Ward C R, Storey B T, Kopf G S
Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia 19104-6080.
J Biol Chem. 1992 Jul 15;267(20):14061-7.
Zona pellucida (ZP)-induced acrosomal exocytosis in mammalian spermatozoa is thought to be mediated by signal transduction cascades similar to those found in hormonally responsive cells. In order to characterize this process further, we have examined the role of GTP-binding regulatory proteins (G proteins) in coupling sperm-ZP interaction to intracellular second messenger systems in mouse sperm. An in vitro signal transduction assay was developed to assess ZP-G protein dynamics in sperm membrane preparations. Guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), a poorly hydrolyzable analogue of GTP, bound to these membranes in a specific and concentration-dependent fashion which reached saturation at 100 nM. Incubation of the membrane preparations with heat-solubilized ZP resulted in a significant increase in specific GTP gamma S binding in a concentration-dependent fashion with a half-maximal response at 1.25-2 ZP/microliters. Solubilized ZP also caused a significant increase in high affinity GTPase activity in the membranes over basal levels. Mastoparan increased specific GTP gamma S binding to the sperm membranes and stimulated high-affinity membrane GTPase activity to levels consistently greater than that seen with the solubilized ZP. Mastoparan, together with solubilized ZP, gave the same level of stimulation of GTP gamma S binding as mastoparan alone. Pertussis toxin completely inhibited the ZP-stimulated GTP gamma S binding, but only decreased mastoparan-stimulated GTP gamma S binding by 70-80%. Purified ZP3, the ZP component which possesses quantitatively all of the acrosomal exocytosis-inducing activity of the intact ZP, stimulated GTP gamma S binding to the same level as solubilized ZP; ZP1 and ZP2 did not stimulate GTP gamma S binding. ZP from fertilized eggs (ZPf), which does not possess acrosome reaction-inducing activity, also failed to stimulate GTP gamma S binding to sperm membranes. These data demonstrate the direct activation of a Gi protein in sperm membrane preparations in response to the ZP glycoprotein, ZP3, that induces the acrosome reaction. These data imply that Gi protein activation is an early event in the signal sequence leading to sperm acrosomal exocytosis.
透明带（ZP）诱导哺乳动物精子发生顶体反应，被认为是由类似于激素反应细胞中的信号转导级联介导的。为了进一步表征这一过程，我们研究了GTP结合调节蛋白（G蛋白）在小鼠精子中将精子与ZP相互作用偶联到细胞内第二信使系统中的作用。开发了一种体外信号转导测定法，以评估精子膜制剂中的ZP - G蛋白动态。鸟苷5'-3 - O -（硫代）三磷酸（GTPγS）是一种难以水解的GTP类似物，以特定的浓度依赖性方式与这些膜结合，在100 nM时达到饱和。将膜制剂与热溶解的ZP一起孵育导致特定GTPγS结合以浓度依赖性方式显著增加，在1.25 - 2 ZP/微升时达到半数最大反应。溶解的ZP还导致膜中高亲和力GTP酶活性比基础水平显著增加。马斯托帕兰增加了精子膜上特定GTPγS的结合，并将高亲和力膜GTP酶活性刺激到始终高于溶解的ZP所观察到的水平。马斯托帕兰与溶解的ZP一起，对GTPγS结合的刺激水平与单独使用马斯托帕兰相同。百日咳毒素完全抑制了ZP刺激的GTPγS结合，但仅使马斯托帕兰刺激的GTPγS结合降低了70 - 80％。纯化的ZP3是ZP的组成部分，其定量地具有完整ZP的所有顶体反应诱导活性，刺激GTPγS结合到与溶解的ZP相同的水平；ZP1和ZP2不刺激GTPγS结合。来自受精卵的ZP（ZPf）不具有顶体反应诱导活性，也未能刺激精子膜上的GTPγS结合。这些数据证明了精子膜制剂中的Gi蛋白响应诱导顶体反应的ZP糖蛋白ZP3而被直接激活。这些数据表明Gi蛋白激活是导致精子顶体反应的信号序列中的早期事件。
