Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Tiger puffer, Takifugu rubripes, a commercially important long-distance migratory fish, return to specific spawning grounds for reproduction. To clarify reproductive neuroendocrine system of the tiger puffer, the changes in the expression levels of the genes encoding three gonadotropin-releasing hormones (GnRHs), gonadotropin-inhibitory hormone (GnIH), GnIH receptor (GnIH-R), kisspeptin and kisspeptin receptor in the brain and gonadotropin (GTH) subunits, growth hormone (GH) and prolactin (PRL) in the pituitary were examined in the tiger puffer captured in the wild at different reproductive stages, namely immature and mature fish of both sexes, and post-ovulatory females that were obtained by hormonal treatment. The amounts of three gnrh mRNAs, gnih, gnih-r, fshb and lhb were substantially increased in the mature fish compared to the immature fish, especially in the females, and these augmented expressions were drastically decreased in the post-ovulatory females. gh expression showed a slight increase in the mature males. In contrast, kiss2, kiss2r and prl did not show significant changes in the males but significantly decreased in the post-ovulatory females. The present results demonstrate the expression dynamics of the hypothalamo-pituitary-gonadal axis genes associated with the reproductive conditions and the possible involvement of the GnRH/GnIH/GTH system in the regulation of the sexual maturation and spawning in the wild tiger puffer.

DOI： 10.1016/j.ygcen.2020.113660

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DOI： 10.1016/j.ygcen.2019.06.004

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Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

DOI： 10.1111/jpi.12594

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/jpi.12594

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Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.

DOI： 10.1016/j.ygcen.2017.06.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

The seasonal, daily and lunar control of reproduction involves photoperiodic, circadian and lunar changes in the activity of kisspeptin, gonadotropin-inhibitory hormone (GnIH) and gonadotropin-releasing hormone (GnRH) neurons. These changes are brought through complex networks of light-, time- and non-photic signal-dependent control mechanisms, which are mostly unknown at present. The grass puffer, Takifugu alboplumbeus, a semilunar spawner, provides a unique and excellent animal model to assess this question because its spawning is synchronized with seasonal, daily and lunar cycles. In the diencephalon, the genes for kisspeptin, GnIH and their receptors showed similar expression patterns with clear seasonal and daily oscillations, suggesting that they are regulated by common mechanisms involving melatonin, circadian clock and water temperature. For implications in semilunar-synchronized spawning rhythm, melatonin receptor genes showed ultradian oscillations in expression with the period of 14.0–15.4 h in the pineal gland. This unique ultradian rhythm might be driven by circatidal clock. The possible circatidal clock and circadian clock in the pineal gland may cooperate to drive circasemilunar rhythm to regulate the expression of the kisspeptin, GnIH and their receptor genes. On the other hand, high temperature (over 28 °C) conditions, under which the expression of the kisspeptin and its receptor genes is markedly suppressed, may provide an environmental signal that terminates reproduction at the end of breeding period. Taken together, the periodic regulation of the kisspeptin, GnIH and their receptor genes by melatonin, circadian clock and water temperature may be important in the precisely-timed spawning of the grass puffer.

DOI： 10.1016/j.ygcen.2018.04.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Water temperature is an environmental factor of primary importance that influences reproductive function in fish. To understand the molecular and physiological mechanisms underlying the regulation of reproduction by temperature, we examined changes in expression of genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r) and three gonadotropin-releasing hormones (gnrhl, gnrh2 and gnrh3) in the brain and genes encoding gonadotropin (GTH) subunits (gpa, fshb and lhb) in the pituitary of grass puffer exposed to a low temperature (14 degrees C), normal temperature (21 degrees C) and high temperature (28 degrees C) for 7 days. In addition, the plasma levels of cortisol were examined after exposed to three temperature conditions. The gonadosomatic index was significantly decreased in both low and high temperature conditions. The levels of kiss2 and kiss2r mRNAs were significantly decreased at both low and high temperature conditions compared to normal temperature (control) condition. gnrhl but not gnrh2 were significantly decreased in both temperature conditions, while gnrh3 showed a decreasing tendency in low temperature. Consequently, the levels of fshb and lhb mRNAs were significantly decreased in both low and high temperature conditions. Interestingly, the plasma levels of cortisol were significantly increased in low temperature but remain unchanged in high temperature, suggesting that the fish were under stress in the low temperature conditions but not in the high temperature conditions. Taken together, the present results indicate that anomalous temperature have an inhibitory effect on reproductive function through suppressing kiss2/kiss2r/gnrhl/fshb and lhb expression and these changes may occur in a normal physiological response as well as in a malfunctional stress response. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2016.11.012

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© 2016, Akadémiai Kiadó, Budapest, Hungary. Radionuclide concentrations in wharf roaches inhabiting coastal areas of Honshu, Japan, were investigated in October 2011 and June 2012. Relative high concentrations of 110mAg (2.1–127 Bq kg-wet−1), 134Cs (2.6–61 Bq kg-wet−1), and 137Cs (3.5–92 Bq kg-wet−1) were detected in specimens from the eastern Honshu areas. Significantly lower 137Cs concentrations (0.7–1.6 Bq kg-wet−1) were detected in specimens from western and northern Honshu. The decay-corrected 137Cs concentration was significantly inversely correlated with the distance from the Fukushima Dai-ichi Nuclear Power Plant. Thus, wharf roach may serve as a good bioindicator for monitoring radioactive contamination of its habitats.

DOI： 10.1007/s10967-016-4879-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2015.09.008

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC FISHERIES SCIENCE  

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In an exposure test, wharf roach (Ligia exotica Roux) was exposed to perfluorooctane sulfonate (PFOS )via diet (0.1 mg/g-diet) for 2 days. 1 day after cessation of exposure, concentration of PFOS was increased to 1019 ± 144 ng/g-wet and still detected to 377 ± 321 ng/g-wet at 12 days after the exposure. Biological half-life of PFOS in wharf roach was estimated to be 11.9 days. In a field study, wharf roach were collected from 14 coastal sites in Japan. PFOS were detected from each one individual in Tsuyazaki, Fukuoka Pref. (215 ng/g-dry) and Taira, Nagasaki Pref. (601 ng/g-dry). These results suggested that wharf roach could be a promised biomonitoring species for PFOS in coastal area.

DOI： 10.15017/1543577

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Other Link： http://hdl.handle.net/2324/1543577

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Pain-modulatory neuropeptides, PQRFamide (PQRFa) peptides, have recently been implicated in the regulation of reproduction in fish. As a first step toward investigating the role of PQRFa peptides on reproductive function in the grass puffer Takifugu niphobles, which is a semilunar spawner, we cloned genes encoding PQRFa peptide precursor (pqrfa) and its two types of receptors (pqrfa-rl and pqrfa-r2), and examined changes in their expression levels in the brain and pituitary over several months during the reproductive cycle. The grass puffer PQRFa peptide precursor of 126 amino acid residues contains two putative PQRFa peptides, PQRFa-1 and PQRFa-2, which correspond to NPFF and NPAF in other vertebrates, respectively. The grass puffer PQRFa-R1 and PQRFa-R2 consist of 426 and 453 amino acid residues, respectively, and contain distinct characteristics of G-protein coupled receptors. These three genes were exclusively expressed in the brain and pituitary. The expression levels of pqrfa and pqrfa-rl were significantly increased during the late stage of sexual maturation, but low in the spawning fish just after releasing sperms and eggs. Therefore, the grass puffer PQRFa peptide may have a role in the late stage of sexual maturation before spawning via PQRFa-R1. In contrast, the pqrfa-r2 expression showed maximum levels in the spawning fish and in the post-spawning period. The present results provide fundamental data suggesting that the grass puffer PQRFa peptide may have multiple roles in the control of reproduction that are dependent on the reproductive stages. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2014.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Research Foundation  

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel1a1.4, mel1a1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0-15.4 h, suggesting the presence of circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

DOI： 10.3389/fnins.2015.00009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In seasonally breeding animals, the circadian and photoperiodic regulation of neuroendocrine system is important for precisely-timed reproduction. Kisspeptin, encoded by the Kiss1 gene, acts as a principal positive regulator of the reproductive axis by stimulating gonadotrophin-releasing hormone (GnRH) neurone activity in vertebrates. However, the precise mechanisms underlying the cyclic regulation of the kisspeptin neuroendocrine system remain largely unknown. The grass puffer, Takifugu niphobles, exhibits a unique spawning rhythm: spawning occurs 1.5-2h before high tide on the day of spring tide every 2weeks, and the spawning rhythm is connected to circadian and lunar-/tide-related clock mechanisms. The grass puffer has only one kisspeptin gene (kiss2), which is expressed in a single neural population in the preoptic area (POA), and has one kisspeptin receptor gene (kiss2r), which is expressed in the POA and the nucleus dorsomedialis thalami. Both kiss2 and kiss2r show diurnal variations in expression levels, with a peak at Zeitgeber time (ZT) 6 (middle of day time) under the light/dark conditions. They also show circadian expression with a peak at circadian time 15 (beginning of subjective night-time) under constant darkness. The synchronous and diurnal oscillations of kiss2 and kiss2r expression suggest that the action of Kiss2 in the diencephalon is highly dependent on time. Moreover, midbrain GnRH2 gene (gnrh2) but not GnRH1 or GnRH3 genes show a unique semidiurnal oscillation with two peaks at ZT6 and ZT18 within a day. The cyclic expression of kiss2, kiss2r and gnrh2 may be important in the control of the precisely-timed diurnal and semilunar spawning rhythm of the grass puffer, possibly through the circadian clock and melatonin, which may transmit the photoperiodic information of daylight and moonlight to the reproductive neuroendocrine centre in the hypothalamus.

