Analysis of sperm carbohydrate recognition molecules involved in fertilization

已有研究表明，多碳水化合物识别系统在配子相互作用中起作用。本项目对猪精子上与受精有关的碳水化合物识别分子进行了研究，得到了以下结果：1)多价低聚糖探针细胞化学分析表明，猪精子头部表达了两种识别N-乙酰乳糖胺结构和Le^x结构的分子。2)这些碳水化合物识别分子在获能精子上比在未获能精子上检测得更强，但在顶体反应的精子上检测不到。3)将洗涤剂溶解的猪精子质膜应用于涂有这两种低聚糖探针的塑料孔中，4)亲和层析表明一个分子(70 KDa)与N-乙酰乳糖胺结构具有亲和力。测定了该蛋白N末端的部分氨基酸序列和蛋白水解肽片段。在此基础上，克隆了编码该蛋白的CDNA，并测定了其核苷酸序列，为进一步在分子水平上进行受精分析奠定了基础。

It has been shown that a multiple carbohydrate recognition system is working in the gamete interaction. In this project, carbohydrate recognition molecules on boar sperm involved in fertilization were investigated and the following results were obtained.1) Cytochemical analysis using multivalent oligosaccharide probes indicated that two kinds of molecules, one recognizing the N-acetyllactosamine structure and the other recognizing the Le^x structure, are expressed on boar sperm heads.2) These carbohydrate recognition molecules are detected more strongly on capacitated sperm than on uncapacitaed sperm, but not detected on acrosome-reacted sperm.3) When the detergent-solubilized plasma membranes of boar sperm were applied to plastic wells coated with either of the two kind of oligosaccharide probes, several molecules bound to the wells and binding was inhibited by addition of corresponding oligosaccharides.4) A molecule (70 kDa) was shown to have an affinity to the N-acetyllactosamine structure by affinity chromatography. Partial amino acid sequences of N-terminal portion and proteolytic peptide fragments of the protein were determined. On the basis of the sequence, CDNA encoding the protein could be cloned and its nucleotide sequence was determined.These results will help us further analysis of fertilization on the molecular level.