Cellular and biophysical interactions to build oocyte-follicle complexes - Project Summary How do animals make eggs? Sexual reproduction is necessary for the continuity of most animal species and requires the production of egg cell precursors called oocytes. As individuals are delaying having children until later in life and may be subject to genetic and disease-related stressors, a substantial fraction of the population will be impacted by fertility challenges. The goal of this proposal is therefore to determine fundamentally how oocytes develop, which may inform new reproductive therapies. Oocytes develop in the ovary in complexes called follicles, where they are enveloped in somatic granulosa cells that support their development. This proposal aims to determine how oocytes interact with these granulosa cells to build follicles, and how oocytes and granulosa cells communicate with each other across the thick extracellular layers separating them. Studying folliculogenesis in mammals is challenging because oogenesis occurs only once in a hard-to-access fetal window. In contrast to mammals, the sea star Patiria miniata produces new oocytes in existing ovaries and regenerates entire ovaries throughout its adult lifespan. P. miniata is an echinoderm, a member of the sibling group to chordates, and thus is more closely related to mammals than many other invertebrate models. P. miniata follicle cells also share substantial gene expression and structural features with mammalian granulosa cells. These features, in addition to our ability to culture ovary explants long-term in the lab, make sea star oogenesis an accessible, genetically tractable, and biomedically relevant model. Aim 1 of this proposal defines the structure, function, and dynamics of microtubule-based projections (OMPs) that emanate from the oocyte toward the follicle cell, under the hypothesis that they serve as signaling organelles. This is done by microinjecting oocytes with fluorescent microtubule probes and GFP-fusion constructs for candidate structural and signaling proteins to determine their localization. This aim also defines the life history of OMPs, how their dynamics are affected by the presence of follicle cells, and their functional importance for oocyte developmental competency. Aim 2 investigates the cellular dynamics and forces required for follicle formation, using live and fixed imaging of follicle-building. It further asks what cytoskeletal components are involved in building a follicle and how this is affected by the physical properties of the ovary extracellular matrix. This is done using live imaging, a coupled in vivo – in vitro approach, staining of cytoskeletal and ECM components, and inhibitor and reconstitution experiments. Together, this work will uncover the physical basis of follicle formation and provide a framework for future studies of how follicle formation changes during aging. This proposal also takes advantage of the unique expertise and biological and microscopy resources available at the Marine Biological Laboratory in Woods Hole.
