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

哺乳动物卵子受精时，细胞内Ca~(2+)浓度([Ca^&lt；2+&gt；]_i)的反复瞬时升高称为Ca^&lt；2+&gt；振荡，并触发卵子的激活。[Ca~(2+)]_i的升高主要是由于内质网钙离子主要通过三磷酸肌醇(IP_3)受体释放所致。有证据表明，精子与卵子融合时，胞质中的精子因子进入卵质是导致细胞内Ca~(2+)振荡的原因。钙振荡诱导因子是卵子激活蛋白(EAP)。磷脂酶C-Zeta(PLCζ)是一种新的IP_3产生酶同工酶。本研究旨在对PLCζ进行表征，以确定PLCζ在受精过程中是否起到EAP的作用。得到了以下结果。1)注射由杆状病毒/SF9细胞表达系统合成的重组PLCζ在低浓度下诱导小鼠卵内钙振荡。2)可编程逻辑控制器ζ对可编程逻辑控制器活性具有很高的钙敏感性，以至于…静息细胞在~100 nm处有较多的酶活性，适合于在受精时引入可能的EAP。3)N-末端Ef-Hand结构域1(Ef1)和Ef2对ζ活性起重要作用，EF3是其高钙敏感性的原因。4)C2结构域与PI(3)P有较强的亲和力，但对PI(5)P的亲和力较弱。5)编码PLCζ的核糖核酸与荧光蛋白“Venus”融合后可引起Ca~(2+)振荡。表达的PLCζ相当于单个精子所含的估计量。6)表达的PLCζ移位到形成的原核中。7)核定位信号位于PLCζ分子的X-Y连接区。一个包含中间折叠的高度协调的三维结构被认为不仅是Ca^&lt；2+&gt；振荡诱导活动所必需的，也是核转位能力所必需的。这些结果支持了可编程逻辑控制器ζ作为电化学反应的有力候选者。为了确定PLCζ在受精时是否起到卵泡刺激素的作用，制备了PLCζ基因敲除小鼠，并制备了PLCζ转基因小鼠，用于在敲除小鼠的精子中保存PLCζ。较少

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