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M Suzuki, K Kubokawa, H Nagasawa, and A Urano


We determined the nucleotide sequences of cDNAs encoding precursors of vasotocin (VT) from two cyclostomes, the lamprey Lampetra japonica and the hagfish Eptatretus burgeri, for estimation of their phylogenetic relationships. Although only 47% similarity was found between the VT cDNAs, the predicted VT precursors of the lamprey and the hagfish were both composed of a signal peptide, VT, Gly-Lys-Arg and a neurophysin, as has been shown for precursors of vasopressin (VP) family hormones, including VP, VT and molluscan conopressin.

The central region of the lamprey neurophysin was very similar to those of previously characterized gnathostome neurophysins. Conspicuously, all the positions of 14 Cys residues were conserved in the lamprey neurophysin. The C-terminal region did not have a distinctive Leu-rich core segment, which is always found in the glycopeptide (copeptin) moiety of VP precursors. In contrast, the hagfish neurophysin showed at least two insertions and one deletion in the conserved central region including 14 Cys residues, but contained a potential N-linked glycosylation site and had a high proportion of Leu residues in the C-terminal region, like the neurophysin of another hagfish, Eptatretus stouti.

The evolutionary relationships of the precursors of VP family hormones among the lamprey, hagfish, gnathostomes and a mollusc were estimated by a maximum likelihood method. The phylogenetic tree with the highest bootstrap probability showed that the lamprey VT precursor is more closely related to the gnathostome VT and VP precursors than to the hagfish VT precursors.

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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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S Hiraoka, H Ando, M Ban, H Ueda, and A Urano


We analyzed changes in the hypothalamic levels of vasotocin (VT) and isotocin (IT) mRNA in chum salmon during spawning migration to the Ishikari river. The fish were caught at Atsuta, a fisherman's village facing the Ishikari bay, and at Chitose, an upstream branch of the Ishikari river. The former are referred to as sea water (SW) fish, and the latter as freshwater (FW) fish. The levels of VT and IT mRNA in the forebrains were determined by quantitative Northern blot analysis using single-stranded DNA with the same mRNA sequences as the standards. Levels of VT mRNA were higher in the FW males than the FW females, although no such difference was seen in the SW fish. Changes in the levels of VT mRNA were markedly different in males and females. In the males, no significant differences were seen in the levels of VT-I and VT-II mRNA between the SW and FW fish. However, in the females, the levels of VT mRNA in the FW fish were significantly lower than those in the SW fish. Changes in the levels of IT-I and IT-II mRNA were essentially similar in the males and females. These results suggest that the control of VT gene expression is different in males and females during spawning migration, although the neuroendocrine mechanism is not known.

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M Ashihara, M Suzuki, K Kubokawa, Y Yoshiura, M Kobayashi, A Urano, and K Aida


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.

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S Taniyama, T Kitahashi, H Ando, M Ban, H Ueda, and A Urano

Changes in the levels of pituitary mRNAs encoding GH, prolactin (PRL) and somatolactin (SL) were determined in pre-spawning chum salmon (Oncorhynchus keta) caught at a few key points along their homing pathway in 1994 and 1995. Furthermore, we analyzed relationships between expression of pituitary-specific POU homeodomain transcription factor (Pit-1/GHF-1) and GH/PRL/SL family genes. In 1994, seawater (SW) fish and matured fresh-water (FW) fish were sequentially captured at two points along their homing pathway, the coast and the hatchery. In addition to these two points, maturing FW fish were captured at the intermediate of the two points in 1995. The levels of hormonal mRNAs were determined by a quantitative dot blot analysis using single-stranded sense DNA as the standard. Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis. In 1994, the levels of GH/PRL/SL family mRNAs except for PRL mRNA in the male FW fish were 1.8-4 times higher than those in the SW fish. In 1995, the level of PRL mRNA was somewhat sharply elevated in the maturing FW fish soon after entry into the FW environment, while that of SL mRNA was gradually increased during upstream migration from the coast to the hatchery. The levels of 3 kb Pit-1/GHF-1 mRNA in the FW fish were higher than those in the SW fish in both 1994 and 1995. The present results indicate that expression of genes for the GH/PRL/SL family and Pit-1/GHF-1 is coincidentally enhanced in homing chum salmon. Moreover, the present study suggests that expression of the SL gene is elevated with sexual maturation, whereas that of PRL gene is elevated with osmotic change during the final stages of spawning migration.