Sperm histone code and epigenetic-mediated subfertility

目前普遍认为，精子特异性表观遗传密码是在精子发生过程中建立的，并传递给卵母细胞。畸变可能是导致IVF/ICSI结局降低的特发性男性不育的原因之一。在先前的研究中，已经分别应用ChIP芯片HG 18启动子阵列和ENCODE阵列在来自生育供体和生育力低下患者的射精精子中鉴定了与组蛋白H4在赖氨酸12处乙酰化（H4 K12 ac）相关的基因启动子以及与组蛋白H3在赖氨酸9处乙酰化（H3 K9 ac）相关的外显子序列。本研究的主要目的是：1）应用Re-ChIP技术，阐明H4 K12 ac相关基因启动子是否与其他组蛋白如H3 K9 ac、H3 K4 me 3和H3 K27 me 3或鱼精蛋白共定位; 2）确定鱼精蛋白-1在生育力低下患者和供者中的结合序列; 3）应用焦磷酸测序技术分析H4 K12 ac和鱼精蛋白-1相关发育基因启动子区CpG二核苷酸甲基化状态; 4）分析H4 K12 ac和H3 K9 ac在父系印迹位点上的可能作用：5）采用原位杂交、免疫荧光结合共聚焦显微镜技术分析H4 K12 ac相关基因在小鼠受精卵父系原核中的表达; 6）对来自H4 K12 ac ChIP芯片和鱼精蛋白-1 ChIP测序的自身数据与来自人植入前胚胎/胚胎干细胞的转录组和在牛模型中产生的数据进行基于全基因组生物信息学的数据比较，所述牛模型提供了用于早期发育的父本贡献的候选基因（coop，项目7）。

It is now generally accepted that a sperm-specific epigenetic code is established during spermiogenesis and transmitted to the oocyte. Aberrations may represent one reason for idiopathic male infertility resulting in decreased IVF/ICSI outcome. In a previous study, gene promoters associated with histone H4 acetylated at lysine 12 (H4K12ac) and also exonic sequences associated with histone H3 acetylated at lysine 9 (H3K9ac) have been identified in ejaculated spermatozoa from fertile donors and subfertile patients applying ChlP-on-chip HG18 promoter array and ENCODE array, respectively. In the present study, the following aims will be addressed: 1) To clarify whether H4K12ac-associated gene promoters colocalize with other histones, such as H3K9ac, H3K4me3 and H3K27me3, or protamine applying Re-ChIP; 2) To determine binding sequences for protamine-1 iri fertile donors and subfertile patients; 3) To analyze the methylation status of CpG dinucleotides within gene promoters of developmental genes associated with H4K12ac and protamine-1 applying pyrosequencing; 4) To analyze the possible role of H4K12ac and H3K9ac on paternal imprinted loci; 5) To analyze expression of H4K12ac-associated genes in the paternal pronucleus of mouse zygote using in-situ hybridization and immunofluorescence in combination with confocal microscopy; 6) To perform a genome-wide bioinformatic-based data comparision of own data from H4K12ac ChlP-on-chip and protamine-1 ChlPsequencing with transcriptomes from human preimplantation embryos/embryonic stem cells and data generated in bovine model providing paternal contributed candidate genes for eariy development (coop, with project 7).