Carroll D J, Albay D T, Terasaki M, Jaffe L A, Foltz K R
Department of Molecular, Cellular and Developmental Biology and the Marine Science Institute, University of California at Santa Barbara, 93106-9610, USA.
Dev Biol. 1999 Feb 15;206(2):232-47. doi: 10.1006/dbio.1998.9145.
At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.
在受精过程中，海胆卵会经历一系列激活事件，包括钙离子动作电位、内质网释放钙离子、细胞内pH值升高、精子原核形成、丝裂原活化蛋白激酶去磷酸化以及DNA合成。为了研究这些事件中哪些可能是由磷脂酶Cγ（PLCγ）激活引发的，磷脂酶Cγ可产生第二信使肌醇三磷酸（IP3）和二酰基甘油，我们使用PLCγ的重组SH2结构域作为特异性抑制剂。将海胆卵与由牛PLCγ的两个串联SH2结构域组成的GST融合蛋白共同注射，并分别注射：（1）钙离子绿色葡聚糖以监测细胞内游离钙离子，（2）BCECF葡聚糖以监测细胞内pH值，（3）俄勒冈绿dUTP以监测DNA合成，或（4）荧光素70 kDa葡聚糖以监测核膜形成。显微注射PLCγ的串联SH2结构域会产生浓度依赖性的钙离子释放抑制作用，并且还会抑制受精后的皮质颗粒胞吐作用、细胞质碱化、丝裂原活化蛋白激酶去磷酸化、DNA合成以及卵裂。然而，注射PLCγSH2结构域蛋白并不能阻止钙离子动作电位、精子进入以及精子原核形成。显微注射对照蛋白——磷酸酶SHP2的串联SH2结构域，对钙离子释放、皮质颗粒胞吐作用、DNA合成或卵裂没有影响。在关键精氨酸处发生点突变的PLCγSH2结构域显微注射后不抑制受精时的钙离子释放，这一发现进一步表明了PLCγSH2结构域抑制作用的特异性。此外，PLCγSH2融合蛋白不会抑制因显微注射IP3、霍乱毒素、环ADP核糖或环鸟苷酸而引起的钙离子释放。这些结果表明，PLCγ在海胆卵的多个受精事件中起关键作用，包括钙离子释放和DNA合成，但动作电位、精子进入和雄原核形成在没有PLCγ激活或钙离子增加的情况下也能发生。
