- Dynamic DNA methylation of ovaries during pubertal transition in gilts. - Previous studies have suggested that the disrupted DNA methylation results in the delayed puberty. - In this study, using gilts as a pubertal model, the genome-wide DNA methylation were profiled to explore their dynamics during pubertal transition across Pre-, In- and Post-puberty.. - Results: During pubertal transition, the follicles underwent maturation and luteinization, coupled with the significant changes in the mRNA expression of DNMT1 and DNMT3a . - DNA methylation levels of In-puberty were higher than that of Pre- and Post-puberty at the locations of genes and CpG islands (CGIs). - Analysis of the DNA methylation changes identified and 17,694 differentially methylated CpGs (DMCs) for the. - Conclusions: During pubertal transition in gilts, the DNA methylation changes of ovaries were likely to affect the transcription of genes related to PI3K-Akt signaling pathway, GnRH signaling pathway, and Insulin secretion. - In female mammals, the initiation of puberty indicates the sexual and ovarian maturation with the acquisition of the capacity to undergo fertilization and propagate the species [8–10]. - Moreover, disrupted DNA methylation results in the delayed puberty [12, 13]. - Never- theless, few investigations have focused on the dynamics and changes of DNA methylation in ovaries during the pubertal transition in mammals.. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - In pigs, puberty is defined as the emergence of the first estrous and follicular maturation [26], and early puberty is selected to shorten the generation interval [27, 28].. - To explore the DNA methylation changes during the pubertal transition, we first detected the mRNA expres- sion levels of DNMT1, DNMT3a and DNMT3b across the Pre-, In- and Post-pubertal ovaries (Fig. - DNA methylation levels of ovarian tissues during pubertal transition. - The DNA methylation levels of CpGs presented a bimodal distribution in Pre-, In- and Post-puberty ovaries, but the distributed features could be clearly distinguished from each other at the hyper-methylated peaks (Fig. - The average methylation levels of the ovarian methylomes were 44.35. - 2b and Table 1), and the average methylation levels of In-puberty were higher than that of Pre- and Post- puberty in the gene-related and CGI-related regions (Fig. - Additionally, the average methy- lation levels of the CpGs located in upstream and CGI regions showed the lowest methylated levels, compared with those in the other gene- or CGI-related regions (Fig. - Genome-wide DNA methylation in ovarian methylomes The Pearson’s correlations between the ovarian methylomes and the density of CpGs, genes and CGIs per 1 Mb window were explored and shown in Fig. - and these ovarian methylomes were positively correlated with the densities of the CGIs (0.26, P <. - in the Pre-, In-and Post- pubertal methylomes, respectively), but negatively corre- lated with the densities of the genes. - in the Pre-, In-and Post-pubertal methylomes, respectively).. - 2 DNA methylation levels of ovarian methylomes during the pubertal transition. - (a) Distribution of the methylation levels in CpGs. - Average methylation levels of CpGs in ovarian methylomes (b), CGI-related regions (c) and gene-related regions (d). - Additionally, these ovarian methylomes were weakly posi- tively correlated with the density of the CpGs (0.16, P <. - DNA methylation levels in gene and CGI locations. - To investigative the DNA methylation changes of genic regions in these ovarian methylomes, the methylation patterns of all genes, HCP genes, and LCP genes were profiled in Fig. - levels of the introns were higher than those of the exons (Fig. - To determine the changes in the ovarian methylomes at CGI regions, the methylation patterns were profiled in the locations of different genomic CGIs (Fig. - The enriched KEGG pathways included the Autophagy-animal, mTOR signaling pathway, Apoptosis, and Insulin resistance (Additional file 1: Figure S1b), which were closely associ- ated with the developments of the ovaries and follicles.. - The densities of the CpGs (track 4), genes (track 5) and CGIs (track 6) in a and b were also quantified per 1 Mb window. - The labels outside of track 1 represent the chromosomes in the pig genome. - The methylated patterns of the Upstream- (Fig. - 5f ) were profiled to further explore the methylation changes in the ovarian methylomes at the CGI locations. - the lowest DNA methylation level (Fig. - Moreover, the DNA methylation level could not be separately distin- guished among the methylomes in