Mammalian Gamete Surface Organization and Function

描述（由申请人提供）：精卵融合发生在受精过程中形成受精卵的关键时刻。最近的进展表明配子表面的关键蛋白质与膜融合有关。目前的证据表明：(1) 精子 ADAM 可能在融合中发挥作用，(2) 融合需要卵子四跨膜蛋白 CD9 和卵子 GPI 锚定蛋白。 ADAM 蛋白可能具有细胞间粘附活性或蛋白酶活性，我们认为这些都是正常受精所必需的。尽管基因敲除研究表明融合不需要 ADAM 1、2 和 3，但其他 ADAM 家族成员可能也发挥作用。我们将测试不同的 ADAM 是否在精子和卵子质膜的融合中发挥作用。我们还将测试一种已知的蛋白酶 ADAM 24 是否能够产生多精受精的膜阻断作用。由于基因删除研究表明融合需要卵子 CD9，因此我们重点关注 CD9 的作用机制及其在脂质微结构域中的定位。已知 CD9 具有可溶性配体，我们将测试精子是否具有 CD9 结合的相关跨膜配体。在组织培养系中，CD9 与表达 CD9 的细胞表面上的许多其他蛋白质结合。我们将测试卵子 CD9 相关蛋白是否与精子表面结合。基因敲除研究还表明，鸡蛋 GPI 锚定蛋白是融合所必需的。我们建议鉴定关键的 GP1 锚定蛋白并分析其作用模式。由于多种蛋白质似乎在融合中发挥作用，我们询问它们的活动如何协调。我们建议，为了在融合中发挥作用，活性卵分子必须组织成脂质微结构域。因此，这些研究旨在结合分子和细胞水平，为配子膜融合过程带来新的见解。

DESCRIPTION (provided by applicant): Sperm-egg fusion occurs at a critical moment in fertilization at which the zygote is formed. In recent progress key proteins on the gamete surfaces have been implicated in membrane fusion. Current evidence indicates: (1) sperm ADAMs may have a role in fusion and (2) the egg tetraspanin CD9 and egg GPI-anchored proteins are required for fusion. ADAM proteins potentially have either cell-cell adhesion activity or protease activity and we propose that each of these is required for normal fertilization. Although gene knockout studies have shown that ADAMs 1, 2 and 3 are not required for fusion, it is possible that other ADAM family members have a role. We will test if a different ADAM acts in fusion of the sperm and egg plasma membranes. We will also test if a known protease, ADAM 24, acts to create the membrane block to polyspermy. Since gene deletion studies have shown that egg CD9 is required for fusion, we focus on CD9's mechanism of action and its localization to lipid microdomains. CD9 is known to have a soluble ligand and we will test if sperm have a related transmembrane ligand to which CD9 binds. In tissue culture lines, CD9 associates with many other proteins on the surface of a CD9-expressing cell. We will test if an egg CD9-associated protein binds to the sperm surface. Gene knockout studies have also shown that egg GPI-anchored proteins are required for fusion. We propose to identify the critical GPl-anchored protein(s) and analyze its mode of action. Since multiple proteins appear to play a role in fusion, we ask how their activities are coordinated. We propose that to function in fusion the active egg molecules must be organized into lipid microdomains. Thus, these studies are designed to combine molecular and cellular levels to bring new insight into the process of gamete membrane fusion.