Lipid Signaling During Fertilization

描述（由申请人提供）：存在许多与受精相关的膜事件：磷脂代谢、精子顶体反应期间的膜融合、精卵融合期间的膜融合、皮质颗粒胞吐期间的膜融合以及微绒毛的变化。令人惊讶的是，只有磷脂酶C激活（PLC）（其导致肌醇1，4，5-三磷酸和sn 1，2-DAG的产生）、钙释放和蛋白激酶C激活已被广泛研究。在过去的15年里，我的实验室使用专业的脂质分析设备和与Avanti Polar Lipids开发的协议，对其他脂质信号通路进行了一系列独特的研究。我们最近的出版物（选自其他文章ASBMB今天）表明磷脂酶D和自分泌运动因子在NIH认可的模型系统（非洲爪蟾）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，并详细说明它们在诱导受精事件中的作用。我们将抑制自分泌运动因子的活性，定量溶血磷脂酸的水平（通过HPLC/ELSD或质谱法测量），并检查对非洲爪蟾受精事件的影响。在最后两个具体的目标，我们将检查磷脂酶D（催化磷脂酸形成）在受精过程中。我们还提供了大量的初步结果，证明增加磷脂酸（PA）的生产涉及受精事件，如DAG的升高，Src，PLC的激活，以及IP 3和细胞内钙的升高。我们将使用已证实的方法来量化（Kd）LPA、PA和Src之间的亲和力。我们将改变脂肪酶D活性（通过使用组成型活性或显性负PLD）或吗啉代对爪蟾磷脂酶D的信息。这些研究将利用我们在过去20年中获得的脂质信号和非洲爪蟾生物学方面的专业知识和专用设备。 公共卫生相关性：虽然在受精过程中有许多脂质事件，但它们尚未得到很好的研究。该提案的资金将支持关于自分泌运动因子和磷脂酶D在受精中的作用的第一项研究。这项工作应该提供深入了解受精的机制，并可能导致识别与人类不育相关的缺陷。

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.