Quantifying germ cell lineage dynamics in mammalian oogenesis

PROJECT SUMMARY/ABSTRACT Infertility affects 10% of people with ovaries between the ages of 15-44 in the United States, and reduced oocyte number in the ovarian reserve is a leading factor underlying reduced fertility in cases of premature ovarian insufficiency and age-related fertility decline. In mammals, including humans, many oocyte precursors (oogonia) are initially generated in the developing ovary, but the vast majority are culled before or shortly after birth through the process of fetal oocyte attrition (FOA). This yields a much smaller pool of primordial follicles responsible for maintaining ovarian fertility throughout reproductive life: the ovarian reserve. While migrating through the early embryo, early germ cells accrue cellular heterogeneity which manifests as variable genomic demethylation and epigenetic reprogramming, DNA damage and genotoxicity, meiotic entry timing, and exposure and response to signaling cues. This heterogeneity and the massive reduction in oocyte population during FOA have led many to speculate that FOA functions as a selective quality control filter for only the highest quality oocytes to enter the ovarian reserve. However, whether developing germ cell lineages are actively selected to survive based on earlier cellular heterogeneity remains unknown. I propose to quantify lineage dynamics during mammalian oogenesis to characterize germ cell selection and determine the functional importance of early germ cell heterogeneity for shaping the developing ovarian reserve. I hypothesize that heterogeneous early germ cells undergo non-neutral clonal selection during FOA to generate an oocyte pool with shared transcriptomic and chromatin accessibility features. Leveraging in vivo single-cell inducible lineage tracing with genetic barcodes, single-cell multi-omics profiling, and repeat clonal sampling throughout in vitro oocyte differentiation, this proposal will 1) characterize the nature of germ cell selection during FOA through clone size distribution analysis, 2) profile the extent of transcriptomic and epigenomic heterogeneity in the germline before and selection, 3) identify cell states of early germ cells that predict and actively drive successful oocyte differentiation and survival. This approach will identify essential mechanisms for oocyte development and survival that can be applied to improve human in vitro oogenesis protocols and inform the selection of high-quality oocytes for use in clinical assisted reproductive technologies. I have developed a rigorous training plan with my mentors Dr. Diana Laird (sponsor) and Dr. Dan Wagner (co-sponsor) to empower my continued development as a scientist and clinician. With Dr. Laird’s expertise in germ cell development, mouse genetics, and quantitative imaging, Dr. Wagner’s knowledge of single-cell lineage tracing with molecular recorders, cell fate specification, and bioinformatics analyses, and consultation from Drs. Nowakowski, Avagyan, and Bowling, I am well equipped to develop the interdisciplinary skillset needed to execute this project. The support provided by this F30 NRSA predoctoral fellowship award will ensure that I receive the training and mentorship needed to complete the proposed research.