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

Abstract

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.