Identification and characterization of two distinct GnRH receptor subtypes in a teleost, the medaka Oryzias latipes.

We report the identification and characterization of two distinct GnRH receptor (GnRH-R) subtypes, designated GnRH-R1 and GnRH-R2, in a model teleost, the medaka Oryzias latipes. These seven-transmembrane receptors of the medaka contain a cytoplasmic C-terminal tail, which has been found in all other nonmammalian GnRH-Rs cloned to date. The GnRH-R1 gene is composed of three exons separated by two introns, whereas the GnRH-R2 gene has an additional intron and therefore consists of four exons and three introns. The GnRH-R1 and GnRH-R2 genes, both of which exist as single-copy genes in the medaka genome, were mapped to linkage groups 3 and 16, respectively. Inositol phosphate assays using COS-7 cells transfected with GnRH-R1 and GnRH-R2 demonstrated that they had remarkably different ligand sensitivities, although both receptors showed highest preference for chicken-II-type GnRH. Phylogenetic analysis showed the presence of three paralogous lineages for vertebrate GnRH-Rs and indicated that neither GnRH-R1 nor GnRH-R2 is the medaka ortholog to mammalian GnRH-Rs that lack a cytoplasmic tail. This, together with an observation that medaka-type GnRH had low affinity for GnRH-R1 and GnRH-R2, suggests that a third GnRH-R may exist in the medaka.

Structural and expression analyses of gonadotropin Ibeta subunit genes in goldfish (Carassius auratus).

Gonadotropin (GTH) is a pituitary glycoprotein hormone that regulates gonadal development in vertebrates. In teleosts, it is considered that two types of GTH, GTH I (follicle-stimulating hormone-like GTH) and GTH II (luteinizing hormone-like GTH), are produced in the pituitary, and their molecules are comprised of common alpha and distinct beta subunits. In this study, we describe the complete structure and 5'-flanking regulatory region of two distinct genes encoding GTH Ibeta in goldfish, Carassius auratus. The two goldfish GTH Ibeta genes, gfGTHIbeta-1 and gfGTHIbeta-2, span 1719 and 1545 base pairs (bp) nucleotides, respectively, and there is a high sequence identity (92.1%) between the coding regions. Both genes consist of three exons separated by two introns as in mammalian FSH beta genes. The locations of the first intron and second intron showed a well-conserved pattern similar to those of mammalian FSH beta genes. Inspection of the 5'-flanking region of the gfGTHIbeta-1 and gfGTHIbeta-2 (approximately 1.4 and 1.1kb, respectively) revealed the presence of several putative cis-acting elements, including the gonadotrope-specific element, gonadotropin-releasing hormone responsive element, and half steroid hormone responsive elements. Interestingly, some of their elements were located contiguously between -187 and -124bp upstream from a TATAA sequence. Reverse transcription polymerase chain reaction confirmed that these two genes are expressed in the pituitary of individual fish. These results, taken together, demonstrate that there are at least two functional genes encoding GTH Ibeta, probably due to the tetraploidy of goldfish. The unique locations of the cis-acting elements in the GTH Ibeta genes suggest they may be involved in the expression of the goldfish GTH Ibeta gene.

Two differing precursor genes for the salmon-type gonadotropin-releasing hormone exist in salmonids.

