Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom.
Mol Biol Cell. 2012 Jan;23(2):371-80. doi: 10.1091/mbc.E11-08-0687. Epub 2011 Nov 23.
Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.
精子特异性磷酯酶 C ζ (PLCζ) 通过在哺乳动物卵子中触发与受精时无法区分的细胞内 Ca(2+) 振荡来激活胚胎发育。体细胞 PLC 同工酶通过水解质膜中的磷脂酰肌醇 4,5-二磷酸 (PI(4,5)P(2)) 产生肌醇 1,4,5-三磷酸介导的 Ca(2+) 释放。在这里，我们研究了精子 PLCζ 在小鼠卵子中靶向的质膜 PI(4,5)P(2) 的亚细胞来源。通过用绿色荧光蛋白标记的 PH 结构域监测卵子质膜 PI(4,5)P(2)，我们表明 PLCζ 对卵母细胞膜的 PI(4,5)P(2) 损失影响最小，与对照 PLCδ1 形成对比，尽管 PLCζ 在引发 Ca(2+) 振荡方面的效力要高得多。通过质膜靶向肌醇多磷酸-5-磷酸酶 (Inp54p) 特异性耗尽该 PI(4,5)P(2) 池阻断了 PLCδ1 介导的 Ca(2+) 振荡，但不能阻断由 PLCζ 或精子刺激的 Ca(2+) 振荡。PI(4,5)P(2)、PLCζ 和无催化活性的 PLCζ (ciPLCζ) 的免疫定位显示它们在卵子皮层内的不同囊泡结构中共定位。这些囊泡在 PLCζ 注射后显示 PI(4,5)P(2) 减少。表达 ciPLCζ 融合的 Inp54p 靶向耗尽囊泡 PI(4,5)P(2) 抑制了由 PLCζ 或精子触发的 Ca(2+) 振荡，但未能影响由 PLCδ1 介导的 Ca(2+) 振荡。与体细胞 PLC 不同，我们的数据表明精子 PLCζ 通过水解内部 PI(4,5)P(2) 库来诱导 Ca(2+) 动员，这表明哺乳动物受精的机制包括一种新型的磷酯酰肌醇信号通路。
