Genome-wide DNA Methylation Dynamics Of Prepubertal Porcine Testis

The testis is the organ that produces sperm and androgen for male mammals.The proliferation and functional maturation of testicular somatic cells at prepuberty is of great significance to the formation of testis and the spermatogenesis.DNA methylation is the basis for the establishment and maintenance of genomic epigenetic status,and plays an important role in the regulation of testis development.As a good model and economic animal,the pig has important research value in many fields.Therefore,in order to investigate the changes of DNA methylation in prepubertal porcine testis,we generated the first genome-wide DNA methylation profile of prepubertal porcine testis(samples from pigs aged 1,2,and 3 months,denoted as T1,T2 and T3)using MeDIP-seq.In the profile,over 190 million high quality reads(filtered)were generated,and the average aligned rate was 82.94%.There was an overall downtrend of methylation during development,which was clear in promoter regions but less so in gene body regions.In addition,the methylation level at T3 was clearly lower than that at T1 and T2(P-value<0.01),and the downtrend of methylation levels was remarkable in the T1 Vs.T3 and T2 Vs.T3 contrasts(P-value<0.05),but was not in the T1 Vs.T2 contrast(P-value>0.05).These results indicate that DNA methylation has an important role in prepubertal development of porcine testis,with an obvious downtrend of methylation levels from T1 to T3.Thousands of differentially methylated regions(DMRs)were identified among the three prepubertal time points(3,931 in T3 vs.T1,1,599 in T3 vs.T2,1,534 in T2 vs.T1),the majority of which showed decreasing methylation levels over time.GO and KEGG Pathway analyses revealed that many differentially methylated genes were linked with cell proliferation,energy metabolism,steroids and cholesterol synthetic and metabolic process,vitamin A metabolic process,and negative regulation of the response to oxidative stress,indicating that prepubertal development plays an important role in cell proliferation,structural formation and functional maturation of porcine testis.In addition,PPAR signaling pathway and TGF-beta signaling pathway play regulatory roles in the testicular somatic cells development.The methylation levels and their changes of eight porcine imprinted genes:DIRAS3,IGF2,IGF2R,MEST,NAP1L5,NNAT,PEG10 and PLAGL1,were detected in this study.All these eight imprinted genes showed low methylation levels,with "unmethylated" or"partially methylated" status.The methylation levels of NNAT and PLAGL1 promoters increased from T1 to T3;the methylation levels of NAP1L5 and IGF2R promoters decreased from T1 to T2 but increased from T2 to T3;while the methylation levels of DIRAS3,IGF2,MEST and PEG10 promoters increased from T1 to T2 but decreased from T2 to T3.Besides,we only aligned 4 gene bodies from the methylation profile,and the methylation levels of IGF2R and MEST gene bodies decreased from T1 to T3,while the methylation levels of PLAGL1 and PEG10 gene bodies increased from T1 to T2 but decreased from T2 to T3.It can be postulated from the results that these eight imprinted genes may be active during the development of prepubertal porcine testis,and that DNA methylation may have a role in regulation of imprinted genes.This study provides some scientific guides for revealing the regulatory mechanisms of DNA methylation on the prepubertal development of porcine testis,and it is conducive to our in-depth exploration of mammalian testis development and the spermatogenesis,and gives new insights into genetic breeding research and the treatment of male sterility.