Elucidating the Role of YAP and TAZ in the Aging Human Ovary

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PROJECT SUMMARY Ovarian senescence begins about age 30 and over the next 10 to 15 years is manifested by infertility. An expanded knowledge of the underlying molecular mechanisms that dictate granulosa cell function will facilitate our understanding of the health disparities of age and infertility. Over the past half century, the number of first- time mothers of advanced maternal aged women 35 years and older has increased more than five-fold. With more women entering the workforce, changes in social structure, advanced education and increased use of contraception, this trend will likely continue to increase. There is a constant bi-directional communication between the oocytes and granulosa cells of the follicle, which is required for development and maturation of high-quality follicles. The Hippo signaling is known to regulate organ size. Our published results established the role of Hippo signaling, and its downstream effector molecules, YAP and TAZ, as critical regulators of granulosa cell function and fertility. Notably, our preliminary analysis identified YAP/TAZ activity to be downregulated in advanced aged women (≥ 40 years) undergoing IVF and having low oocyte yields. We hypothesize that YAP and TAZ orchestrate crucial transcriptional events that regulate development of high-quality follicles. However, there is a gap in knowledge in understanding the actual mechanism of how these transcription factors regulate granulosa cell function, especially in aging women undergoing IVF. The overarching goal of this proposal is to understand YAP- and TAZ-mediated transcription and chromatin architecture in granulosa cells of women with advanced age. A drawback of using primary patient granulosa samples are low sample numbers which is a major roadblock to progress. The major innovation of this proposal is the ability to simultaneous profile chromatin and transcription regulation in the same patient sample thus providing greater in-depth understanding of the role of YAP and TAZ transcription factors in granulosa cell function. Our research strategy includes two specific aims. In Specific Aim 1. We will define the transcriptional targets of YAP and TAZ in granulosa cells by using CUT&RUN, a micrococal nuclease technique which can isolate specific protein-DNA complexes and distinguish direct TF binding from low numbers of granulosa cells. Our analysis includes granulosa cells obtained from IVF patients with poor response (< 6 oocytes retrieved) and good response (> 17 oocytes retrieved) in women of advanced maternal age (≥ 40 yrs old). We will also employ ATAC-seq to determine chromatin accessibility in these patient samples. In Aim 2, we will modulate YAP and TAZ in human granulosa cells and define their resultant transcriptomic profile. Genes identified in this study will be functionally validated by analysis of granulosa cell proliferation, differentiation, and senescence. These novel insights will help to better understand age-related decline in fertility and the role of the Hippo signaling pathway effectors YAP and TAZ in the ovary.