O-011 A live imaging system to study the spatiotemporal control of ovulation

Study question What can we learn about the spatiotemporal and cellular dynamics of ovulation by performing high resolution live microscopy of the entire process? Summary answer We identified three distinct, highly synchronous phases of ovulation - expansion, contraction, and rupture - and investigated their regulatory mechanisms. What is known already There have been studies performing lower-resolution live imaging, but not confocal or two-photon microscopy, of some parts of ovulation. Most of what we knew comes from fixed and knockout studies, which have shed light on key pathways such as Endothelin signaling and Hyaluronic acid. These studies identified molecular regulators of follicle rupture but lacked temporal resolution to capture dynamic processes. Some in vivo imaging studies provided insights into vascular changes and oocyte release, but real-time tracking of follicle expansion, contraction, and rupture remained unexplored. Our study bridges this gap by offering a high-resolution, continuous view of ovulation dynamics. Study design, size, duration We conducted a live-imaging study using isolated mouse ovarian follicles to analyze ovulation dynamics. The study included over 100 follicles observed over 16-24 hours to capture the full sequence of ovulation. High-resolution confocal and two photon imaging allowed us to describe cellular dynamics, observe oocyte maturation inside the intact follicle and quantify the volume of the follicles over time. We additionally generated a single-cell RNA sequencing dataset of ovulating follicles to identify new pathways. Participants/materials, setting, methods Female C57BL/6J, CAG-TAG, and Oct4-GFP mice (23-28 days old) were used. Ovarian antral follicles (300-500 µm) were isolated and cultured in optimized media under controlled conditions. Live imaging of ovulation was performed using confocal and two-photon microscopy, capturing follicle expansion, contraction, and rupture. Pharmacological perturbations were applied to investigate regulatory mechanisms. Follicle volume changes were quantified using image segmentation and 3D reconstruction. Main results and the role of chance We established a live imaging system to study ovulation in isolated mouse follicles, revealing three distinct and highly synchronous phases: follicle expansion, contraction, and rupture. Expansion is driven by hyaluronic acid secretion, creating an osmotic gradient that induces fluid influx and follicle swelling. Contraction follows, initiated by smooth muscle cells surrounding the follicle, generating hydrostatic pressure. Once a threshold is exceeded, the follicle wall rapidly stretches and ruptures, releasing the oocyte and cumulus complex. This ex vivo system faithfully recapitulates ovulatory dynamics, validated through single-cell RNA sequencing and immunofluorescence analyses. Notably, ovulatory timing was consistent across follicles, independent of size, indicating an intrinsic regulatory mechanism. The role of protease activity, specifically MMP2, in rupture was confirmed, while vasoconstriction, a potential in vivo contributor, remains unexplored due to the absence of vasculature in isolated follicles. The study provides mechanistic insights into ovulation and offers a powerful model for pharmacological and genetic perturbations. Limitations, reasons for caution Although isolated follicles replicate ovulatory processes well, they expand isotropically, unlike in vivo follicles, which protrude outward from the ovary. The model lacks vasculature, preventing assessment of vasoconstriction. While protease activity is crucial for rupture, other ovarian factors may contribute in vivo. Findings should be interpreted considering these physiological differences. Wider implications of the findings Our live imaging system provides unprecedented insights into ovulation dynamics, enabling precise mechanistic studies and drug testing. Understanding ovulation at this resolution may inform anovulatory phenotypes, improve fertility treatments and contraceptive development. The ex vivo follicle model offers a versatile platform for studying reproductive biology and broader tissue remodeling processes. Trial registration number No