MOLECULES WHICH MEDIATE MURINE SPERM/ZONA BINDING

精子与周围透明带的紧密结合 卵子是受精过程中的关键事件。因此， 负责这种结合的分子的鉴定是 根本性的重要性。人们普遍认为，在老鼠身上， 精子质膜上的受体与非还原蛋白结合 透明带糖蛋白-3上特异性O-连接糖链末端的研究 (中新社3)。目前的争论集中在受体(S)的身份上 精子及其结合的葡聚糖的结构(S)。获得洞察力 关于精子结合的配体的结构，我们分析了 一系列已知结构的小分子多聚糖的活性 竞争性精子-透明带结合试验。结果显示，一个贝塔- 半乳糖封端但不是β-C1cNAc封端的三糖是低的 活性竞争性抑制物(ED50=42muM)。增加了一个 非还原末端α3-半乳糖基为β-半乳糖基 活跃度增加了8倍。然而，添加α-3-岩藻糖 残基为β-半乳糖基或α-半乳糖基三糖 生产了一种高效的竞争性抑制剂(ED50约为 两种多糖均为0.45微米)。最后，用成对的混合实验 竞争性抑制剂在饱和时表现出相加效应。 岩藻糖化、半乳糖封端的多糖和β-半乳糖封端的多聚糖 被添加到精子和卵子中。这些结果引出了假设 岩藻糖化、半乳糖封端的多糖对 精子上的受体，β-半乳糖具有较低的亲和力 对于受体，这两类多糖结合的方式不同 精子表面的受体。为了检验这些假设，我们建议 合成并放射性标记两种新糖蛋白，并使用这些 探针直接确定它们是否与不同的受体结合。 我们还将研究多糖结构的变化如何影响其 对这些受体的亲和力。接下来，我们建议测试该功能 通过检测这些受体的表达是否受到影响 通过获能，无论它们是介导顶体反应还是 受精所需的。然后我们将鉴定多糖 精子表面的受体通过交联新糖蛋白 与它们各自的受体结合，并检测交叉连接的蛋白质。 最后，使用抗体、糖基转移酶、糖苷酶和 具有糖链结构特异性的凝集素，我们建议 以确定含有特定岩藻糖基化、半乳糖封端的葡聚糖 和/或带有β-半乳糖帽的末端出现在ZP3上。完成 这些实验将提供对 小鼠精子表面分子的结构和功能 调节受精过程中关键事件的透明带。

The tight binding of a spermatozoan to the zona pellucida surrounding an egg is a critical event in fertilization. Thus, the identification of the molecules responsible for this binding is of fundamental importance. It is generally agreed that in the mouse, receptors on the sperm plasma membrane bind to the nonreducing termini of specific O-linked glycans on zona pellucida glycoprotein-3 (ZP3). Current debate centers on the identity of the receptor(s) on sperm and the structure(s) of the glycans they bind. To gain insight into the structures of the ligands bound by sperm, we analyzed the activities of a series of small glycans of known structure in a competitive sperm-zona binding assay. Results showed that a beta- Gal-capped but not a beta-C1cNAc-capped trisaccharide was a low activity competitive inhibitor (ED50=42muM). Addition of a nonreducing terminal alpha3-Gal to the beta-Gal-capped glycan increased activity 8-fold. However, additional of an alpha3-fucose residue to either the beta-Gal- or the alpha-Gal-capped trisaccharide produced a highly effective competitive inhibitor (ED50 approximately 0.45muM for both glycans). Lastly, mixing experiments with pairs of competitive inhibitors demonstrated additive effects when saturating does of fucosylated, Gal-capped glycans and a beta-Gal-capped glycan were added to sperm and eggs. These results lead to the hypotheses that fucosylated, Gal-capped glycans have high affinity for a receptor on sperm, that beta-Gal-capped glycans have a lower affinity for a receptor and that these two classes of glycans bind different receptors on the sperm surface. To test these hypotheses, we propose to synthesize and radiolabel two neoglycoproteins, and use these probes to directly determine whether they bind separate receptors. We will also examine how changes in a glycan's structure affects its affinity for these receptors. We next propose to test the function of these receptors by examining whether their expression is affected by capacitation, whether they mediate the acrosome reaction or are required for fertilization. We will then identify the glycan receptors on the sperm surface by cross linking the neoglcoproteins to their respective receptors and examining the cross-linked protein. Finally, using antibodies, glycosyltransferases, glycosidases and lectins with defined specificities for glycan structures, we propose to determine whether glycans with specific fucosylated, Gal-capped and/or beta-Gal-capped termini are present on ZP3. Completion of these experiments will provide substantial insight into the structures and functions of the molecules on murine sperm and the zona pellucida that mediate critical events in fertilization.