Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation

Abstract

Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRα, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRα in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRα cDNA from a goldfish ovarian cDNA library. 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%), zebrafish (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 MIH (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 MIH induction of oocyte maturation in teleosts.