Ionomycin

Oocyte activation induced by calcium oscillations is an important process in normal fertilization and subsequent embryogenesis. In the clinical-assisted reproduction, artificial oocyte activation (AOA) is an effective method to improve the clinical outcome of patients with null or low fertilization rate after ICSI. However, little is known about the effect of AOA on preimplantation embryo development in cases with normal fertilization by ICSI. Here, we used ionomycin at different concentrations to activate oocytes after ICSI with normal sperm and evaluated energy metabolism and preimplantation embryo development. We found that a high concentration of ionomycin increased the frequency and amplitude of calcium oscillation patterns, affecting the balance of mitochondrial energy metabolism, leading to increased reactive oxygen species (ROS) and decreased ATP. Eventually, it increases DNA damage and decreases blastocyst formation. In addition, the addition of vitamin C to the culture medium ameliorated the increase in ROS and DNA damage and rescued the abnormal embryo development caused by excessive ionomycin activation. This study provides a perspective that the improper application of AOA may have adverse effects on preimplantation embryo development. Thus, clinical AOA treatment should be cautiously administered.

Ionomycin is a calcium ionophore that induces release of calcium ions (Ca(2+)) from cellular storage to cytoplasm and Ca(2+) influx from the outside of the cell. We investigated the effect of ionomycin on endoplasmic reticulum (ER)-Golgi transport in the vesicular stomatitis virus glycoprotein (VSV-G) system. Ionomycin inhibited transport of VSV-G in a concentration-dependent manner in baby hamster kidney (BHK) cells and HeLa cells. Half-maximum inhibition was observed at 5 μM. The inhibitory effect of ionomycin was not dependent on the cytoplasmic portion. Chelation of Ca(2+) in culture medium did not affect transport efficiency, but co-incubation with ionomycin completely shut off transport. These findings highlight the importance of Ca(2+) release from cellular storage. Because the inhibitory effect of ionomycin was expected to be dependent on mutual interaction of VSV-G and the ER chaperone calnexin, we further investigated interaction kinetics. In HeLa cells but not BHK cells the interaction of VSV-G and calnexin was prolonged in the presence of ionomycin. Taken together, the present results indicate that, by releasing Ca(2+) from cellular storage, ionomycin inhibits ER-Golgi transport by interfering with the release of VSV-G from calnexin in HeLa cells. A mechanism of cell type-dependent ER-Golgi transport regulation was revealed.

Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOXTMincreased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.