Drosophila as a model to dissect the rise and role of calcium in egg activation.

项目摘要 一个分化的成熟卵母细胞被“激活”成为全能的，并开始开始胚胎 通过增加其细胞质游离Ca 2+水平的发展。在哺乳动物中， 增加最初是由精子引起的，它刺激Ca 2+从 内部存储。然而，最近的研究表明，随后的Ca 2+振荡 哺乳动物卵子激活所必需的钙离子需要从卵细胞中流入， 通过未知的渠道和机制，环境进入卵母细胞。 尽管果蝇并不被认为是这些过程的良好模型 因为果蝇的卵在受精前就被激活了，我们最近的研究表明， 卵的激活还涉及局部Ca 2+内流。这种涌入之后是一波 增加了钙离子在卵细胞质中的流动， 哺乳动物卵母细胞所使用的。我们发现，果蝇中最初的Ca 2+内流发生在 在卵母细胞的两极，通过机械敏感通道-最有可能是保守的 TRP家族中的通道。我们建议建立果蝇作为一个新的模式， 卵激活的钙基阶段。我们将使用这个模型来识别 介导精子非依赖性Ca 2+内流和导致Ca 2+通道 只在蛋杆处开放。我们还将确定增加的Ca 2+是否 介导通常伴随卵子的卵母细胞磷酸化蛋白质组的变化 激活，以及这些是否需要Ca 2+信号转导钙调磷酸酶 磷酸化蛋白质组变化。我们希望我们的研究结果能够推广到 哺乳动物卵子激活的机制。此外，保守的蛋白质与Ca 2 +- 依赖性磷酸化修饰将是未来测试的候选者， 评估人类ART条件、卵母细胞质量和辅助诊断不孕症 无法应对现有的ART方法。

Project Summary A differentiated, mature oocyte is “activated” to become totipotent and to begin embryo development through an increase in its cytoplasmic free Ca2+ levels. In mammals, this increase is initially induced by the fertilizing sperm, which stimulates release of Ca2+ from internal stores. However, recent studies have shown that subsequent Ca2+ oscillations that are essential for mammalian egg activation require influx of external Ca2+ from the environment into the oocyte, through as yet unknown channels and mechanisms. Although Drosophila had not been considered a good model for these processes because its eggs activate prior to fertilization, our recent work has shown that Drosophila egg activation also involves a local Ca2+ influx. This influx is followed by a wave of increased Ca2+ that sweeps through the egg cytoplasm, using mechanisms like those used by mammalian oocytes. We showed that the initial Ca2+ influx in Drosophila occurs at the oocyte poles, through mechanosensitive channels – most likely conserved channels in the TRP family. We propose to establish Drosophila as a new model for the Ca2+-based phase of egg activation. We will use this model to identify the channels that mediate sperm-independent Ca2+ influx and the conditions that cause Ca2+ channels to be open only at the egg poles. We will also determine whether the increased Ca2+ mediates the changes to the oocyte phosphoproteome that normally accompany egg activation, and whether the Ca2+ signal transducer calcineurin is required for these phosphoproteome changes. We expect the results of our studies to be generalizable to mechanisms in mammalian egg activation. Moreover, conserved proteins with Ca2+- dependent phosphomodifications will be candidates for future testing as markers for assessing human ART conditions, oocyte quality and aiding the diagnosis of infertilities that cannot respond to current ART methods.