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

## TL;DR

This study uses proteomics to uncover how Hu sheep achieve high fertility through enhanced signaling in their reproductive system.

## Contribution

The study is the first multi-tissue proteomic investigation linking the cAMP signaling pathway to high reproductive performance in Hu sheep.

## Key findings

- Hu sheep show accelerated testicular development and earlier spermatogenesis initiation.
- The cAMP signaling pathway is consistently active across hypothalamus, pituitary, and testes in Hu sheep.
- Differentially expressed proteins in testes are enriched in spermatogenesis and blood–testis barrier pathways.

## Abstract

Reproductive efficiency is a cornerstone of sustainable livestock production. This study investigated the molecular basis for the superior fertility of Hu sheep, a prolific indigenous Chinese breed, by performing a comprehensive proteomic comparison of the key reproductive tissues—hypothalamus, pituitary, and testes—between Hu sheep and three conventional meat breeds. Our analysis revealed that Hu sheep exhibit accelerated testicular development and earlier initiation of spermatogenesis. A central finding was the consistent and heightened activity of the cAMP signaling pathway across all three tissues in Hu sheep, indicating enhanced communication within their reproductive axis. Furthermore, we identified several tissue-specific proteins with altered abundance, implicating improved neuroendocrine regulation, antioxidant defense, and blood–testis barrier integrity. These results suggest a potential mechanistic framework for the high fecundity of Hu sheep and identify candidate molecular targets for future functional validation and offer potential molecular targets for enhancing reproductive performance in sheep through breeding or management strategies.

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.