Bornslaeger E A, Poueymirou W T, Mattei P, Schultz R M
Exp Cell Res. 1986 Aug;165(2):507-17. doi: 10.1016/0014-4827(86)90603-8.
Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.
由环磷酸腺苷（cAMP）依赖性蛋白激酶介导的蛋白质磷酸化对于维持小鼠卵母细胞的减数分裂阻滞至关重要。为了评估由钙/磷脂依赖性蛋白激酶（蛋白激酶C）介导的蛋白质磷酸化是否也可能抑制减数分裂的恢复，我们用该酶的激活剂处理卵母细胞。活性佛波酯12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）和4β - 佛波醇12,13 - 十二烷酸酯（4β - PDD）抑制了生发泡破裂（GVBD），蛋白激酶C的一种更天然的激活剂sn - 1,2 - 二辛酰甘油（diC8）也有同样作用。一种无活性的佛波酯4α - 佛波醇12,13 - 十二烷酸酯（4α - PDD）则不抑制GVBD。然后我们研究了蛋白激酶C激活剂是否抑制cAMP调节的、调控减数分裂恢复的信号通路中的某一步骤。TPA不抑制卵母细胞cAMP中与成熟相关的减少。在存在TPA的情况下，显微注射的cAMP依赖性蛋白激酶的热稳定蛋白抑制剂未能诱导GVBD。TPA和diC8都部分抑制了与减数分裂恢复紧密相关的卵母细胞磷蛋白代谢的特定变化；这些试剂还诱导了特定卵母细胞蛋白的明显磷酸化。这些结果表明，蛋白激酶C激活剂可能通过在cAMP依赖性蛋白激酶活性降低的下游起作用，但在减数分裂恢复可能所需的卵母细胞磷蛋白代谢变化之前起作用，从而抑制减数分裂的恢复。最后，我们比较了二丁酰环磷腺苷（db - cAMP）和蛋白激酶C激活剂对GVBD后极体排放的影响。TPA、4β - PDD或diC8，但不是4α - PDD或db - cAMP，以剂量依赖性方式抑制极体排放。TPA或4β - PDD抑制极体排放的卵母细胞的形态和细胞学特征与用diC8处理的卵母细胞不同。前者30%至60%呈圆形，有一团染色体但无纺锤体；其余的形状膨胀，有中期I纺锤体。然而，所有用diC8处理的卵母细胞都是圆形的，染色体分散，且无纺锤体。这些结果表明，与减数分裂恢复相反，极体排放是由蛋白激酶C的激活而非cAMP依赖性蛋白激酶的激活所抑制。
