Characterization of N6-methyladenosine in the testis tissue of cattle-yak

N6-methyladenosine (m6A) is the most prominent mRNA modification in eukaryotes, and its potential regulatory role has recently been identified in mammals, plants, and yeast. However, how m6A methylation regulates spermatogenesis remains unknown. In this study, cattle-yak testis tissue was used as the experimental material, and the m6A map was generated through preliminary experiments and methylated RNA immunoprecipitation sequencing. Only spermatogonia and Sertoli cells were observed in cattle-yak testis tissue. Experiments examining the expression of methylation-related enzymes and the overall methylation level showed that the methylation level in the testis of the cattle-yak was slightly lower than that of the sexually mature yak, but significantly higher than that of the pre-sexually mature yak. Annotation analysis indicated that differentially methylated peaks were most frequently concentrated in exonic regions, followed by 3'UTR and finally 5'UTR regions. Through enrichment analysis of differentially expressed genes and differentially methylated corresponding genes, GO analysis of T-vs-Y group mainly involved spermatogenesis, including cytoskeleton, actin binding, etc. KEGG analysis showed that the differential genes were mainly enriched in actin cytoskeleton regulation and MAPK signaling pathway. GO analysis of the T-vs-M group mainly involved protein ubiquitination, ubiquitin ligase complexes, ubiquitin-dependent protein catabolism and endocytosis. KEGG analysis mainly involved apoptosis and Fanconi anemia pathways. This study will lay the foundation for elucidating the molecular mechanism of m6A in male sterility of cattle-yak. Detection of m6A in the testis tissue of cattle-yak

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是真核生物中最普遍的信使RNA（messenger RNA，mRNA）修饰形式，其潜在的调控功能近年来已在哺乳动物、植物与酵母中被证实。然而，m6A甲基化如何调控精子发生的分子机制仍未阐明。本研究以牛犏牛睾丸组织为实验材料，通过前期实验及甲基化RNA免疫共沉淀测序构建了其m6A修饰图谱。牛犏牛睾丸组织中仅观察到精原细胞与支持细胞两类细胞。针对甲基化相关酶的表达水平及整体甲基化水平的检测结果显示，牛犏牛睾丸组织的甲基化水平略低于性成熟牦牛，但显著高于性未成熟牦牛。注释分析表明，差异甲基化峰最富集于外显子区域，其次为3'非翻译区（3'UTR），最后为5'非翻译区（5'UTR）。通过对差异表达基因及差异甲基化关联基因开展富集分析：T-vs-Y组的基因本体（Gene Ontology，GO）分析主要涉及精子发生相关生物学过程，包括细胞骨架、肌动蛋白结合等；京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）分析显示，差异基因主要富集于肌动蛋白细胞骨架调控通路与丝裂原活化蛋白激酶（MAPK）信号通路。T-vs-M组的GO分析主要涉及蛋白质泛素化、泛素连接酶复合物、泛素依赖的蛋白质分解代谢及胞吞作用；KEGG分析则主要涉及细胞凋亡与范可尼贫血通路。本研究为阐明m6A在牛犏牛雄性不育中的分子调控机制奠定了坚实的实验基础。牛犏牛睾丸组织的m6A检测。