DOI： 10.1111/jne.12165

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2 d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the deputation period, but they were still detected 12 d after exposure ceased (TBT 290 +/- 140 ng/g; dibutyltin 1280 +/- 430 ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4 d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9 +/- 16.5 ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3 +/- 6.2 ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ecoenv.2013.06.019

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Grass puffer, Takifugu niphobles, exhibits unique spawning behavior: it spawns on beach in semilunar cycles during spring tide in early summer. The fish aggregate at certain seashore locations several hours before high tide every two weeks. To explore the molecular and neuroendocrine mechanisms underlying the regulation of the lunar-related spawning rhythm, seasonal and cyclic variations in gene expression for hypothalamic neuropeptides related to reproduction were examined by quantitative real-time PCR. The expression levels of genes for gonadotropin-releasing hormone, kisspeptin, LPXRFamide peptide and PQRFamide peptide in the hypothalamus varied differently depending on reproductive stage and gender, suggesting their specific roles in reproduction. In the spawning period, the expression levels of LPXRFamide peptide and its receptor genes showed diurnal and circadian variations in association with the expression of four subtypes of melatonin receptor genes. Together with the nocturnal secretion of melatonin from the pineal gland, melatonin may play an important role in transmitting the photoperiodic information of moonlight to the reproductive neuroendocrine center in the hypothalamus of grass puffer. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2012.07.027

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Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

Grass puffer (Takifugu niphobles) exhibits a unique spawning rhythm: it spawns on the beach in semilunar cycles during spring tide in early summer. To explore the molecular and neuroendocrine mechanisms underlying the regulation of the semilunarsynchronized spawning rhythm, cyclic variations in melatonin secretion from the pineal gland and in the expression of four subtypes of melatonin receptor genes in the diencephalon were examined. The hypothalamic expression of genes for neuropeptides related to reproduction and their receptors were also examined during the reproductive cycle. The secretion of melatonin from the pineal gland showed diurnal and circadian variations with a significant peak in the nighttime. The nocturnal secretion of melatonin and the synchronous rise in the expression of four subtypes of melatonin receptor genes during the nighttime indicate that the action of melatonin on the hypothalamus is highly dependent on time and light. In the hypothalamus, the expression of genes for gonadotropin-releasing hormone, kisspeptin, LPXRFamide peptide and PQRFamide peptide and their receptors showed seasonal variations with increased expression in the spawning period. In particular, the expression of LPXRFa and LPXRFa-R genes showed diurnal and circadian variations in association with the expression of the four subtypes of melatonin receptor genes, indicating that the action of LPXRFa is most probably cyclic due to the regulation by melatonin. Although the link between the lunar cycle and the melatonin signal is missing at present, melatonin may play an important role in transmitting the photoperiodic information of moonlight to the reproductive neuroendocrine center in the hypothalamus of grass puffer. © 2013 Nova Science Publishers, Inc.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Full length cDNA and gene encoding ghrelin precursor and mature ghrelin peptide were identified from the stomach of Pacific bluefin tuna, Thunnus orientalis, which has unique metabolic physiology and high commercial value at fishery markets. Quantitative expression analysis was conducted for the gastric ghrelin and pepsinogen 2 genes during the early stage of somatic growth from the underyearling to yearling fish. The full length cDNA of bluefin tuna ghrelin precursor has a length of 470 bp and the deduced precursor is composed of 107 amino acids. The ghrelin gene is 1.9 kbp in length and has a 4 exon-3 intron structure. The major form of mature ghrelin in the stomach was an octanoylated 20-amino acid peptide with C-terminal amidation, while overall 12 different forms of ghrelin peptides, including short form of 18-amino acid peptide and seven kinds of acyl modifications were identified. The expression profiles of the gastric ghrelin and pepsinogen 2 genes showed no significant changes related to the early growth stages. The present results suggest that digestive physiology has already been functional in this growth stage of the juvenile bluefin tuna and ghrelin may have a role in the sustained digestive and metabolic activities. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2012.04.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Neuropeptide tyrosine (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in rodents. However, the involvement of NPY in feeding behavior has not well been studied in fish. Therefore, we investigated the role of NPY in food intake using a tiger puffer (Takifugu rubripes) model. We observed the distribution of NPY-like immunoreactivity in the brain. Neuronal cell bodies containing NPY were located in the telencephalon, hypothalamus, mesencephalon, and medulla oblongata, and their nerve fibers were also found throughout the brain. We cloned two cDNAs, encoding NPYa and NPYb orthologs, respectively, from the brain, and also confirmed two genes encoding these NPYs in the Takifugu genome database. We examined the distribution of these transcripts in the brain using real-time PCR. Levels of NPYa mRNA in the telencephalon, mesencephalon and hypothalamus were much higher than in the medulla oblongata and cerebellum, whereas levels of NPYb mRNA in the medulla oblongata were higher than in other regions. We also examined prandial effects on the expression level of these transcripts in the telencephalon and hypothalamus. NPYa mRNA levels in the hypothalamus, but not in the telencephalon, obtained from fish fasted for one week were higher than those in fish that had been fed normally. The level was decreased at 2 h after feeding. Levels of NPYb mRNA were not affected by prandial conditions. These results suggest that NPY is present throughout the brain, and that NPYa, but not NPYb, in the hypothalamus is involved in the feeding regulation in the tiger puffer.

DOI： 10.2108/zsj.28.882

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Language：English   Publisher：VERSITA  

The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin (GTH) secretion. This review focuses on a family of neuropeptides, LPXRFamide (LPXRFa) peptides, which have been implicated in the regulation of GTH secretion. LPXRFa acts on the pituitary via a G protein-coupled receptor, LPXRFa-R, to enhance gonadal development and maintenance by increasing gonadotropin release and synthesis. Because LPXRFa exists and functions in several fish species, LPXRFa is considered to be a key neurohormone in fish reproduction control. The precursors to LPXRFamide peptides encoded plural LPXRFamide peptides and were highly divergent in vertebrates, particularly in lower vertebrates. Tissue distribution analyses indicated that LPXRFamide peptides were highly concentrated in the hypothalamus and other brainstem regions. In view of the localization and expression of LPXRFamide peptides in the hypothalamo-hypophysial system, LPXRFamide peptide in fish increase GTH release in vitro and in vivo. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of LPXRFa, a newly discovered key neurohormone.

DOI： 10.2478/s11535-011-0048-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-time in vivo imaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.

DOI： 10.1523/JNEUROSCI.6804-10.2011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa-R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real-time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa-R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle-stimulating hormone beta- and luteinising hormone beta-subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa-R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa-R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa-R system in the regulation of lunar-synchronised spawning of grass puffer.

DOI： 10.1111/j.1365-2826.2010.02081.x

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The hypothalamic neurosecretory cells (NSCs) which produce and release neurohypophysial hormones are involved in controls of diverse physiological phenomena including homeostatic controls of unconscious functions and reproduction. The far and wide distribution of neurosecretory processes in the discrete brain loci and the neurohypophysis is appropriate for coordination of neural and endocrine events that are required for the functions of NSCs. The presence of dye couplings and intimate contacts among NSCs supports harmonious production and release of hormone to maintain the plasma level within a certain range which is adequate for a particular physiological condition. Neurosecretory cells integrate diverse input signals from internal and external sources that define this particular physiological condition, although reactions of NSCs vary among different species, and among different cell types. An input signal to NSC is received by specific receptors and transduced as unique intracellular signals, important for the various functions of neurohypophysial hormones. Orchestration of multiple intracellular signaling systems, activities of which are individually modulated by input signals, determines the rates of synthesis and release of hormone through regulation of gene expression. The first step of gene expression, i.e., transcription, is amenable for diverse reaction of NSCs, because the 5' upstream regions of genes encoding neurohypophysial hormones are highly variable. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.09.016

