DIA Proteomics Reveals the Mechanism of cAMP Signaling Pathway-Mediated HPT Axis in Regulating Spermatogenesis of Hu Sheep.

OBJECTIVE

Although Hu sheep are renowned for their high fecundity, the multi-tissue regulatory networks governing spermatogenesis, particularly within the hypothalamic-pituitary-testicular (HPT) axis, remain poorly understood. This study aimed to elucidate these mechanisms by performing a comparative proteomic analysis of the HPT axis in Hu sheep and three other breeds.

METHODS

We utilized data-independent acquisition (DIA) proteomics to analyze hypothalamic, pituitary, and testis tissues from 36 samples across four breeds. The experimental workflow included protein extraction, enzymatic digestion, LC-MS/MS, and subsequent bioinformatic analyses, complemented by histological examination.

RESULTS

Hu sheep exhibited accelerated testicular development and an earlier onset of spermatogenesis. Comprehensive proteomic profiling identified a total of 10,528 proteins, with 771 differentially expressed proteins (DEPs) detected in the testis. These testicular DEPs were significantly enriched in pathways related to spermatogenesis, the blood-testis barrier, and steroid hormone biosynthesis. Notably, the cAMP signaling pathway was consistently enriched across all three tissues, underscoring its pivotal role in regulating spermatogenesis. Protein-protein interaction (PPI) network analysis further highlighted hub proteins, such as MET, suggesting their potential involvement in somatic cell functions and the spermatogenic microenvironment. Key findings were validated by Western blot analysis.

CONCLUSION

This study is the first multi-tissue proteomic investigation proposing a model in which the high reproductive performance of Hu sheep is potentially linked to the efficient, coordinated regulation of spermatogenesis-related proteins and signaling pathways-particularly in the testis. These findings offer novel insights into the molecular mechanisms of male reproduction in sheep and identify potential targets for future research and breeding applications.