Tibetan sheep serve as an ideal animal model for investigating adaptation to high-altitude hypoxia and have developed highly distinctive reproductive regulatory mechanisms during long-term evolution. In the male reproductive system, Leydig cells (LCs) are central to testosterone synthesis and the maintenance of microenvironmental homeostasis. Insulin-like growth factor 1 (IGF1) plays a critical role in maintaining cellular development and testicular function. However, the molecular mechanisms by which IGF1 mediates LC proliferation and maintenance of the cellular microenvironment in Tibetan sheep remain poorly understood. This study aimed to systematically elucidate the molecular regulatory networks through which IGF1 controls Leydig cell proliferation, metabolic homeostasis, and extracellular matrix (ECM) remodeling in Tibetan sheep testes by integrating multi-omics analyses with functional validation.

In this study, Tibetan sheep testicular LCs were used as an in vitro model. Cellular interventions were performed by constructing an IGF1 overexpression vector and designing specific knockdown sequences. At the functional phenotypic level, DNA synthesis rates were quantitatively evaluated using EdU assays, and the expression levels of classical cell-cycle markers were detected. At the molecular level, high-throughput transcriptome sequencing (RNA sequencing) and label-free quantitative proteomics were simultaneously performed on treated cell samples. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were stringently screened, followed by GO/KEGG pathway enrichment analysis and protein-protein interaction network topology analysis to systematically characterize downstream cooperative pathways regulated by IGF1.

Cellular functional assays demonstrated that IGF1 overexpression significantly promoted DNA synthesis in Tibetan sheep testicular LCs and markedly upregulated the expression of cell-cycle markers. Conversely, IGF1 knockdown inhibited these proliferation-related processes. Integrated transcriptomic and proteomic analyses showed that the differentially expressed genes and proteins were significantly enriched in key signaling pathways associated with cell-cycle progression, DNA replication, extracellular matrix remodeling, energy metabolic homeostasis, and anti-apoptotic processes. Interaction network analysis revealed a collagen-enriched core ECM functional module and identified several key regulatory factors closely associated with mitochondrial function and cellular stress responses. These findings indicate that ECM structural components are highly coordinated with the peptide growth factor IGF1 through synergistic signaling interactions.

The results of this study confirm that IGF1 not only directly regulates the proliferation of testicular LCs but also plays a central role in remodeling the microenvironment essential for cell survival. Under plateau stress conditions, the close coordination between IGF1 and ECM macromolecular components may provide robust physical structural support for Leydig cells and coordinate their metabolic homeostasis, thereby effectively counteracting hypoxic stress from the external environment. In conclusion, this study establishes IGF1 as a central regulatory factor governing Leydig cell proliferation and ECM reprogramming in Tibetan sheep testes. These findings provide a new theoretical perspective and potential molecular targets for further elucidating the mechanisms underlying the maintenance of superior male reproductive adaptation and fertility in extreme high-altitude environments.

Supplementary material is available in the online version of this article at http://doi.org/10.63823/ARP0260007 and is accessible for authorized users.