Salmon gonadotropin-releasing hormone (sGnRH) is considered to have an important role in the control of reproduction in salmonid fish. As a basis for understanding the physiological functioning of sGnRH at the molecular level, we characterized the nucleotide sequences of two types of cDNAs encoding the precursors of sGnRH in sockeye salmon (ss), Oncorhynchus nerka, by a cloning strategy based on reverse transcription-PCR. The two types of cDNAs are referred to as ss-pro-sGnRH-I and -II, and consisted of 435 and 481 bases respectively. Both precursors are predicted to contain a signal peptide, the hormone and a GnRH-associated peptide that is attached to the hormone via a Gly-Lys-Arg sequence. The presence of two types of mRNAs hybridizing with either cDNA was confirmed by Northern blot analysis of brain RNA from sockeye salmon, masu salmon, O. masou, and rainbow trout, O. mykiss. The ss-pro-sGnRH-I cDNA had 97.2% and 82.8% overall identity with sGnRH cDNA from masu salmon and putative sGnRH cDNA deduced from the gene of the Atlantic salmon, Salmo salar respectively, whereas the ss-pro-sGnRH-II cDNA had 80.0% and 91.2% overall identity with the former and the latter respectively. The nucleotide sequences of ss-sGnRH-I and -II cDNAs showed less similarity (79.3%). These results indicated that each salmonid species possesses two differing sGnRH genes. The results of Southern blot analysis using genomic DNA extracted from individuals support this evidence in sockeye salmon, masu salmon and rainbow trout.

Medaka (Oryzias latipes) FTZ-F1 potentially regulates the transcription of P-450 aromatase in ovarian follicles: cDNA cloning and functional characterization.

Our previous findings suggest the activity of cytochrome P-450 aromatase (P-450arom), the enzyme which converts testosterone to estradiol-17beta, in the ovarian follicle of medaka (Oryzias latipes) is regulated at the transcriptional level. In this study, we cloned a cDNA encoding a FTZ-F1-like protein (mdFtz-F1) from ovarian follicles of medaka. In vitro translated mdFTZ-F1, and nuclear extract from medaka ovarian follicles, formed complexes with oligonucleotide probes containing putative orphan nuclear receptor binding motifs, which are present in the promoter region of the medaka P-450arom gene. The expression pattern of mdFtz-F1 transcripts during oogenesis coincides with that of P-450arom transcripts. Transfection assays further suggest a potential transcriptional regulatory activity of mdFTZ-F1 upon the medaka P-450arom promoter. Taken together, these results suggest a potential role of mdFTZ-F1 in the transcriptional regulation of P-450arom in the ovarian follicle of medaka.

Molecular Cloning and Characterization of a cDNA Encoding the Retinal Arylalkylamine N-Acetyltransferase of the Rainbow Trout, Oncorhynchus mykiss.

Melatonin synthesis in the retina as well as in the pineal gland exhibits daily variations with higher levels during the dark phase of light-dark cycles. To analyze the molecular mechanism of melatonin synthesis in the retina, we have cloned, sequenced and characterized a putative cDNA for arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), a rate-limiting enzyme in melatonin production, from the retina of the rainbow trout (Oncorhynchus mykiss). The trout AANAT cDNA (1,585 bp) contains an open reading frame encoding 240 amino acid protein (predicted molecular weight, 27,420) that is 51-65% identical to avian and mammalian AANAT. The trout retinal AANAT protein contains motifs A and B that are conserved among the N-acetyltransferase superfamily and eight potential phosphorylation sites. Southern blot analysis demonstrated that the protein is expressed by a single copy gene. A single AANAT transcript (1.6 kb) was detected in the retina but not in the liver by Northern blot analysis. The levels of AANAT mRNA in the retina exhibited day-night changes with 3.3-fold increase at night. These results indicate that in the rainbow trout retina, the activity of AANAT and thus melatonin synthesis are regulated at least in part at the transcriptional level.

Fish gonadotropin and thyrotropin receptors: the evolution of glycoprotein hormone receptors in vertebrates.

We have cloned and characterized, for the first time in fish, two different gonadotropin receptors (GTHR) and a single thyrotropin receptor (TSHR) from amago salmon (Oncorhynchus rhodurus) and Nile tilapia (Oreochromis niloticus). Phylogenetic analyses and intron/exon structure suggest that the two GTHRs in fish are comparable to tetrapod follicle stimulating hormone and luteinizing hormone receptors. Temporal and spatial expression patterns, examined by Northern blot analysis and in situ hybridization, paralleled those seen in mammals and birds. Consequently, genetic and functional divergence of two GTHRs and TSHR probably occurred before the teleost and tetrapod split.

