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

项目摘要 部分由于社会经济原因，在工业化国家，父母年龄普遍增加， 国家众所周知，父母年龄大与几个风险因素有关， 基因组完整性，受损的器官维护，增加突变（可能导致睾丸癌），改变 生殖系中的表观基因组，以及生育能力的降低。重要的是，这些生殖细胞的改变可以通过 对后代，导致几种疾病/综合征（如软骨发育不全，Apert，努南）的风险较高 和科斯特洛）在他们的孩子。然而，人类睾丸衰老的详细分子机制是 被低估了先前对人类生殖系和生态位的研究涉及生理学方法，但 没有揭示生殖系发育的详细分子和基因组驱动因素，或者哪些方面恶化 在老化过程中。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 表达，染色质和DNA甲基化（DNAme）全基因组，并提供了重大的新机会， 衰老对生殖系和体细胞生态位细胞影响的分子和机制理解。在 该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 为了获得人类生殖系如何对衰老生态位做出反应的机械理解，以及这是如何发生的， 关系到个人及其后代的健康。我们先前的研究建立了许多生殖系的原则， 转录表观遗传关系和遗传，提供了第一个单细胞分析青春期和成人 睾丸，并描述了精子DNA的第一个年龄依赖性变化。该项目旨在大大扩展我们的 先前的工作，通过利用我们强大的睾丸采集管道（66对整个睾丸保存， 获取）来检查从年轻成年人到老年男性的数千个生殖系和小生境细胞， 为了验证以下假设：人类男性生殖细胞的转录、开放染色质和DNAme状态 生殖细胞（精原细胞和发育中的配子）在衰老过程中发生变化， 衰老的利基。具体来说，我们将利用单细胞基因组学技术来检查转录，开放 来自年龄分层的男性（20-29、30-39等）的睾丸细胞的染色质和DNAme谱。我们的目标是 了解衰老对生殖细胞（AIM 1）和体细胞生态位细胞（AIM 2）的影响。重要的是，我们将 还应用了精原细胞异种移植和体外类器官培养等功能实验， 旨在测试生殖系或睾丸小生境细胞的发育潜力。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， 足够的样本，以分层每个年龄范围的高和低BMI。综合来看，通过确定潜在的驱动因素， 对于人类睾丸衰老，我们的目标是改变与年龄相关的病理学（例如不孕症和 癌症），并获得由高龄父亲引起的疾病/病症的深刻分子理解。

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.