In brief Proper development of ovarian follicles, comprised of an oocyte and surrounding somatic cells, is essential to support female fertility and endocrine health. Here, we describe a method to isolate single oocytes and somatic cells from the earliest stage follicles, called primordial follicles, and we characterize signals that drive their activation. Abstract Primordial 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 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, TGFB1, 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.
Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we demonstrated that compared to freshly-harvested follicles, vitrified follicles had normal follicle and oocyte reproductive outcomes. However, it is unknown whether vitrification preserves molecular signatures of folliculogenesis, which is the primary research focus in this study. Six fresh and six vitrified antral follicles grown from eIVFG were collected on day 8 for the whole single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles had similar transcriptomic profiles to fresh follicles. There were 35 differentially expressed genes between vitrified and fresh follicles, however, none of those genes have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system, providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.
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