Cytoplasmic and Nucleic Calcium Oscillations in Immature Mouse Oocytes: Evidence of Wave Polarization by Confocal Imaging

Abstract

Using confocal laser scanning microscopy and time-lapse reconstruction, we monitored [Ca 2+] i in living mouse oocytes loaded with the calcium-sensitive fluorescent dyes fluo-3/AM or NuCa Green (specific for nuclear calcium) at the time of meiosis reinitiation. Our data confirm that spontaneous Ca 2+ oscillations occurred in most of the immature fully grown mouse oocytes at least during the first half hour after release of the folliele and these oscillating oocytes underwent GVB in a large proportion after 1 h of culture. These spontaneous oscillations were disrupted when GVB was inhibited by dbcAMP and suppressed in the absence of external calcium. They were dependent on intracellular InsP 3-sensitive Ca 2+ stores since they were inhibited by heparin, an InsP 3-receptor antagonist, or thapsigargin, which depletes InsP 3-sensitive Ca 2+ stores. A relation appeared between the germinal vesicle chromatin appearance and the oocyte's ability to exhibit calcium oscillations. NuCa Green-loaded oocytes exhibited clear fluorescence oscillations in the nuclear region which lead us to discuss the implication of the nuclear calcium in the meiotic process. Moreover, the observation of a clear polarization of the Ca 2+ waves in 30% of the oocytes permits us to hypothesize the existence of a predetermined localization for the initial point of the calcium wave.