PROJECT SUMMARY 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. My work on ovarian stiffness laid the foundation of this proposal where I will test the overarching hypothesis that the age-associated and spatially-dependent increase in ovarian stiffness creates a physical environment that impacts follicle development and oocyte quality through activation of mechanotransduction pathways in the follicle. This hypothesis will be tested in three specific aims. First, I will determine the subcellular features that define ovarian stiffness by performing a 3D spatio-temporal architecture map of the ovarian stiffness in an age and estrous cycle dependent manner. Second, I will investigate how stiffness affects follicle development and oocyte competency at the transcriptional and cellular level. I will establish an in vitro system which enables precise control of the physical environment. Third, I will explore the mechanism by which the follicle integrates the physical cues and whether the dysregulation of this mechanism accelerates reproductive aging. I will investigate whether follicles from reproductively young and old mice have the same capacity to respond to physical cues through the activation of mechanotransduction pathways, focusing on YAP1. I will complement these studies with in vitro loss-of-function approaches and a YAP1 engineered animal model. Overall, this research will define the ovary’s mechanical properties as a novel regulatory mechanism of reproductive aging. Finally, the re...