CARBOHYDRATE DETERMINANTS IN MAMMALIAN FERTILIZATION

描述：(改编自申请者摘要)这项计划的总体目标 建议是确定特定的碳水化合物决定因素在 哺乳动物精子在受精过程中能识别的透明带。 将使用一些生化和细胞生物学方法来 准确地确定低聚糖的确切性质 糖蛋白ZP3是精子区黏附所必需的 诱导顶体胞吐。增溶法定量分析 和125I标记的透明带糖蛋白来表征 ZP3与其互补受体S在小鼠精子上的相互作用 表面已被开发出来。这些化验将被用来探索 精子识别中的特异性分子相互作用 并以使用先前的细胞结合不可能的方式 化验。使用这种新的分析方法，定义的单糖，简单 将低聚糖与复合低聚糖进行比较，以确定 参与精子黏附的潜在碳水化合物部分 透明带。具体地说，含有末端的寡糖 将对与精子区粘连有关的糖类进行测试。通过 改变这些低聚糖的组成和复杂性， 精子ZP3受体上的碳水化合物识别结构域将是 检查过了。此外，ZP3糖蛋白将被化学解剖以 量化O-连接和N-连接的寡糖的贡献， 以及ZP3上特定的末端单糖，与精子区结合。 这些低聚糖将首先使用凝集素亲和力进行分析 用气相色谱和质谱法测定特定糖类和 ZP3甘氨酸辅蛋白中的寡糖含量。这项研究将产生 有关大分子组成的重要信息 哺乳动物精子-卵子的识别和黏附可能对两者都有影响 为了开发避孕药具和了解具体情况 可能导致不孕症的分子损伤。

DESCRIPTION: (Adapted from applicant's abstract) The overall goal of this proposal is to determine the specific carbohydrate determinants within the zona pellucida that are recognized by mammalian sperm during fertilization. A number of biochemical and cell biological approaches will be used to precisely determine the exact nature of the oligosaccharides on the glycoprotein ZP3, which are necessary for sperm-zone adhesion and the induction of acrosomal exocytosis. Quantitative assays using solubilized and 125I-labeled zone pellucida glycoproteins to characterize the interaction between ZP3 and its complementary receptor(s) on the mouse sperm surface have been developed. These assays will be used to probe the specific molecular interactions involved in the recognition between sperm and egg in a manner which is not possible using previous cell binding assays. Using this new assay, defined monosaccharides, simple oligosaccharides, and complex oligosaccharides will be compared to identify potential carbohydrate moieties which are involved in sperm adhesion to the zona pellucida. Specifically, oligo-saccharides which contain terminal sugars that have been implicated in sperm-zone adhesion will be tested. By varying both the composition and the complexity of these oligosaccharides, the carbohydrate recognition domains on the sperm's ZP3 receptor will be examined. In addition, the ZP3 glycoprotein will be chemically dissected to quantify the contributions of both O-linked and N-linked oligosaccharides, as well as specific terminal monosaccharides on ZP3, in sperm-zone binding. These oligosaccharides will be initially analyzed using lectin affinity chromatography and mass spectrometry to determine the specific sugars and oligosaccharide mass in the ZP3 gly-coprotein. This research will yield important information about the composition of macromolecules involved in mammalian sperm-egg recognition and adhesion and may have implications both for the development of contraceptives and for understanding specific molecular lesions which may lead to infertility.