Kline D, Kopf G S, Muncy L F, Jaffe L A
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Dev Biol. 1991 Feb;143(2):218-29. doi: 10.1016/0012-1606(91)90072-b.
Activation responses of the frog egg at fertilization include the release of calcium from intracellular stores and the opening of calcium-dependent chloride channels, which produce the fertilization potential. To investigate the presence of guanine nucleotide-binding proteins (G-proteins), and their role in initiation of these events in the egg of the frog Xenopus laevis, we assayed for pertussis and cholera toxin substrates, and applied activators and inhibitors of G-proteins. Pertussis toxin catalyzed the [32P]ADP ribosylation of a Mr 40,000 component, but no cholera toxin substrates were demonstrated. Injection of greater than or equal to 25 pmole of guanosine-5'-O-(3-thiotriphosphate) GTP-gamma-S), an activator of G-proteins, produced a change in membrane potential that mimicked the fertilization potential and also caused cortical granule exocytosis and cortical contraction. Injections of up to 600 pmole of guanosine 3':5'-cyclic monophosphate or 9 nmole of guanosine-5'-(beta-gamma-imido)triphosphate did not active eggs. The membrane potential response to GTP-gamma-S injection showed the same peak and chloride dependence as the fertilization potential, although the duration of the GTP-gamma-S response was somewhat greater. GTP-gamma-S did not activate eggs if the calcium rise was prevented by prior injection of the calcium chelator BAPTA. Injection of up to 200 ng of cholera toxin did not activate eggs. However, eggs were activated by applying 1 nM serotonin to eggs that had been injected with a specific mRNA for the serotonin 1c receptor, a member of the class of receptors that act by way of G-proteins. Egg activation in response to either sperm or serotonin was not inhibited by pertussis toxin, under experimental conditions where approximately 80-90% of the toxin substrate was ADP-ribosylated. These results support the hypothesis that sperm activate Xenopus eggs at fertilization by way of a pertussis and cholera toxin-insensitive G-protein.
蛙卵受精时的激活反应包括从细胞内储存库释放钙以及打开钙依赖性氯通道，从而产生受精电位。为了研究鸟嘌呤核苷酸结合蛋白（G蛋白）的存在及其在非洲爪蟾卵中引发这些事件的作用，我们检测了百日咳毒素和霍乱毒素底物，并应用了G蛋白的激活剂和抑制剂。百日咳毒素催化了一个分子量为40,000的成分的[32P]ADP核糖基化，但未检测到霍乱毒素底物。注射大于或等于25皮摩尔的鸟苷-5'-O-(3-硫代三磷酸)（GTP-γ-S），一种G蛋白激活剂，会引起膜电位变化，模拟受精电位，还会导致皮质颗粒胞吐和皮质收缩。注射高达600皮摩尔的鸟苷3':5'-环一磷酸或9纳摩尔的鸟苷-5'-(β-γ-亚氨基)三磷酸都不会激活卵。对GTP-γ-S注射的膜电位反应显示出与受精电位相同的峰值和对氯的依赖性，尽管GTP-γ-S反应的持续时间稍长。如果预先注射钙螯合剂BAPTA阻止钙升高，GTP-γ-S就不会激活卵。注射高达200纳克的霍乱毒素不会激活卵。然而，通过向注射了5-羟色胺1c受体特异性mRNA的卵施加1纳摩尔5-羟色胺来激活卵，5-羟色胺1c受体是通过G蛋白起作用的受体类别中的一员。在大约80-90%的毒素底物被ADP核糖基化的实验条件下，百日咳毒素不会抑制对精子或5-羟色胺的卵激活。这些结果支持了这样一种假设，即精子在受精时通过一种对百日咳毒素和霍乱毒素不敏感的G蛋白激活非洲爪蟾卵。
