PROJECT SUMMARY/ABSTRACT – Project 2(Davis) Ovarian senescence begins about age 30 and over the next 10 to 15 years is manifest by infertility and a diminished ovarian reserve reflected in decreased Anti-Mullerian hormone levels, decreased expression of the biologically active hypo-glycosylated follicle stimulating hormone (FSH) and elevated serum levels of the less active fully glycosylated FSH. The process of ovarian aging may lead to modifications within the transcriptome of ovarian cells that reduce the chances of an older woman having her own biological children. The investigators of this P01 have established that hypo-glycosylated FSH has greater bioactivity when examined in physiologically relevant models in vitro and in vivo. It is established that FSH activates a variety of cell signaling pathways, but how FSH glycoforms regulate the granulosa cell transcriptome is not understood. Missing from previous studies is an understanding of the role that crucial transcription factors like CREB and YAP1 play in relaying the actions of FSH glycoforms to control granulosa cell proliferation, apoptosis, differentiation, and senescence. Our central hypothesis is that FSH18/21 and FSH24 differentially regulate the key transcription factors CREB and YAP1 to modulate granulosa cell function. We hypothesize that aging promotes an inherent dysregulation of key transcriptional events that regulate follicular development. This project makes use of aging mouse models and in vitro models using granulosa cells recovered from young and older reproductive age women and mice. This project will determine the effect of FSH glycoforms on transcriptional programs of granulosa cells of young and advanced reproductive age mice and women. We will employ genetic loss and gain of function of CREB and YAP1 and conduct RNA-seq for an in-depth gene expression analysis on their role in the response to FSH glycoforms. We will use a state-of-the-art approach (CUT&RUN) to identify differential binding patterns of CREB and YAP1 in granulosa cells from young and advanced reproductive aged women. We will also perform ATAC-seq on these same samples to profile chromatin accessibility between young and older individuals. RNA-seq will be performed to capture the entire transcriptome and bioinformatics will assemble the individual omics approaches allowing a comprehensive understanding of the impact of aging on the response to FSH glycoforms. We will conduct an integrative bioinformatics analysis to build a comprehensive understanding of the genomic landscape and target genes and cellular pathways regulated by FSH glycoforms as a function of age. Successful completion of these aims will reveal unique ovarian transcriptomic signatures induced by FSH glycoforms as a function of age using a combination of in vitro and in vivo approaches. Employment of mouse and human aging models coupled with transcriptomics and bioinformatics analysis will reveal new knowledge that informs approaches to ...