Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.     

Identification of rod- and cone-specific phosducins in teleost retinas

Phosducin (PD) is a regulatory protein of vertebrate phototransduction cascades. In mammalian retina, it has been thought that only one kind of PD commonly exists in both rods and cones. However, we have found two kinds of PD (OIPD-R and OIPD-C) in the retina of a teleost, medaka (Oryzias latipes). In situ hybridization and immunohistochemical analysis demonstrated that OIPD-R and -C are selectively expressed in rods and cones, respectively. The antiserum against medaka PDs recognized two kinds of proteins in bluegill (Lepomis macrochirus) retina. These results suggest that rod- and cone-specific PDs exist in teleost retinas, probably creating differences in light adaptation between rods and cones.     

A third photoreceptor-specific GRK found in the retina of Oryzias latipes (Japanese killifish)

We previously reported that the teleost fish medaka (Oryzias latipes, Japanese killifish), possesses two kinds of G protein-coupled receptor kinases (GRKs) in the retina with different localizations: GRK7 (OlGRK-C) in cones and GRK1 (OlGRK-R1) in rods. To further clarify the diversity of teleost photoreceptor GRKs, we sought other medaka GRKs. We found an additional cDNA that encodes a second retina-specific GRK1 (OlGRK-R2). In situ hybridization experiments demonstrated that OlGRK-R2 mRNA is selectively expressed in rods. Sequence analysis of the Fugu rubripes genomic database unveiled a larger diversity of GRKs than previously expected. We also describe the light-dependent regulation of GRK1, a phenomenon that has not been found in other species. Immunocytochemical analysis indicated that OlGRK-R2 is localized in rod outer segments, independent of light condition. OlGRK-R1 is localized in the rod inner segments and synaptic termini of dark-adapted eyes, and moves to rod outer segments after light adaptation. Our studies suggest that the two medaka GRKs are not functionally redundant, and demonstrate a complicated light-dependent regulation of GRK1 in vivo.     

Diverse forms of guanylyl cyclases in medaka fish -- their genomic structure and phylogenetic relationships to those in vertebrates and invertebrates

Fish species such as medaka fish, fugu, and zebrafish contain more guanylyl cyclases (GCs) than do mammals. These GCs can be divided into two types: soluble GCs and membrane GCs. The latter are further divided into four subfamilies: (i) natriuretic peptide receptors, (ii) STa/guanylin receptors, (iii) sensory-organ-specific membrane GCs, and (iv) orphan receptors. Phylogenetic analyses of medaka fish GCs, along with those of fugu and zebrafish, suggest that medaka fish is a much closer relative to fugu than to zebrafish. Analyses of nucleotide data available on a web site (http://www.ncbi. nlm.nih.gov/) of GCs from a range of organisms from bacteria to vertebrates suggest that gene duplication, and possibly chromosomal duplication, play important roles in the divergence of GCs. In particular, the membrane GC genes were generated by chromosomal duplication before the divergence of tetrapods and teleosts.     

Genomic structure and expression of the soluble guanylyl cyclase alpha2 subunit gene in the medaka fish Oryzias latipes

A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).     

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.     

Cloning of Protamine cDNA of the Medaka (Oryzias latipes) and Its Expression during Spermatogenesis

Protamines or sperm specific basic proteins are highly basic low molecular weight proteins that substitute histones in the chromatin of sperm during spermatogenesis. They condense sperm DNA into a highly compact, stable and inactive complex. In this study, cDNA of protamine of the medaka, Oryzias latipes , was cloned to elucidate the molecular mechanisms involved in spermatogenesis. A medaka testis cDNA library constructed in lambda gt 11 showed 2.78X10⁶ independent recombinants. Several positive clones were obtained by immunoscreening with polyclonal antiserum against medaka protamine. Sequencing showed that one of these positive clones, named MP-1, encoded arginine clusters characteristic of protamine. The putative amino acid sequence of MP-1 revealed a remarkable extent of homology with other fish protamines, such as 71% identity with thynnin Y, a sperm specific basic protein isolated from the bluefin tuna, Thunnus thynnus . Northern hybridization using a MP-1 cDNA probe showed that MP-1 mRNA is present exclusively in the testes and that it gave three detectable bands: a major band of 280 b, and two others of 400 b and 500 b. In situ hybridization of a complementary RNA probe (digoxigenine-UTP-labeled MP-1 RNA) revealed that MP-1 mRNA is localized in some secondary spermatocytes and spermatids, but not in primary spermatocytes or spermatogonia. These results differ from those obtained in studies on the rainbow trout by solution hybridization and in situ hybridization.