Clotteau G, Dubé F
Centre Océanographique de Rimouski, Département d'Océanographie, Université du Québec à Rimouski, Canada.
Mol Reprod Dev. 1996 Sep;45(1):78-86. doi: 10.1002/(SICI)1098-2795(199609)45:1<78::AID-MRD11>3.0.CO;2-T.
Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K(+)-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with 35S-methionine or 32P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K(+)-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K+ or serotonin (12-15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes.
硬壳蛤（Spisula solidissima）的卵母细胞在减数分裂成熟的前期I停滞，直至受精。我们在不同程度上模拟正常精子诱导的激活过程，分析了磷酸化蛋白的模式。使用高钾海水、佛波酯TPA、血清素或它们的组合来分析其对生发泡破裂（GVBD）和蛋白磷酸化的影响。卵母细胞预先加载35S-甲硫氨酸或32P-磷酸盐，通过聚丙烯酰胺凝胶电泳和放射自显影分析标记蛋白的模式。当比较高钾激活的卵母细胞中磷酸化蛋白的模式与合成蛋白的模式时，发现这两个过程在很大程度上彼此无关。TPA诱导的激活比高钾或血清素诱导的激活慢（GVBD需60分钟）（高钾或血清素诱导GVBD需12 - 15分钟），但对蛋白磷酸化抑制剂6 - 二甲基氨基嘌呤同样敏感，并且导致磷酸化蛋白出现的定性模式相似，但动力学较慢，反映出GVBD较慢。当血清素和TPA都添加到卵母细胞中时，GVBD的动力学处于中间水平（30分钟），磷酸化蛋白的出现也是如此。最后，评估了血清素、TPA或两者激活的卵母细胞中H1激酶活性的发展动力学。与磷酸化蛋白的总体模式相似，组蛋白H1激酶活性增加的程度相似，但动力学反映了每种情况下GVBD的动力学。总之，不同的人工试剂利用不同的途径进行激活，导致GVBD的动力学不同，但在所用试剂典型的延迟后，磷酸化蛋白的总体模式相似。这表明最初可能使用多种途径来激活卵母细胞，但这些不同的途径最终会合并为一个共同的途径，导致磷酸化过程具有高度保守和受调控的序列。
