(A-29) Understanding hormone-controlled tissue remodeling and ovarian follicle rupture using spatiotemporal transcriptomics

The rupture of ovarian follicles during ovulation is a crucial and intricate process essential for procreation, yet the molecular mechanisms behind this process are not fully understood. In this study, we used high-resolution spatial transcriptomics to reveal the cell-type-specific molecular programs driving follicle maturation and rupture during hormone-induced ovulation. We classified ovarian follicles and performed a time-course analysis to characterize dynamics of cell-type-specific transcriptional programs that change with follicular maturation before and after hormone stimulation. We identified a novel Inhbb and Kctd14 double-positive granulosa cell state associated with preantral-to-antral transition during early follicular development. Our data further revealed novel mural granulosa cell-specific dynamics of gene transcription immediately following hormone stimulation and associated with follicle rupture. We also identified preovulatory stage-specific cell-cell interactions between oocytes and cumulus granulosa cells. Lastly, we report spatially-localized mesenchyme-specific gene expression programs that are distinct to the base versus apex of the rupturing follicles. Collectively, our work provides valuable insights into the spatiotemporal regulation of cellular and molecular processes associated with ovulation.

We treated one-month old female mice with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovulation. We extracted immature and preovulatory ovaries from eight different time points and mounted tissues sections from each sample on a 3mm x 3mm spatially-indexed monolayer of 10 μm beads to perform near single-cell resolution spatial transcriptomics (Seeker Platform, Curio Bioscience). We prepared sequencing libraries from the mRNA molecules captured on indexed beads and sequenced them on an Illumina NextSeq 2K sequencer. Using STARsolo1, we then aligned the sequencing data to the mouse genome to obtain spatial gene expression count matrices for each tissue section.

We conducted a study to investigate the regulation of cellular and molecular processes involved in ovulation in mice (Fig. 1A). Using high-resolution spatial transcriptomics, we  create a detailed map of cell types in ten ovaries collected at eight different points, spanning from immaturity to follicle rupture (Fig. 1B-D). 

Our analysis of granulosa cells in immature ovaries revealed an enrichment of Inhbb+ Kctd14+ granulosa cells in the center of small antral follicles, similar to cumulus granulosa cells found in preovulatory follicles (Fig. 2A-E). In contrast, Inhbb+ Kctd14- cells located along the follicle wall resembled mural cells in preovulatory follicles, suggesting that double-positive proliferating granulosa cells play a role in preantral-to-antral transition (Fig. 2D-E). Furthermore, we investigated temporal gene expression changes to understand the molecular mechanisms driving follicle maturation. We found that rupture-associated markers such as Edn2 and Adamts1 were upregulated, and novel genes, including Fabp4, Ldhd, and Apln, were spatiotemporally-associated with Edn2 in preovulatory ovaries (Fig. 3A-C). Additionally, we observed a gradual decrease in the expression of genes associated with the negative regulation of apoptosis, such as Grem1, Grem2, and Comp, in mural granulosa cells within the preovulatory follicles, highlighting the significance of apoptosis in regulation of follicle maturation (Fig. 3D-F). To understand the dynamics of molecular interactions between oocytes and surrounding cumulus cells, we analyzed ligand-receptor pairs within preovulatory follicles and found them to be temporally-patterned during ovulation (Fig. 4A-B). Additionally, we characterized remodeling-associated programs in stromal tissues during ovarian follicle rupture. Analysis of gene expression changes in the mesenchyme revealed three distinct time-dependent signatures: neurotrophic signaling genes (early), extracellular structure organization (intermediate), and myofibril assembly and muscle contraction (late) (Fig. 5A-C). These observations suggest that mesenchymal cells have temporally-defined roles throughout ovulation. Moreover, we found that spatially-restricted tissue remodeling occurs at the basal and apex sides of ovulating follicles (Fig. 5D-E). 

In summary, our study provides valuable insights into the spatiotemporal regulation of cellular and molecular processes associated with ovulation in mice. These findings contribute to a better understanding of follicle maturation, ovulation, and the complex interactions between different cell types involved in this process.

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