Characterization and Expression of the Nuclear Progestin Receptor in Zebrafish Gonads and Brain1

The zebrafish nuclear progestin receptor (nPR; official symbol PGR) was identified and characterized to better understand its role in regulating reproduction in this well-established teleost model. A full-length cDNA was identified that encoded a 617-amino acid residue protein with high homology to PGRs in other vertebrates, and contained five domains characteristic of nuclear steroid receptors. In contrast to the multiplicity of steroid receptors often found in euteleosts and attributed to probable genome duplication, only a single locus encoding the full-length zebrafish pgr was identified. Cytosolic proteins from pgr-transfected cells showed a high affinity (Kd = 2 nM), saturable, single-binding site specific for a native progestin in euteleosts, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). Both 17,20beta-DHP and progesterone were potent inducers of transcriptional activity in cells transiently transfected with pgr in a dual luciferase reporter assay, whereas androgens and estrogens had little potency. The pgr transcript and protein were abundant in the ovaries, testis, and brain and were scarce or undetectable in the intestine, muscle, and gills. Further analyses indicate that Pgr was expressed robustly in the preoptic region of the hypothalamus in the brain; proliferating spermatogonia and early spermatocytes in the testis; and in follicular cells and early-stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV) in the ovary. The localization of Pgr suggests that it mediates progestin regulation of reproductive signaling in the brain, early germ cell proliferation in testis, and ovarian follicular functions, but not final oocyte or sperm maturation

Cloning expression and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes

The structures of membrane receptors mediating rapid nongenomic ctions of steroids have not been identified. We describe the cloning f a cDNA from spotted seatrout ovaries encoding a protein that satisfies the following seven criteria for its designation as a steroid embrane receptor: plausible structure tissue specificity cellular istribution steroid binding signal transduction hormonal regulation nd biological relevance. For plausible structure computer modeling predicts that the protein has seven transmembrane domains typical of G protein-coupled receptors. The mRNA (4.0 kb) is only detected in the brain and reproductive tissues on Northern blots. Antisera only detect the protein (40 kDa) in plasma membranes of reproductive tissues. The recombinant protein produced in an Escherichia coli expression system has a high affinity (Kd 30 nM) saturable displaceable single binding site specific for progestins. Progestins alter signal transduction pathways activating mitogenactivated protein kinase and inhibiting adenylyl cyclase in a transfected mammalian cell line. Inhibition of adenylyl cyclase is pertussis toxin sensitive suggesting the receptor may be coupled to an inhibitoryGprotein. Progestins and gonadotropin up-regulate bothmRNA and protein levels in seatrout ovaries. Changes in receptor abundance in response to hormones and at various stages of oocyte development its probable coupling to an inhibitory G protein and inhibition of progestin induction of oocyte maturation upon microinjection of antisense oligonucleotides are consistent with the identity of the receptor as an intermediary in oocyte maturation. These characteristics suggest the fish protein is a membrane progestin receptor mediating a ‘‘nonclassical’’ action of progestins to induce oocyte maturation in fish. Originally published Proceedings of the National Academy of Sciences Vol. 100 No. 5 Mar 200