Analysis of sperm carbohydrate recognition molecules involved in fertilization

研究表明，配子相互作用中存在多种碳水化合物识别系统。本课题研究了与受精有关的猪精子的碳水化合物识别分子。1)利用多价低聚糖探针进行细胞化学分析发现，猪精子头上表达了两种分子，一种是n -乙酰乳胺结构，另一种是Le^x结构。2)这些碳水化合物识别分子在失能精子上比在失能精子上被检测到更强，但在顶体反应的精子上没有被检测到。3)将洗涤剂溶解的猪精子质膜涂于两种低聚糖探针任一种的塑料孔上，添加相应的低聚糖抑制了几种分子与孔的结合。4)亲和层析表明，一个分子（70 kDa）与n -乙酰乳胺结构具有亲和关系。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。根据该序列克隆出编码该蛋白的CDNA，并确定其核苷酸序列。这些结果将有助于我们在分子水平上进一步分析施肥。

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.