Lefèvre B, Pesty A, Testart J
Institut National de la Santé et de la Recherche Médicale (I.N.S.E.R.M) Unité 355, Clamart, France.
Exp Cell Res. 1995 May;218(1):166-73. doi: 10.1006/excr.1995.1144.
Using confocal laser scanning microscopy and time-lapse reconstruction, we monitored [Ca2+]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 Ca2+ oscillations occurred in most of the immature fully grown mouse oocytes at least during the first half hour after release of the follicle 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 InsP3-sensitive Ca2+ stores since they were inhibited by heparin, an InsP3-receptor antagonist, or thapsigargin, which depletes InsP3-sensitive Ca2+ 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 Ca2+ waves in 30% of the oocytes permits us to hypothesize the existence of a predetermined localization for the initial point of the calcium wave.
利用共聚焦激光扫描显微镜和延时重建技术，我们在减数分裂重新启动时，对加载了钙敏荧光染料fluo-3/AM或NuCa Green（对核钙具有特异性）的活小鼠卵母细胞中的[Ca2+]i进行了监测。我们的数据证实，在卵泡释放后的至少前半小时内，大多数未成熟的完全生长的小鼠卵母细胞中会发生自发的Ca2+振荡，并且这些振荡的卵母细胞在培养1小时后大部分会经历生发泡破裂（GVB）。当dbcAMP抑制GVB时，这些自发振荡会被破坏，并且在没有外部钙的情况下会受到抑制。它们依赖于细胞内对肌醇三磷酸（InsP3）敏感的钙库，因为它们会被肝素（一种InsP3受体拮抗剂）或毒胡萝卜素（可耗尽对InsP3敏感的钙库）抑制。生发泡染色质外观与卵母细胞表现出钙振荡的能力之间似乎存在关联。加载NuCa Green的卵母细胞在核区域表现出明显的荧光振荡，这使我们讨论核钙在减数分裂过程中的作用。此外，在30%的卵母细胞中观察到Ca2+波的明显极化，这使我们能够假设钙波起始点存在预定的定位。
