Transcriptome-wide m6A profiling reveals mRNA post-transcriptional modification of boar sperm during cryopreservation

In present study, we first tried to reveal the transcriptome-wide m6A methylation pattern in boar fresh sperm (Fs) and frozen-thawed sperm (Fts) through MeRIP-seq, which demonstrates occurrence the diverse m6A modification patterns during cryopreservation. Results: the expression of m6A modification enzymes were significantly dysregulated in sperm during cryopreservation. Furthermore, the m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. In Fts, a total of 1754 differentially methylated m6A genes containing 1928 differentially methylated peaks were identified as compared to Fs. Bioinformatics analysis revealed the role of these genes containing significantly altered m6A peaks (DMMGs) were significantly enriched in terms of metabolism and transcription, as well as a number of DMMGs were annotated to ubiquitin mediated proteolysis, AMPK, mTOR and MAPK signaling pathways. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. Conclusion: Our study is first to reveal the dynamic m6A modification in mRNAs of boar sperm during cryopreservation, and provides a new evidence regarding the potential roles of m6A in modulating sperm motility, apoptosis and metabolism. Keywords: N6-methyladenosine (m6A); Boar sperm; Cryopreservation; MeRIP-seq Three Duroc boars were used in this study. A total of 5 sperm-rich fractions were collected from each boar using the gloved-hand technique during the autumn-winter period (n=15). Then, the fresh ejaculates were equally divided into two parts and one was immediately exposed to liquid nitrogen and then stored for RNA extraction (Fresh sperm, Fs). While, the other part (Frozen-thawed sperm, Fts) was cryopreserved according to our previously described procedure (PMID: 24974820). Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to evaluate the differences in m6A methylation and gene expression between boar fresh sperm (Fs) and frozen-thawed sperm (Fts).