Dual-FRET imaging of IP and Ca revealed Ca-induced IP production maintains long lasting Ca oscillations in fertilized mouse eggs.

In most species, fertilization induces Ca transients in the egg. In mammals, the Ca rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP generated by PLCζ induces Ca release from the intracellular Ca store through IP receptor, termed IP-induced Ca release. Here, we developed new fluorescent IP sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca and IP, using IRIS-2s as the IP sensor, we developed a new single fluorophore Ca sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP concentration ([IP]) starts to increase before the onset of the first Ca wave. [IP] stayed at the elevated level with small peaks followed after Ca spikes through Ca oscillations. We detected delays in the peak of [IP] compared to the peak of each Ca spike, suggesting that Ca-induced regenerative IP production through PLC produces small [IP] rises to maintain [IP] over the basal level, which results in long lasting Ca oscillations in fertilized eggs.