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

在低等脊椎动物中，卵母细胞的成熟是由成熟诱导类固醇（maturation-inducing steroid，MIS）触发的，其作用于位于卵母细胞膜上的受体并诱导卵母细胞胞质中促成熟因子的激活。在卵母细胞成熟过程中，伴随着减数分裂周期的进行，卵母细胞发生了剧烈的形态学变化，其中发生在前期/中期的卵母细胞核膜破裂（germinatingvesiclebreakdown，GVBD）通常被认为是卵母细胞成熟的标志。我们从金鱼卵巢cDNA文库中克隆了膜促性腺激素受体（mPR）cDNA。通过RACE-PCR从金鱼卵巢中获得4个mPR亚型的克隆。其中，对mPRα进行了进一步表征。北方印迹分析表明，在卵巢中存在一个主要的2.6 kb转录本，其编码一个354个氨基酸的蛋白质，该蛋白质与seatrout（81%）、zebrafi 关于我们 sh（93%）和人（55%）mPRαs。使用多克隆金鱼mPRα抗体的Western印迹分析显示金鱼卵巢膜中预测分子量（40 kDa）的主要免疫反应条带。计算机模拟预测，推导的蛋白质有七个跨膜结构域，典型的G蛋白偶联受体。在体外用促性腺激素处理完全生长的卵黄发生晚期卵泡封闭的卵母细胞可增加mPRα蛋白水平。用促性腺激素处理后，观察到mPRα蛋白水平与卵母细胞对MIS（成熟能力）的反应中经历GVBD的能力之间的相关性。将mPRα反义寡核苷酸注射到金鱼卵母细胞中，可阻断促性腺激素对卵母细胞成熟能力的诱导和mPRα蛋白的上调。用金鱼mPRα蛋白获得的这些结果与以前用斑点石斑鱼获得的结果相似，进一步支持了mPRα在MIS诱导硬骨鱼卵母细胞成熟中起中介作用的假设，用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表达的金鱼mPRα对[^3H]-17，20 β-DHP具有高亲和力、有限容量、可置换的特异性结合。各种类固醇和EDCs与这两种受体制剂的相对结合亲和力相似，支持mPRα是体内MIS受体的观点。

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