Determining the Role of the Conserved TRPM Ion Channel in Egg Activation, Using the Drosophila Model

摘要 生育需要成熟的卵母细胞具备转化为胚胎的能力。这种“卵子激活” 随着卵母细胞内钙离子的增加而启动。在哺乳动物中，受精的精子会触发局部的上升 细胞质中的钙离子，能从内部储藏物中释放出一波钙离子，并在卵子中移动。 然后，通过TRPM7通道从外部环境摄取钙离子，从而发生钙离子振荡。 果蝇卵的激活也始于局部钙离子的上升，通过钙离子释放引发一波 内部商店。最初的Ca~(2+)升高是由于从外部环境吸收了Ca~(2+)。最近，我们发现 TRPM是果蝇TRPM7的同源基因，它介导了这种钙摄取。因此，钙离子通过TRPM- 经络是从苍蝇到哺乳动物生殖的基础，剖析经络的机制和作用具有重要意义。 与人类不孕不育诊断及其缓解的相关性。然而，TRPM通道在卵母细胞中是如何运作的， 而钙离子的升高如何与使卵母细胞有能力发育的大分子变化有关？ 在任何有机体中都是未知的。果蝇的遗传优势和巨大的卵子使其成为理想的模式系统 用它来确定这样的机制，并识别对所有动物的这种转变至关重要的分子。我们 我们将通过两个目标来实现这一目标： 首先，我们将调查为什么TRPM渠道会导致局部而不是全球钙的上升： 通道位于卵母细胞极部，还是均匀分布在卵母细胞质膜周围 而且只在极地被激活？除了揭示TRPM通道在卵子激活过程中如何激活外， 我们的结果与这些通道在其他几个与医学相关的生物学中如何在当地发挥作用有关 流程。根据我们发现哪一种假设是正确的，我们未来的研究将确定TRPM如何 在卵子发生过程中定位于极点，或者，非定位通道如何被激活以仅起作用 本地的。 其次，我们将跟进我们的惊人发现，与老鼠的情况类似，尽管有卵 激活异常TRPM缺陷的果蝇卵母细胞可以启动早期胚胎发育，尽管 后来的发展变得不正常。我们将研究TRPM介导的钙内流与血管内流的关系。 卵子激活过程中发生的母体必需蛋白质的磷酸化调节。我们和其他人有 研究表明，钙激活的磷酸酶钙调神经磷酸酶对苍蝇和青蛙的卵子激活都是必不可少的。 利用果蝇特有的遗传和生化优势，我们随后展示了钙调神经磷酸酶的活性 调节卵子中细胞周期调节因子和翻译启动因子的磷酸状态的变化 激活。我们建议在这里确定这些磷酸蛋白质组的变化是否依赖于TRPM- 介导的钙内流。如果他们这样做，我们未来的研究将致力于确定如何在 没有这种磷酸化调节。如果基于钙调神经磷酸酶的磷酸蛋白质组的变化不依赖于 关于TRPM介导的Ca~(2+)内流，我们此时将探讨激活钙调神经磷酸酶的Ca~(2+)的来源。

SUMMARY Fertility requires that a mature oocyte become competent to transform into an embryo. This “egg activation” initiates with increases of Ca2+ in the oocyte. In mammals, the fertilizing sperm triggers a local rise in cytoplasmic Ca2+ that induces a wave of Ca2+ release from internal stores that moves across the egg. Oscillations in Ca2+ then occur via Ca2+ uptake from the external environment through TRPM7 channels. Drosophila egg activation also begins with a local rise in Ca2+ that induces a wave via Ca2+ release from internal stores. The initial Ca2+ rise is due to uptake of Ca2+ from the external environment. Recently, we found that TRPM, the Drosophila ortholog of TRPM7, mediates this Ca2+ uptake. Thus Ca2+ influx through a TRPM- channel is fundamental to fertility from flies to mammals, and dissecting its mechanism and effects is of relevance to human infertility diagnosis and its alleviation. However, how TRPM channels operate in oocytes, and how the Ca2+ rise relates to the macromolecular changes that make the oocyte competent to develop, is unknown in any organism. Drosophila’s genetic advantages, and large eggs, make it the ideal model system with which to determine such mechanisms and identify molecules critical for this transition in all animals. We will do so via two aims: First, we will investigate why TRPM channels cause a local rather than global calcium rise: are the channels localized at the oocyte poles or are they uniformly distributed around the oocyte plasma membrane and only activated at the poles? In addition to revealing how TRPM channels activate during egg activation, our results are relevant to how these channels act locally in several additional medically-relevant biological processes. Depending on which hypothesis we find to be correct, our future studies will determine how TRPM is localized to the poles during oogenesis or, alternatively how a non-localized channel is activated to act only locally. Second, we will follow up our surprising discovery that, similar to the situation in mouse, despite egg activation abnormalities TRPM-deficient Drosophila oocytes can initiate early embryogenesis, although development later becomes abnormal. We will investigate the relationship of TRPM-mediated Ca2+ influx to the phospho- modulation of essential maternal proteins that occurs during egg activation. We and others have shown that the Ca2+-activated phosphatase calcineurin is essential for egg activation in both flies and frogs. Exploiting genetic and biochemical advantages unique to Drosophila, we then showed that calcineurin activity regulates changes in the phospho-state of cell cycle regulators and translation initiation factors during egg activation. We propose here to determine whether these phosphoproteome changes depend on TRPM- mediated Ca2+ influx. If they do, our future studies will aim to identify how development can initiate in the absence of this phosphomodulation. If the calcineurin-based changes to the phosphoproteome do not depend on TRPM- mediated Ca2+ influx, we will investigate the source of the Ca2+ that activates calcineurin at this time.