Dual-FRET imaging of IP(3) and Ca(2+) revealed Ca(2+)-induced IP(3) production maintains long lasting Ca(2+) oscillations in fertilized mouse eggs

In most species, fertilization induces Ca(2+) transients in the egg. In mammals, the Ca(2+) rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP(3) generated by PLCζ induces Ca(2+) release from the intracellular Ca(2+) store through IP(3) receptor, termed IP(3)-induced Ca(2+) release. Here, we developed new fluorescent IP(3) 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(2+) and IP(3), using IRIS-2s as the IP(3) sensor, we developed a new single fluorophore Ca(2+) sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP(3) concentration ([IP(3)]) starts to increase before the onset of the first Ca(2+) wave. [IP(3)] stayed at the elevated level with small peaks followed after Ca(2+) spikes through Ca(2+) oscillations. We detected delays in the peak of [IP(3)] compared to the peak of each Ca(2+) spike, suggesting that Ca(2+)-induced regenerative IP(3) production through PLC produces small [IP(3)] rises to maintain [IP(3)] over the basal level, which results in long lasting Ca(2+) oscillations in fertilized eggs.