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Kisspeptins are a family of neuropeptides encoded by Kiss1 and Kiss2 genes, and participate in neuroendocrine regulation of gonadotropin-releasing hormone (GnRH) secretion through activating their receptor, Kiss1r (or GPR54). Bioinformatic analyses have shown that there is a single gene for each kisspeptin (Kiss2) and its receptor (Kiss1r) in pufferfish, the function of which has yet to be elucidated. We cloned these two genes in grass puffer, which spawns on beach with semilunar cycles, and examined changes in their expression levels in the brain and pituitary at different reproductive stages over the spawning season. The Kiss2 precursor of 104 amino acid residues contains a putative kisspeptin peptide (SKFNLNPFGLRF). Kiss1r consists of 377 amino acid residues containing distinct characteristics of G-protein coupled receptors. Kiss2 and Kiss1r genes were expressed extensively in the brain, pituitary and gonads. The amounts of Kiss2 and Kiss1r mRNAs were significantly elevated during the spawning period in the brain and pituitary of both sexes. There were strong positive correlations between the amounts of Kiss2 and Kiss1r mRNAs in the brain and pituitary over the spawning season. Significant positive correlations were also observed between the amounts of Kiss2/Kiss1r mRNAs and GnRH1 mRNA in the brain. The present results indicate that the Kiss2/Kiss1r system most probably plays an important role in the regulation of reproductive function in the spawning period of grass puffer, possibly through the stimulation of GnRH1 secretion. Furthermore, Kiss2 may have a local action in the pituitary. (c) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.07.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Our previous studies suggested the importance of gonadotropin-releasing hormones (GnRHs) for initiation of spawning migration of chum salmon, although supporting evidence had been not available from oceanic fish. In farmed masu salmon, the amounts of salmon GnRH (sGnRH) mRNAs in the forebrain increased in the pre-pubertal stage from winter through spring, followed by a decrease toward summer. We thus hypothesized that gene expression for GnRHs in oceanic chum salmon changes similarly, and examined this hypothesis using brain samples from winter chum salmon in the Gulf of Alaska and summer fish in the Bering Sea. They were classified into sexually immature and maturing adults, which had maturing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GnRH mRNAs were determined by real-time PCRs. The amounts of sGnRH mRNA in the maturing winter adults were significantly larger than those in the maturing summer adults. The amounts of sGnRH and chicken GnRH mRNAs then peaked during upstream migration from the coast to the natal hatchery. Such changes were observed in various brain loci including the olfactory bulb, terminal nerve, ventral telencephalon, nucleus preopticus parvocellularis anterioris, nucleus preopticus magnocellularis and midbrain tegmentum. These results suggest that sGnRH neurons change their activity for gonadal maturation prior to initiation of homing behavior from the Bering Sea. The present study provides the first evidence to support a possible involvement of neuropeptides in the onset of spawning migration. Crown Copyright 2010 Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.05.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

The grass puffer (Takifugu niphobles) exhibits unique spawning behavior. Large numbers of fish aggregate to coastal spawning beds several hours before high tide during the spring tide. In order to examine the environmental and physiological regulation of this semilunar-synchronized spawning rhythm, the aggregating and spawning behaviors of the grass puffer were observed in the field, and in an aquarium without tidal changes. The fish aggregated to a spawning bed in the rising tidal phases both in the morning and evening during the spring tide, and several days after the spring tide. Spawning occurred on several days when large numbers of fish (200-1000) aggregated to the spawning bed. The timing of aggregation and spawning was tightly connected to the tidal changes; aggregation occurred 2-3 h before high tide, and spawning occurred 2 h before high tide. In the aquarium, in which a slope was constructed with pebbles, small groups of mature fish aggregated on the slope only in the rising tidal phases during and after the spring tide, when the fish aggregated in the field. However, there was no spawning in the aquarium. The aggregating behavior observed in the aquarium without tidal changes suggests that the semilunar reproductive rhythm is endogenously maintained with surprising precision during the spawning period in grass puffer.

DOI： 10.2108/zsj.27.559

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around new moon and full moon from spring to early summer. To investigate the role of gonadotropin-releasing hormone (GnRH) in the reproductive function, genes encoding three types of GnRHs, namely seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II) and salmon GnRH (sGnRH), were cloned and changes in their mRNA amounts were examined over the spawning season. In addition, changes in the pituitary gonadotropin subunit mRNAs and the plasma steroid hormones were examined over the spawning season. Fishes were assessed at four reproductive stages, i.e., in December (early maturation), in April (maturing), in May (spawning), and in July (post-spawning). Moreover, spawning fish just after releasing eggs and sperm were taken at a spawning bed. The amounts of sbGnRH mRNA were substantially elevated in May and the spawning fish in both sexes, concomitant with considerable elevations of follicle-stimulating hormone and luteinizing hormone beta subunit mRNAs and plasma estradiol-17 beta (E(2)) and testosterone (T) levels. There were strong positive correlations between the sbGnRH mRNA and the plasma E(2) and T levels over the spawning season in both sexes. The amounts of cGnRH-II mRNA showed no noticeable changes except for an increase in the post-spawning females. The amounts of sGnRH mRNA in the males were significantly increased in May, but they were low in the spawning males. In the females, sGnRH mRNA increased from the maturing stage and reached a maximum in the post-spawning stage, in which a positive correlation with the plasma cortisol levels was observed. These specific changes suggest that the expression of three types of GnRH genes is differentially regulated during the spawning season, and sex steroids may be important for the differential expression of GnRH genes. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.01.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na(+),K(+)-ATPase activity in the coastal SW fish and the plasma Na(+) levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.01.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

An increase in activity of the pituitary-gonadal axis (PG-axis) and gonadal development are essential for the onset of spawning migration of chum salmon from the Bering Sea. In the Bering Sea, fish with larger body sizes initiated gonadal development and commenced spawning migration to the natal river by the end Of Summer. We thus hypothesized that insulin-like growth factor-I (IGF-I), a somatotropic signal that interacts with the PG-axis, can be one of such factors responsible for the onset of migration, and examined changes in plasma levels and hepatic expression of IGF-I gene in oceanic and homing chum salmon in 2001-2003. The plasma IGF-I levels and corresponding body sizes in maturing adults, which had developing gonads, were significantly higher than those in immature fish in all years examined. Such increase in the plasma IGF-I levels in maturing fish was observed even in the Gulf of Alaska during February 2006, while coincident increase was not observed in the hepatic amounts of IGF-I mRNA. In autumn, the plasma IGF-I levels in homing adults decreased during upstream migration in the Ishikari River-Ishikari bay water system in Hokkaido, Japan. In conclusion, the plasma IGF-I levels increased with gonadal development when chum salmon migrated from the winter Gulf of Alaska to the summer Bering Sea. Circulating IGF-I may interact with the PG-axis and promote gonadal development that is inseparable from the onset of spawning migration. Circulating IGF-I levels were thereafter lowered in accordance with final maturation during upstream migration in the breeding season. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2009.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

To examine possible roles of vasotocin (V(T)), isotocin (IT), prolactin (PRL) and growth hormone (GH) in osmoregulation of a stenohaline marine teleost, tiger puffer (Takifugu rubripes), changes in expression levels of these genes following hypoosmotic challenge, were examined in two experiments. Fish were transferred from 100% seawater (SW) to 33% SW, 10% SW and fresh water (FW), and left for 3 days in experiment L In experiment II, fish were transferred to FW, and left for I day. Changes in plasma osmolality, concentrations of Na(+) and Cl(-), and Na(+)/K(+)-ATPase activity in the gills and kidney were examined. Changes in the absolute amounts of VT, IT, PRL and GH mRNAs were determined by real-time PCR. In experiment 1, almost all fish survived over 3 days of acclimation. The plasma parameters decreased on day 1, and remained at similar levels until day 3. The renal Na(+)/K(+)-ATPase activity significantly increased in 10% SW and FW on day 1. The amounts of VT and IT mRNAs tended to decrease in the hypoosmotic conditions on day 1. The amounts of PRL mRNA significantly increased in the hypoosmotic conditions, whereas those of GH mRNA decreased in FW. In experiment II, the amount of VT mRNA significantly decreased in FW concomitantly with the changes in PRL and GH mRNAs. The present results suggest that the hyperosmotic responses may be regulated by neuroendocrine factors such as VT, PRL and GH in tiger puffer, as in case of euryhaline teleosts. Particularly, the present study first shows that the expression of VT gene may be down-regulated following hypoosmotic challenge in the stenohaline marine fish. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2009.07.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Multiple subtypes of melatonin receptors are expressed in neural and peripheral tissues to mediate melatonin actions on the regulation of circadian rhythms in vertebrates. To elucidate molecular basis of "circa" rhythms in the grass puffer Takifugu niphobles, which spawns synchronously with semilunar cycles, tissue distribution of four melatonin receptor subtype mRNAs (Mel(1a) 1.4, Mel(1a) 1.7, Mel(1b), and Mel(1c)) were examined, and diurnal and circadian changes in their absolute amounts were examined in the retina, diencephalon, and optic tectum. Mel(1a) 1.4, Mel(1a) 1.7, and Mel(1b) mRNAs were widely distributed in various brain regions, retina, pituitary, and peripheral tissues, whereas Mel(1c) mRNA was mainly detected in the nervous tissues and pituitary. All subtype genes showed diurnal expressions with one or two peaks during nighttime. When the fish were reared under constant darkness, the retinal expressions of Mel(1a) 1.7, Mel(1b,) and Mel(1c) genes were markedly diminished but still showed circadian variations. In contrast, increased and synchronized expressions of the four subtype genes were noticeable with one peak at circadian time 18 in the diencephalon. The circadian expression profiles in the optic rectum were different among the subtypes. The present results suggest that melatonin receptor gene expression is regulated by circadian clock and light, but the effects of light are different among the tissues. The synchronized expressions of the four subtype genes in the diencephalon may be related to the exertion of reproductive rhythmicity in this puffer species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2009.06.076

