Regulation of capacitative calcium entry in porcine oocytes

描述（由申请人提供）：在哺乳动物受精过程中，精子利用重复的钙信号来激活卵母细胞的发育程序。受精的精子触发细胞内储存的钙的释放，随后钙通过卵母细胞细胞膜的通道流入。尽管储存耗尽所产生的钙流入对于持久信号的产生至关重要，但调节这种容性钙进入的机制在很大程度上是未知的。该建议的总体目标是确定猪卵母细胞中容性钙进入的调节机制。我们的目的是研究STIM1在容性钙进入中的作用。在体细胞中，STIM1被认为是一种钙传感器，用于检测细胞内储存的钙水平，在初步实验中，我们证明猪卵母细胞也表达STIM1同源物。在这里，我们建议通过定量RT-PCR来确定卵母细胞成熟过程中STIM1基因表达的动态。通过使用荧光标记探针，将显示STIM1在储存耗尽后从细胞质溶胶转移到质膜，并将提供数据，表明sirna下调其功能可抑制电容性钙进入。通过提出的实验获得的信息将有助于开发新的卵母细胞激活方法，从而提高核移植技术的效率。这被认为是大规模生产转基因大型动物的关键。这些具有特定基因修饰的动物被认为是许多人类疾病的极有价值的模型：它们有助于更好地了解疾病机制，也有助于制定治疗干预的新战略。通过拟议的实验获得的信息将有助于有效地生产转基因大型动物，用作人类疾病的模型。通过使用具有特定遗传改变的动物，将有可能更好地了解疾病的机制并开发新的有效疗法。

DESCRIPTION (provided by applicant): During fertilization in mammals, the sperm utilizes repetitive calcium signals to activate the oocyte's developmental program. The fertilizing sperm triggers a release of calcium from the intracellular stores that is followed by a calcium influx through channels in the oocyte plasma membrane. Although a calcium influx generated by store depletion is vital for the generation of the long-lasting signal, the mechanisms that regulate this capacitative calcium entry are largely unknown. The overall goal of the proposal is to define the regulatory mechanisms of capacitative calcium entry in porcine oocytes. Our aim is to investigate the role of STIM1 in capacitative calcium entry. In somatic cells, STIM1 has been suggested to serve as a calcium sensor to detect calcium levels in the intracellular stores and in preliminary experiments we demonstrated that porcine oocytes also express a STIM1 homolog. Here we propose to determine the dynamics of STIM1 gene expression during oocyte maturation by means of quantitative RT-PCR. By using a fluorescently labeled probe, STIM1 will be shown to translocate from the cytosol to the plasma membrane after store depletion and data will be presented that downregulation of its function with siRNAs inhibits capacitative calcium entry. The information obtained through the proposed experiments will help in the development of novel oocyte activation methods that will increase the efficiency of nuclear transfer technology. This is believed to be critical for the large scale production of transgenic large animals. Such animals with specific genetic modifications are regarded as extremely valuable models for a great number of human diseases: they help to better understand disease mechanisms and also, to develop new strategies for therapeutic interventions. The information obtained through the proposed experiments will contribute to the effective production of transgenic large animals to be used as models for human diseases. By using the animals with specific genetic alterations it will be possible to better understand the mechanism of diseases and develop new and effective therapies.