| Objective: To explore the expressions of DNMTs?MBPs mRNA as well as the change of the total DNA methylation level and build the genomic DNA methylation pattern in the hypothalamus of pubertal rat.Methods:(1)10 d,25 d,35 d,puberty(which is evidenced in females by vaginal openingon 39.8 d)and adult(diestrum,56.2 d)female SD rat were used in our study,the hypothalamus was collected to detecte the expression levels change of DNMTs mRNA and MBPs mRNA by using real-time fluorescence quantitative PCR and total DNA methylation level by colorimetry.(2)The DNA methylation pattern of hypothalamus in rat at 25 d and puberty(as aboved)was analysed by using Reduced Representation Bismulfite Sequencing(RRBS)technology,and the related genes and signal pathways involved in the changes of DNA methylation were obtained by GO and KEGG in puberty rat.Results:(1)The expressions of DNMT1 mRNA in the hypothalamus of rat at 10 d,25 d and 56.2 d were significantly lower than at 35 d and 39.8 d(P<0.05),and was higher at 56.2d compared to the rat at 10 d and d 25 d(P<0.05),but no significant difference was found(P>0.05)in rat between 25 d and 10 d,35 d and 39.8 d.DNMT3 A mRNA of hypothalamus reached the peak in the rat at 10 d(P<0.05),then reduced in animals from 25 d to 56.2 d and remained in a relatively stable level(P>0.05).The expression of DNMT3 B mRNA was the highest in the rat at 35 d(P<0.05),then reduced at 39.8 d and 56.2 d but still significantly higher than the animals at 10 d and 25 d(P<0.05),but there were not significant difference in the rats between 10 d and 25 d,39.8 d and 56.2 d(P>0.05).(2)MBD1,MBD2,MBD3 and MBD4 mRNA were the lowest in the rat at 10 d(P<0.05).MBD1 mRNA in the animals at 39.8 d was significantly higher than at 25 d,35 d and 56 d(P<0.05),respectively.No significant difference was found in the rat between 25 d,35 d and 56 d(P>0.05).MBD2 mRNA increased in the rat from 25 to 56.2 d d and remained in a relatively stable level(P>0.05).MBD3 mRNA of hypothalamus was the highest in the rat at 35 d(P<0.05),but there were not significant difference in the rat between 25 d,39.8 d and 56 d(P>0.05).MBD4 mRNA elevated in the animals at 25 d(P<0.05),the highest expression was found in the rat at 35 d,39.8 d and 56 d(P<0.05)but no significant difference was observed among them(P>0.05).The expression of MECP2 mRNA in the rat at 10 d and 25 d were lower than at 35 d and 39.8 d(P<0.05).No significant difference was found in the animal among 10 d,25 d,56.2 d and 35 d,39.8 d and 56.2 d(P>0.05).(3)No significant difference was found in the total DNA methylation level of hypothalamus in the rat during the pubertal development(P>0.05).(4)The three types of methylation C bases proportion of promoter in the hypothalamus were 87.79% CG,3.05% CHG,9.16% CHH and 88.35% CG,3.21% CHG,88.35% CHH for CGI in prepubertal rats,and 90.78% CG,2.13% CHG,7.09% CHH,88.59% CG,88.59% CHG,8.35% CHH for CGI in pubertal animals.The percentage of methylation for CG was the highest and mCG was the most in high methylation status for the CGI.(5)DNA methylation differences presented in GnRH signaling pathways,oocyte meiotic pathways and other gene ontology and pathways in promoter and CGI of hypothalamus in the rat at puberty.Conclusion:(1)The expressions of DNMT1,DNMT3 B,MBD1,MBD3,MBD4 and MECP2 mRNAs increase but DNMT3 A and MBD2 mRNAs don't change in the hypothalamus of pubertal rats.(2)The total DNA methylation of hypothalamus is not different in the rat during pubertal development.(3)The proportion of methylated CG increase but CHG and CHH decrease in the promoter and CGI of hypothalamus in the pubertal rat.The percentage of methylation in CG is the highest and mCG in the CGI is the most high methylation status.(4)DNA methylation of hypothalamus is different beween prepuberty and puberty.There are many different genes and signaling pathways in CGI and promoter of hypothalamus such as GnRH signaling pathways and oocyte meiotic pathways etc,which indicates that many signaling pathways involve in the regulation of puberty onset. |
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