Schatten G, Bestor T, Balczon R, Henson J, Schatten H
Eur J Cell Biol. 1985 Jan;36(1):116-27.
The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.
针对海胆（Lytechinus variegatus和Arbacia punctulata）卵激活离子序列过程中发生的细胞内pH值（pHi）变化，研究了受精过程中微管介导的卵子内运动的调节机制。通过抗微管蛋白免疫荧光显微镜检测精子星体和有丝分裂器形成过程中的微管组装，并利用延时视频微分干涉相差显微镜研究精子和卵细胞核的微管介导迁移。通过对作为荧光素二乙酸酯掺入的细胞质荧光素进行荧光分析，验证细胞内pH值的操作。对卵激活的离子序列进行如下操作：i）在受精时阻断pHi变化或将受精卵的pHi降低至未受精时的值；ii）将未受精卵的pHi人为提高至受精时的值；iii）在先前已阻止受精时pHi变化的受精卵中，人为提高或允许正常的pHi变化。抑制pHi变化的受精卵不组装微管，也不经历正常的微管介导运动。在精子星体组装后将pHi降低至未受精水平的受精卵中，未检测到运动。如果随后允许细胞内pH值升高，则会发生导致分裂和发育的正常运动事件，但会因pH值升高受阻的时间而延迟。微管介导的事件发生在细胞内pH值升高的卵子中，即使是在pH值人为升高的未受精卵中。这些结果表明，卵子微管的形成和正常功能直接或间接地由受精过程中发生的细胞内pH值变化引发。
