"To Survive or Die" - Adamts9 in Folliculogenesis and Germ Cell Loss

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Development and maintenance of an ovarian follicle, and thus a functional ovary is a remarkable tissue remodeling processes that requires involvements of various proteases. However, our knowledge concerning the enzymes that are responsible for, and underlying mechanisms are incomplete. Our long-term objective is to identify molecules and pathways that affect female fertility, and therefore to advance knowledge on the regulators for reproductive processes and fertility in female animals. The specific aims of this grant application are to elucidate functions, processes, interacting molecules and pathways, and targets of an overlooked metalloprotease, i.e., Adamts9 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 9) in the development and maintenance of an ovarian follicle. Our published or preliminary results strongly indicate Adamts9 has a critical role in survival and programmed death of an ovarian follicle. We hypothesize that oocyte originated Adamts9 enzyme is vital for normal follicle development and survival by varying turnover of extracellular matrix (ECM) proteins therefore increase or decrease of intra-follicular signaling. We will test this hypothesis via a comprehensive examination of status of ovarian follicles, i.e., healthy vs. dying follicles in wildtype or various knockouts, targeted molecules and pathways using tradition approaches including but not limited to confocal microscope imaging, assays of apoptosis, proliferation & Western blots, staining of RNA or proteins. We will also apply advanced tools using in vivo fluorescence reporters or molecular biosensors as well as utilizing unbiased approaches, e.g., single cell RNA sequencing (scRNAseq) and mass spectrometry. To elucidate cell- or molecule-specific events, we have established or obtained various knockouts, reporter or biosensor lines for targeted cells or signaling molecules, which allow us to characterize detailed changes of germ cells, gonad somatic cells, or targeted molecules at a high resolution. Using the advantages of these in vivo systems and fluorescence reporters, we will characterize and elucidate Adamts9 dependent ECM turnover, gene expression, signaling molecule changes, pathways, and processes in development and maintenance of an ovarian follicle. For the first time, functions, and mechanisms of Adamts9, particularly in folliculogenesis, will be comprehensively characterized in a vertebrate model, which will fill a knowledge gap, i.e., how a germ cell originated enzyme such as Adatmts9, regulates its ECM environments, thus determines survival or programmed death of an ovarian follicle. New knowledge gained will be appreciated for understanding or designing new treatments for reproductive disorders such as infertility, gonadal dysgenesis, early menopause, primary ovarian insufficiency (POI) and polycystic ovarian syndrome (PCOS). This AREA grant will also provide critical research opportunities and training for diverse undergraduates at a university mainly serving rural areas and disadvantage populations, and also address NIH mission for biomedical workforce enhancement.