Paternal age effects on the sperm epigenome and its impact on the next generation

父亲年龄对精子表观基因组的影响及其对下一代的影响

• 批准号: 422212342
• 负责人: Professor Dr. Thomas Haaf
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422212342?language=en
• 关键词: Paternal; age; effects; sperm; epigenome

Compared to numerous studies on epigenetic programming by maternal factors, little is known about the influence of paternal factors on the next generation. Aim of this proposal is to identify evolutionarily conserved sperm methylation patterns which are influenced by paternal age and to find out to which extent these epigenetic changes are transmitted into the next generation. To detect epigenetic signatures for semen quality, paternal age and outcome of assisted reproductive technologies (ART) in the human sperm methylome, we will perform reduced representation bisulfite sequencing (RRBS) on four groups of 24 carefully matched (for known confounding factors) sperm samples each, with normal and abnormal semen parameters, respectively, either leading or not leading to a pregnancy/live birth. For a subset of 12 samples per group, hydroxymethylation will be studied by oxidative (ox) bisulfite sequencing. To identify similar age-related methylation changes in non-human primates and the bovine model, RRBS and RRoxBS will be performed on sperm samples from 16 common marmosets of different age classes and bovine sperm samples collected at young and old ages, respectively from the same 12 bulls. Identical correlations between sperm methylation and paternal age in three species will point to functionally relevant genes/pathways susceptible to paternal ageing. The 10 most promising candidate genes each for predicting paternal age and/or ART outcome will be validated by bisulfite pyrosequencing in an independent replication cohort of up to 1000 human sperm samples. To study transmission of human age-related sperm methylation changes to the offspring, informative cord blood samples conceived by IVF/ICSI with the analysed sperm samples will be identified. Deep bisulfite sequencing combined with SNP typing allows one to distinguish between paternal and maternal allele methylation and to correlate sperm methylation with paternal allele-methylation (in diploid somatic cells). In many aspects including embryo development and gestation, the bovine model is more similar to the human than rodents. Bovine blastocyst embryos will be produced by in vitro maturation and fertilization of slaughterhouse oocytes with sperm samples from three bulls at young and old age, respectively. Bisulfite pyrosequencing and Taqman assays will reveal possible paternal age effects on embryonal rDNA methylation and expression. RNA sequencing of individual blastocysts will be used to compare the transcriptomes of embryos conceived by sperm from young vs. old bulls. Collectively, our study will provide comprehensive information on the association of the sperm methylome with paternal age, ART outcome, embryogenesis and transmission to the offspring epigenome. Such data are urgently needed to better assess the epigenetic safety of ART.

与大量研究母体因素对表观遗传编程的影响相比，父系因素对下一代的影响知之甚少。本研究的目的是确定受父亲年龄影响的进化保守的精子甲基化模式，并找出这些表观遗传变化在多大程度上传递给下一代。为了检测人类精子甲基组中精液质量、父亲年龄和辅助生殖技术（ART）结果的表观遗传特征，我们将对四组24个精心匹配（已知混杂因素）的精子样本进行减少代表性亚硫酸盐测序（RRBS），每组精子样本分别具有正常和异常的精液参数，导致或不导致怀孕/活产。对于每组12个样品的子集，羟甲基化将通过氧化亚硫酸氢盐测序来研究。为了确定非人灵长类动物和牛模型中类似的年龄相关甲基化变化，RRBS和RRoxBS将分别对来自16个不同年龄段的常见狨猴的精子样本和来自同一12头公牛的年轻和老年牛的精子样本进行RRBS和RRoxBS。在三个物种中，精子甲基化和父亲年龄之间的相同相关性将指向对父亲衰老敏感的功能相关基因/途径。10个最有希望预测父亲年龄和/或ART结果的候选基因将通过亚硫酸盐焦磷酸测序在多达1000个人类精子样本的独立复制队列中进行验证。为了研究人类年龄相关的精子甲基化变化对后代的传播，将鉴定通过体外受精(IVF) /ICSI （ICSI）获得的信息丰富的脐带血样本和分析的精子样本。深度亚硫酸盐测序结合SNP分型可以区分父亲和母亲的等位基因甲基化，并将精子甲基化与父亲的等位基因甲基化联系起来（在二倍体体细胞中）。在包括胚胎发育和妊娠在内的许多方面，牛模型比啮齿动物更接近人类。牛囊胚胚将分别通过体外成熟和屠宰场卵母细胞与三头幼年公牛和老年公牛的精子样本受精而产生。亚硫酸氢盐焦磷酸测序和Taqman分析将揭示可能的父亲年龄对胚胎rDNA甲基化和表达的影响。单个囊胚的RNA测序将用于比较由年轻公牛和老年公牛的精子孕育的胚胎的转录组。总的来说，我们的研究将提供精子甲基组与父亲年龄、抗逆转录病毒治疗结果、胚胎发生和传递给后代表观基因组之间关系的全面信息。为了更好地评估抗逆转录病毒治疗的表观遗传安全性，迫切需要这些数据。