Effect Of DNA Methylation On The Onset Of Puberty In Female Goat

The aim of this study is to explore the expression variation of DNMTs and MBPs mRNAs and the level of genome-wide DNA methylation of hypothalamus in prepubertal and pubertal goats to discover a group of candidate genes which are regulated by the DNA methylation.Then the function of candidate genes in puberty onset were analysedto provide a new perspective to understand the mechanism of puberty onset.The hypothalamus of female goats in prepuberty(2.5-3 mounths and weight9.60±2.36kg)and puberty(4.5-5 mounths and weight 17.43±1.63kg)were used in our study.Real-time fluorescence quantitative PCR was used to detecte the expression of DNMTs and MBPs mRNAs.Whole-genome bisulfite sequencing were used to analysis DNA methylation of hypothalamus to build the genomic DNA methylation pattern.The genes with different methylation levels in prepuberty and puberty were screened out by comparing the methylation level of different genes,and GO and KEGG pathway analysis were used to screen the candidate genes.By in vitro culture of hypothalamic primary nerve cells,siRNA interference method was used to knock down the expression of candidate gene to study its effect on the expression of GnRH,GnIH and KISS1 mRNA.Results are listed below:(1)From prepuberty to puberty,the expression of DNMT1,MBD2 and MeCP2 mRNA increased significantly(P<0.05),and the expression of DNMT3 A and MBD3 mRNA decreased(P<0.05),while the expression of DNMT3 B and MBD1 mRNA showed no significance(P>0.05).(2)During puberty onset,the methylation of the hypothalamus occurred mainly at the CG site,and the whole genome methylation showed a hypermethylation state and remained essentially unchanged in prepuberty and puberty.(3)In each chromosome,the methylation level also showed a similar state in both periods.In the different gene content,the methylation level of 5'URT and intron was the lowest and the level of exon methylation was the highest and the methylation level of the promoter region showed a decreasing pattern.(4)By analyzing the methylation differential region,267 DMRs were obtained,and thelength was distributed within 2000 bp.The methylation level of DMR was at a low level in prepuberty and puberty.Analysis of the genes that had different methylation revealed that 55 genes were hypermethylaed and 62 genes were hypomethylated and methylation mainly occurred in the intron region.By GO and KEGG pathway analysis,Oxytocin signaling pathway,Estrogen signaling pathway and GnRH signaling pathway were enriched and we screened eight candidate genes and six genes that generated absolute methylation.(5)The expression of GnRH increased and the expression of GnIH decreased while KISS1 mRNA showed no significance with the expression of GRID1 in the hypothalamic primary nerve cells by siRNA interference.From prepuberty to puberty,(1)The expression of DNMTs and MBPs mRNA are changed and DNMT1,MBD2 and MECP2 mRNA upregulate,and DNMT3 A and MBD3 mRNA down-regulate.(2)The genome methylation level of the hypothalamus remains unchanged.(3)DNA methylation in the hypothalamus occurs mainly at the CG site.(4)For the different genomic functional regions,the methylation level is different.The lowest methylation is in 5'UTR and exons and the highest methylation is in the intron while the promoter methylation level gradually decrease.(5)A number of genes related to puberty onset are screened out,such as GRID1,PTPRN2.The expression of GnRH and GnIH mRNAs are changed with GRID1 siRNA interference.