Cloning and identification of a membrane progestin receptor in goldfish ovaries and evidence it is an intermediary in oocyte meiotic maturation.
Previously, a cDNA clone encoding a protein that satisfies the criteria for its designation as a membrane progestin receptor, mPRalpha, was discovered in spotted seatrout ovaries. Moreover, preliminary evidence was obtained for a role for mPRalpha in maturation-inducing hormone (MIH) induction of oocyte maturation in this species. Here, we describe the cloning of the mPRalpha cDNA from a goldfish ovarian cDNA library. Northern blot analysis indicates the presence of a major 2.6kb transcript in ovaries that encodes a 354 amino acid protein which shows high sequence identity with seatrout (81%), zebrafish (93%), and human (55%) mPRalphas. Western blot analysis using a polyclonal goldfish mPRalpha antibody shows a major immunoreactive band of the predicted molecular weight (40kDa) 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 mPRalpha protein levels. A correlation between mPRalpha 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 mPRalpha blocked both the induction of oocyte maturational competence and mPRalpha protein upregulation by gonadotropin. These results with the goldfish mPRalpha protein are similar to those obtained previously with spotted seatrout, further supporting the hypothesis that the mPRalpha acts as an intermediary in MIH induction of oocyte maturation in teleosts.
Tokumoto
Mika
M
Department of Biology and Geosciences, Faculty of Science, National University Corporation, Shizuoka University, Shizuoka 422-8529, Japan.
Nagahama
Yoshitaka
Y
Thomas
Peter
P
Tokumoto
Toshinobu
T
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006054:Goldfish; D006801:Humans; D016200:Maturation-Promoting Factor / Q000502:physiology; D008540:Meiosis; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000254:growth & development*; D010053:Ovary / Q000737:chemistry*; D011980:Receptors, Progesterone / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D011980:Receptors, Progesterone; D016200:Maturation-Promoting Factor