Dynamic Changes Of DNA Methylation And Transcriptome Expression In Porcine Ovaries During Aging

Among the major challenges in women’s reproductive health,the incidence trends of the age-related decline in reproductive performance and various pathologies such as premature ovarian insufficiency(POI)and polycystic ovary syndrome(PCOS)is increasing year by year.However,studies about ovarian nature in women are difficult,due to the ethical issues.Therefore,pig could be a good model for the study of women ovarian aging or disease due to their similarities to humans in anatomic,physiologic,and biochemical.Female natural ovarian aging is delineated by menarche and menopause-the two key time points during woman’s life that open and close the reproductive window,respectively.Thus,we used pig as a model animal and performed genome-wide DNA methylation and transcriptome-wide RNA expression analysis via high-throughput sequencing of ovaries from young(180 days,puberty stage)and old(8 years,reproductive exhaustion stage)pigs as the menarche and menopause.The following results were obtained:1.We constructed the genome-wide DNA methylation profile.Analysis of the sequence context of cytosines showed methylation occurred in Cp G and non-Cp G(CHG and CHH)context,gene methylation occurred mainly in the CDS area of the gene and both the gene body region and the surrounding intergenic region were heavily methylated.There was a sharp decrease in TSS and TTS.Whereas,the methylation level between them had no obvious change.In addition,we found the methylation level across the chromosomes was negatively correlated with the chromosomal length(Pearson’s r =-0.636,P = 1.844×10-9),and a strong positive correlation was observed with GC content(r = 0.903,P < 2.2×10-16),gene number(r = 0.398,P < 2.2×10-16),Cp G island(CGI)ratio(r = 0.792,P < 2.2×10-16)and the density of repeat regions(r = 0.126,P < 2.2×10-16),respectively;2.422 DMRs were identified between the two developmental stages,and 303 of these DMRs were showed up-methylated expression while 119 DMRs were exhibited down-methylated expression during OP compared to YP.These results,suggesting that the dynamical DMRs level in both ovarian developmental stages.Furthermore,we found that 146 of these 422 DMRs were overlapped at gene body regions while only 12 of these DMRs were located in the gene promoter regions.The GO enrichment of DMRs were involved in apoptosis metabolic pathway,embryonic development and immune system regulation.More DMRs were located in gene bodies(146)than in promoters(12),which may conclude that gene bodies are more susceptible to change as compared to promoters during the aging process,methylation within a gene body may indicate the presence of alternative promoters.To explore whether intragenic methylation affects gene expression,using the RNA-Seq data,we performed a correlation analysis of DMR-m RNA pairs and observed a significant negative correlation(r =-0.179,P = 5.72×10-7)between the changes of methylation levels in gene bodies and gene expression levels.3.According to the screening criteria chosen(P ≤ 0.05),we found 2243 differential expression genes(DEGs),95 differentially expressed mi RNAs(DEMs),248 differentially expressed lnc RNAs(DELs)and 116 differentially expressed circ RNAs(DECs)in both stages of ovarian development.According to the GO enrichment,we found these differential expression RNAs were related to reproductive function including defense response to virus,G-protein coupled receptor signaling pathway,apoptotic signaling pathway,female pregnancy,fertilization process,embryonic development and ovulation cycle.4.The combined analysis of DNA methylation and RNAs identified,8,845(34.9%)and 9,663(38.2%)genes of the entire gene number in swine genome were methylated and expressed m RNA and mi RNA.Besides,fewer genes were methylated or expressed,and 600 genes were no methylated or no expressed.Similarly,methylation,and expression of lnc RNA and mi RNA had the largest number of genes.These results indicated that a large number of genes were tended to combination regulation among DNA methylation and transcriptome expression and the combination regulation pattern may be more prior to old females.To further highlight the potential coordinated control roles of DNA methylation and several RNA species involved in ovarian aging,we overlapped GO terms of genes related to DMRs and four differentially expressed RNA species,we found some intersection of apoptotic process,and signaling pathways involved in ovarian aging cycle such as embryonic development,fertilization,female pregnancy and reproduction.5.Based on the theory of ce RNA,we construct a network with differentially expressed m RNA,mi RNA,lnc RNA and circ RNA which including 4 mi RNAs(mi R-125 b,mi R-504,mi R-92b-5P,mi R-9),4 genes(FADD,EGFL7,PER2,CELSR1),3 lnc RNAs(MSTRG62621,MSTRG114143,MSTRG167556)and 2 circ RNAs(circ000675,circ13607).And the co-expression analysis revealed RNA network regulated by FADD,MSTRG.114143 and mi R-92b-5p as well as two ways of PER2 and mi R-92b-5p regulation are CELSR1,PER2 and circ13607 compete mi R-125 b and mi R-92b-5p,respectively.While,mi R-92b-5p and PER2 did not share a same MRE,suggesting that the co-expression relationship between both may be mediated by other mediators.This result also revealed there may be other regulatory factors and more complex relationships within the RNAs regulation.In this study,we investigated the joint analysis of DNA methylation and RNAs on genomic level and transcriptome level of porcine ovary at puberty stage and reproductive exhaustion stage with WGBS,RNA-seq and correlation analysis.And construct a regulation network based on the co-expression and theory of ce RNA,but also reveals the interaction of DNA and transcription,and mechanism of RNAs mutual regulation and coordination regulation.These results will provide the basic data for understanding and studying the aging process in ovary and its molecular mechanisms.