Identification of sperm-derived egg-activating protein at fertilization of mammalian eggs

成熟的哺乳动物卵在第二次减数分裂中期停止，并通过受精恢复减数分裂，导致第二极体和雌雄原核的形成。EGG的激活是通过细胞内Ca~(2+)浓度的反复升高(Ca~(2+)&gt；振荡)来诱导的。本研究旨在鉴定精子源性卵子激活蛋白(EAP)在小鼠卵子中引起Ca~(2+)振荡。1)从金黄地鼠精子或猪睾丸提取液，用不同的层析方法进行分离，并将其注入小鼠卵子中，测定其诱发振荡的活性。在纯化过程中失去了活性，这表明在CD9-(一种卵子表面蛋白)缺陷的小鼠卵子中可能存在两种EAP2功能的代偿成分，精子-卵子融合有缺陷，没有发生钙离子反应。通过INJE…在小鼠卵子中表达CD9，恢复了精卵融合的能力3)注射小鼠精子而不是圆形精子细胞可诱导卵子激活。EAP被认为在小鼠的圆形精子细胞和精子之间的阶段表达。受精后，HAP活性从卵子细胞质集中到雄性和雌性原核。4)肌动蛋白细丝参与精卵融合和精子并入卵子。Dfficile梭菌毒素B抑制Rho家族小G蛋白参与精子掺入。5)有报道表明新型磷脂酶C(PLCζ)是EAP的有力候选者。6)利用杆状病毒/Sf9细胞系统合成PLCζζ。重组磷脂酶Cζ诱导卵细胞内钙振荡。在体外，ζ的PLC活性在静息细胞的Ca~(2+)浓度范围内表现出较高的活性。5和6支持PLCζ作为EAP的有力候选者。较少

Mature mammalian eggs are arrested at the metaphase of the second meiosis, and resume meiosis by fertilization, leading to formation of the second polar body and male and female pronuclei. Egg activation is induced by repetitive increase in intracellular Ca^<2+> concentration (Ca^<2+> oscillation). The present study aimed to identify the sperm-derived egg activating protein (EAP) that induces Ca^<2+> oscillations in mouse eggs. We obtained the following results.1)Extract from the hamster sperm or boar testis was separated using various chromatography and Ca^<2+> oscillation-inducing activity of each fraction was assayed by injecting it into mouse eggs. The activity was lost during purification, suggesting that there may be two compensatory components for the function of EAP.2)In CD9-(an egg surface protein) deficient mouse eggs, sperm-egg fusion is defective, and no Ca^<2+> response occurs. The ability of sperm-egg fusion was found to be restored by expressing CD9 in mouse eggs by inje … More ction of CD9 RNA.3)Egg activation is induced by injection of a mouse spermatozoon but not a round spermatid. EAP is thought to be expressed at the stage between the round spermatid and the sperm in the mouse. After fertilization the HAP activity is concentrated to the male and female pronucleus from the egg cytoplasm.4)Actin filaments are involved in sperm-egg fusion and sperm incorporation into the egg. The involvement of Rho family small G proteins in sperm incorporation was demonstrated by inhibition with Clostridium dfficile toxin B.5)A report showed that novel type of phospholipase C (PLCζ) is a strong candidate of EAP. Expression of PLCζ fused with a fluorescent protein induced Ca^<2+> oscillations and is accumulated into the pronucleus.6)PLCζ was synthesized using baculovirus/Sf9 cell system. Recombinant PLCζ induced Ca^<2+> oscillations in eggs. In vitro PLC activity of PLCζ was active at the Ca^<2+> concentration of resting cells. 5 and 6 supported PLCζ as a strong candidate of EAP. Less