Stricker S A
Department of Biology, University of New Mexico, Albuquerque 87131.
Dev Biol. 1996 Jun 15;176(2):243-63. doi: 10.1006/dbio.1996.0131.
To analyze fertilization-induced calcium dynamics in a protostome worm, unfertilized oocytes of the nemertean Cerebratulus lacteus were co-injected with calcium green (CG) and rhodamine (Rh) dextrans for dual-channel confocal imaging of early development. Based on CG/Rh ratioed images collected every 800 msec, fertilization elicits a "cortical flash" of elevated free calcium that spreads rapidly around the oocyte without propagating as a point-source wave. A similar calcium transient occurs in unfertilized oocytes treated with KCl to depolarize the oolemma, and the fertilization-induced cortical flash is eliminated if cobalt is used to block calcium channels, collectively indicating that fertilization initially triggers an influx of calcium ions through voltage-gated calcium channels in the oolemma. However, within minutes after producing a cortical flash, C. lacteus oocytes begin to display a series of point-source, oscillating waves of elevated free calcium that are propagated at about 15 micron/sec. The first two calcium waves arise at the site of sperm fusion and typically fail to reach the antipode, but after sperm incorporation, the waves spread globally throughout the ooplasm and typically shift their origin to a pacemaker region in the vegetal cortex. About 10 oscillations with an average duration of 3.3 +/- 1.2 min are generated for approximately 60-100 min postfertilization as meiotic maturation is completed, and such waves continue to occur in cobalt-containing seawater or calcium-free seawater. Thus, wavelike calcium oscillations induced by fertilization are apparently dependent upon internal calcium stores, which in turn may contain IP3-insensitive and/or IP3-sensitive receptors based on experiments using ryanodine, caged IP3, and heparin. Unfertilized oocytes also display repetitive calcium waves following intracytoplasmic injections of whole sperm, and such oscillations are eliminated if the sperm suspensions are boiled prior to injection, suggesting the possible presence of a heat-labile sperm component that can elicit wavelike oscillations during fertilization.
为了分析一种原口动物蠕虫受精诱导的钙动力学，将海产纽形动物乳突脑纹虫（Cerebratulus lacteus）的未受精卵母细胞与钙绿（CG）和罗丹明（Rh）葡聚糖共同注射，用于早期发育的双通道共聚焦成像。基于每800毫秒收集的CG/Rh比率图像，受精引发游离钙升高的“皮质闪光”，其在卵母细胞周围迅速扩散，而不会作为点源波传播。在用氯化钾处理使卵膜去极化的未受精卵母细胞中也会出现类似的钙瞬变，如果使用钴来阻断钙通道，受精诱导的皮质闪光就会消失，这共同表明受精最初通过卵膜中的电压门控钙通道触发钙离子内流。然而，在产生皮质闪光后的几分钟内，乳突脑纹虫卵母细胞开始显示一系列游离钙升高的点源振荡波，其传播速度约为15微米/秒。前两个钙波出现在精子融合的部位，通常无法到达对极，但在精子并入后，这些波在整个卵质中全局传播，并且通常将其起源转移到植物皮质中的一个起搏区域。在受精后约60 - 100分钟减数分裂成熟完成时，会产生约10次振荡，平均持续时间为3.3 +/- 1.2分钟，并且在含钴海水或无钙海水中也会继续出现这种波。因此，受精诱导的波状钙振荡显然依赖于内部钙库，根据使用ryanodine、笼锁IP3和肝素的实验，这些钙库可能含有IP3不敏感和/或IP3敏感受体。未受精卵母细胞在胞质内注射完整精子后也会显示重复性钙波，如果在注射前将精子悬液煮沸，这种振荡就会消失，这表明可能存在一种热不稳定的精子成分，在受精过程中可引发波状振荡。
