Project Summary Ovarian follicles are the functional multicellular units of the ovary responsible for a woman’s fertility and ovarian endocrine function. Currently, young women and prepubertal girls diagnosed with cancer and facing ovo-toxic treatments have limited options to preserve their fertility, with cryopreservation of ovarian tissue prior to chemotherapy being the most promising route. Only primordial and early-stage primary follicles survive cryopreservation. To grow and mature, follicles have to be isolated and cultured in groups, because they die if cultured individually. Culture of isolated early follicles as groups has limited translational potential, because of different developmental stages and varying quality of follicles in the cohort, thus emphasizing the need to develop approaches to successfully culture early follicles individually. The low success rates of in vitro follicle development are attributed to the complex and poorly understood paracrine and autocrine signaling between the cells in a follicle, neighboring follicles and their microenvironment. Our overall research objectives are: (a) to identify key factors essential for activation and growth of early stage follicles in vitro, (b) to establish networks and functional relationships between secreted factors, downstream receptors and transcription factors, and (c) to create a standardized in vitro culture system that promotes growth and maturation of early follicles individually. The novelty and the significance of the proposed research are in the discovery of key factors that control and direct the earliest stages of ovarian follicle development. Application of a systems biology approach to study dynamic processes in complex multicellular organoid structures, such as follicles, has the potential to translate to human follicles and study the development and interplay in other tissue organoids and embryos.