Female fertility is dictated by the number of oocyte-containing primordial follicles within the ovary. These follicles are established early in development and their selective activation represents the definitive first step towards oocyte maturation and ovulation. Although evidence supports crosstalk between the oocyte and its supportive somatic granulosa cells, the intrinsic molecular mechanisms that regulate ovarian follicle activation are largely uncharacterised.
In this study, we used single-cell RNA sequencing (scRNAseq) to examine the transcriptional profile of mouse embryonic and neonatal ovaries at three timepoints: embryonic day (E) 18.5, postnatal day (PND) 4 and PND7. These timepoints correspond with the major developmental events of primordial follicle formation and activation. In total, 24,810 cells were sequenced with high confidence, and we identified five distinct clusters of granulosa cells. Bioinformatic subcluster analysis revealed a distinct cluster of pregranulosa cells that appeared to be undergoing follicle activation, evident by the subtle change in known granulosa cell genes including Cdkn1b (p27kip1), Amh and Inhbb. Additionally, this cluster was uniquely differentiated by the expression of Tnni3, Slc18a2, Fam13a and Klf2, all of which are novel to follicle activation. To understand the role of Cdkn1b/p27kip1 (a cell cycle inhibitor) as a potential regulator of these novel activation genes, we performed transcriptomic analysis on Cdkn1b-/- knockout mice. The scRNAseq gene expression signature of activating granulosa cells was observed precociously in Cdkn1b-/- ovaries, suggesting that p27kip1 may act as an important repressor of the genes expressed in activating pregranulosa cells.
This dataset provides the foundations for characterising a genetic network that regulates follicle activation. These findings will reveal key insights into how the ovarian reserve is established and how this is dysregulated in female infertility disorders.