Aging affects all tissues and is associated with functional deterioration. Each tissue has specific aging kinetics, and the female reproductive system is the first to age. Female reproductive aging is associated with a decrease in oocyte quality and quantity as well as a reduction in the ovarian hormones, which accelerates women physiologic aging. Reproductive transitions, such as reproductive aging, are a priority of the Fertility and Infertility branch of the National Institutes of Health, and thus my proposed research is tightly aligned with the mission of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. A major contributor to the age-associated reduction of female fertility is the decrease in oocyte quality due to an increase in oocyte aneuploidy, but our work and others have demonstrated that other factors, such as the tissue microenvironment, might contribute to the age-associated reduction in oocyte quality. Physical cues from the tissue environment are major regulators of cell behavior. In the ovary, stiffness is relevant for normal follicle development but also associated with pathological conditions. In mice, stiff environments maintain primordial follicles in a quiescent state. However ovarian stiffness is also a characteristic of polycystic ovarian syndrome in humans. In my postdoctoral work I pioneered the use of instrumental indentation to measure the biomechanical properties of the ovary and I found that mice ovaries become stiffer with advanced reproductive age. During the K99 phase of this award, I utilized in vitro follicle culture and alginate gels to demonstrate that the age-associated increase in ovarian stiffness impacts folliculogenesis and oocyte quality. My work on ovarian stiffness and folliculogenesis laid the foundation of this R00 application where I will test the overarching hypothesis that the age-associated and spatially-dependent increase in ovarian stiffness creates a physical environment that the follicle senses through the activation of mechanotransduction pathways. This hypothesis will be tested in three specific aims. First, I will determine the subcellular features that define ovarian stiffness by performing a 3D spatiotemporal architecture map of the ovarian stiffness in an age and estrous cycle-dependent manner using a stiffness mapping system optimized during the K99 phase. Second, I will employ an in vitro system that enables precise control of the physical environment. In the K99 phase, I discovered that increased levels of

The stiffness of the mammalian ovary increases significantly with age, yet the spatial and temporal dynamics of this tissue stiffness, how it impacts follicular development, and how mechanical signals from the ovary are transmitted to the follicle have not been explored. In this R00 application I examine how the increase in the biomechanical properties of the ovary with age are spatially and hormonally regulated and how they influence follicle transcriptome through the YAP1 pathway. Overall, the proposed studies will