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S Hyodo

To study the ontogeny of the vasotocin (VT) system and its contribution to anuran metamorphosis, VT mRNA levels were determined by Northern blot analysis in metamorphosing bullfrog tadpoles. Effects of osmotic stimulation on VT mRNA levels were also analyzed in order to follow the development of osmotic responsiveness of VT neurons. The intensity of hybridization signals for VT mRNA gradually increased during prometamorphic development. The increase became marked thereafter until metamorphic climax. Plasma osmolality and hematocrit remained unchanged before metamorphosis, and increased after metamorphic climax, indicating that climactic tadpoles in a semi-terrestrial environment were in a dehydrated condition. These increases correlated well with the increase in VT mRNA level. Immersion of tadpoles in 30% seawater (approximately 350 mOsmol) for 3 days increased plasma osmolality at all stages. No significant changes were observed in the VT mRNA level in response to this treatment during premetamorphic stages. The VT mRNA levels were significantly higher in the treated tadpoles after preclimax stages. Hyperosmotic treatment also increased hematocrit until early metamorphic climax, but did not alter it in tadpoles at late metamorphic climax. These results suggest that the responsiveness of VT-producing neurons to hyperosmotic or hypovolemic stimulation, or both, is established by the time of the metamorphic climax in bullfrog. The marked increase in VT mRNA levels at metamorphic climax stages of intact individuals is probably induced by dehydration. VT-stimulated water absorption and reabsorption in the target organs probably prevented the increase in hematocrit at late metamorphic climax. Thus VT may contribute importantly to osmoregulatory mechanisms in relation to adaptation to a semi-terrestrial habitat through the metamorphosis.

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A Kawakoshi, S Hyodo, A Yasuda, and Y Takei

In teleost fish and tetrapods, the natriuretic peptide (NP) family consists of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and VNP (ventricular natriuretic peptide) that are secreted from the heart, and C-type natriuretic peptide (CNP) that is found in the brain. However, CNP is the only NP identified in the heart and brain of elasmobranchs, suggesting that it is the ancestral type of the NP family and that ANP, BNP and VNP appeared later in the vertebrate phylogeny. To delineate more clearly the molecular evolution of this hormone family, we determined the sequence of NP molecule(s) in evolutionarily the oldest vertebrate group, the cyclostomes. We have cloned a novel NP cDNA from the heart and brain of hagfish, Eptatretus burgeri, using the RACE method and degenerate primers that amplify all known types of NP cDNAs. The novel NP, named EbuNP after the scientific name of this hagfish, appears to be the only NP in the heart and brain, as no other NP cDNAs were amplified even after specific removal of the cloned EbuNP mRNA from the mRNA pool, except for a minor alternatively spliced EbuNP cDNA with a truncated 3'-untranslated sequence. The EbuNP was equally similar to known NPs but was not considered to be a CNP because of the presence of a C-terminal tail sequence. The EbuNP gene was abundantly expressed in the cardiac atrium, ventricle, portal heart and brain but scarcely in the intestine; no expression was observed in the gill and kidney. Mass spectrometry of affinity-purified EbuNP in plasma, heart and brain revealed a 68 amino acid peptide circulating in the blood and stored in the heart, which is cleaved at the typical cleavage signal of a processing enzyme, furin, as observed in mammalian BNP. The C-terminal Gly residue was used for amidation as is the case in eel ANP. The immunoreactive EbuNP was not detected in the brain, suggesting the presence of a different processing form in the brain. These results show that the molecular evolution of the NP family in vertebrates is more complex than previously thought.

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M. Suzuki, S. Hyodo, M. Kobayashi, K. Aida, and A. Urano


Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou.

The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8·3–15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions.

An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.

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A Kawakoshi, S Hyodo, K Inoue, Y Kobayashi, and Y Takei

The natriuretic peptide (NP) family is composed of three members: atrial, brain/ventricular and C-type NPs (ANP, BNP/VNP and CNP respectively) in tetrapods and teleostean fish, but only CNP in elasmobranch fish. In order to trace the process of divergence of the NP family in early vertebrate evolution, we attempted to detect NPs in the primitive ray-finned fish, the sturgeon (Acipenser transmontanus). Unexpectedly, we isolated four distinct NP cDNAs from the heart and brain of this chondrostean fish. The single NP from the brain was CNP, as judged from the lack of C-terminal 'tail' sequence extending from the intramolecular ring. Two of the three cardiac NPs were ANP and VNP, as judged by the presence of an amidation signal at its C-terminus (ANP) and a long and conserved C-terminal tail sequence (VNP) respectively. The third cardiac NP was most probably BNP because it possessed all the features characteristic of BNP including: (1) the presence of dibasic amino acids within the intramolecular ring; (2) the presence of AUUUA repeats in the 3'-untranslated region of its mRNA; (3) equivalent expression of its mRNA in the atrium and ventricle and appreciable expression in the brain. Based on the sturgeon BNP sequence, we further isolated BNP cDNA from the heart of tilapia and pufferfish for the first time in teleostean fish. Phylogenetic analysis of the precursors showed that newly identified NPs belong to each group of the four NPs. The current identification of both VNP and BNP in the sturgeon clearly showed that BNP and VNP are coded by distinct genes, and that the NP family consists of at least four members in the ray-finned fish. VNP has not been molecularly identified in mammals but its presence is suggested from physiological studies; heterologous fish VNP exhibited more potent vasorelaxant activity than homologous mammalian ANP in the isolated coronary artery of dogs.

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T Sakamoto, M Amano, S Hyodo, S Moriyama, A Takahashi, H Kawauchi, and M Ando

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression is unclear even in mammals. To study the possible presence of PrRP–PRL axes not only in the brain-pituitary but also in peripheral organs, the expression patterns of PrRP, PRL and growth hormone (GH) were characterized in amphibious euryhaline mudskippers (Periophthalmus modestus). PrRP mRNA is abundantly expressed not only in the brain but also in the liver, gut and ovary, while less abundant expression was also detected in the skin and kidney. Corresponding to the distribution of PrRP mRNA, PRL mRNA was also detectable in these organs. During adaptation to different environments, the changes in mRNA levels of PrRP paralleled those in PRL in the brain-pituitary, liver and gut in an organ-specific manner. Brain PrRP mRNA and the pituitary PRL mRNA increased under freshwater and terrestrial conditions (P<0.05); expression of PrRP and PRL in the gut of freshwater fish was higher (P<0.05) than those in sea-water fish although there were no changes in fish kept out of water; no significant change was seen in the liver. Expressions of GH were not correlated with PrRP. In the gut, PrRP and PRL appear to be co-localized in the mucosal layer, especially in the mucous cells. Thus, PrRP may also be a local modulator of extrapituitary PRL expression and the PrRP–PRL axes in various organs may play an organ-specific role during environmental adaptation.