Dynamic Methylomes Of Hypothalamus-Pituitary-Ovary Axis During The Onset Of Puberty In Gilts

In gilts,the initiation of puberty marks the sexual maturation,and the pubertal age is closely associated with its reproductive capacity.Previous studies demonstrated that DNA methylation got involved in the regulation of the onset of mammalian puberty and suggested that the epigenetic modifications in Hypothalamus-Pituitary-Ovary Axis(HPO)might be one of the important molecular mechanism for inititing puberty in pigs.In this study,the Landrace × Yorkshire crossed gilts on Pre-Puberty,In-Puberty and Post-Puberty were selected to compare and describe the dynamics of HPO methylomes during the onset of puberty.Firstly,we bioinformatically predicted distribution characteristics of the different MspI-digested fragment sizes of 40-110 bp,110-220 bp and 40-220 bp in the porcine genome,respectively.Reduced representation bisulfite sequencing(RRBS)libraries were built with these fragment sizes from the same porcine DNA sample to display the technical assessments.Based on the performances of these three RRBS libraries,the appropriate fragment size was selected to build the reduced representation genome and methylome.Secondly,the biological characterisations of porcine methylomes were profiled.Thirdly,the dynamics of DNA methylation and functional molecular network were explored in HPO methylomes.Lastly,based on the functional relationship of differentially methylated genes(DMGs)in a distance matrix,the candidate genes were selected,and the expression levels of mRNA of several candidate genes were detected among HPO tissues.All the bioinformatic work and calculations were finished by R and Perl scripts.The main results were:(1)The performances of MspI-digested fragment sizes of 40-110 bp,110-220 bp and 40-220 bp were technically evaluted in the porcine genome,and 110-220 bp were selected to build the reduced representation genome and methylome.In this methylome,DNA methylation level was negatively correlated with the gene desity;DNA methylation level and density of CpGs was negatively at around transcription start site;genes with high CpG density promoter showed the distinctly methylated pattern from genes with low CpG density promtoer;The densities and DNA methylation of CpGs were negatively correlated at both locations of CpG islands(CGIs)and genes;moreover,the methylation interactions between CGIs and genes showed similar pattern in the CpG context but tissue-specific pattern in the CpH context.(2)The methylation levels of ovarian methylomes were decreasing during the onset of puberty;the methylation levels of pituitary methylomes were also decreaseing,but the methylation differences between In-Puberty and Post-Puberty were larger than that between Pre-Puberty and In-Puberty;on the Post-Puberty,the methylation levels of hypothalamic methylomes were lower than that on the Pre-Puberty but higher that on the In-Puberty.Futhermore,the differentially methylated CpGs were overrepresented in CGI shores,CGI shelves and intergenic regions but underrepresented in the CGI,upstream and exonic regions.(3)Basing on distances of the functional relationship of DMGs in a matrix,297,279 and 273 DMGs were selected as the candidate genes for the hypothalamic,pituitary and ovarian tissues,respectively.These candidate genes,which were highly overlapped among hypothalamic,pituitary and ovarian tissues,included LHB,AR,MMP2,BMP7,BMP6,FGFR1,IGF1 R and so on,which were highly associated with the initiation of the mammalian puberty.Furthermore,the relative mRNA levels of several candidate genes signifcantly expressed in hypothalamic,pituitary and ovarian tissues during the onset of puberty,respectively.(4)Among the HPO methylomes,the DNA methylation level was the highest in hypothalamus and was the lowest in ovary.Moreover,the methylation differences among HPO methylomes were enlarged during the onset of puberty.Basing on the distance matrix of functional relationships among DMGs,which were overlapped in HPO tissues,159 DMGs were selected as the candidate genes.These candidate genes included LHB,AR,MMP2,BMP7,BMP6,IGF1 R,FGF family,WNT family and so on,which were highly associated with pathways of Estrogen signaling,GnRH signaling,MAPK signaling,Wnt signaling,etc.The main conclusions were(1)the performances of 110-220 bp were better than that of 40-110 bp and 40-220 bp in porcine genome;(2)in the porcine methylome,genes with high CpG density promoter showed the distinctly methylated pattern from genes with low CpG density promoter.Moreover,the methylation interactions between CGIs and genes showed similar pattern in the CpG context but tissue-specific pattern in the CpH context;(3)the different dynamics of HPO methylomes suggested that DNA methylation got involved in the initiation of puberty in pigs,and there would be a complex molecular mechanism,linking with the onset of puberty,in each tissues of HPO axis;(4)among HPO axis,the analysis of functional relationships in the distance matrix recommended that 159 genes,including LHB,AR,MMP2,BMP7,BMP6 and IGF1 R,regulated the onset of puberty in gilts.These analyses provided valuable insights into the epigenetic mechanism for initiating puberty in gilts.