Duality of gonadotropin in a primitive teleost, Japanese eel (Anguilla japonica).

The duality of gonadotropin (GTH) is well-established in relatively modern teleosts. In primitive teleosts such as eel and catfish, however, only a single GTH (GTH-II) has been isolated and characterized. Therefore, the objective of this study was to clarify the duality of GTH, particularly the presence of GTH-I in primitive teleosts. We attempted to obtain a cDNA encoding the beta subunit of GTH-I from Japanese eel, Anguilla japonica, as a representative primitive teleost species. Rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) was used to amplify GTH-Ibeta cDNA prepared from immature male Japanese eel pituitaries, and the obtained PCR products were subcloned and sequenced. A degenerate PCR primer was designed based on a highly conserved region between teleost GTH-Ibeta and mammalian FSHbeta. DNA sequence analysis of the cloned PCR products confirmed the presence of the predicted complete coding region as well as the 5' and 3' untranslated regions. The deduced amino acid sequence from these clones showed high homology to goldfish GTH-Ibeta (60%), whereas the identity between Japanese eel GTH-Ibeta and GTH-IIbeta was lower (42%). Phylogenetic analysis confirmed that Japanese eel GTH-Ibeta belonged to the teleost GTH-Ibeta group. These results provide a definitive proof of the presence of two types of GTHs (GTH-I and GTH-II) in Japanese eel, as has been shown in other teleosts. The duality of GTHs is applicable for teleosts in general. Northern blot analysis showed the transcripts of Japanese eel GTH-Ibeta and GTH-IIbeta to be 1200 and 750 bases, respectively. GTH-Ibeta gene was expressed in immature fish, while GTH-IIbeta gene was expressed in spermiating males and ovulated females, suggesting that two GTHs are differentially expressed at different sexual stages and may play separate roles in the reproductive process in Japanese eel.

Molecular cloning of cDNA encoding two types of pituitary gonadotropin alpha subunit from the goldfish, Carassius auratus.

Two types of cDNA encoding the alpha subunit of pituitary gonadotropin (GTH) were cloned by polymerase chain reaction (PCR) for goldfish pituitary cDNA library. The goldfish GTH-alpha subunit cDNAs, designated as alpha 1 and alpha 2, encode 117 and 118 amino acids, respectively, including a 23-amino-acid signal peptide. These two types of cDNAs showed high homology in nucleotide and amino acid sequence, but deletion of a triplet nucleotides was present in alpha 1. Deduced amino acid sequences represented differences in four residues between alpha 1 and alpha 2 subunits. Analyses of goldfish genomic DNA revealed that each individual goldfish possesses two distinct genes relevant to GTH-alpha 1 and -alpha 2 subunits. Results of reverse transcriptase-PCR analysis suggest that these two GTH-alpha subunit genes are expressed at different levels without relation to sex and gonadal maturity.

Molecular cloning of the cDNAs encoding two gonadotropin beta subunits (GTH-I beta and -II beta) from the goldfish, Carassius auratus.

Two types of cDNAs (GTH-I beta and -II beta) that encode the beta subunit of goldfish gonadotropin were cloned from a goldfish pituitary cDNA library. The nucleotide sequence of GTH-I beta cDNA was 616 bp long, encoding 130 amino acids, and that of GTH-II beta was 552 bp long, encoding 140 amino acids. These two types of goldfish GTH beta subunits showed an identity of 52% in the nucleotide sequence and 38% in the amino acid sequence. When compared to mammalian GTH beta subunits, goldfish GTH-I beta showed higher homology to FSH-beta s than LH-beta s, and goldfish-II beta to LH-beta s. Genomic Southern blot analysis revealed that cyprinid species harbored genes which were homologous to goldfish GTH-I beta and -II beta, demonstrating the duality of GTH in cyprinid fish as has been shown in other teleost fish. Northern blot analysis of GTH-I beta and -II beta mRNAs in goldfish at differing stages of ovarian maturity (immature, maturing, mature, and regressed) showed that both GTH-I beta and -II beta mRNA levels increased with the progression of maturity and declined in regressed stage. These results suggested that gonadal development in the goldfish was regulated by the two GTHs.

