Integration of mRNA and MiRNA analysis reveals the molecular mechanisms of testicular development in newborn and adult sheep

Fecundity limits sheep industry development, but crossbreeding can help. Yet, mechanisms of testis development in Southdown × Hu F₁, especially differential mechanisms at key stages, are rarely studied. Recent miRNA-mRNA analyses have enhanced our understanding of testicular sexual development stages. We conducted histological, physiological, and transcriptomic analyses on neonatal (M0) and adult (Y1) testis tis-sues of Southdown × Hu F₁. Results showed Y1 testes were fully developed with more germ cells than M0. miRNA-seq identified 405 differentially expressed miRNAs (DEMIs) (43 existing, 212 known from 175 families, and 150 novel), and mRNA-seq found 9794 differentially expressed mRNAs (DEMs). miRNA-mRNA integration analysis yielded 20 miRNA-target gene pairs (20 DEMIs and 145 DEMs). Key pathways like Rap1 signaling, ECM-receptor interaction, and MAPK were identified. miR-29a, miR-544-z, and miR-128-z were found to regulate ITGB1, MAP3K13, and Rap1 respectively, along with key genes KITLG, GATA4, and CDC42 in testicular development, affecting male reproduction. This study clarifies testicular development mechanisms and offers candidate genes for molecular breeding.