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Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike the orexigenic action in rodents, via the melanocortin system with suppression of neuropeptide Y (NPY) mRNA expression. We therefore investigated the neuronal relationship between MCH- and NPY-containing neurons in the goldfish brain, using a double-immunofluorescence method and confocal laser scanning microscopy. MCH- and NPY-like immunoreactivities were distributed throughout the brain. In particular, MCH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These observations suggest that MCH-containing neurons provide direct input to NPY-containing neurons in the NPPv of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide, inhibiting the orexigenic activity of NPY. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2008.10.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Four subtypes of melatonin receptor genes (Mel(1a) 1.4, Mel(1a) 1.7, Mel(1b), and Mel(1c)) are considered to be expressed to mediate various physiological functions of melatonin in goldfish (Carassius auratus). To examine their tissue distribution and diurnal changes in expression levels, we cloned partial gene fragments for these melatonin receptor subtypes, and established specific RT-PCR and quantitative real-time PCR systems. Mel(1a) 1.4 and Mel(1b) were predominantly expressed in various neuronal and peripheral tissues, while Mel(1a) 1.7 and Mel(1c) were expressed in the restricted tissues. All subtype genes were expressed in the optic tectum, diencephalon, mesencephalon, vagal lobe, retina and spleen. The real-time PCR analyses showed that significant differences among time were observed for Mel(1a) 1.4 in the optic tectum and for Mel(1a) 1.7 and Mel(1b) in the retina. In the retina, the levels of Mel(1a) 1.7 and Mel(1b) mRNAs showed diurnal changes with one peak at ZT24. The present results show differential distribution of four subtypes of melatonin receptor mRNAs in the neuronal and peripheral tissues. However, the expressions of all subtype genes of the retinorecipient brain regions and retina reinforce the role of the melatonin receptor in processing visual information. Furthermore, the present study demonstrates diurnal expressions of the major subtype genes, i.e. Mel(1a) 1.4 in the optic tectum and Mel(1a) 1.7 in the retina. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2008.09.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

The activity of the pituitary-gonadal axis ( PG axis) in pre-migratory and homing chum salmon was examined because endocrine mechanisms underlying the onset of spawning migration remain unknown. Pre-migratory fish were caught in the central Bering Sea in June, July and September 2001, 2002 and 2003, and in the Gulf of Alaska in February 2006. They were classified into immature and maturing adults on the basis of gonadal development. The maturing adults commenced spawning migration to coastal areas by the end of summer, because almost all fish in the Bering Sea were immature in September. In the pituitaries of maturing adults, the copy numbers of FSH beta mRNA and the FSH content were 2.5- to 100-fold those of the immature fish. Similarly, the amounts of LH beta mRNA and LH content in the maturing adults were 100- to 1000-fold those of immature fish. The plasma levels of testosterone, 11-ketotestosterone and estradiol were higher than 10nmoll(-1) in maturing adults, but lower than 1.0nmoll(-1) in immature fish. The increase in the activity of the PG-axis components had already initiated in the maturing adults while they were still in the Gulf of Alaska in winter. In the homing adults, the pituitary contents and the plasma levels of gonadotropins and plasma sex steroid hormones peaked during upstream migration from the coast to the natal hatchery. The present results thus indicate that the seasonal increase in the activity of the PG axis is an important endocrine event that is inseparable from initiation of spawning migration of chum salmon.

DOI： 10.1242/jeb.021352

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

When, where, and how oceanic churn salmon initiate spawning migration is unknown although gonadal development and elevation of the activity of the pituitary-gonadal axis (PG-axis) are essential. Insulin-like growth factor-I (IGF-I) is a somatotropic signal that interacts with the PG-axis for gametogenesis. We thus examined the plasma level of IGF-I in immature and maturing chum salmon in the Bering Sea and the Gulf of Alaska. The maturing adults which had maturing gonads left the Bering Sea for the natal river by the end of summer, because almost all fish were immature in September. The plasma level of IGF-I and corresponding body size in the maturing adults were two- to threefold that of immature fish. The plasma IGF-I level correlated positively with the pituitary contents of follicle-stimulating hormone and the plasma levels of 11-ketotestosterone and estradiol-17 beta. Therefore, the plasma level of IGF-I increased with elevation of the PG-axis activity prior to the initiation of spawning migration from the Bering Sea. Circulatory IGF-I from visceral organs may inform the status of body growth to the PG-axis for gonadal development that is inseparable from decision of chum salmon whether to initiate homing behavior from the Bering Sea or not to initiate spawning migration by the coming spawning season.

DOI： 10.1111/j.1749-6632.2008.03668.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, which possesses some structural variants. A molecular form known as chicken GnRH II ([His(5) Trp(7) Tyr(8)] GnRH, cGnRH II) is widely distributed in vertebrates, and has recently been implicated in the regulation of sexual behavior and food intake in an insectivore, the musk shrew. However, the influence of cGnRH II on feeding behavior has not yet been studied in model animals such as rodents and teleost fish. In this study, therefore, we investigated the role of cGnRH II in the regulation of feeding behavior in the goldfish, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV-injected cGnRH II at graded doses, from 0.1 to 10 pmol/g body weight (BW), induced a decrease of food consumption in a dose-dependent manner during 60 min after treatment. Cumulative food intake was significantly decreased by ICV injection of cGnRH II at doses of 1 and 10 pmol/g BW during the 60-min post-treatment observation period. ICV injection of salmon GnRH ([Trp(7) Leu(8)] GnRH, sGnRH) at doses of 0.1-10 pmol/g BW did not affect food intake. The anorexigenic action of cGnRH II was completely blocked by treatment with the GnRH type I receptor antagonist, Antide. However, the anorexigenic action of cGnRH II was not inhibited by treatment with the corticotropin-releasing hormone (CRH) 1/2 receptor antagonist, alpha-helical CRH((9-41)), and the melanocortin 4 receptor antagonist, HS024. These results suggest that, in the goldfish, cGnRH II, but not sGnRH, acts as an anorexigenic factor, as is the case in the musk shrew, and that the anorexigenic action of cGnRH II is independent of CRH- and melanocortin-signaling pathways. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yhbeh.2008.01.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpb.2006.11.033

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Vasotocin (VT) has been shown to influence various aspects of social and sexual behaviors in a broad range of vertebrate species, but less is known about the mechanisms through which this peptide modulates behavior. Additionally, much less is known about roles of isotocin (IT) in regulation of behavior. Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around the spring tide and is conducted by a group of 10-60 individuals, of which one is female. As a first step toward investigating the roles of VT and IT in this species' spawning behavior, we determined the structures of the VT and IT genes from grass puffer using! the genome resources of the closely related tiger puffer and green puffer. We then used these sequences to develop real-time PCR assays and examined changes in expression of the VT and IT genes over the spawning season. The structures of VT and IT genes are well conserved among three puffer species. Particularly, the sequence similarities between grass and tiger puffers were very high not only in the coding region (85-99%), but also in the non-coding regions (92-98%) that include the 5'-upstream regions. The levels of expression of VT gene increased in the brain of pre-spawning females. The levels of VT mRNA in the spawning females tended to be higher than that in the spawning males. In contrast, the levels of IT mRNA did not show such variation. The present results suggest that VT gene expression augments in the brain of females during the spawning period. The unique spawning behavior of grass puffer provides a useful model for studying the molecular mechanism of sexual behavior utilizing the genome resources of tiger puffer. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2007.07.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGIF-I on expression of genes encoding glycoprotein alpha (GP alpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GP alpha, FSH beta, and LH beta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.