cDNA cloning of two gonadotropin beta subunits (GTH-Ibeta and -IIbeta) and their expression profiles during gametogenesis in the Japanese flounder (Paralichthys olivaceus).

To clarify the profiles of two distinct gonadotropin (GTH-I and -II) mRNA levels during gametogenesis in a multiple spawner, the Japanese flounder (Paralichthys olivaceus), the cDNAs encoding GTH-Ibeta and -IIbeta from the pituitary gland have been cloned and sequenced. The nucleotide sequence of GTH-Ibeta was 542 bp long, encoding 120 amino acids, and that of GTH-IIbeta was 554 bp long, encoding 145 amino acids. In females, Northern blot analysis has revealed that relative mRNA levels of GTH-Ibeta and -IIbeta were low in immature fish, showed a gradual increase with ovarian development, and reached the highest level at the maturation stage. Both GTH-Ibeta and -IIbeta mRNA levels were highly correlated with gonadosomatic index (GSI) values and with circulating estradiol-17beta and testosterone (T) levels. In males, the mRNA levels of GTH-Ibeta increased with the increase in GSI values and in circulating 11-ketotestosterone and T levels, whereas the mRNA levels of GTH-IIbeta did not show any correlation with GSI values and with circulating steroid levels, suggesting a difference in regulatory mechanisms of GTH-I and -II synthesis in males. The similar changes in GTH-Ibeta and -IIbeta mRNA levels during oogenesis are considered to be characteristic of GTH synthesis in multiple spawners, differing from the differential changes reported in annual spawners such as salmonids.

Recombinant goldfish activin B stimulates gonadotropin-Ibeta but inhibits gonadotropin-IIbeta expression in the goldfish, Carassius auratus.

It is well documented that the pituitary in teleosts produces two gonadotropins, namely gonadotropin-I (GTH-I) and gonadotropin-II (GTH-II), which may regulate different phases of the reproductive cycle. However, unlike in mammals, very little is known about the differential regulation of the two GTHs in fish. Using goldfish as a model, the present study demonstrates, for the first time, that activin, a protein factor that plays a critical role in the differential regulation of mammalian FSH and LH, has opposite effects on GTH-Ibeta and GTH-IIbeta mRNA expression. Recombinant goldfish activin B stimulates GTH-Ibeta but significantly suppresses GTH-IIbeta mRNA levels in a dose-dependent manner in cultured goldfish pituitary cells. Administration of recombinant human follistatin completely abolished the effects of activin, thus demonstrating the specificity of the activin activities. The novel opposite effects of activin on the two goldfish GTHs make goldfish a very unique vertebrate model for activin studies. The present study not only contributes to our understanding of the mechanisms that control the temporal expression patterns of the two GTHs during the fish reproductive cycle, but also provides important information on the evolution of gonadotropin regulation in vertebrates.

Seasonal changes in mRNA levels of gonadotropin and thyrotropin subunits in the goldfish, Carassius auratus.

