A 3D in vitro mouse ovarian follicle culture system preserves molecular signatures of ovulation for studying ovulation biology and novel non-hormonal discovery

We previously established an alginate hydrogel-based 3D encapsulated in vitro follicle growth (eIVFG) system to culture mouse and human immature follicles in vitro. The alginate encapsulation maintained the 3D architecture of follicles and support their growth from the secondary to the antral stage to acquire maturation. Upon the stimulation of human chorionic gonadotropin (hCG), a LH analog, the grown antral follicle from eIVFG was able to rupture, undergo cumulus cell expansion, and ovulate a fertilizable metaphase II (MII) oocytes. We have also demonstrated that mouse follicles cultured using eIVFG preserved dynamic transcriptomic profile of many key genes that are essential for gonadotropin-dependent folliculogenesis, such as genes related to gonadotropin hormone receptors and ovarian steroidogenesis. However, it is unknown whether these follicles preserve molecular signatures of ovulation, which would enable this system to serve as a truly scalable and highly controllable system for nominating therapeutic and contraceptive candidates. In the present study, we treated mouse mature follicles from eIVFG with hCG to induce in vitro ovulation. Follicles were collected at 0, 1, 4, and 8 hours post-hCG for single-follicle RNA sequencing (RNA-seq) analysis. Our results showed that follicles grown from eIVFG preserve key ovulatory genes and signaling pathways. Overall design: After 8 days of eIVFG culture, follicles were stimulated for ovulation by hCG, and we collected time-course samples for RNA-seq analysis.Among all 46 samples collected in two batches, 9, 13, 13 and 11 follicles were collected at 0, 1, 4, and 8 hours post-hCG treatment, resepectively.