DOI： 10.2108/zsj.25.88

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Pacific salmon employ a semelparous reproductive strategy where sexual maturation is followed by rapid senescence and death. Cortisol overproduction has been implicated as the central physiologic event responsible for the post-spawning demise of these fish. Cortisol homeostasis is regulated through the action of hormones of the hypothalamus-pituitary-interrenal (HPI) axis. These include corticotropin-releasing factor (CRF) and urotensin-I (UI). In the present study, masu salmon (Oncorhynchus masou) were assayed for changes in the levels CRF-I and UI mRNA transcripts by quantitative real-time PCR (qRT-PCR). These results were compared to plasma cortisol levels in juvenile, adult, and spawning masu salmon to identify specific regulatory factors that appear to be functionally associated with changes in cortisol levels. Intramuscular implantation of GnRH analog (GnRHa) capsules was also used to determine whether GnRH influences stress hormone levels. In both male and female masu salmon, spawning fish experienced a 5- to 7-fold increase in plasma cortisol levels relative to juvenile non-spawning salmon. Changes in CRF-I mRNA levels were characterized by 1-2 distinctive short-term surges in adult masu salmon. Conversely, seasonal changes in UI mRNA levels displayed broad and sustained increases during the pre-spawning and spawning periods. The increases in UI mRNA levels were positively correlated (R-2 = 0.21 male and 0.26 female, p < 0.0001) with levels of plasma cortisol in the pre-spawning and spawning periods. Despite the importance of GnRH in sexual maturation and reproduction, the administration of GnRHa to test animals failed to produce broad changes in CRF-I, Ul or plasma cortisol levels. These findings suggest a more direct role for UI than for CRF-I in the regulation of cortisol levels in spawning Pacific salmon. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2007.03.013

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DOI： 10.2187/bss.22.3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Photoperiod is one of the major environmental cues that initiate smolting, but its mechanisms of action are not clear. We therefore examined possible effects of increased daylength on the development of seawater tolerance and gill Na+, K+-ATPase (NKA) in sockeye salmon (Oncorhynchus nerka). Seawater tolerance and related biological changes were assessed in terms of plasma sodium concentration after 24 h seawater challenge tests (plasma Na), gill NKA activity, expression of two isoform genes encoding gill NKA alpha subunits (alpha 1a and alpha 1b), and plasma levels of cortisol and growth hormone (GH). Juvenile fish transferred to an artificial long-day (16 h light/8 h dark cycle) in December showed lower plasma Na and higher gill NKA activity during winter than fish with held on a short-day (8 h light/16 h dark cycle). The amount of alpha 1b mRNA in the long-day group was higher than that in the short-day group, whereas the amount of alpha 1a mRNA in the long-day group was lower than that in the short-day group. Furthermore, the plasma levels of GH and cortisol in the long-day group increased earlier than in the short-day group. These results indicate that the increased daylength stimulates seawater tolerance, possibly through induction of the gill NKA alpha 1b gene by elevated GH and cortisol. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquaculture.2007.10.001

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Pacific salmon migrate from ocean through the natal river for spawning. Information on expression of genes encoding osmoregulatory hormones and migratory behavior is important for understanding of molecular events that underlie osmoregulation of homing salmon. In the present article, regulation of gene expression for osmoregulatory hormones in pre-spawning salmon was briefly reviewed with special reference to neurobypophysial hormone, vasotocin (VT), and pituitary hormones, growth hormone (GH) and prolactin (PRL). Thereafter, we introduced recent data on migratory behavior from SW to FW environment. In pre-spawning chum salmon, the hypothalamic VT mRNA levels increased in the males, while decreased in the females with loss of salinity tolerance when they were kept in SW. The amounts of GH mRNA in the pituitary decreased during ocean migration prior to entrance into FW. Hypo-osmotic stimulation by SW-to-FW transfer did not significantly affect the amount of PRL mRNA, but it was elevated in both SW and FW environments along with progress in final maturation. Behaviorally, homing chum salmon continued vertical movement between SW and FW layers in the mouth of the natal river for about 12 It prior to upstream migration. Pre-spawning chum salmon in an aquarium, which allowed fish free access to SW and FW, showed that individuals with the lower plasma testosterone (T) and higher estradiol-17 beta (E2) levels spent longer time in FW when compared with the SW fish. Taken together, neuroendocrine mechanisms that underlie salt and water homeostasis and migratory behavior from SW to FW may be under the control of the hypothalamus-pituitary-gonadal axis in pre-spawning salmon. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2007.01.010

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Expression of genes encoding gonadotropin (GTH) subunits in the salmon pituitary was regulated by salmon gonadotropin-releasing hormone (sGnRH) and sex steroid hormones in a reproductive stage-dependent manner, probably through DNA-binding transcription factors. Direct effects of these hormones on expression of genes encoding salmon fushi tarazu factor 1 homolog (sFF1-I) and estrogen receptor alpha (ER alpha) were therefore examined by use of primary pituitary cell cultures of masu salmon at different reproductive stages. Pituitaries were collected in March (before initiation of gonadal maturation), in May (early maturing), in July (late maturing), and in September (spawning period). Amounts of sFF1-I and ERa mRNAs in the pituitary cells were determined by real-time polymerase chain reactions after a treatment with sGnRH, estradiol-17 beta (E2), testosterone (T) or 11-ketotestosterone (11KT). The amounts of sFF1-I mRNA were elevated by E2 in the males, and by sGnRH and T in the females before initiation of gonadal maturation and at the early maturing stage. The amounts of ER alpha mRNA in the early maturing females were elevated by sGnRH. Effects of sGnRH were not significant at the late maturing and spawning stages. The amounts of ER alpha mRNA in the spawning males were halved by 11KT and E2, and those of sFF1-I and ER alpha mRNAs in the late maturing females were decreased by T and 11KT. These results indicated that responsiveness of sFF1-I and ER alpha genes to sGnRH and sex steroid hormones is seasonally variable in relation to reproductive stages. Expression of sFF1 and ER alpha genes should be stimulated at the early stages of gonadal maturation prior to increases in the amounts of GTH subunit mRNAs, while attenuated after the late maturing period when stored amounts of GTH subunit mRNAs reached near the maximum. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2007.02.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The two principal endocannabinoids, N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG), are synthesized from arachidonic acid (AA) and AA is released as they are degraded. Therefore, the function of endocannabinoids is closely linked to AA, but the exact relationships have not been clarified, especially with respect to endocannabinoid metabolism. In the present study, oil rich in AA was administered (0, 100, 200 and 300 ml) orally to male mice for 7 days. Phospholipase D (PLD), fatty acid amide hydrolase (FAAH), diacyl-glycerol lipase (DAGL), monoacyl-glycerol lipase (MAGL) and cannabinoid 1 (CB1) receptor mRNA expressions were determined in the whole brain. No changes in the expression of any gene investigated were detected following AA treatment. However, it was demonstrated that the expression of the CB1 receptor was positively correlated with PLD, FAAH and DAGL expression. This suggests that expression of the CB1 receptor is closely coordinated with that of the enzymes which synthesize its ligands.

DOI： 10.1080/10284150701250671

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Effects of insulin-like growth factor I (IGF-I) and salmon gonadotropin-releasing hormone (sGDRH) on expression of gonadotropin (GTH) subunit genes were examined using primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Fishes were assessed at three reproductive stages, i.e., in April (early maturation), in June (maturing), and in September (spawning). Amounts of GTH subunit mRNAs in pituitary cells were determined using real-time PCR after incubation with IGF-I and/or sGnRH. IGF-I alone had almost no effects on three GTH subunit mRNAs in both sexes, except for decrease in follicle-stimulating hormone (FSH) beta mRNA in males in June. sGnRH alone was effective in stimulation of FSH beta and luteinizing hormone (LH) beta gene expression in males in April. Thereafter it had no significant effects on GTH subunit mRNAs, although in September it tended to increase FSH beta and LH beta mRNAs in females. Co-administered IGF-I counteracted the sGnRH-induced expression of FSH beta and LH beta genes in males in April, but not in females in September. These results suggest that IGF-I is involved in direct regulation of GTH subunit genes during sexual maturation. In particular, IGF-I differently modulates sGnRH-induced GTH subunit gene expression, depending on reproductive stages. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2006.04.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, -R2, -R3, -R4, and -R5, was examined in the brain of masu salmon (Oncorhynchus masou). In addition, responses of these genes to GnRH were examined in a GnRH analog (GnRHa) implantation experiment. Brain samples were collected one week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single forebrains was determined by real-time PCR assays. Among the five genes, R4 and R5 were dominantly expressed in both sexes. R1, R4, and R5 mRNAs showed similar changes throughout the experimental period in both sexes. Levels tended to be high in winter and low in the pre-spawning season, followed by elevations in the spawning period. The mRNA levels had weak to moderate negative correlations with the plasma level of estradiol-17 beta (E2) in females. The effects of GnRHa on msGnRH-R mRNAs were not apparent for all the subtypes. These results indicate that the msGnRH-R1, -R4, and -R5 genes are synchronously expressed during sexual maturation. There was a trend toward decreased levels of their expression prior to the spawning period and then increased levels at spawning, possibly causing GnRH target neurons to sensitize to a GnRH stimulus. Furthermore, E2 may be involved in msGnRH-R gene expression in the brain of female masu salmon during sexual maturation.

DOI： 10.2108/zsj.22.1331

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Cetaceans are well adapted to their hyperosmotic environment by properly developed osmoregulatory ability. A question here is how they regulate water and mineral balances in marine habitats. In the present study, we determined blood and urine levels of various chemicals involved in osmoregulation, compared them with those in artiodactyls, and characterized the values in the whales. Blood and urine samples obtained from baleen whales of common minke (Balaenoptera acutorostrata), sei (B. borealis), and Bryde's whales (B. brydei), and toothed whales of sperm whales (Physeter macrocephalus) were analyzed for osmolality, major electrolytes, urea, steroid hormones and glucose. The urine osmolality and Na+ concentrations in the cetaceans were much higher than those in the cattle. Furthermore, the cetaceans had 5 to 11-fold urea in plasma than the cattle, and 2 to 4-fold urea in urine. There were no significant difference in the plasma concentrations of corticosteroids between the cetaceans and the cattle. The present results indicate that the osmoregulatory parameters seem to be not affected by the reproductive stage and sex steroid hormones. The concentrations of urea in plasma and urine of the baleen whales were higher than those of the sperm whales, indicating a possibility that their osmoregulatory mechanisms may be correlated to their feeding habits. The present results suggest that cetaceans have unique osmoregulatory mechanisms by which they excrete strongly hypertonic urine to maintain fluid homeostasis in marine habitats.

