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 ag...