Single cell transcriptomics reveals cell populations with unique molecular identities over the time course of ovulation in vivo

The objective of this study was to develop a single cell atlas of the mouse ovary across a time course of ovulation. A time course of in vivo ovulation was performed using CD-1 mice (n=3 per timepoint). Superovulation was induced with gonadotropins and ovaries were dissected at three timepoints post ovulation induction: 0h, 4h (peak expression of genes known to regulate ovulation), and 12h (point at which approximately half of the cumulus-oocyte-complexes [COCs] have undergone ovulation). One ovary from each mouse was collected at each time point. Pooled ovaries were dissociated into a single-cell suspension and subsequently analyzed using single cell sequencing. Following unbiased clustering of single cell data, differential expression, trajectory, and gene ontology analyses (DEA, TA, and GO, respectively) were performed to determine uniquely expressed genes and pathways across cell clusters. Single-cell analysis revealed 16 cell clusters (n = 25,714 cells) throughout ovulation, including epithelial, endothelial, stroma, theca, luteal, granulosa, and immune cells. Stroma, theca, and luteal cells (SC, TC, and LC, respectively) clustered separately by time, demonstrating unique identities specific to early and late stages of ovulation. Of note, cumulus cell (CC) clusters specific to ovulation were also detected. TA and GO analysis of top differentially expressed genes revealed that these cell types undergo distinct molecular changes that likely permit critical shifts in function. Early CC were enriched in genes that positively regulate EGF signaling, possibly stimulating COC expansion. Genes enriched in late CC negatively regulate cell motility/migration, suggesting loss of the migratory capability of CC at the conclusion of ovulation. Early SC were enriched in genes promoting angiogenesis within the extracellular matrix (ECM), likely accommodating transfer of factors such as immune cells and steroid hormones. Notably, genes enriched in late SC were involved in depolarization of voltage-gated calcium channels, a currently understudied process in the ovary. Gene enrichment in early TC suggested involvement in steroidogenic processes localized to the mitochondria, a known location of steroid biosynthesis. Importantly, GO analysis revealed a potential novel role for late TC in regulating metal homeostasis in the mitochondria. Like early TC, genes enriched in early LC were also involved in steroid production within the mitochondria. However, late LC were enriched in genes that exhibit positive regulation of stress fiber/actin filament assembly and cell secretion in the ECM, possibly reflecting the tissue remodeling and progesterone secretion that occurs with luteinization. This study is the first to evaluate gene expression and localization in the mouse ovary throughout ovulation with single cell technology and describes both novel cell types and time-dependent molecular changes within known cell types.