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

哺乳动物受精的分子方法及其在基因转移到受精卵中的应用

• 批准号: 02454523
• 负责人: MORI Tsuneatsu
• 金额: $ 3.07万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454523/
• 关键词: Zona pellucida; Sugar chain of Zona pellucida; vitelline membrane; CD4 of vitelline membrane; P56^<lck> of vitelline membrane; MHC class II molecule of sperm; DNA binding molecule of sperm; Signal transduction in fertilization; 透明帯中性糖鎖; 透明帯酸性糖鎖 /

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.

采用单克隆抗体技术，获得了6株抗猪透明带膜蛋白（ZP）的杂交瘤细胞株。这些单克隆抗体识别的表位进行了化学表征。其中4个识别了猪ZP蛋白的一级结构，2个识别了猪ZP蛋白与人ZP蛋白相似的空间结构。此外，对猪ZP的碳水化合物结构的生化分析表明，猪ZP中相当数量的N-连接的中性寡糖以N-乙酰葡萄糖胺残基终止，提示这些糖链可能是精子上半乳糖基转移酶的受体。我们发现了精子后部MHC-II类分子的表达及其与外源DNA结合的可能作用，相应的精子表面MHC-II类分子，卵细胞卵黄膜上可明确观察到与p56 ~+相关的CD 4分子<lck>。此外，在小鼠受精融合阶段，精子头部后部与卵子卵黄之间的粘附是由精子的MHCII类分子和卵子的CD 4分子介导的，它们的信号通过p56介导进入卵子激活<lck>。通过使用RT/PCR技术获得小鼠卵中对应于胸腺细胞中CD 4基因和lck基因部分的RNA转录物。PCR方法，然后对PCR产物进行测序。推测的序列可能分别编码CD 4跨膜区和p56 N端区的氨基酸序列<lck>。这些结果表明，类似于免疫T细胞中的信号转导复合物在小鼠卵中以转录水平表达。

项目成果

Mori,T.et.al.: "生殖・腫瘍免疫と糖鎖" 蛋白質 核酸 酵素. (1992)

Mori, T.et.al.：“复制/肿瘤免疫和糖链”蛋白质、核酸、酶 (1992)。

森 庸厚.他: "生殖系列-親から子への生命の流れ 第七回「大学と科学」公開シンポジウム構演収録集" 「大学と科学」公開シンポジウム組織委員会(クバプロ), (1993)

Tsuneatsu Mori 等人：“生殖系——生命从父母到孩子的流动——第七届“大学与科学”公共研讨会录音集”“大学与科学”公共研讨会组织委员会（Kubapro）， (1993)

Mori,E.,et al.: "Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida gycoproteins" Biochemistry. (1991)

Mori,E.,et al.：“与猪透明带糖蛋白天冬酰胺相关的中性寡糖结构”生物化学。

Mori.T.et.al.: "Studies of mammalian implantation in vitro (I)-Production of laminin from human decidual cell line and its effects on murinetrophoblastic outgrow" J.Reorod.Dev.38. 285-292 (1992)

Mori.T.et.al.：“哺乳动物体外植入研究 (I) - 从人蜕膜细胞系产生层粘连蛋白及其对小鼠滋养层生长的影响”J.Reorod.Dev.38。

森 庸厚他: "生殖・腫瘍免疫と糖鎖" 蚕白質・核酸・酸素(増刊号). 37. 1772-1782 (1992)

Tsuneatsu Mori 等人：“繁殖/肿瘤免疫和糖链”蚕白质、核酸和氧气（特刊）37。1772-1782（1992）。