Multi-omics analyses identify PGAM2-associated energy-metabolism signatures linked to bull testis size divergence and spermatogenic potential

Testicular development and spermatogenic capacity are important determinants of male reproductive potential and breeding value in livestock. However, the molecular features associated with natural variation in testicular size remain incompletely understood. In this study, large-testis (BT) and small-testis (ST) bulls from Blcattle Black cattle were used as a comparative model. Histomorphological evaluation, reproductive hormone assays, transcriptomic, proteomic, and metabolomic analyses, and functional assays in primary Sertoli-cell-enriched testicular somatic cell cultures were integrated to identify molecular signatures associated with testicular size divergence and spermatogenic potential. Compared with BT testes, ST testes showed disorganized seminiferous tubule architecture, vacuolar changes in the spermatogenic epithelium, reduced interstitial-cell distribution, and lower serum FSH, LH, and testosterone levels. In contrast, BT testes exhibited larger testicular morphometric indices, higher gonadosomatic index, more intact seminiferous tubule structure, and increased reproductive hormone levels. Multi-omics analyses revealed distinct molecular signatures between the two groups. ST testes were enriched for immune-inflammatory responses, interferon-related signaling, oxidative/stress-response pathways, extracellular matrix remodeling, and cytoskeletal regulation, whereas BT testes were enriched for spermatogenesis, meiosis, flagellar assembly, steroidogenesis-related metabolic features, and glycolytic energy-metabolism programs. Integrated transcriptome–proteome analysis identified several candidate hub genes, including PGAM2, GAPDH, TKTL2, LDHC, AK1, and SIL1. Among these candidates, PGAM2 was prioritized because it showed concordant mRNA–protein upregulation in BT testes, was involved in repeatedly enriched glycolysis/gluconeogenesis pathways, was identified as a hub molecule in the protein–protein interaction network, and was located in a WGCNA module associated with testicular phenotypic traits. Functional assays in primary Sertoli-cell-enriched testicular somatic cell cultures showed that PGAM2 overexpression or knockdown affected glycolytic activity, proliferation- and apoptosis-related markers, nutrient-support-related gene expression, F-actin organization, and blood–testis barrier-related gene expression. This study identifies energy-metabolism reprogramming, particularly glycolytic metabolism, as a major molecular feature associated with testicular size divergence in Blcattle Black bulls. PGAM2 was prioritized as a candidate glycolytic hub linked to the larger-testis phenotype and spermatogenesis-related molecular signatures. These findings provide a multi-omics resource and candidate molecular targets for future studies of metabolic regulation in bovine testicular development and male reproductive potential.