Characterization and functional analysis of phospholipase C-zeta, a candidate of mammalian egg-activating sperm factor

细胞内Ca^<2+>浓度（[Ca^<2+>]_i）的短暂增加称为Ca^<2+>振荡，发生在哺乳动物卵子受精时，并触发卵子激活。每一次[Ca^<2+>]_i的升高是由于Ca^<2+>主要通过肌醇1,4,5-三磷酸（IP_3）受体从内质网释放所致。有证据表明，Ca^<2+>的振荡是由精卵融合时细胞质精子因子驱动进入卵浆引起的。Ca^<2+>振荡诱导因子是卵细胞活化蛋白（EAP）。目前EAP的有力候选是ip_3产生酶的一种新型同工酶，磷脂酶c - ζ (PLCζ)。本研究旨在表征PLCζ，并确认PLCζ是否在受精过程中作为EAP起作用。得到了以下结果：1)注射杆状病毒/Sf9细胞表达系统合成的重组PLCζ，可诱导低浓度的小鼠卵细胞Ca^<2+>振荡。2) PLCζ对PLC活性具有很高的Ca^<2+>-敏感性，因此在~ 100 nM [Ca^<2+>]_i的静息细胞中，More酶可以被激活，适合于在受精时将假定的EAP引入卵子。3) n端EF-hand结构域1 （EF1）和EF2对PLCζ活性很重要，EF3是其高Ca^<2+>灵敏度的原因。4) C2结构域对PI(3)P有较强的亲和力，对PI(5)P有较弱的亲和力。5)通过注射编码PLCζ的RNA与荧光蛋白“Venus”融合诱导Ca^<2+>振荡。表达的PLCζ对应于单个精子中所含的估计量。6)表达的plc - ζ易位到形成的原核中。7)核定位信号位于PLCζ分子的X-Y连接子区。一个高度协调的三维结构，包括中间的折叠，被认为不仅需要Ca^<2+>振荡诱导活性，而且需要核易位能力。这些结果支持PLCζ作为EAP的有力候选。为了证实PLCζ是否在受精过程中作为EAP起作用，我们制备了PLCζ基因敲除小鼠，并制备了PLCζ基因转基因小鼠，以便在敲除小鼠的精子中保留PLCζ。少

Repetitive transient increase in intracellular Ca^<2+> concentration ([Ca^<2+>]_i) called Ca^<2+> oscillations occur at fertilization of mammalian eggs, and trigger egg activation. Each [Ca^<2+>]_i rise is due to Ca^<2+> release from the endoplasmic reticulum mainly through inositol 1,4,5-trisphosphate (IP_3) receptors. Evidence indicates that Ca^<2+> oscillations are caused by cytosolic sperm factor driven into the ooplasm upon sperm-egg fusion. The Ca^<2+> oscillation-inducing factor is the egg-activating protein (EAP). A current strong candidate of EAP is a novel isozyme of IP_3-producing enzyme, phospholipase C-zeta (PLCζ). This study aims to characterize PLCζ and confirm whether PLCζ operates as EAP at fertilization. The following results were obtained. 1) Injection of recombinant PLCζ that was synthesized by baculovirus/Sf9 cell expression system induced Ca^<2+> oscillations in mouse eggs at low concentrations. 2) PLCζ has such a high Ca^<2+>-sensitivity of PLC activity that the … More enzyme can be active in resting cells at 〜100 nM [Ca^<2+>]_i, suitable for a putative EAP to be introduced into the egg at fertilization. 3) The N-terminal EF-hand domain 1 (EF1) and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca^<2+> sensitivity. 4) C2 domain has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P. 5) Ca^<2+> oscillations were induced by injection of RNA encoding PLCζ fused with a fluorescent protein "Venus". The expressed PLCζ corresponded to the estimated amount contained in a single spermatozoon. 6) Expressed PLCζtranslocated into the formed pronucleus. 7) The nuclear localization signal was located at the X-Y linker region of PLCζ molecule. A highly coordinated three-dimensional structure involving a folding in the middle is thought to be required for not only Ca^<2+> oscillation-inducing activity but also nuclear translocation ability. These results support PLCζ as a strong candidate of EAP. To confirm whether PLCζ operates as EAP at fertilization, PLCζ-knockout mice were produced, and PLCζ-transgenic mice were prepared for reserve PLCζ in the knockout mice sperm. Less