Characterization of N⁶-methyladenosine in cattle-yak testis tissue

N⁶-methyladenosine (m⁶A) is the most common form of eukaryotic mRNA modification, and it has been shown to exhibit broad regulatory activity in yeast, plants, and mammals. The specific role of m⁶A methylation as a regulator of spermatogenesis, however, has yet to be established. In this experiment, through a series of preliminary studies and methylated RNA immunoprecipitation sequencing, the m⁶A map of cattle-yak testicular tissue was established as a means of exploring how m⁶A modification affects cattle-yak male infertility. Cattle-yak testis tissues used in this study were found to contain sertoli cells and spermatogonia. Relative to sexually mature yak samples, those isolated from cattle-yak testis exhibited slightly reduced levels of overall methylation, although these levels were significantly higher than those in samples from pre-sexually mature yaks. Annotation analyses revealed that differentially methylated peaks were most concentrated in exonic regions, with progressively lower levels of concentration in the 3'-untranslated region (UTR) and 5'-UTR regions. To further explore the role of such m⁶A modification, enrichment analyses were performed on differentially methylated and differentially expressed genes in these samples. For the cattle-yaks vs. 18-months-old yaks group comparisons, differentially methylated genes were found to be associated with spermatogenesis-related GO terms related to the cytoskeleton and actin-binding, as well as with KEGG terms related to the regulation of the actin cytoskeleton and the MAPK signaling pathway. Similarly, enrichment analyses performed for the cattle-yaks vs. 5-years-old yaks comparison revealed differentially methylated genes to be associated with GO terms related to protein ubiquitination, ubiquitin ligase complexes, ubiquitin-dependent protein catabolism, and endocytotic activity, as well as with KEGG terms related to apoptosis and the Fanconi anemia pathway. Overall, enrichment analyses for the cattle-yaks vs. 18-months-old yaks comparison were primarily associated with spermatogenesis, whereas those for the cattle-yaks vs. 5-years-old yaks comparison were primarily associated with apoptosis.