Comparison of in vitro and in vivo ovulation in mouse using single-nuclei RNA-seq   — ASN Events
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Comparison of in vitro and in vivo ovulation in mouse using single-nuclei RNA-seq   (#495)

Harin Natarajan Ganesh 1 , Hannah M VanBenschoten 2 , Jiyang Zhang 3 , Shuo Xiao 3 , Brittany A Goods 1 2
  1. University of Melbourne, Melbourne, VIC, Australia
  2. Thayer College of Engineering, Dartmouth College, Hanover, NH, USA
  3. Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ, USA

Background
 Ovulation involves coordinated, time-dependent shifts in cell behavior, but whether in vitro culture faithfully recapitulates this remains unclear. Encapsulated in vitro follicle growth (eIVFG) is widely used to model ovulation, yet how it compares to in vivo at single-cell resolution is unknown, limiting optimization of culture conditions and confidence in eIVFG's fidelity. This study compares cellular composition and transcriptomic profiles between in vivo and eIVFG ovulation time-course datasets.

Methods
 Single-cell RNA-sequencing (10x Genomics Chromium) was performed on eIVFG follicles (n=3 mice) collected at Days 0, 4, and 8, pre- and post-hCG (n=25 follicles pooled/condition). Cell-typing used Seurat-based clustering, SARGENT annotation, and canonical markers from Huang et al.

Results
 We identified 122,376 single cells across the eIVFG time-course, resolving into nine major cell types (Cumulus, Granulosa, Luteal, Theca, Stroma, Epithelial, Endothelial, Oocyte, Myeloid). Composition shifted over time: cumulus subclusters marked by early hCG-response genes (Ptgs2, Cxcl1/2) peaked at 4 hours (~35%, ~29%), then declined. An Amh+ granulosa subcluster dominated Day 0 (~77%) but was absent elsewhere; a distinct granulosa subcluster peaked at 8 hours (~35%), and a stromal subcluster (Col1a1, Lum) emerged at Day 0 (~20%). Oocytes remained rare throughout; by Day 4-8, composition became more even, with no dominant cluster. Compared to an in vivo dataset from Huang et al., early cumulus and stromal signatures matched in timing and marker-identity, while the early, dominant Amh+ population diverged from the late, minority in vivo state (1).

Conclusion
 This work provides a comprehensive single-nuclei transcriptome comparison of in vivo and in vitro ovulation with defined cell types. Ovulation in eIVFG is marked by transient, synchronized shifts in cumulus and granulosa cell states around hCG stimulation, followed by diversification by Day 4-8. Future pathway-analyses and metabolic inference will inform improvements to in-vitro conditions and clarify how metabolism drives cellular transitions across ovulation.

  1. Huang R, Kratka CE, Pea J, McCann C, Nelson J, et al. (2025) Single-cell and spatiotemporal profile of ovulation in the mouse ovary. PLOS Biology 23(6): e3003193. https://doi.org/10.1371/journal.pbio.3003193