Deciphering RNA modification dynamics during spermatogenesis and sperm maturation

Emerging evidence highlights the pivotal roles of RNA modifications in affecting male fertility and offspring health. However, the dynamic landscapes of RNA modification during spermatogenesis and sperm maturation are still incompletely characterized. Here, we profiled 27 RNA modifications across total RNA, tRNA-enriched fractions, and small RNA fractions in developing mouse testes (P7-P56), isolated spermatogenic cells, and epididymal sperm by applying a liquid chromatography-tandem mass spectrometry-based RNA modification quantification platform, deciphered the developmental dynamics of RNA modification signature and coordinative inter-modification cross-talk. Through integrative analyses of RNA modification signatures among spermatogenic cells in the testis and spermatids at different maturation stages in the epididymis, we found that most RNA modifications were gradually reprogrammed from the testis to the epididymis, and finally declined in mature sperm. Specifically, some RNA modifications (e.g., m⁶A and m⁵U) were enriched in meiotic prophase II spermatocytes and round spermatids, while others (e.g., m⁵C and m²G) exhibited high abundance in caput sperm. Additionally, using a type 2 diabetes mellitus mouse model, we found that sperm RNA modification signatures are dynamically reprogrammed during epididymal maturation in response to diabetes, leading to a remarkable alteration in sperm RNA modification signatures and inter-modification correlation networks. Collectively, our study delineates the dynamic landscape of RNA modifications during spermatogenesis and maturation, uncovering its origins and trajectory alterations in the type 2 diabetes mellitus mouse model. This provides novel insights into both the underpinned regulatory mechanisms of RNA modification-dependent spermatogenesis and epigenetic remodeling of sperm during epididymal maturation.