the regions of the Upstream-CGIs (Fig. - 5b) but could be separately dis- tinguished in the other genomic CGIs (Fig. - 5 Methylation changes in CGI locations in the ovarian methylomes. - Up2k is denoted as the 2 kb upstream regions of the CGIs. - Down2k is denoted as the 2 kb downstream regions of the CGIs. - Changes in ovarian methylomes during pubertal transition To explore the methylation changes during the pubertal transition in the ovaries, the consistently hypermethylated CpGs (HyperCs) and hypomethylated CpGs (HypoCs) were counted across these ovarian methylomes (Fig. - In total, 16.47 and 7.33% of the detected CpGs lo- cated CGI and upstream regions, respectively, were HyperCs, which were lower than those detected in the other gene- and CGI-related regions (Fig. - However, 64.43 and 75.53% of the detected CpGs located in CGIs and upstream regions, respectively, were HypoCs, which were more than those in other gene- and CGI-related re- gions (Fig. - Interestingly, in the comparisons between the CGIs and upstream regions, the HyperCs were more frequently located at CGIs, but the HypoCs were more likely to locate at upstream regions (Fig. - Additionally, 225 consistently increased methylation (IncrmCs) and 154 consistently decreased methylation (DecrmCs) were identi- fied across Pre-, In- and Post-puberty, representing 0.01 and 0.009% of the detected CpGs, respectively (Fig. - Dynamics in ovarian methylomes during pubertal transition. - and 17,694 DMCs, representing and 2.11% of the detected CpGs with at least eight reads, were identified in Pre- vs. - 6 Changes in ovarian methylomes across Pre-, In- and Post-puberty. - Distribution of the consistently increasing (c) and decreasing (d) methylated CpGs. - The Pearson’s correlation coefficients between the densities of the DMCs and densities of the CpGs were and 0.83 for Pre- vs. - The densities of the DMCs were highly correlated with the densities of the CGIs for Pre- vs.. - Moreover, these DMCs were differentially distributed in the CGI- and gene-related regions, and were observed to be hypomethylated in Post- but hypermethylated in Pre- and In-puberty (Fig. - In total, 598 genes were related to at least one DMR, and these DMR genes were enriched in the PI3K-Akt sig- naling pathway, MAPK signaling pathway, mTOR signaling pathway, GnRH signaling pathway, Insulin secretion, corti- sol synthesis and secretion (Additional file 1: Figure S3).. - We found that the average methylation levels of the ovarian methylomes (Fig. - In the present study, the mRNA expression of DNMT1 and DNMT3a in ovaries significantly changed in the ovaries across the Pre-, In- and Post-stage (Fig. - We found that the Post-stage displayed the lowest DNA methylation levels, and the DNA methylation level of In-stage was observed to be a little higher than Pre- stage in all genes (Fig. - 4c,d), which is in line with others studies [21, 33], and the methylation levels in in- tronic regions were higher than those in the exonic re- gions (Fig. - 4b), especially around the TSSs and TESs across the gene bodies, which might have be explained by the appearance of the CpG density, which was lower in the introns than that in the exons [34]. - Moreover, the average DNA methylation level of the CGIs in the Pre- stage was lower than that in In-stage but higher than that in Post-stage (Fig. - Furthermore, 69.95% of the related genes in these specific CGIs whose methylation level in the Pre- was lower than that in the In- but higher than that in the Post-pubertal stage were HCP genes, and only 30.05% of the related genes in these spe- cific CGIs were LCP genes. - The HypoCs tended to be found in the CGIs, up- stream and exonic regions rather than in other CGI- and gene-related regions (Fig. - The HyperCs were depleted in the CGIs and upstream regions but oc- curred more frequently in other CGI-and gene-related regions (Fig. - In total and 75.65% of the detected CpGs located in CGIs, upstream and exonic re- gions were HypoCs or HyperCs across these ovarian methylomes (Fig. - Moreover, the DecrmCs and IncrmCs were more likely to be depleted in the CGIs, upstream and exonic regions (Fig. - The DMCs were also depleted in the CGIs, upstream and exonic regions, but occurred more frequently in the other CGI- and gene-related regions (Fig. - How- ever, the DMRs were likely to occur in the CGI, CGI shores, introns, exon regions but were depleted from the CGI shelves, upstream, downstream and intergenic re- gions (Fig. - We found that the mRNA of MMP2 of the GnRH signaling pathway and Estrogen. - signaling pathway, which displayed a peak at the ovulation-luteogenesis