Comprehensive analysis of the transcriptome-wide m6A Methylome in sheep testicular development.

N6-methyladenosine (m6A) modification of RNA is a critical post-transcriptional modification, that dynamically contributes to testicular development and spermatogenesis. Nevertheless, the investigation into the role of m6A in testicular development of sheep remains insufficient. Herein, we conducted a comprehensive analysis of the m6A transcriptome landscape in the testes of F1 hybrid Southdown × Hu sheep across M0 (0 months old, newborn), M3 (3 months old, sexually immature), M6 (6 months old, sexually mature), and Y1 (1 years old, adult). By profiling the m6A signatures across the transcriptome, we observed distinct differences in m6A modification patterns during sheep testicular development. Our cross-analysis of m6A and mRNA expression revealed that the expression of 743 genes and their m6A modification were concurrent. Notably, the combined analysis of the two comparative groups, M0 vs. M6 and M0 vs. Y1, exhibited a positive correlation, with 30 candidate genes each located within the largest protein-protein interaction network. Intriguingly, eight key hub genes (VEGFA, HDAC9, ZBTB40, KDM5B, MTRR, EAPS1, TSSK3, and BMP4) were identified to be associated with the regulation of sheep testicular development and spermatogenesis. These findings contribute to a deeper understanding of the dynamic role of m6A modification in sheep testicular biology. This study to map RNA m6A modification in sheep testes at different ages, providing novel insights into m6A topology and the molecular mechanisms associated with spermatogenesis in Southdown × Hu sheep F1 hybrids and laying the foundation for further investigations of mammalian spermatogenesis.