Lipid Signaling During Fertilization

描述(申请人提供)：有许多与受精有关的膜事件：磷脂代谢，精子顶体反应期间的膜融合，精子-卵子融合期间，皮质颗粒胞吐期间，以及微绒毛的变化。令人惊讶的是，只有磷脂酶C激活(PLC)(导致肌醇1，4，5-三磷酸和sn-1，2-DAG的产生)、钙释放和蛋白激酶C激活被广泛研究。在过去的15年里，我的实验室使用了专门的脂类分析设备和使用Avanti Polar脂类开发的方案，对其他脂类信号通路进行了一系列独特的研究。我们最近的出版物(选自ASBMB Today的其他文章)提出了磷脂酶D和自体趋化蛋白在NIH认可的模式系统(非洲爪哇)受精中的作用。我们的长期目标是获得受精过程中激活的脂质信号通路的调查，并详细说明它们在受精事件诱导中的作用。我们将抑制自体趋化蛋白的活性，量化溶血磷脂酸的水平(通过高效液相色谱/蒸发光散射光谱分析或质谱仪测量)，并研究其对非洲爪哇受精事件的影响。在后两个具体目标中，我们将检测受精过程中的磷脂酶D(催化磷脂酸的形成)。我们还提供了大量的初步结果，证明磷脂酸(PA)的产生增加与受精事件有关，如DAG的升高，Src，PLC的激活，以及IP3和细胞内钙的升高。我们将使用成熟的方法来量化(Kd)LPA、PA和Src之间的亲和力。我们将改变脂肪酶D的活性(通过使用结构性活性或显性阴性的PLD)或吗啉来对抗非洲爪哇的磷脂酶D信息。这些研究将利用我们在过去20年中在脂质信号和非洲爪哇生物学方面获得的专业知识和专门设备。 公共卫生相关性：虽然在受精过程中有许多脂质事件，但它们还没有得到很好的研究。这项提案的资金将支持关于自体趋化素和磷脂酶D在受精过程中的作用的第一项研究。这项工作应该有助于深入了解受精的机制，并可能导致识别与人类不孕不育有关的缺陷。

DESCRIPTION (provided by applicant): There is numerous membrane events associated with fertilization: phospholipids metabolism, membrane fusion during the sperm acrosome reaction, during sperm-egg fusion, during cortical granule exocytosis, and changes in microvilli. Surprisingly, only phospholipase C activation (PLC)(which leads to production of inositol 1,4,5- trisphosphate and sn 1,2-DAG, calcium release and protein kinase C activation, has been widely studied. Over the 15 years, my lab has used specialized lipid analysis equipment and protocols developed with Avanti Polar Lipids to undertake a unique series of studies on other lipid signaling pathways. Our recent publication (selected over other articles by ASBMB Today) suggests roles for phospholipase D and autotaxin in fertilization in an NIH-recognized model system (Xenopus laevis). Our long term goal is obtain a survey of lipid signaling paths activated during fertilization and detail their role in the induction of fertilization events. We will inhibit autotaxin activity, quantify the levels of lysophosphatidic acid (measured by HPLC/ELSD or mass spectrometry) and examine the effect upon fertilization events in Xenopus. In the last two specific aims, we will examine phospholipase D (which catalyzes phosphatidic acid formation) during fertilization. We also provide extensive Preliminary Results demonstrating that increased phosphatidic acid (PA) production is involved in fertilization events such as the elevation of DAG, the activation of Src, PLC, and elevation of IP3 and intracellular calcium. We will use proven methods to quantify (Kd) the affinity between LPA, PA and Src. We will alter lipase D activity (by use of constitutively active or dominant negative PLD) or morpholinos against Xenopus phospholipase D message. These studies will make use of our expertise and specialized equipment in lipid signaling and Xenopus biology obtained over the past 20 years. PUBLIC HEALTH RELEVANCE: Although there are many lipid events during fertilization, they have not been well studied. Funding of this proposal will support the first study on the role of autotaxin and phospholipase D in fertilization. This work should provide insight into the mechanism of fertilization and may lead to identification of defects associated with human infertility.