DOI： 10.2108/zsj.22.1245

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Five types of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, R2, R3, R4, and R5, are expressed in the brain and pituitary of masu salmon (Oncorhynchus masou). In the present study, seasonal changes in the expression of these five genes were examined in the pituitary to elucidate their roles in GnRH action during growth and sexual maturation. In addition, the seasonal variation of these genes in response to GnRH was examined in a GnRH analog (GnRHa) implantation experiment. Pituitary samples were collected I week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single pituitaries was determined by real-time PCR assays. Among the five genes, R4 was predominantly expressed in the pituitaries. In the immature fish, the amount of GnRH-R mRNA varied with seasons and subtypes. In the pre-spawning period, R1 and R4 mRNAs in both sexes and R2 and R3 mRNAs in the females increased 4- to 20-fold and then decreased in the spawning season. The effects of GnRHa treatment were significantly different in both sexes. In the females, GnRHa tended to elevate the expression of all the subtypes of GnRH-R genes in various stages during the experimental period, whereas it had almost no apparent effects in the males. These results indicate that the expression of the five GnRH-R genes is seasonally variable and may be related to the responses of the pituitary hormone genes to GnRH, and the regulation of GnRH-R genes by GnRH is different in both sexes. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2005.04.001

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Expression of genes encoding growth hormone (GH), prolactin (PRL), and somatolactin (SL) in growing and maturing salmon was stimulated by gonadotropin-releasing hormone (GnRH) analog during particular periods of the life cycle. GnRH therefore appears to directly and/or indirectly regulate gene expression for GH, PRL, and SL in combination with the pituitary-gonadal axis, such as sex steroid hormones. Direct effects of salmon GnRH (sGnRH), estradiol-17 beta (E2), testosterone, and 11-ketotestosterone (11KT) on the amounts of GH, PRL, and SL mRNAs were thus examined using primary pituitary cell cultures of masu salmon at the four reproductive stages. We also determined the amounts of mRNA encoding pituitary specific POU homeodomain transcription factor (Pit-1) by real-time polymerase chain reactions. The amounts of GH, PRL, and SL mRNAs in the control cells elevated with gonadal maturation, coincidently with those of Pit-1 mRNA. sGnRH at 1.0 nM elevated the amounts of all mRNAs examined in the pre-spawning females, whereas significant effects were not observed with 100 nM sGnRH at any reproductive stages. Sex steroid hormones had no significant effects before initiation of gonadal maturation and at the maturing stage. In the males, E2 tended to decrease the amounts of SL mRNA in the pre-spawning stage. In the females, E2 and 11KT increased the amounts of PRL and SL mRNAs in the pre-spawning stage, but halved those of PRL mRNA in the spawning stage. The amounts of Pit-1 mRNA changed coincidently with those of PRL and SL mRNAs at all examined stages. The effects of E2 alone were abolished by 100 nM sGnRH. The present results indicated that both sGnRH and steroid hormones directly modulate synthesis of Pit-1, and further expression of PRL and SL genes. sGnRH may indirectly regulate GH/PRL/SL family hormone genes through the pituitary-gonadal axis, particularly in the late stage of gametogenesis. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2005.03.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Our previous studies suggested that salmon gonadotropin-releasing hormone (sGnRH) neurons regulate both final maturation and migratory behavior in homing salmonids. Activation of sGnRH neurons can occur during upstream migration. We therefore examined expression of genes encoding the precursors of sGnRH, sGnRH-I, and sGnRH-II, in discrete forebrain loci of prespawning chum salmon, Oncorhynchus keta. Fish were captured from 1997 through 1999 along their homing pathway: coastal areas, a midway of the river, 4 km downstream of the natal hatchery, and the hatchery. Amounts of sGnRH mRNAs in fresh frozen sections including the olfactory bulb (OB), terminal nerve (TN), ventral telencephalon (VT), nucleus preopticus parvocellularis anterioris (PPa), and nucleus preopticus magnocellularis (PM) were determined by quantitative real-time polymerase chain reactions. The amounts of sGnRH-II mRNA were higher than those of sGnRH-I mRNA, while they showed similar changes during upstream migration. In the OB and TN, the amounts of sGnRH mRNAs elevated from the coast to the natal hatchery. In the VT and PPa, they elevated along with the progress of final maturation. Such elevation was also observed in the rostroventral, middle, and dorsocaudal parts of the PM. The amounts of gonadotropin II beta and somatolactin mRNAs in the pituitary also increased consistently with the elevation of gene expression for sGnRH. These results, in combination with lines of previous evidence, indicate that sGnRH neurons are activated in almost all the forebrain loci during the last phases of spawning migration, resulting in coordination of final gonadal maturation and migratory behavior to the spawning ground. (c) 2005 Wiley Periodicals, Inc.

DOI： 10.1002/neu.20125

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/cne.20473

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

Gonadotropin-releasing hormone (GnRH) plays a central role in the control of reproductive function in vertebrates. In salmonids, salmon GnRH (sGnRH) secreted by preoptic GnRH neurons regulates gonadal maturation through stimulation of synthesis and release of pituitary gonadotropins (GTHs). In addition, several lines of our evidence indicate that sGnRH is involved in spawning behavior, and serves to integrate the gonadal maturation with the reproductive behavior. A growing number of studies show that the effects of GnRH are mediated by multiple subtypes of GnRH receptors, successive multiple signaling pathways, and finally multiple transcription factors which act cooperatively to stimulate transcription of GTH subunit genes. This complex regulatory system of the action of GnRH may serve as a molecular basis of divergent physiological strategies of reproductive success in various vertebrate species. In this article, recent data on the molecular mechanisms of action of GnRH are reviewed with special reference to the regulation of synthesis and release of GTHs in the pituitary of salmonids to elucidate the multifunctional action of GnRH.

DOI： 10.2108/zsj.22.379

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Xenoestrogens such as 4-nonylphenol (4-NP) have been shown to affect the parr-smolt transformation, but their mechanisms of action are not known. We therefore examined effects of 4-NP and estradiol-17 beta (E2) on expression of estrogen receptor (ER) alpha gene in the liver, gill, pituitary and brain of sockeye salmon to elucidate molecular mechanisms of 4-NP and E2 and developmental differences in response during smolting. Fish were treated twice within a week with 4-NP (15 and 150 mg/kg BW), E2 (2 mg/kg BW) or only vehicle at three stages of smolting, pre-smolting in March, early smolting in April and late smolting in May. The absolute amounts of ER alpha mRNA were determined by real-time PCR. The basal amounts of ER alpha mRNA peaked in April in the liver, gill and pituitary. In March, E2 extensively increased the amounts in the liver, while 4-NP had no effects at this stage. In contrast, 4-NP (but not E2) decreased liver ER alpha mRNA in April. 4-NP also decreased the amount of ER alpha mRNA in the gill in April. In the pituitary, 4-NP increased ER alpha mRNA in March but decreased it in May. There were no significant effects in the brain. Changes in basal ER alpha mRNA observed in this study indicate that estrogen responsiveness of tissues may change during salmon smolting. Furthermore, 4-NP and E2 have different effects on expression of ERa gene in the liver and gill during smolting, and the response is dependent on smolt stage. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cca.2005.01.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Detailed distribution of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase (sGC) was examined in the brain of rainbow trout by in situ hybridization. In addition, distribution of nitric oxide synthase (NOS) was mapped in adjacent parallel sections by neuronal NOS (nNOS) immunocytochemistry and NADPH-diaphorase (NADPHd) histochemistry. Following application of digoxigenin-labeled riboprobes for sGC alpha and beta subunit mRNAs, we found comparatively intense hybridization signals in the telencephalon, preoptic area, thalamus, hypothalamus, pretectum and tegmentum. Both nNOS immunocytochemistry and NADPHd histochemistry showed extensive distribution of nitroxergic neurons in various brain areas, although various degrees of dissociation of nNOS immunoreactivity (ir) and NADPHd staining were detected. In comparison with sGC subunit mRNAs, nNOS signals were more widely distributed in many neurons, including parvocellular neurons in the preoptic area, nucleus anterior tuberis in the hypothalamus, periventricular neurons in the optic tectum, most of the rhombencephalic neurons and pituitary cells. However, wide overlaps of sGC mRNA-containing neurons and nNOS-positive neurons were observed in the olfactory bulb, telencephalon, preoptic area, thalamus, hypothalamus, pretectum, optic tectum, tegmentum and cerebellum. The widespread overlapping in sGC subunit mRNAs and nNOS distribution suggests a role for sGC in various neuronal functions, such as processing of olfactory and visual signals and neuroendocrine function, possibly via NO/cGMP signaling in the brain of rainbow trout. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2004.03.063

