Molecular approach to mammalian fertilization and its application to gene transfer into fertilized egg

用单抗技术获得了6株能产生抗猪卵透明带(ZP)单抗的杂交瘤细胞株。对这些单抗识别的表位进行了化学表征。此外，对猪ZP糖结构的生化分析表明，猪ZP中相当数量的N-连接的中性寡糖被N-乙酰氨基葡萄糖残基终止，提示这些糖链可能是精子上半乳糖基转移酶的受体。随着对ZP上精子受体的研究，我们发现MHC II类分子在精子后部表达，以及外源DNA与精子结合的可能作用。对应于精子上MHC II类分子与p56^&lt相关的CD4分子；在卵黄膜上明显观察到LCK&gt；。此外，精子头部后部与卵子卵黄之间的黏附是由精子的MHC-II类分子和卵子的CD4分子介导的，它们之间的信号在小鼠受精融合阶段通过p56^；lt；lck&gt；转导到卵子激活。推导出的序列分别编码跨膜氨基酸序列和p56；lt；lck&gt；N-末端氨基酸序列，分别编码CD4的胞质区和N端区。这些结果表明，与免疫T细胞相似的信号转导复合体在小鼠卵子中的转录水平上表达。

By means of monoclonal technology, we obtained six hybridoma cell lines producing mAbs to the porcine zona pellucida (ZP). The epitopes recognized by these mAbs were chemically characterized. Four of them recognized a porcine ZP-specific primary structure of protein and two of them recognized a steric structure of the protein similar in both porcine and human ZPs.Furthermore, the biochemical analysis of carbohydrate structure of porcine ZP revealed that considerable quantities of N-linked, neutral oligosaccharides in porcine ZP were terminated with N-acetylglucosamine residues, suggesting the possible roles of these sugar chains as the acceptors for the galactosyltransferase on sperm.Following the studies of sperm acceptor on ZP, we found the expression of MHC class II molecule on the posterior region of sperm and its possible roles of foreign DNA binding to sperm.Corresponding to the MHC class II molecule on sperm, CD4 molecule associated with p56^<lck> was definitely observed on the vitelline membrane of egg. Besides, the adherence between the posterior region of sperm head and the vitellus of egg was mediated by MHC class II molecule of sperm and CD4 molecule of egg and their signal into egg activation was transduced through p56^<lck> at the fusion step of murine fertilization.Actually, RNA transcript in murine eggs corresponding to parts of the CD4 gene and lck gene in thymocytes were obtained by using RT/PCR methods followed by the sequencing of PCR products. The deduced sequences potentially encode the aminoacid sequence of transmembrane to cytoplasmic domain of CD4 and that of N-terminal domain of p56^<lck> respectively. These results demonstrated that the signal transducing complex similar to that in immune T cells are expressed at the transcriptional levels in murine eggs.