Recapitulation of folliculogenesis in vitro is of interest for both preserving human fertility and assessing the influence of chemical contaminants on ovarian health. Although live offspring can be produced from oocytes recovered from murine follicles grown in vitro, the larger size and protracted duration of folliculogenesis of large mammals likely necessitates more complex culture requirements. The domestic cat will be used as a study model because this species shares a number of anatomical and reproductive characteristics including follicle size and oocyte nuclear configuration with the human. The proposed project will take advantage of ?organ-on-chip? microfluidic technology combined with somatic cell co-culture and my experience with 3-dimensional hydrogels to recapitulate, as possible, in vivo conditions in an ?ovary-on-a-chip?. Specifically, I propose to quantify the rigidity of the domestic cat ovary via micropipette aspiration and atomic force microscopy and identify biomimetic densities of alginate hydrogel that provide optimal structural support to primordial follicles. I will determine the influence of rigidity and medium flow rate on in vitro folliculogenesis, and identify via tandem mass spectrometry and RNA sequencing technologies, proteins and genes involved in follicle activation in culture. Comparison of the dynamic system with traditional, static culture will allow me to improve understanding of the contributions of structural rigidity (hydrogel density), medium flow (microfluidic chip), and the paracrine environment (produced via co-culture) on follicle survival and activation in vitro (Aim 1). I then propose to evaluate the epigenetic impacts (methylation of histones, key imprinted genes) of long-term culture in this system compared with traditional culture, as well as determine the utility of the ?ovary-on-a-chip? in ovarian toxicology studies, using the active form of the chemotherapeutic cyclophosphamide in proof-of-concept studies (Aim 2). Together, these studies advance understanding of contributions of the microenvironment on follicle development toward the goal of an in vitro system for enhancing fertility preservation and reproductive toxicology.
Improved understanding of the mechanisms of large mammalian ovarian folliculogenesis, and the ability to grow follicles in vitro long-term is necessary for the future of female fertility preservation. The proposed studies use a microfluidic ?ovary-on-a-chip? to evaluate the contributions of structural rigidity, medium flow rate, and somatic cell co-culture to elucidate the mechanisms of primordial follicle survival and activation in vitro, as well as determine the utility of this dynamic system for application to ovarian toxicology studies.