Analysis of sperm carbohydrate recognition molecules involved in fertilization

研究表明，在配子相互作用中存在着一个多糖识别系统。本课题对猪精子上与受精有关的糖识别分子进行了研究，得到以下结果：1）多价寡糖探针细胞化学分析表明，猪精子上存在两种糖识别分子，一种识别N-乙酰乳糖胺结构，另一种识别Le^x结构，在公猪精子头部表达。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更强烈地被检测到，但在顶体反应的精子上未检测到。3）当将洗涤剂溶解的猪精子质膜应用于涂覆有两种寡糖探针中的任一种的塑料威尔斯孔时，几种分子与威尔斯孔结合，并通过加入相应的寡糖抑制结合。4）一种分子（70 kDa）通过亲和层析显示对N-乙酰乳糖胺结构具有亲和性。测定了该蛋白N端部分和蛋白水解肽片段的部分氨基酸序列。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，为进一步从分子水平上研究受精作用奠定了基础。

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.