Molecular, Epigenetic and Genomic Approaches to Understand Mechanisms of Aging in the Human Testis

项目总结摘要 部分由于社会经济原因，在工业化国家中，父母年龄增长是一个普遍趋势。 国家。众所周知，父母年龄较高与几个风险因素有关，包括年龄偏低 基因组完整性，器官维护受损，突变增加(可能导致睾丸癌)，改变 生殖系中的表观基因组，以及生育力的降低。重要的是，这些生殖系的改变可以传播 对后代来说，导致几种疾病/综合征(如软骨发育不全、Apert、Noonan)的风险更高 和科斯特洛)在他们的孩子身上。然而，人类睾丸衰老的详细分子机制是 被高度低估了。之前关于人类生殖系和生态位的研究涉及到生理学方法，但 没有揭示生殖系发育的详细分子和基因组驱动因素，也没有揭示哪些方面恶化 在衰老过程中。最近出现的强大的单细胞基因组学方法使我们能够检查基因 全基因组的表达、染色质和DNA甲基化(DNAME)，并为 对衰老对生殖系和体细胞龛细胞影响的分子和机制的理解。在……里面 这项建议，通过利用单细胞基因组学、分子和细胞生物学方法的组合，我们的目标是 为了从机制上理解人类生殖系如何老化以应对老化的生态位，以及这是如何 与个人及其后代的健康有关。我们以前的研究确立了许多生殖系的原则 转录-表观遗传关系和遗传，首次提供了青春期和成年期的单细胞分析 并首次描述了精子DNAME随年龄的变化。该项目旨在极大地扩展我们的 之前的工作，通过利用我们强大的睾丸采集管道(66对完整的睾丸在进行中保存 获取)来检查从年轻人到老年男性的数千个生殖系和利基细胞， 为了检验以下假设：人类男性的转录、开放染色质和DNAME状态 生殖系(精原细胞和发育中的配子)在衰老过程中发生变化，并对 老化的利基市场。具体地说，我们将利用单细胞基因组学技术来检测转录，打开 年龄分层男性(20-29，30-39等)的睾丸细胞染色质和DNAME图谱。我们的目标是 了解衰老对生殖系(AIM 1)和体巢细胞(AIM 2)的影响。重要的是，我们会 还应用了包括精原细胞异种移植和体外器官培养在内的功能实验， 目的是测试生殖系或睾丸壁龛细胞的发育潜力。此外，作为BMI(身体质量 指数)通常随着年龄的增长而增加，我们还将评估观察到的变化与BMI的相关性，就像我们所做的那样 足够的样本根据高BMI和低BMI对每个年龄范围进行分层。总而言之，通过确定潜在驱动因素 对于人类睾丸老化，我们的目标是改变与年龄相关的病理(例如，不育症和 癌症)，并对父性高龄引起的疾病/障碍有深入的分子理解。

PROJECT SUMMARY ABSTRACT Due partly to socio-economic reasons, there is a general trend of increased parental age in the industrialized countries. Advanced parental age is known to be associated with several risk factors including decreased genome integrity, impaired organ maintenance, increased mutations (which may cause testicular cancer), altered epigenome in the germline, and reduction in fertility. Importantly, these germline alterations can be transmitted to the offspring, resulting in higher risks for several diseases/syndromes (e.g. Achondroplasia, Apert, Noonan and Costello) in their children. However, the detailed molecular mechanism underlying human testis aging is highly understudied. Prior work on human germline and niche has involved physiological approaches, but has not revealed the detailed molecular and genomic drivers of germline development, or which aspects deteriorate during aging. The recent advent of powerful single-cell genomics approaches allows us to examine gene expression, chromatin and DNA methylation (DNAme) genome-wide, and offer major new opportunities for a molecular and mechanistic understanding of the impact of aging on both germline and somatic niche cells. In this proposal, by utilizing a combination of single cell genomics, molecular and cell biology approaches, we aim to gain a mechanistic understanding of how human germline ages in respond to the aging niche, and how this is related to individuals’ health and that of their offspring. Our prior studies established many principles of germline transcription-epigenetic relationships and inheritance, provided the first single-cell analyses of pubertal and adult testes, and described the first age-dependent changes in sperm DNAme. This project aims to greatly extend our prior work, by utilizing our robust testis acquisition pipeline (66 pairs of whole testes preserved with ongoing acquisition) to examine thousands of germline and niche cells from young adults through men of advanced age, to test the following Hypothesis: The transcription, open chromatin and DNAme status of human male germline (spermatogonia and developing gametes) changes during aging, and responds to changes in the aging niche. Specifically, we will utilize single cell genomics techniques to examine the transcription, open chromatin and DNAme profiles of testicular cells from age stratified males (20-29, 30-39 etc). We aim to understand the impact of aging on the germline (AIM 1) and somatic niche cells (AIM 2). Importantly, we will also apply functional experiments including spermatogonia xenotransplantation and in vitro organoid culturing, aiming to test the developmental potential of germline or testicular niche cells. Furthermore, as BMI (body mass index) often increases with age, we will also assess the correlations of observed changes with BMI, as we have sufficient samples to stratify each age range by high and low BMI. Taken together, by identifying potential drivers for human testis aging, we aim to transform the management of age-associated pathology (e.g. infertility and cancer) and gain a deep molecular understanding of the diseases/disorders caused by advanced paternal age.