Integrated 4D-DIA proteomic and transcriptomic profiling delineates the molecular trajectory of yak testicular development

Abstract Background Normal testicular development is essential for sustaining male fertility in livestock, yet the underlying molecular regulatory mechanisms remain poorly understood, particularly in environmentally adapted breeds such as the yak. In this study, we employed an integrated 4D-DIA proteomic and transcriptomic strategy to systematically characterize testicular tissues of yaks at 6 months old (Y6M), 18 months old (Y18M), and 4 years old (Y48M), with the goal of identifying key proteins potentially involved in testicular development and spermatogenesis. Results A total of 2,901 differentially abundant proteins (DAPs) were identified during three developmental stages. Functional enrichment analysis revealed distinct functional characteristics between the two comparison groups. From Y6M to Y18M, DAPs were significantly enriched in ECM-receptor interaction, the PI3K-Akt signaling pathway, and cell adhesion molecules, whereas DAPs in Y18M vs. Y48M were predominantly enriched in protein digestion and absorption, the PPAR signaling pathway, and additional metabolic pathways. Integrative analysis identified 438 genes exhibiting concordant changes at both the transcript and protein levels in the Y6M vs. Y18M group. Among these, KIFC1, STK31, TOP2A, BECN1, CUL4A, and PBK were highly expressed during puberty and remained elevated through sexual maturity, suggesting potential roles in meiosis and cell cycle regulation. Protein–protein interaction (PPI) network analysis further identified several hub nodes, including ITGB1, TOP2A, PBK, KIF11, MCM3, and BECN1, which are postulated to play central roles in the regulatory network governing testicular development. Conclusions This study establishes a comprehensive proteomic atlas of yak testicular development and provides a preliminary screen of candidate regulatory proteins, offering new perspectives for understanding the molecular basis of yak testicular development and broader insights into mammalian spermatogenesis.