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

成熟的哺乳动物卵子在第二次减数分裂中期被阻止，并通过受精恢复减数分裂，导致第二极体和雄性和雌性原核的形成。卵的激活是由细胞内Ca^2+浓度的反复增加（Ca^2+振荡）引起的.本研究旨在鉴定能诱导小鼠卵细胞内Ca^2+振荡的精源卵激活蛋白（EAP）。结果表明：（1）仓鼠精子和猪睾丸提取物经不同的层析分离后，注入小鼠卵细胞，测定各组分的Ca^2+振荡诱导活性。2）在CD 9-（一种卵表面蛋白）缺陷的小鼠卵子中，精卵融合缺陷，没有Ca^2+反应发生。通过注射CD 9在小鼠卵细胞中表达，发现精卵融合能力得到恢复。 关于我们 3）注射小鼠精子而不是圆形精子细胞可诱导卵活化。EAP被认为在小鼠中圆形精子细胞和精子之间的阶段表达。受精后，HAP活性从卵细胞质集中到雄、雌原核。4）肌动蛋白丝参与精卵融合和精子入卵。Rho家族的小G蛋白参与精子的掺入，并被梭菌毒素B抑制。5）有报道表明新型磷脂酶C（PLC β）是EAP的强有力的候选者。与荧光蛋白融合的PLC β的表达诱导Ca^<2+>振荡并积聚到原核中。6）使用杆状病毒/Sf 9细胞系统合成PLC β。重组PLC可诱导卵细胞内Ca^2+振荡。在体外实验中，PLC的活性在静息细胞的Ca^<2+>浓度下是有活性的。5和6支持PLC作为EAP的强有力的候选者。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