show Abstracthide AbstractPrimordial follicles are the first class of follicles formed in the mammalian ovary and are comprised of an oocyte surrounded by a layer of squamous pre-granulosa cells. This developmental class remains in a non-growing state until individual follicles activate to initiate folliculogenesis. What regulates the timing of follicle activation and the upstream signals that govern these processes are major unanswered questions in ovarian biology. This is partly due to the paucity of data on staged follicle cells since isolating and manipulating individual oocytes and somatic cells from early follicle stages are challenging. To date, most studies on isolated primordial follicles have been conducted on cells collected from animal-age- or oocyte size-specific samples, which encompass multiple follicular stages. Here, we report a method for collecting primordial follicles and their associated oocytes and somatic cells from neonatal murine ovaries using liberase, DNase I, and Accutase. This methodology allows for the identification and collection of follicles immediately post-activation enabling unprecedented interrogation of the primordial-to-primary follicle transition. Molecular profiling by single-cell RNA sequencing (scRNA-seq) revealed that processes including organelle disassembly and cadherin binding were enriched in oocytes and somatic cells as they transitioned from primordial to the primary follicle stage. Furthermore, targets including WNT4, TGFß, FOXO3, and a network of transcription factors were identified in the transitioning oocytes and somatic cells as potential upstream regulators that collectively may drive follicle activation. Taken together, we have developed a more precise characterization and selection method for studying staged-follicle cells, revealing several novel regulators of early folliculogenesis. Overall design: Single oocytes and somatic cells from CD-1 mice at various stages of ovarian follicle development were collected. Eight to twelve P6 ovaries were used to obtain all oocytes and somatic cells. In this study we included samples from the the primordial , transitioning from primordial to primary, primary, and secondary stages of follicular development. For the single oocytes, we included 14 primordial stage, 19 transitioning to primary, 20 primary, and 7 secondary replicates. There were a greater number of replicates for somatic cells with 40 primordial, 47 tranisitioning to primary, 56 primary, and 51 secondary being included in our study.