MOLECULES WHICH MEDIATE MURINE SPERM/ZONA BINDING

介导鼠精子/透明带结合的分子

• 批准号: 6182616
• 负责人: WILLIAM Wallace WRIGHT
• 金额: $ 24.14万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182616
• 关键词: acrosome; affinity chromatography; carbohydrate receptor; carbohydrate structure; chemical structure function; crosslink; fertilization; fluorescent dye /probe; fucose; hexosan; immunofluorescence technique; laboratory mouse; protein localization; receptor binding; sperm; sperm capacitation; synthetic protein; western blottings; zona pellucida glycoproteins

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.

精子与周围透明细胞的紧密结合 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 至关重要。 人们普遍认为，在小鼠中， 精子质膜上的受体与非还原性 透明质酸糖蛋白-3上特异性O-连接聚糖末端 （ZP3）。 目前的争论集中在受体的身份上， 精子及其结合的聚糖结构。 获得洞察力 精子结合配体的结构，我们分析了 活性的一系列小聚糖的已知结构， 竞争性精子结合试验。 结果显示，beta- Gal-封端的而不是β-C1 cNAc-封端的三糖是低的 活性竞争性抑制剂（ED_（50）= 42 μ M）。 添加 非还原性末端α 3-Gal连接至β-Gal加帽聚糖 活性增加8倍。 然而，另外的α 3-岩藻糖 β-Gal-或α-Gal-封端三糖的残基 产生了高效的竞争性抑制剂（ED 50约为 0.45两种聚糖均为μ M）。 最后，混合实验与对 竞争性抑制剂在饱和时表现出加和效应 岩藻糖基化、半乳糖苷封端聚糖和β-半乳糖苷封端聚糖的剂量 添加到精子和卵子中。这些结果导致的假设 岩藻糖基化的Gal-加帽聚糖对 精子上的受体，β-Gal-加帽聚糖具有较低的亲和力 而这两类聚糖结合不同的 精子表面的受体。 为了验证这些假设，我们建议 合成并放射性标记两种新糖蛋白， 探针来直接确定它们是否结合单独的受体。 我们还将研究聚糖结构的变化如何影响其 对这些受体的亲和力。 接下来我们将测试函数 通过检测这些受体的表达是否受到 通过获能，无论它们介导顶体反应还是 需要受精。 然后我们将鉴定聚糖 通过交联新糖蛋白在精子表面的受体 与它们各自的受体结合并检测交联蛋白。 最后，使用抗体、糖基转移酶、糖苷酶和 凝集素与确定的特异性聚糖结构，我们建议 以确定具有特定岩藻糖基化、Gal-封端的 和/或β-Gal封端的末端存在于ZP 3上。 完成 这些实验将提供实质性的洞察力， 小鼠精子表面分子的结构和功能， 介导受精过程中关键事件的透明细胞。