Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs

Abstract

In most species, fertilization induces Ca²⁺ transients in the egg. In mammals, the Ca²⁺ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP₃ generated by PLCζ induces Ca²⁺ release from the intracellular Ca²⁺ store through IP₃ receptor, termed IP₃-induced Ca²⁺ release. Here, we developed new fluorescent IP₃ 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 Ca²⁺ and IP₃, using IRIS-2s as the IP₃ sensor, we developed a new single fluorophore Ca²⁺ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP₃ concentration ([IP₃]) starts to increase before the onset of the first Ca²⁺ wave. [IP₃] stayed at the elevated level with small peaks followed after Ca²⁺ spikes through Ca²⁺ oscillations. We detected delays in the peak of [IP₃] compared to the peak of each Ca²⁺ spike, suggesting that Ca²⁺-induced regenerative IP₃ production through PLC produces small [IP₃] rises to maintain [IP₃] over the basal level, which results in long lasting Ca²⁺ oscillations in fertilized eggs.