Comparative analysis of mRNA and circRNA expression profiles and interaction networks in testicular tissues of Mongolian horses before and after sexual maturity.

Mongolian horses, celebrated for their resilience and adaptability, undergo marked physiological transformations during sexual maturation. This study employed whole-transcriptome sequencing to conduct a comparative analysis of mRNA and circular RNA (circRNA) expression profiles, along with their interactive regulatory networks, in testicular tissues of Mongolian horses pre- and post-sexual maturity. Histological examinations using hematoxylin-eosin (H.E.) and 4’,6-diamidino-2-phenylindole (DAPI) staining revealed distinct morphological differences between sexually immature and mature testicular tissues. Employing DESeq2 analysis on the whole-transcriptome data, we identified 10,317 differentially expressed genes (5,280 upregulated, 5,033 downregulated) and 356 differentially expressed circRNAs (263 upregulated, 93 downregulated) across the two developmental stages. Real-time PCR validation is supportive of the reliability of the RNA-seq findings. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses elucidated that the differentially expressed genes predominantly contribute to reproductive development, gamete generation, and diverse signaling pathways. Additionally, annotation analysis of host genes corresponding to the differentially expressed circRNAs demonstrated congruent expression trends between selected host genes and their associated circRNAs. Notably, this study explored the regulatory network interconnecting circRNAs, microRNAs (miRNAs), and mRNAs, underscoring a critical role for circRNAs in miRNA-mediated gene expression modulation. These findings provide novel mechanistic insights into the molecular underpinnings of testicular development in stallions, using the Mongolian horse as a model. Specifically, we identified the circRNA eca_circ_0005223 and its host gene TGFBRAP1 as key regulators, with their high expression in immature testes suggesting a pivotal role in the TGF-β signaling pathway during early development. Furthermore, we validated a functional ceRNA regulatory axis comprising eca_circ_0008614, eca-miR-432, and the target gene AASDH, revealing a novel post-transcriptional mechanism governing spermatogenesis. These findings provide novel mechanistic insights into the molecular underpinnings of testicular development in Mongolian horses and establish a foundational framework for future investigations into the specific functions of these genes and circRNAs within the context of whole-transcriptome dynamics.