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.

Sertoli cells are essential for testicular development and spermatogenesis, but the post-transcriptional mechanisms regulating their function in sheep remain incompletely understood. This study investigated the regulatory relationship between miR-132-y and Yes-associated protein 1 (YAP1), a core effector of the Hippo pathway, in primary Sertoli cells isolated from Southdown × Hu F1 sheep. Target prediction and dual-luciferase reporter assays supported a direct interaction between miR-132-y and the YAP1 3′ untranslated region. YAP1 overexpression was associated with increased CCK-8-based cell viability and altered mRNA expression of selected viability-associated, YAP1-related, and Sertoli cell function-associated genes, whereas YAP1 silencing showed opposite trends. Conversely, miR-132-y overexpression reduced YAP1 mRNA abundance and was associated with decreased CCK-8-based cell viability and corresponding transcriptional changes, while miR-132-y inhibition produced the opposite pattern. Rescue experiments showed that ectopic YAP1 expression partially attenuated miR-132-y-associated changes. Overall, these findings provide in vitro, cell-based evidence that miR-132-y targets YAP1 at the transcript level and is associated with viability-related transcriptional responses in sheep Sertoli cells.