【作者】 陈曦；

【导师】 余树民；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2017， 硕士

【摘要】 睾丸是雄性哺乳动物产生精子和雄激素的器官,性成熟前睾丸体细胞的增殖与功能成熟对睾丸结构的形成以及精子发生均具有重要的意义。DNA甲基化(DNA methylation)是建立和维持基因组表观遗传状态的基础,在睾丸发育过程中发挥着一定的调控作用。猪作为主要的经济动物和理想的模式动物,具有重要的研究价值,然而目前关于出生后到性成熟前这一阶段猪睾丸基因组甲基化的研究却很少。因此,为了探究性成熟前猪睾丸组织中DNA甲基化的变化特征,本文选取1、2、3月龄三个时间点的猪睾丸实质组织,利用MeDIP-seq的方法成功构建了性成熟前猪睾丸全基因组DNA甲基化图谱,研究该发育阶段猪睾丸基因组甲基化变化以及相关功能基因的甲基化情况。本研究共计获得约19.04 Gb高质量测序数据,将超过190 M的clean reads比对到猪基因组上,平均比对率为82.94%。我们发现随着发育时间推进,基因组DNA甲基化水平整体呈现出下降的趋势,启动子(promoter)甲基化水平也表现出降低的趋势,而Gene body的甲基化水平变化不大;另外,3月龄的平均甲基化水平明显低于1、2月龄(P<0.01),而1、2月龄间平均甲基化水平的差异不显著(P>0.05)。该结果表明猪睾丸发育中DNA甲基化的变化是广泛且动态的,并且逐渐降低的甲基化水平在性成熟前睾丸发育过程中具有一定的调控作用。通过对差异甲基化区域(DMRs)的鉴定和功能富集分析发现,1、3月龄间鉴定得到最多的DMRs(3931个),2、3月龄间次之(1599个),1、2月龄间最少(1534个);DMRs的甲基化水平也整体呈现出下降的趋势。差异甲基化基因显著富集与细胞增殖,能量代谢,类固醇、胆固醇的合成与代谢,视黄醇的代谢,氧化应激负调控等相关功能及通路中(Q-value<0.05),表明性成熟前这一阶段的发育在猪睾丸细胞增殖、结构形成、功能成熟中发挥重要的作用;其中富集到的PPAR信号通路和TGF-β信号通路在睾丸体细胞的发育中发挥一定调控作用。从全基因组DNA甲基化图谱中比对得到8个猪印记基因的甲基化状态:DIRAS3、IGF2、IGF2R、MEST、NAP1L5、NNAT、PEG10以及PLAGL1,发现它们的甲基化程度均不高,表现为“非甲基化”或“部分甲基化”,提示这8个印记基因在性成熟前猪睾丸发育过程中均具一定活性。NAAT、PLAGL1启动子甲基化水平在1到3月龄之间保持增长,NAPIL5、IGF2R启动子甲基化水平在1到2月龄下降,2到3月龄增长,DIRAS3、IGF2、MEST、PEG10启动子甲基化水平在1到2月龄增长,2到3月龄下降;图谱中仅比对到其中4个基因的Gene body甲基化状态,IGF2R、MEST Gene body甲基化水平在1到3月龄保持下降,PLAGL1、PEG10 Gene body甲基化水平在1到2月龄增长,2到3月龄下降。以上结果提示DNA甲基化对印记基因发挥一定的调控作用。本文研究结果揭示了性成熟前猪睾丸基因组DNA甲基化呈现动态变化,甲基化水平表现出下降趋势,为明确DNA甲基化对性成熟前猪睾丸发育的调控机制提供了一定的科学依据,有利于我们深入探索哺乳动物睾丸发育及精子发生,并为遗传育种研究和治疗雄性不育等方面拓展新的思路。更多还原

【Abstract】 The testis is the organ that produces sperm and androgen for male mammals.The proliferation and functional maturation of testicular somatic cells at prepuberty is of great significance to the formation of testis and the spermatogenesis.DNA methylation is the basis for the establishment and maintenance of genomic epigenetic status,and plays an important role in the regulation of testis development.As a good model and economic animal,the pig has important research value in many fields.Therefore,in order to investigate the changes of DNA methylation in prepubertal porcine testis,we generated the first genome-wide DNA methylation profile of prepubertal porcine testis(samples from pigs aged 1,2,and 3 months,denoted as T1,T2 and T3)using MeDIP-seq.In the profile,over 190 million high quality reads(filtered)were generated,and the average aligned rate was 82.94%.There was an overall downtrend of methylation during development,which was clear in promoter regions but less so in gene body regions.In addition,the methylation level at T3 was clearly lower than that at T1 and T2(P-value<0.01),and the downtrend of methylation levels was remarkable in the T1 Vs.T3 and T2 Vs.T3 contrasts(P-value<0.05),but was not in the T1 Vs.T2 contrast(P-value>0.05).These results indicate that DNA methylation has an important role in prepubertal development of porcine testis,with an obvious downtrend of methylation levels from T1 to T3.Thousands of differentially methylated regions(DMRs)were identified among the three prepubertal time points(3,931 in T3 vs.T1,1,599 in T3 vs.T2,1,534 in T2 vs.T1),the majority of which showed decreasing methylation levels over time.GO and KEGG Pathway analyses revealed that many differentially methylated genes were linked with cell proliferation,energy metabolism,steroids and cholesterol synthetic and metabolic process,vitamin A metabolic process,and negative regulation of the response to oxidative stress,indicating that prepubertal development plays an important role in cell proliferation,structural formation and functional maturation of porcine testis.In addition,PPAR signaling pathway and TGF-beta signaling pathway play regulatory roles in the testicular somatic cells development.The methylation levels and their changes of eight porcine imprinted genes:DIRAS3,IGF2,IGF2R,MEST,NAP1L5,NNAT,PEG10 and PLAGL1,were detected in this study.All these eight imprinted genes showed low methylation levels,with "unmethylated" or"partially methylated" status.The methylation levels of NNAT and PLAGL1 promoters increased from T1 to T3;the methylation levels of NAP1L5 and IGF2R promoters decreased from T1 to T2 but increased from T2 to T3;while the methylation levels of DIRAS3,IGF2,MEST and PEG10 promoters increased from T1 to T2 but decreased from T2 to T3.Besides,we only aligned 4 gene bodies from the methylation profile,and the methylation levels of IGF2R and MEST gene bodies decreased from T1 to T3,while the methylation levels of PLAGL1 and PEG10 gene bodies increased from T1 to T2 but decreased from T2 to T3.It can be postulated from the results that these eight imprinted genes may be active during the development of prepubertal porcine testis,and that DNA methylation may have a role in regulation of imprinted genes.This study provides some scientific guides for revealing the regulatory mechanisms of DNA methylation on the prepubertal development of porcine testis,and it is conducive to our in-depth exploration of mammalian testis development and the spermatogenesis,and gives new insights into genetic breeding research and the treatment of male sterility.更多还原