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.

Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel.

Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.

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.

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 and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp.

cDNA clones encoding the myogenic regulatory factors (MRFs) myogenin, MyoD and myf-5 were isolated by reverse-transcription polymerase chain reaction from larvae and embryos of the common carp (Cyprinus carpio L.). Myocyte-specific enhancer factor 2 (MEF2) cDNAs were identified from a cDNA library from adult carp. Northern blot analysis showed that MyoD, myf-5 and MEF2C transcripts were present in three-somite embryos, whereas myogenin and MEF2A transcripts were not detected until the 15-somite stage. Intense signals of myogenin and MyoD transcripts were observed even in 1-month-old juveniles. Levels of MyoD, myogenin and MEF2A transcripts declined between 1 and 7 months after hatching, and myf-5 gave only a weak signal in the oldest fish. In contrast, levels of MEF2C transcripts were considerably higher in 7-month-old juveniles than in 1-month-old larvae. mRNAs encoding carp myosin heavy chain and alpha-actin were first detected at approximately the time of the first heartbeat, and levels were maximal in juveniles 1 month post-hatching. The relatively high levels of MRF mRNA in juvenile fish probably reflect the recruitment of new muscle fibres from the satellite cell population. It was concluded that the relative importance of the different members of the MyoD and MEF2 families of transcription factors for muscle differentiation changes during ontogeny in the carp.

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.

Molecular cloning and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp.

cDNA clones encoding the myogenic regulatory factors (MRFs) myogenin, MyoD and myf-5 were isolated by reverse-transcription polymerase chain reaction from larvae and embryos of the common carp (Cyprinus carpio L.). Myocyte-specific enhancer factor 2 (MEF2) cDNAs were identified from a cDNA library from adult carp. Northern blot analysis showed that MyoD, myf-5 and MEF2C transcripts were present in three-somite embryos, whereas myogenin and MEF2A transcripts were not detected until the 15-somite stage. Intense signals of myogenin and MyoD transcripts were observed even in 1-month-old juveniles. Levels of MyoD, myogenin and MEF2A transcripts declined between 1 and 7 months after hatching, and myf-5 gave only a weak signal in the oldest fish. In contrast, levels of MEF2C transcripts were considerably higher in 7-month-old juveniles than in 1-month-old larvae. mRNAs encoding carp myosin heavy chain and -actin were first detected at approximately the time of the first heartbeat, and levels were maximal in juveniles 1 month post-hatching. The relatively high levels of MRF mRNA in juvenile fish probably reflect the recruitment of new muscle fibres from the satellite cell population. It was concluded that the relative importance of the different members of the MyoD and MEF2 families of transcription factors for muscle differentiation changes during ontogeny in the carp.

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.