Alkylating chemotherapies, which are used to treat cancers and blood disorders, cause dose-dependent destruction of germ cells, and may induce fibrosis within gonadal tissues. The primary option for fertility preservation in children with ovaries is to undergo ovarian tissue cryopreservation (OTC), as eggs are not produced until after puberty. Currently, the only method to restore fertility is through autotransplantation of OTC tissue. Although ovarian tissue transplantation in these patients resulted in >140 reported live births, the rate of live birth for ovarian tissue transplantation is 20-30 % and the tissue only produces hormones for an average of 2-5 years. The low rate of successful live births and short duration of restored hormones is attributed to an immediate, increased activation of primordial follicles upon transplantation, which results in depletion (~ 80% loss) of the finite oocyte pool. The key to ovarian graft longevity lies in the balance between maintaining the bank of ovarian primordial follicles while promoting regular activation. The ovarian microenvironment is dynamic and continues to develop after birth and through puberty. It is compartmentalized into an avascular, rigid cortex that contains primordial follicles and a vascular, pliable medulla that contains growing follicles. Proteins of interest were identified after mapping the presence and relative abundance of matrisome proteins across porcine and bovine ovaries. Biochemical and physical attributes of ovarian matrisome proteins were modulated using a protein filtration technique to generate novel ovarian matrisome-derived hydrogels. These hydrogels were used to encapsulate and assay small murine follicle growth. Additionally, an assay was developed to identify the potential role of paracrine-secreted factors from human ovarian interstitial cells on follicle growth. These assays identified that a matrisome glycoprotein and interstitial cell-derived paracrine factors that are only secreted in the presence of follicles, promote follicle growth. Additional evaluation of how variations in these microenvironment properties influence quiescence versus growth are ongoing. This research will support efforts to engineer a bioprosthetic ovary that will support human folliculogenesis and increase the fertility and hormone potential of ovarian tissue transplants.