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2004.02.012

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In the present study, diurnal changes in plasma levels of melatonin and cortisol, and hypothalamic contents of neurohypophysial hormone mRNAs were examined in pre-spawning chum salmon, Oncorhynchus keta. From late November to early December, homing fish were captured at two sites along their migratory pathway on the Sanriku coast, Japan. Fish captured in the seawater (SW) environment were transferred to SW aquaria, and fish captured in the freshwater (FW) environment were to FW aquaria. They were maintained under natural photoperiod of approximately 10L:14D and sacrificed at 4-h interval through 24-h period. Plasma levels of melatonin were determined by radioimmunoassay, while cortisol levels were determined by enzyme immunoassay. Hypothalamic contents of vasotocin and isotocin mRNAs were determined by quantitative dot-blot hybridization assay. The melatonin levels showed weak nocturnal elevations in the SW and FW males, and FW females. The levels were maximal at 22:00 and minimal at 10:00 or 14:00, however the amplitudes were smaller than those reported in the previous studies using immature salmonids. The levels of vasotocin and isotocin mRNAs were higher in the males at all time points. The mRNA levels, however, did not show any diurnal variations in either of group. The same applied to plasma cortisol levels. These results indicate that the diurnal endocrine rhythms were attenuated in pre-spawning chum salmon, in contrast to the prominent diurnal rhythms in immature salmonids. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2004.02.010

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Melatonin and arylalkylamine N-acetyltransferase (AANAT), the rate-limiting enzyme in melatonin synthesis, have taken on special importance in vertebrate circadian biology. Recent identification of genes encoding two AANAT (AANAT(1) and AANAT(2)) and two subtypes of melatonin receptor (Mel-R; Mel(1a) and Mel(1b)) in several fish species has led to rapid advances in characterizing the physiological roles of melatonin. In the present study, partial cDNAs encoding these four genes were cloned from the eye and brain of chum salmon (Oncorhynchus keta). Based on the nucleotide sequences, we developed highly sensitive real-time PCR systems for these four mRNAs. The development of daily rhythmicity in AANAT(1), AANAT(2), Mel(1a), and Mel(1b) transcript levels was examined in the eye and brain of chum salmon during embryonic and post-embryonic stages (from day -9 to day +180). In a parallel experiment, ocular and brain melatonin levels were measured by radioimmunoassay. Parallelism in developmental changes and in circadian rhythms of AANAT mRNAs and melatonin levels in the eye and the brain supports a hypothesis that the developmental increases of nocturnal melatonin levels results partly from the elevated transcription of AANAT genes. Moreover, abundant expression of AANAT and Mel-R mRNAs in the optic tectum, thalamus, hypothalamus, cerebellum, and eye indicates possible roles of melatonin in visual processing and neuroendocrine regulation, through which melatonin might be involved in migratory behavior of chum salmon. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2004.01.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5' end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency chi(2) tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.

DOI： 10.1023/B:EBFI.0000022881.90237.aa

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity. (C) 2003 Wiley Periodicals, Inc.

DOI： 10.1002/neu.10296

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Recent studies have shown that there are multiple genes encoding gonadotropin-releasing hormone receptor (GnRH-R) in single species. In salmonids, however, only a single gene has been identified in the rainbow trout. We therefore isolated partial cDNAs from the brain and the pituitary of masu salmon Oncorhynchus masou by reverse transcription-polymerase chain reaction and 5'-rapid amplification of cDNA ends, using primers corresponding to conserved transmembrane domains (TMs). Five different partial cDNAs were isolated from an individual and termed as msGnRH-R1, R2, R3, R4 and R5. They are divided into two groups, msGnRH-R1, R2, R3 and msGnRH-R4, R5. Two groups share 59-71% nucleotide sequence identities. Phylogenetic analysis showed that the former group is closely related to the goldfish GnRH-R GfA, and the latter to GfB. All five msGnRH-R genes were expressed in the brain and msGnRH-R1, R3 and R5 were expressed in the pituitary. In addition, we found mRNA for msGnRH-R1 in the kidney and ovary, and R2 in the ovary, whereas msGnRH-R5 gene was widely expressed in the muscle, heart, kidney and testis. Differences in the expression of msGnRH-R genes between maturing and spawning fish were observed in the brain and pituitary, except for the constantly expressed msGnRH-R5. A splicing variant of msGnRH-R1 mRNA that is capable of generating a truncated GnRH-R that consists of 5TMs was also expressed in the brain, pituitary and kidney. These results indicate that five different types of putative GnRH-R gene are present and expressed in the brain of masu salmon.

DOI： 10.2108/zsj.20.1117

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Changes in plasma levels of steroid hormones in pre-spawning chum salmon (Oncorhynchus keta) were examined for 6 years in association with sexual maturation. Fish were sampled along their homing pathway from the coastal sea to the spawning ground from 1995 to 2000. Plasma levels of testosterone (T), I I-ketotestosterone (11KT), estradiol-17beta (E-2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), and cortisol were determined by enzyme immunoassays. Sexual maturity was comprehensively estimated by gonadosomatic indices, histology of gonads, nuptial color, spermiation or ovulation ratio. Since the plasma levels of steroid hormones and sexual maturation differed from year to year, they were compared with year-to-year variation of sea surface temperature (SST) of coastal sea to study influence of oceanographic environment on these physiological data. The SST of the migratory route varied among the years, so that we classified the 6 years into cool, intermediate, and warm years. Concerning maturity, the males that returned to the natal hatchery in the warm years were sexually more advanced than those in the cool years. Furthermore, histological data suggested that final oocyte maturation occurred before arrival at the hatchery in one of the warm years, i.e., 1999, while it occurred at the hatchery in one of the intermediate years, i.e., 2000. In the males, T and 11KT levels increased significantly on midway of the homing route in the warm years, whereas they did not show any noticeable changes in the cool years. Furthermore, the levels of T and 11KT on midway of the homing route in the warm years, i.e., 1998 and 1999, were significantly higher than those in one of the cool years, i.e., 1995, in both sexes. In the females, the levels of E-2 decreased during upstream migration. Conversely, those of DHP considerably elevated at spawning ground in all years examined. The levels of cortisol were different from year to year regardless of the SST. The present results showed that there were year-to-year differences in plasma levels of steroid hormones and maturity, and some of them may be influenced by the year-to-year variation of SST. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0016-6480(03)00171-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

The pituitary levels of mRNAs encoding gonadotropin (GTH) subunits (GTH alpha2 and IIbeta), prolactin (PRL), and somatolactin (SL) increased in chum salmon during the last stages of spawning migration. In the-present study, changes in pituitary levels of mRNAs encoding GTH alpha2, Ibeta, and IIbeta; growth hormone (GH); PRL; and SL were examined in homing chum salmon of Sanriku stock to clarify whether the changes are associated with final maturation or freshwater (FW) adaptation. In 1993, fish were caught at four areas: off the coast of Sanriku (off-coast), the mouth of Otsuchi Bay (ocean), inside of Otsuchi Bay (bay), and the Otsuchi River (river). In addition, effects of hypoosmotic stimulation by transition from seawater (SW) to FW were examined in 1994 and 1995. The amounts of mRNAs were determined by dot-blot analyses or real-time polymerase chain reactions. The levels of GTH alpha2 and IIbeta mRNAs in the ocean, bay, and river fish were two to five times those in the off-coast fish, and the levels of SL mRNAs in the bay fish were two to four times those in the off-coast fish. The levels of GH and PRL mRNAs in the ocean and bay fish were significantly lower than those in the off-coast fish, and those in the river fish were three to five times those in the ocean and bay fish. In the SW-to-FW transition experiment in 1994, the levels of GTH alpha2, Ibeta, and lip mRNAs transiently increased, whereas changes were insignificant in 1995. The levels of GH, PRL, and SL mRNAs increased in both SW and FW environments, and no apparent effects of SW-to-FW transition were observed. The present study suggests that in prespawning chum salmon, expression of genes encoding GTH alpha2, IIbeta, and SL elevates with final maturation regardless of osmotic environment. Hypoosmotic stimulation by transition from the SW-to-FW environment is not critical to modulate expression of genes for PRL. PRL gene expression can be elevated in SW fish that were sexually almost matured.

