Characteristic analysis of N6-methyladenine in different parts of yak epididymis

Abstract

Background: The epididymis is essential for sperm maturation. During sperm maturation, markable alterations of the payload of small noncoding RNAs are observed in the epididymis, which indicated the role of epigenetic alterations in sperm maturation. However, the N⁶-Methyladenosine (m⁶A) modification profile of the epididymis remains unelucidated. Therefore, in this study, we assessed the m⁶A modification levels in the caput, corpus, and cauda of the yak epididymis using a combination of methylated RNA immunoprecipitation and RNA sequencing.

Results: The m⁶A levels were significantly increased in the corpus of the epididymis. Functional enrichment analysis of differentially methylated RNA (DMR) between the corpus and caput group revealed the significant enrichment of DMRs in the gap junction, ErbB signaling pathway, and mTOR signaling pathway, which participate in cell communication and sperm maturation. In addition, the DMRs of cauda-vs-corpus group were enriched in apoptosis, the FoxO signaling pathway, the PI3K-Akt signaling pathway, and the tumor necrosis factor signaling pathway that were associated with sperm autophagy, oxidative stress, and sperm maturation. Furthermore, we identified the key genes exhibiting significant changes in m⁶A levels but with no differences in RNA levels, including YY1-associated factor 2, forkhead box J2, and forkhead box O1. This finding indicated that m⁶A modifications affect these genes during translation, thereby participating in sperm maturation.

Conclusions: In summary, we generated the m⁶A profile of the yak epididymis, which will aid in further elucidating the maturation process of sperm and reveal more information related to male infertility.

Keywords: N 6-Methyladenosine; Epididymis; Reproductive physiology; Sperm maturation; Yak.

Fig. 1

Histological observation of the epididymal regions and measurement of m⁶A levels. A Histological observation of the epididymal caput, corpus, and cauda (15×). B m⁶A levels in the different epididymal regions. C Expression of m⁶A reader-related genes in among different regions of epididymal. D Expression of m⁶A writer and eraser-related genes in among different regions of epididymal

Fig. 2

Distribution characteristics and visualization analysis of m⁶A in among different regions of epididymal. A Number of m⁶A peaks with different methylation degrees in the epididymal caput, corpus, and cauda. B Statistics of the enrichment positions of the m⁶A peak in among different regions of epididymal. C Peaks annotation and classification in the gene functional element regions. D m⁶A peak enrichment of the top motif. E Site distribution map of the m⁶A peaks compared with the yak reference genome. F Visual analysis of the m⁶A peaks in SRD5 A1 and SP1

Fig. 3

Differentially methylated peak (DMP) and enrichment analyses of the yak epididymal caput, corpus, and cauda. A Number of DMPs in the corpus-vs-caput and cauda-vs-corpus comparison groups. B Volcano maps of the distribution of DMPs in the two comparison groups. C KEGG enrichment analysis of DMPs. D GO enrichment analysis. GO enrichment analysis showing the top 10 enrichment entries in each ontology

Fig. 4

Differentially expressed genes (DEGs) in the yak epididymal caput, corpus, and cauda. A Distribution map of the FPKM values of the epididymal caput, corpus, and cauda genes. B Number of DEGs in the corpus-vs-caput and cauda-vs-corpus comparison groups. C Volcano map showing the distribution of DEGs. D GO enrichment analysis. E KEGG enrichment analysis. F GSEA of the screened pathways

Fig. 5

Combined analysis and differential expression verification of m⁶A-seq and RNA-seq data. A Nine-quadrant distribution plot showing the combined analysis of m⁶A and RNA data. The bright-colored dots indicate genes with significant differences in both m⁶A and RNA, whereas gray dots indicate the genes with no significant differences. B Validation of the differentially expressed genes identified via RNA-seq