 2274 differential genes, 222 differential proteins and 117 co-expression genes in the cauda epididymis of yak before and after sexual maturity by RNA-seq and proteomics techniques, which included TGFBI, COL1A1, COL1A2, COL3A1, COL12A1, SULT2B1, KRT19, and NPC2. These high abundance genes are mainly related to cell growth, differentiation, adhesion and sperm maturation, and are mainly enriched via extracellular matrix receptor interaction, protein differentiation and absorption, and lysosome and estrogen signaling pathways. The abnormal expression of these genes may lead to the retardation of epididymal cauda development and abnormal sperm function in yak. In conclusion, through single and combined analysis, we provided a theoretical basis for the development of the yak epididymal cauda, sperm maturation, and screening of key genes involved in the regulation of male yak reproduction.

Spermatogenesis is a complex process that involves proliferation and differentiation of diploid male germ cells into haploid flagellated sperm and requires intricate interactions between testicular somatic cells and germ cells. The cellular heterogeneity of this process presents a challenge in analyzing the different cell types at various developmental stages. Single-cell RNA sequencing (scRNA-seq) provides a useful tool for exploring cellular heterogeneity. In this study, we performed a comprehensive and unbiased single-cell transcriptomic study of spermatogenesis in sexually mature 4-year-old yak using 10× Genomics scRNA-seq. Our scRNA-seq analysis identified six somatic cell types and various germ cells, including spermatogonial stem cells, spermatogonia, early-spermatocytes, late-spermatocytes, and spermatids in yak testis. Pseudo-timing analysis showed that Leydig and myoid cells originated from common progenitor cells in yaks. Moreover, functional enrichment analysis demonstrated that the top expressed genes in yak testicular somatic cells were significantly enriched in the cAMP signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and ECM receptor interactions. Throughout the spermatogenesis process, genes related to spermatogenesis, cell differentiation, DNA binding, and ATP binding were expressed. Using immunohistochemical techniques, we identified candidate marker genes for spermatogonial stem cells and Sertoli cells. Our research provides new insights into yak spermatogenesis and the development of various types of cells in the testis, and presents more reliable marker proteins for in vitro culture and identification of yak spermatogonial stem cells in the later stage.