| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
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
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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
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