transition phase in pubertal rat [36], expressed the highest in the ovaries of Post-puberty in gilts (Fig. - the mRNA level of IGF1R, from Ovarian steroidogenesis pathway, which is essential for ste- roidogenesis and follicle survival in the ovarian granulosa cells of mice [41], displayed the highest at Post-puberty (Fig. - the mRNA level of TAC3, which accelerated fol- licle development and enhanced estradiol production in the ovaries of zebrafish [42], exhibited the highest at the In- puberty (Fig. - These observations indicated that DNA methylation changes might affect the transcription and ex- pression of genes associated to the follicle development, and thus produce an effect on the ovarian mature during pubertal transition in gilts.. - Collectively, during the pubertal transition in gilts, the ovaries underwent maturation, coupling with the signifi- cant changes in the mRNA expression of DNMT1 and DNMT3a. - DNA methylation levels of In-puberty were higher than that of Pre- and Post-puberty at the location of genes and CGIs. - Moreover, the DNA methylation changes were stage-specific and were likely to affect the transcription of genes related to PI3K-Akt signaling pathway, GnRH signaling pathway, and Insulin secretion that were highly associated with the reproduction devel- opmental processes.. - The pigs were fed the same diet ad libitum and reared under the same conditions in the same environments. - Pubertal signs were checked and recorded twice daily at 09:00 and 15:30 by inspection of the vulva and assessment of the standing reflex for these 25 gilts. - and another three gilts in the dioestrus phase, 14 days after the day exhibiting the first estrus and standing reflex, were selected as the Post- pubertal gilts. - The gilts were fed the same diet daily and reared in the same conditions and environments. - The relative expression levels of the mRNAs were quantified using Maxima SYBR Green qRT-PCR Master Mix (2×) (Thermo Scien- tific) and THUNDERBIRD SYBR qPCR Mix (Toyobo) on a LightCycler Real-Time PCR system. - Tissue Kit (Qiagen, Beijing), and then, after checking on the quality of the extracted DNA, one library was built for each pigs based on previously pub- lished RRBS studies . - After the DNA methylation calling by Bismark [45] for these nine RRBS datasets CpGs covered by at least five reads that coexisted across all nine ovarian methylomes remained for further analysis. - The methyla- tion level of the CpGs was calculated as the methylated reads divided the total covered reads. - To profile the DNA methylation patterns at the gene and CGI locations, the gene locations were divided into 20, 40 and 20 bins for 5 kb upstream region of the transcrip- tion start sites (TSSs), gene body and 5 kb downstream region of transcription end sites (TESs), respectively, and the CGI locations were divided into 20, 20 and 20 bins for 2 kb upstream region, CGIs and 2 kb down- stream region, respectively. - The upstream region was 5 kb upstream region of the TSS. - The exon was defined as the integration of the 5′UTR, CDS and 3′UTR arranging from the TSS to the TES. - The downstream region was defined as the 5 kb downstream region of the TES. - The intergenic region was denoted as the outside regions of the upstream, exonic, intronic and downstream regions. - The processes and procedures of the CGI annotation have been described in detail in our previous studies [16].. - According to our previous study, the porcine genes could be separated into two classes: HCP and LCP genes Table 3 Primers used for RT-PCR in the present study. - “t.test”, the enrichment was tested using a two-tailed Fisher’s exact test with the function of “fisher.test”, and the Pearson’s correlation coefficient was tested with the function of “cor.test” in R “stats” package (https://www.. - Animal care and all experiments were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised June 2004) and approved by the Animal Care and Use Committee of the South China Agricultural University, Guangzhou, China (approval number SCAU . - Genome-wide DNA methylation analysis of the porcine hypothalamus-pituitary-ovary axis.. - Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation. - Genome-wide DNA methylation patterns and transcription analysis in sheep muscle. - Orphan CpG islands identify numerous conserved promoters in the mammalian genome. - The role of changing pulse frequency in the regulation of ovulation. - Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing. - A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters
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