Characterization of steroid membrane receptor in fish oocyte and its role in the induction of oocyte maturation

鱼类卵母细胞类固醇膜受体的表征及其在诱导卵母细胞成熟中的作用

• 批准号: 17570175
• 负责人: TOKUMOTO Toshinobu
• 金额: $ 2.18万
• 依托单位: Shizuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570175/
• 关键词: membrane progestin receptor alpha; mPRα; oocyte maturation; diethylstilbestrol; DES; endocrine disrupting chemicals; goldfish; プロゲスチン膜受容体; mPR; 内分泌かく乱物質

Oocyte maturation in lower vertebrates is triggered by maturation-inducing steroid (MIS), which acts on receptors located on the oocyte membrane and induces the activation of maturation-promoting factor in the oocyte cytoplasm. During the course of maturation, oocytes undergo drastic morphological changes associated with progression of the meotic cell cycle, among which breakdown of the oocyte nuclear envelope (germinal vesicle breakdown, GVBD) occuring at the prophase/metaphase transition is usually regarded as a hallmark of the progress of oocyte maturation. We conducted the cloning of the membrane progestin receptor (mPR) cDNAs from a goldfish ovarian cDNA library. Four clones for mPR subtypes were obtained by RACE-PCR from goldfish ovary. Among these further characterization was performed on mPRα. Northern blot analysis indicates the presence of a major 2.6 kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafi … More sh (93%) and human (55%) mPRαs. Western blot analysis using a polyclonal goldfish mPRα antibody shows a major immunoreactive band of the predicted molecular weight (40 kDa) in goldfish ovarian membranes. Computer modeling predicts that the deduced protein has seven transmembrane domains, typical of G protein-coupled receptors. Treatment of full grown, late vitellogenic stage follicle-enclosed oocytes in vitro with gonadotropin increased mPRα protein levels. A correlation between mPRα protein levels and the ability of oocytes to undergo GVBD in response to the MIS (maturational competence) was observed after treatment with gonadotropin. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRα blocked both the induction of oocyte maturational competence and mPRα protein upregulation by gonadotropin. These results with the goldfish mPRα protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRα acts as an intermediary in MIS induction of oocyte maturation in teleosts.Further evidence was obtained by using active recombinant protein expressed in human cultured cells. It was demonstrated that both the ovarian plasma membrane receptor in goldfish and goldfish mPRα expressed in transfected cells displayed high affinity, limited capacity, displaceable specific binding for [^3H]-17,20β-DHP. The relative binding affinities of various steroids and EDCs to these two receptor preparations were similar, supporting the idea that mPRα is a MIS receptor in vivo Less

低等脊椎动物的卵母细胞成熟是由成熟诱导类固醇（MIS）触发的，它作用于卵母细胞膜上的受体，诱导卵母细胞细胞质中成熟促进因子的激活。在成熟过程中，卵母细胞经历了与减数分裂细胞周期进展相关的剧烈形态变化，其中卵母细胞核膜的破裂（生发囊泡破裂，GVBD）发生在前期/中期，通常被认为是卵母细胞成熟进程的标志。我们从金鱼卵巢cDNA文库中克隆了膜黄体酮受体（mPR） cDNA。采用RACE-PCR技术从金鱼子房中获得了4个mPR亚型克隆。其中对mPRα进行了进一步的表征。Northern blot分析表明，在卵巢中存在一个2.6 kb的转录本，该转录本编码一个354个氨基酸的蛋白，与seatrout（81%）、斑马鱼…More sh（93%）和人类mPRαs（55%）序列高度一致。使用多克隆金鱼mPRα抗体进行Western blot分析，发现金鱼卵巢膜中存在预测分子量（40 kDa）的主要免疫反应带。计算机模型预测，推导出的蛋白质具有7个跨膜结构域，是典型的G蛋白偶联受体。用促性腺激素处理成熟的卵黄细胞晚期卵泡封闭卵母细胞可提高mPRα蛋白水平。在促性腺激素治疗后，观察到mPRα蛋白水平与卵母细胞对MIS（成熟能力）的反应能力之间的相关性。微量注射含有mPRα反义寡核苷酸的金鱼卵母细胞，可阻断促性腺激素诱导卵母细胞成熟能力和上调mPRα蛋白的作用。金鱼mPRα蛋白的这些结果与之前在斑点seatrout中获得的结果相似，进一步支持了mPRα在MIS诱导硬骨鱼卵母细胞成熟过程中起中介作用的假设。通过在人培养细胞中表达的活性重组蛋白获得了进一步的证据。结果表明，金鱼卵巢质膜受体和转染细胞中表达的金鱼mPRα对[^3H]-17,20β- dhp均表现出高亲和力、容量有限、可置换的特异性结合。各种类固醇和EDCs对这两种受体制剂的相对结合亲和力相似，支持了mPRα在体内较少是MIS受体的观点

项目成果

Interactions of diethylstilbestrol (DES) and DES analogues with membrane progestin receptor α (mPRα) and the correlation with their nongenomic progestin activities

己烯雌酚 (DES) 和 DES 类似物与膜孕激素受体 α (mPRα) 的相互作用及其与非基因组孕激素活性的相关性

• 发表时间: 2007
• 期刊: Endocrinology (in press)
• 作者: Satoh;A.;Sakamaki;K.;Ide;H.;Tamura;K.;Celina Haraguchi;Toshinobu Tokumoto
• 通讯作者: Toshinobu Tokumoto

内分泌撹乱性物質のスクリーニング方法

内分泌干​​扰物筛查方法

• 发表时间: 2005

Interactions of diethylstilbestrol (DES) and DES analogues with membrane progestin receptor α(mPRα) and the correlation with their nongenomic progestin activities

己烯雌酚 (DES) 和 DES 类似物与膜孕激素受体 α (mPRα) 的相互作用及其与非基因组孕激素活性的相关性

• 发表时间: 2007
• 期刊: Endocrinology (In press)
• 作者: Satoh;A.;Sakamaki;K.;Ide;H.;Tamura;K.;Celina Haraguchi;Toshinobu Tokumoto;Celina Haraguchi;Toshinobu Tokumoto;Celina Haraguchi;Toshinobu Tokumoto
• 通讯作者: Toshinobu Tokumoto

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

• DOI: 10.1002/pmic.200600055
• 发表时间: 2006-07-01
• 期刊: PROTEOMICS
• 影响因子: 3.4
• 作者: Horiguchi, Ryo;Dohra, Hideo;Tokumoto, Toshinobu
• 通讯作者: Tokumoto, Toshinobu

Degradation of nucleoproteins by ubiquitin-proteasome system in rat spermatids and human sperm

大鼠精子细胞和人精子中泛素蛋白酶体系统对核蛋白的降解

• 发表时间: 2007
• 期刊: Journal of Histochemistry and Cytochemistry (in press)
• 作者: Satoh;A.;Sakamaki;K.;Ide;H.;Tamura;K.;Celina Haraguchi
• 通讯作者: Celina Haraguchi