Active surveillance of oocyte quality is moderated by the cGAS-STING pathway

About This Grant

PROJECT SUMMARY The ovarian follicle, comprised of an oocyte surrounded by somatic granulosa and theca cells, is the functional unit of the ovary. Folliculogenesis, the process of follicle growth, is highly dependent on bi-directional communication between the granulosa cells and the oocyte to promote oocyte growth, survival, and quality. Individuals are born with a non-renewable source of follicles, indicating a need for tight regulation of follicle activation and growth to prioritize the removal of poor-quality oocytes and the fertilization of high-quality oocytes that give rise to the next generation. The cGAS-STING pathway is a mediator of the innate immune response and functions by detecting cytosolic dsDNA and eliciting an inflammatory response. Through RNA in situ hybridization, we have shown that the machinery for the cGAS-STING pathway is present in intact preantral follicles with distinct localization. cGAS mRNA is enriched in the oocyte, and STING mRNA is predominantly in the granulosa and theca cells. Importantly, cGAS expression increases significantly in the oocyte between the primary and secondary follicle stages. Our overarching hypothesis is that the cGAS-STING pathway functions as an active surveillance mechanism to regulate the removal of poor-quality oocytes from the active growing pool. This prevents damaged oocytes from being fertilized and contributing to the next generation. I will utilize three validated cGAS-STING pathway inducers: Q-VD-OPH/ABT-737, MC-LR, and diABZI to selectively activate different components of the pathway and study the downstream consequences. We expect that pathway activation will cause granulosa cells to acquire an inflammatory signature, which disrupts bi-directional communication due to altered granulosa cell function and compromises oocyte quality and survival. In Aim 1, I will test different pathway inducers to investigate follicle survival and growth, and oocyte quality in an in vitro encapsulated follicle growth system, as well as the downstream inflammatory response through western blots, immunofluorescence, ELISAs and single-cell RNA-Seq compared to vehicle-treated control follicles. In Aim 2, I will isolate follicle compartments to study pathway responsiveness and utilize cGAS and STING knockout mice to generate reconstituted chimeric follicles to further determine the cell-type specific function and responsiveness of cGAS and STING when treated with pathway inducers. This proposal will elucidate the function of the cGAS-STING pathway in ovarian follicles and determine whether its modulation regulates removal of poor-quality oocytes from the active growing pool. This work may provide novel mechanistic insight into how follicles respond to environmental exposures such as MC-LR. Overall, the comprehensive intellectual infrastructure of Northwestern’s Center for Reproductive Science and Northwestern’s Feinberg School of Medicine and my interdisciplinary mentoring team spanning cGAS-STING biology, genomic instability, toxicology, and reproductive science will provide me with the support and unprecedented skillsets to mature into an independent scientist investigating at the interface of reproductive science and public health.