Seasonal changes in the mRNA levels of glycoprotein alpha, gonadotropin (GTH) Ibeta and IIbeta, and thyrotropin (thyroid-stimulating hormone (TSH)) beta subunits in the pituitary of goldfish were quantified by Northern blot analysis and laser densitometry. Reproductive development and thyroid activity were monitored by measuring gonadosomatic index, plasma GTH II, testosterone (T), estradiol, 11-ketotestosterone, and thyroid hormones (T4 and T3). Plasma GTH II and steroids showed characteristic increases, while plasma thyroid hormones levels, in general, decreased in association with the reproductive period. In females, the mRNA levels of the alpha, GTH Ibeta, and GTH IIbeta subunits increased synchronously during early spawning period (April) and then decreased during ovarian regression (August). In males, the levels of the alpha and GTH IIbeta subunits showed changes similar to those in females, but the GTH Ibeta mRNA levels showed only a small increase during the late spawning period (May). In both sexes, TSHbeta mRNA levels were high during winter to early spring (February and April) and low during late spring to summer (May and August). These results suggest that in goldfish the gonadotropins may be synthesized synchronously in order for asynchronous gametogenesis to take place. Additionally, the data suggest a negative feedback relationship between synthesis of the TSHbeta subunit and the thyroid hormones.

Cloning and phylogenetic relationship of red drum somatolactin cDNA and effects of light on pituitary somatolactin mRNA expression.

The nucleotide sequence for red drum somatolactin (SL) cDNA was determined and the expression of pituitary SL mRNA was examined in red drum kept under various light conditions. A full length of SL cDNA (1629 bp) was isolated and characterized from a red drum pituitary cDNA library. The SL cDNA has an open reading frame of 696 nucleotides which encodes a 24-amino-acid signal peptide and a 207-amino-acid mature peptide. Red drum SL shares 58-87% amino acid sequence identity and 56-85% nucleotide sequence identity with other teleost SLs. The characteristic seven cysteine residues and one N-glycosylation site of SL are well conserved in the red drum SL mature peptide. Phylogenetic analysis shows that red drum SL is closely related to seabream SL and is also closely related to lumpfish, flounder, halibut, and sole SLs, whereas SLs of Atlantic cod, chum salmon, rainbow trout, and eel are more distantly related to those of the more advanced teleosts. Two SL transcripts, designated as SL I at 1.8 kb and SL II at 1.3 kb, are expressed in red drum pituitaries and correspond to two polyadenylation signal sites in red drum SL cDNA at nucleotide positions 1554 and 1270. Levels of the SL I mRNA were 2- to 4-fold higher in pituitaries of blind red drum and intact fish kept under constant darkness for 1 week than those in control fish sampled during the light phase of the light-dark cycle. Similarly, pituitary levels of SL II mRNA were 9-fold higher in blind fish and 1.6- to 4-fold higher in intact fish kept under constant darkness than in the control fish. Furthermore, these changes in mRNA levels in pituitaries were accompanied by more than 10-fold increases in SL protein concentrations in plasma. The finding that the absence of light perception for extended periods leads to dramatic increases in SL mRNA expression as well as SL secretion in red drum provides further evidence that illumination levels and SL physiology are intimately related in this species.

Cloning, functional characterization, and expression of thyrotropin receptors in the thyroid of amago salmon (Oncorhynchus rhodurus).

Two thyrotropin receptor cDNAs (sTSH-Ra and sTSH-Rb) were cloned from thyroid tissue of the amago salmon, Oncorhynchus rhodurus. sTSH-Ra and sTSH-Rb showed the highest degrees of sequence homology to mammalian TSH receptors. Functional characterization in COS-7 cells transiently transfected with sTSH-Ra or sTSH-Rb showed the largest increase in cAMP when exposed to bovine TSH. RT-PCR analysis demonstrated that sTSH-Ra and sTSH-Rb were expressed in the basibranchial region, but not in the ovary, testis, liver, kidney or brain. In situ hybridization revealed that sTSH-Ra and sTSH-Rb were exclusively expressed in thyroid follicular epithelial cells of amago salmon undergoing smoltification. These results indicated that the cloned cDNAs encode functional TSH receptor proteins. This is the first report of isolation of TSH receptor molecules from nonmammalian vertebrates.

A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes.

