Epigenetic signatures in the ageing male germ cell.

在西方国家，推迟为人父母的趋势很强。虽然推迟分娩的后果是众所周知的，例如增加了染色体异常的风险，但我们对男性生殖老龄化(即影响男性生殖细胞的衰老)的了解很少。有强有力的证据表明，父亲的年龄对男性生殖细胞功能有显着影响，例如，某些遗传疾病在后代中的患病率增加。然而，我们不知道衰老在多大程度上影响表观遗传特征，特别是DNA甲基化，也不知道不育男性是否存在这些表观突变的附加风险。我们假设生殖老化影响男性生殖细胞的表观遗传特征：与体细胞和精子相比，精原干细胞(SSCs)的年龄不同，反映了这种不同的老化过程。我们将讨论健康男性的生殖系在衰老过程中是否发生显著的表观遗传学、遗传学和细胞变化，以及不育男性的这些动态是否发生改变。从健康的研究男性队列(18-75岁)收集血液和精液样本的充分特征将成为研究老龄化影响的重要工具。将测量年龄匹配的健康和不育男性的血液和精子DNA中的端粒长度(TL)，以确定精子TL是否随年龄变化以及不育对衰老的影响程度。生物/表观遗传年龄可以通过测量基因组中不同CpG位点的DNA甲基化来确定。因此，我们将对健康年龄组的血液、精子和精原DNA进行调查，以确定胚系的年龄是否与体细胞相似或不同。通过对精子表观遗传异质性的研究，我们可以推断精子克隆起源和细胞SSC组成随年龄的变化。在功能水平上，我们将评估SSC种群本身是否随年龄变化。对固定的睾丸组织进行的组织形态计量学分析描绘了其干细胞特征受年龄影响的程度。干细胞的克隆性扩增将用干细胞特异性标记和DNA甲基化标记的免疫组织化学方法进行研究。自然SSCs将从睾丸活检组织中分离出来，并进行RNA图谱和免疫染色。使用丰富的SSCs，我们将研究依赖年龄的表观遗传学变化是仅限于几个基因，还是影响基因组的多个区域。为此，我们将在精原DNA中进行完整的甲基组测序，这将使我们能够确定男性生殖系中与年龄相关的甲基化变化的假定候选基因。这个项目将极大地增加我们对男性生殖老化的了解，并有助于确定与延迟亲子关系相关的风险。

In western countries there is a strong trend for delaying parenthood. While the consequences of postponed maternity, such as an increased risk for chromosomal anomalies, are well known, our knowledge on male reproductive ageing (i.e. ageing affecting the male germ cells) is scant. There is strong evidence for a significant effect of paternal age on male germ cell function e.g. increase in the prevalence of certain genetic disorders in the offspring. However, we do not know to which extent ageing affects epigenetic signatures, especially DNA methylation, and whether infertile men are at an additive risk for these epimutations. We hypothesize that reproductive ageing affects the epigenetic signatures of male germ cells: spermatogonial stem cells (SSCs) age differently when compared to somatic cells and spermatozoa, deriving from these SSCs, reflect this different ageing process.We will address whether noticeable epigenetic, genetic and cellular changes occur in the germ line of healthy men during ageing and if these dynamics are altered in infertile men. A fully-characterized collection of blood and semen samples from a healthy study men cohort (18-75 years) will constitute a vital tool for studying the effects of ageing. Telomere length (TL) will be measured in blood and sperm DNA of age-matched healthy and infertile men to determine if sperm TL changes with age and to which extent infertility affects ageing. Biological/epigenetic age can be determined by measuring the DNA methylation of distinct CpG sites in the genome. Therefore blood, sperm and spermatogonial DNA from our healthy age groups will be investigated to determine whether the germ line ages in a similar or distinct way from somatic cells. By studying the sperm epigenetic heterogeneity we could deduce an age-dependent change in the clonal origin of sperm and in the cellular SSC composition.At the functional level we will evaluate whether the SSC population itself changes with age. A histomorphometric analysis on fixed testicular tissues delineates the extent to which their stem cell features are affected by age. The clonal expansion of SSC will be studied by immunohistochemistry with stem cell specific and DNA methylation markers. Native SSCs will be isolated from testicular biopsies and characterized by RNA profiling and immunostaining .Using the enriched SSCs we head to whether age-dependent epigenetic changes are restricted to only a few genes or affect multiple regions in the genome. For this we will perform a whole-methylome sequencing in spermatogonial DNA, which will allow us to identify putative candidate loci for age-related methylation changes in the male germ line. This project will greatly increase our knowledge of reproductive ageing in men, and help to identify the risks associated with delayed paternity.