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Functional analysis of oocyte-specific gene, c-1, which express during meiosis

减数分裂期间表达的卵母细胞特异性基因 c-1 的功能分析

基本信息

批准号：
14560234
负责人：
MINAMI Naojiro
金额：
$ 1.86万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

In the present study, we report the protein expression and localization of a novel gene (named Oogenesin) in mouse oocytes and embryos. The gene was first isolated based on its elevated expression in 2-cell embryos developed without oviductal tissue, in which embryos cannot develop beyond the 2-cell stage. Molecular cloning of a near full-length cDNA revealed that the novel gene encodes a protein composed of 398 amino acids corresponding to a predicted molecular mass of 46 kDa. A remarkable characteristic of the gene is that it contains a leucine zipper at positions 203-224 and a leucine-rich domain at positions 137-326. In situ hybridization using an RNA probe in sections of the adult ovary resulted in distinct signals in the oocyte of all stages of follicles. Expression analysis of a novel cDNA isolated from various stages of preimplantation embryos revealed that Oogenesin expression abruptly decreased after the 2-cell stage and disappeared after the 4-cell stage.A western blot analy … More sis using an affinity-purified rabbit polyclonal antibody raised against a synthetic peptide revealed that the molecular size of OOGENESIN protein is approximately 46 kDa, which is consistent with the size predicted on the basis of the Oogenesin cDNA. OOGENESIN is expressed predominantly in oocytes and 1-to 4-cell stage embryos, whereas it is expressed only weakly in morula/blastocyst stage embryos. Increasing expression was observed from oocytes to the 1-cell stage and the expression decreased toward the 4-cell stage. Immunohistochemistry detected strong signals in oocytes of secondary and antral follicles, and weak signals in oocytes of primordial and primary follicles. All signals were detected in the cytoplasm of follicular oocytes. However, immunohistochemical analysis of ovulated oocytes and preimplantation embryos revealed that the protein localized in the nuclei at late 1-cell and early 2-cell stage. These results together with the unique structure of the protein suggest that Oogenesin has important roles in the transcriptional activation, especially in zygotic gene activation of mouse embryos as a novel maternal effect gene. Less

在本研究中，我们报道了一个新基因(命名为Ogenesin)在小鼠卵母细胞和胚胎中的蛋白表达和定位。该基因最初是基于其在没有输卵管组织的2-细胞胚胎中表达升高而被分离出来的，在这些胚胎中，胚胎不能发育到2-细胞阶段以上。分子克隆结果表明，该新基因编码一个由398个氨基酸组成的蛋白质，预测的相对分子质量为46 kDa。该基因的一个显著特征是在203-224位含有一个亮氨酸拉链，在137-326位含有一个富含亮氨酸的结构域。在成年卵巢的切片上使用RNA探针进行原位杂交，结果在所有卵泡的卵母细胞中都有不同的信号。从植入前胚胎的不同阶段分离的新基因的表达分析表明，Ogenesin的表达在2-细胞期后突然下降，在4-细胞期后消失。Western印迹分析…进一步用亲和纯化的兔抗合成肽多克隆抗体进行免疫印迹表明，OOGENESIN蛋白的分子大小约为46 kDa，与根据Ogenesin基因预测的大小一致。OOGENESIN主要在卵母细胞和1-4-细胞期胚胎中表达，而在桑拿期/囊胚期胚胎中表达很弱。从卵母细胞到1-细胞期表达增加，到4-细胞期表达降低。免疫组织化学检测到次级卵泡和有腔卵泡卵母细胞呈强信号，原始卵泡和初级卵泡卵母细胞呈弱信号。所有信号均位于卵泡卵母细胞的胞浆中。然而，对排卵卵母细胞和植入前胚胎的免疫组织化学分析表明，该蛋白定位于1-细胞晚期和2-细胞早期的细胞核中。这些结果和该蛋白独特的结构表明，Ogenesin作为一种新的母体效应基因，在转录激活，特别是在小鼠胚胎合子基因的激活中具有重要的作用。较少