Role of Phosphatidic Acid in the Sperm Acrosome Reaction

描述(申请人提供)：顶体反应(AR)存在于所有脊椎动物精子中，是后续受精成功所必需的。AR是位于精子头部前缘的极大顶体的胞吐作用。这种胞吐作用释放了关键的酶，这些酶穿过卵子周围的外套，使精子得以接近。胞吐作用还将关键的精子膜蛋白移动到精子表面，以允许精子和卵子(或卵子表面)结合。因此，AR是受精的关键一步。然而，AR的机制还没有得到很好的描述。这里概述的一系列实验将检验一种脂质，磷脂酸(PA)在AR诱导中的作用。由于激动剂(人工或与蛋壳中发现的天然诱导剂)诱导AR过程中脂质PA的质量增加，有初步证据支持我们的假设。这些结果利用了我们新的脂类分析方法(高效液相色谱-蒸发光散射质量检测)。为了进一步支持，总结了大量关于胞吐作用涉及脂类PA的报道。然而，与AR相关的PA增加可能是结果而不是原因事件。因此，实验将测试用一种成熟的方法降低精子PA水平是否会抑制顶体反应。此外，在添加外源PA后，将测定经历AR的精子的百分比。再一次，所有的方法(使用共聚焦检测非洲爪哇的AR，添加适当水平的商用PA的程序，脂类分析等)都已经制定出来了。非洲爪哇(一种被NIH指定为动物模式生物的物种)20年的经验，非洲爪哇精子不需要获能的事实(AR研究中的一个复杂和变量)，所有的方法学都已经制定出来，减少了人类伦理问题，使我们选择实验模型是合适的。尽管如果我们的AR模型得到支持，未来的研究将涉及人类精子，但这里概述的研究将增加我们对男性配子的了解，并可能导致体外受精或人类生育能力的改善。人工诱导AR可能会使一些不育的男性受孕。

DESCRIPTION (provided by applicant): The acrosome reaction (AR) is found in all vertebrate sperm and is required for the success of subsequent fertilization. AR is the exocytosis of the very large acrosome that is located at the forward edge of the head of the sperm. This exocytosis releases crucial enzymes that cut through the coats surround eggs to allow the approach of the sperm. The exocytosis also moves crucial sperm membrane proteins to the sperm surface to allow for sperm and egg (or egg surface) binding. Thus, the AR is a crucial step to fertilization. However, the mechanism of the AR is not well described. The series of experiments outlined here will examine the role of a lipid, phosphatidic acid (PA) in the induction of the AR. As the mass of the lipid PA increased during agonist (both artificial or with the natural inducer found in egg coats) induction of the AR, there is preliminary evidence supporting our hypothesis. These results utilize our new lipid analysis methodology (HPLC with evaporative light scattering mass detection). In further support, the numerous reports that exocytosis involves the lipid PA are summarized. However, the increase in PA associated with the AR may be a result not a causative event. Thus, experiments will test whether lowering of the sperm PA levels with a proven method will inhibit the acrosome reaction. Furthermore, the percent of sperm undergoing the AR will be determined after addition of exogenous PA. Once again, all methods (use of confocal to detect the Xenopus AR, procedures for the addition of appropriate levels of commercially available PA, lipid analysis, etc) have been worked out. Twenty years of experience with Xenopus laevis (a species that has been designated by the NIH as an animal model organism), the fact that Xenopus sperm do not have to be capacitated (a complication and variable to the study of the AR), that all methodology has been worked out, the reduced human ethical issues make our selection of experimental model appropriate. Although future studies will involve human sperm if our model for the AR is supported, the studies outlined here will increase our knowledge of the male gamete and may lead to improvements of in vitro fertilization or human fertility. Artificial induction of the AR may enable some infertile males to conceive.