During mammalian fertilization, egg Ca²⁺ oscillations are known to play pivotal roles in triggering downstream events such as resumption of the cell cycle and the establishment of blocks to polyspermy. However, viable offspring have not been obtained after monitoring Ca²⁺ oscillations, and their spatiotemporal links to subsequent events are still to be examined. Therefore, the development of imaging methods to avoid phototoxic damage while labeling these events is required. Here, we examined the usefulness of genetically encoded Ca²⁺ indicators for optical imaging (GECOs), in combination with spinning-disk confocal imaging. The Ca²⁺ imaging of fertilized mouse eggs with GEM-, G-, or R-GECO recorded successful oscillations (8.19 ± 0.31, 7.56 ± 0.23, or 7.53 ± 0.27 spikes in the first 2 h, respectively), similar to those obtained with chemical indicators. Then, in vitro viability tests revealed that imaging with G- or R-GECO did not interfere with the rate of development to the blastocyst stage (61.8 or 70.0%, respectively, vs 75.0% in control). Furthermore, two-cell transfer to recipient female mice after imaging with G- or R-GECO resulted in a similar birthrate (53.3 or 52.0%, respectively) to that of controls (48.7%). Next, we assessed the quality of the cortical reaction (CR) in artificially activated or fertilized eggs using fluorescently labeled Lens culinaris agglutinin–fluorescein isothiocyanate. Multicolor imaging demonstrated that the first few Ca²⁺ spikes are sufficient for the completion of the CR and subsequent hardening of the zona pellucida in mouse eggs. These methods provide a framework for studying Ca²⁺ dynamics in mammalian fertilization.

Summary Sentence

A novel method for live-cell imaging revealed the stepwise cortical reaction involved in Ca²⁺ oscillations during egg activation and offers new approaches to analyze egg activation events and subsequent embryonic development.