Multi-omics profiling of the testes in Kazakh horses across sexual maturation

Equus caballus is a species of considerable economic and cultural significance. However, the regulatory networks involved in equine sexual maturation remain unclear and eventually limit its reproduction and utilization. In this study, testicular tissues from eight Kazakh horses at two developmental stages (2 years, representing pre-maturation, and 3 years, representing post-maturation) were analyzed using whole transcriptome sequencing, data-independent acquisition (DIA) proteomics, and untargeted metabolomics. An integrated regulatory network was constructed encompassing ceRNA, mRNA, protein, and metabolite interactions. Transcriptome analysis revealed that significantly changes circRNAs, LncRNAs, including eca-miR-34-5p, eca-miR-34c, eca-miR-449a, eca-miR-486-3p, and target genes such as TRAF1, TRAF3, ALPK3, SLC29A4 and CPNE3. A total of 277 differentially expressed genes were identified at both the mRNA and protein levels, which are primarily involved in biological processes including PI3K-AKT signaling, focal adhesion, extracellular matrix (ECM) receptor interactions, phospholipase D signaling, and ovarian steroidogenesis. LncRNAs and circRNA were found to modulate COL1A1 expression by competitively binding to miR-7844-x and regulating SPARC through interactions with eca-miR-146-5p and eca-miR-328. Integrated proteomic and metabolomic analyses highlighted TAP1, COL14A1, PRDX3, and SEBENP1 central proteins, along with key metabolites such as guanosine, Austocystin B, methanone, and glycerophosphocholine. These molecules were mainly enriched in pathways related to ovarian steroidogenesis, steroid hormone biosynthesis, and propionate metabolism. In conclusion, this study presents a comprehensive regulatory network involving LncRNA, circRNA, miRNA, mRNA, proteins, and metabolites during equine testicular development. The findings provide novel insights into the molecular basis of equine sexual maturation and offer a valuable foundation for improving reproductive regulation and breeding efficiency in horses.