The present study has identified three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain of a teleost, the medaka, by isolation of their cDNAs. This species has a novel GnRH, which is here named medaka-type GnRH (mdGnRH), in addition to two characterized forms, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH). Phylogenetic analysis showed that mdGnRH is a medaka homolog of and seabream-type GnRH (sbGnRH) and mammalian-type GnRH (mGnRH) in other species, and suggested that all vertebrates have three distinct GnRHs. Furthermore, in situ hybridization revealed that the mdGnRH gene is expressed only in neurons clustered within the preoptic area as sbGnRH and mGnRH genes in other species are, while the genes for cGnRH-II and sGnRH are only in the midbrain tegmentum and nucleus olfactoretinalis, respectively. This result suggested that mdGnRH is a hypophysiotropic factor and the other two forms are involved in other physiological events as neuromodulators or neurotransmitters.

Two differing salmon GnRH precursor mRNAs are co-expressed in the brain of sockeye salmon.

The localization of two salmon-type gonadotropin-releasing hormone (sGnRH) precursors, pro-sGnRH-I (short type) and pro-sGnRH-II (long type), was investigated by using in situ hybridization techniques in the brain of the landlocked sockeye salmon, Oncorhynchus nerka. We used 30-mer oligonucleotide probes complementary to pro-sGnRH-I and pro-sGnRH-II cDNA. No significant differences were observed in the localization of sGnRH neurons expressing pro-sGnRH-I and pro-sGnRH-II mRNAs; both were expressed in the olfactory nerve, the olfactory bulbs, the regions between the olfactory bulb and telencephalon, the ventral telencephalon, the preoptic area, and the hypothalamus. Almost all sGnRH neurons examined co-expressed both precursors. The expression of two sGnRH precursors in the same neuron and the wide distribution of such neurons in the brain suggest that there are no functional differences between the two precursors.

The duality of fish gonadotropin receptors: cloning and functional characterization of a second gonadotropin receptor cDNA expressed in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

We previously isolated a cDNA encoding a gonadotropin receptor (sGTH-R) from amago salmon (Oncorhynchus rhodurus) ovarian follicles. In the present study, we cloned a second gonadotropin receptor (sGTH-RI) from the same RNA preparations. Overall sequence homology between sGTH-RI and sGTH-R is 44%. The highest homology occurs with mammalian FSH receptors (49%). Functional characterization examined in COS-7 cells transiently transfected with sGTH-RI showed the largest increase in cAMP production when exposed to salmon GTH I. These results provide the first evidence in any ectothermic vertebrate, the amago salmon, of the duality of gonadotropin receptors.

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.

Isolation and characterization of the goldfish thyrotropin beta subunit gene including the 5'-flanking region.

The complete gene encoding the beta subunit of thyrotropin (thyroid-stimulating hormone, TSH) was isolated from a goldfish genomic library. The goldfish TSHbeta subunit gene, which is approximately 2.0 kilobase pairs (kb) in length, consisted of three exons and two introns. The first intron was much longer (0.89 kb) than the second intron (0.3 kb) as are TSHbeta genes in mammalian species. On the basis of the location of the first intron, the goldfish TSHbeta gene belongs to the mammalian TSHbeta/FSHbeta gene group, which is distinct from the LHbeta group. Inspection of the 5'-flanking and exon 1 regions of the goldfish TSHbeta gene (1.2 kb) revealed the presence of several putative cis-acting elements, including the negative triiodothyronine (T(3))-responsive element (nTRE), Pit-1 element, and GATA-2 element. Comparison of the goldfish sequence with mammalian TSHbeta promoter sequences showed an identical region, nTRE, in the first exon-intron junction region. An in vitro study using dispersed goldfish pituitary cells showed that T(3) treatment (20 ng/ml) suppressed the TSHbeta mRNA level in the cells. These data indicate that (1) the basic structure of TSHbeta genes is highly conserved in vertebrates and that (2) T(3) acts directly on the pituitary and inhibits TSHbeta gene expression in goldfish, probably via the nTRE in the TSHbeta gene.