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The pituitary levels of mRNAs encoding gonadotropin (GTH) subunits (GTH α2 and IIβ), prolactin (PRL), and somatolactin (SL) increased in chum salmon during the last stages of spawning migration. In the present study, changes in pituitary levels of mRNAs encoding GTH α2, Iβ, and IIβ growth hormone (GH)
PRL
and SL were examined in homing chum salmon of Sanriku stock to clarify whether the changes are associated with final maturation or freshwater (FW) adaptation. In 1993, fish were caught at four areas: off the coast of Sanriku (off-coast), the mouth of Otsuchi Bay (ocean), inside of Otsuchi Bay (bay), and the Otsuchi River (river). In addition, effects of hypoosmotic stimulation by transition from seawater (SW) to FW were examined in 1994 and 1995. The amounts of mRNAs were determined by dot-blot analyses or real-time polymerase chain reactions. The levels of GTH α2 and IIβ mRNAs in the ocean, bay, and river fish were two to five times those in the off-coast fish, and the levels of SL mRNAs in the bay fish were two to four times those in the off-coast fish. The levels of GH and PRL mRNAs in the ocean and bay fish were significantly lower than those in the off-coast fish, and those in the river fish were three to five times those in the ocean and bay fish. In the SW-to-FW transition experiment in 1994, the levels of GTH α2, Iβ, and IIβ mRNAs transiently increased, whereas changes were insignificant in 1995. The levels of GH, PRL, and SL mRNAs increased in both SW and FW environments, and no apparent effects of SW-to-FW transition were observed. The present study suggests that in prespawning chum salmon, expression of genes encoding GTH α2, IIβ, and SL elevates with final maturation regardless of osmotic environment. Hypoosmotic stimulation by transition from the SW-to-FW environment is not critical to modulate expression of genes for PRL. PRL gene expression can be elevated in SW fish that were sexually almost matured.

DOI： 10.1385/ENDO:20:1-2:23

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Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/ SL genes, in the pituitaries of growing and maturing masu salmon (Oneorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0016-6480(02)00536-1

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG TOKYO  

We developed an assay method to determine magnitudes of changes in the amount of particular mRNA, as indices of expression of hormone genes in connection with salmon migration. First, we developed a quantitative dot blot analysis in which single-stranded sense cDNA was used as the standard for particular mRNA encoding a certain pituitary hormone. Validation tests showed that cross hybridization of the probe to mRNAs for other pituitary hormones was less than 1%, and intra- and inter-assay coefficients of variations were comparable to or less than those of radio immunoassays. The sensitivity was between 1 to 10 amol. However, this value is not sufficient to determine the levels of neurohormone mRNAs in the discrete brain loci. Hence, we incorporated a quantitative real-time PCR method, and established a reliable protocol yielding the sensitivity of about 100 copies of particular mRNA with satisfactorily ranges of variations.

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The roles of salmon GnRH (sGnRH) and gonadal steroid hormones in regulation of LH synthesis and release were examined in primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Pituitaries were taken from fish at four reproductive stages: in March (initiation of sexual maturation)
May (early maturation)
July (pre-spawning)
and September (spawning period). Amounts of LHβ subunit mRNA in the pituitary cells were determined by real-time PCR, and LH levels in the medium were determined by RIA. sGnRH and gonadal steroids including estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were added to the cultures to examine their direct effects on LH response. sGnRH had no significant effect on LHβ mRNA levels at any stages, although a stimulatory trend was noted in March. In contrast, E2 and T considerably increased LHβ subunit mRNA levels in March and May during initial stages of maturation, and the effects were less pronounced in July and September. On the other hand, sGnRH stimulated LH release at all stages in the males and the effects were most prominent in July and September. E2 and T also stimulated LH release in July and September, but their effects were weaker than that of sGnRH. The present results indicate that sGnRH and gonadal steroids directly regulate LH synthesis and release in masu salmon pituitary cells: sGnRH mainly stimulates LH release in the late stage of sexual maturation
whereas, E2 and T are effective in stimulating LH synthesis at earlier stages of maturation. © 2004 Kluwer Academic Publishers.

DOI： 10.1023/B:FISH.0000030477.87920.2d

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We are conducting a collaborative study to develop molecular markers and baseline information for chum salmon (Oncorhynchus keta) to address stock identification problems and other issues. To date, more than 2,000 individuals representing 46 populations from Japan/Korea, Russia and North America (Alaska, British Columbia and Washington) have been evaluated with mitochondrial (mt) DNA sequence variation and/or at five microsatellite (ms) DNA loci. Analysis of mtDNA disclosed a greater genetic variation in the populations of Japan than those of Russia and North America and a distinct genetic differentiation among the three regional groups of populations, in addition to more than 20 region-specific haplotypes. Similar genetic variation was also found using msDNA, although allelic variation was different among the examined loci. Besides estimation of the genetic differentiation and stock origin of chum salmon, our DNA markers, particularly mtDNA analysis, may provide a useful means for understanding the historical origin and the formation of population structure in this species.

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Ontogenic changes in the expression levels of two salmon gonadotropin-releasing hormone genes (sGnRH-I and -II) were examined in the forebrain region including the ventral telencephalon and preoptic area of masu salmon by competitive reverse transcription-polymerase chain reaction (RT-PCR). Two genes showed similar expression patterns throughout the lifetime in both sexes, although the levels of sGnRH-II mRNA were about 20 times higher than those of sGnRH-I mRNA. In males, the levels of sGnRH mRNAs increased at the beginning of the second year and reached their maximum in the autumn. The levels decreased gradually until the autumn of the third year when fish sexually matured. In females, the levels reached their maximum in the first autumn and fluctuated considerably along with the seasons in the third year. These results suggest that, in the salmon brain, sGnRH genes are activated long before the sexual maturation under sexually different control mechanisms. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3940(01)01942-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Fushi tarazu factor-1 (FTZ-F1) and Pit-1 are major pituitary transcription factors, controlling expression of genes coding for gonadotropin (GTH) subunits and growth hormone/prolactin/somatolactin family hormone, respectively. As a first step to investigate physiological factors regulating gene expression of these transcription factors, we determined their mRNA levels in the pituitaries of chum salmon (Oncorhynchus keta) at different stages of sexual maturation. FTZ-F1 gene expression was increased in males at the stage before spermiation, where the levels of GTH a: and LIP subunit mRNAs were elevated. Pit-1 mRNA showed maximum levels at the final stage of sexual maturation in both sexes, when expression of somatolactin gene peaked. To clarify whether gonadotropin-releasing hormone (GnRH) is involved in these increases in FTZ-F1 and Pit-1 gene expression, we examined effects of GnRH analog (GnRHa) administration on their gene expression in maturing sockeye salmon (Oncorhynchus nerka). GnRHa stimulated Pit-1 gene expression in females only, but failed to stimulate FTZ-F1 gene expression in both sexes. The up-regulated expression of FTZ-F1 and Pit-1 genes at the pre-spawning stages suggest that the two transcription factors have roles in sexual maturation of salmonids. Physiological factors regulating gene expression of FTZ-F1 and Pit-1 are discussed in this review. (C) 2001 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S1096-4959(01)00348-7

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Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) subunit gene expression via G protein-coupled membrane receptors. GnRH-stimulated GTH subunit gene expression is mediated by protein kinase C (PKC) and Ca2+ signaling pathways. Recent numerous reports on signal transduction pathways which are involved in GnRH stimulation of mammalian GTH subunit genes showed differential sensitivity of GTH subunit genes to the two signaling pathways. Our recent studies on salmon GTH (sGTH) II beta subunit gene showed that its stimulation by GnRH is dependent on the PKC pathway. Furthermore, gel retardation and mutagenesis studies suggested that pituitary homeo box 1 (Ptx1) and Sp1 mediate the GnRH-induced PKC signaling on the sGTHII beta gene. However, both PKC and Ca2+ pathways are involved in the GnRH-stimulated GTH alpha and LH beta genes. Different preference to the pathways were often reported in a certain GTH subunit gene in different circumstances, suggesting that molecular targets of the two signaling pathways are different. Ets-related factor and cAMP response element binding protein have been proposed as targets of GnRH signaling on GTH alpha genes. Sp1 and early growth response protein 1 play pivotal roles in GnRH-stimulated LHP gene expression in synergism with steroidogenic factor-1 and Ptx1. Activating protein-1 mediates GnRH-induced PKC signaling to stimulate FSH beta gene expression. Therefore, divergent transcription factors are involved in GnRH stimulation of GTH subunit gene expression, and molecular mechanisms of GnRH stimulation may be partially conserved between sGTH II beta and mammalian LH beta genes. (C) 2001 Elsevier Science Inc. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Effects of the coastal oceanographic conditions on sexual maturation, salinity tolerance, and expression of vasotocin (VT) gene in homing chum salmon, Oncorhynchus keta, were studied by four years of fieldwork and transfer experiments. In fieldwork in 1992 and 1993, fish were sampled at three sites along their migratory pathway on the Sanriku coast, Japan. In transfer experiments in 1994 and 1995, fish captured in the seawater (SW) environment were transferred to SW or freshwater (FW) aquaria and sampled 1 to 4 days later. The distribution of cold and warm currents, which governs the oceanographic conditions of the Sanriku coast, were deduced from the mean sea surface temperature. Maturity of homing fish was estimated by gonadal states. Salinity tolerance was estimated by plasma Na+ levels and mortality in SW environment. Changes in VT gene expression were assessed by a quantitative dot blot analysis of the hypothalamic levels of VT mRNA. Homing fish were fully mature, and showed high plasma Na+ levels and high mortality in the SW environment in 1992 and 1994 when the warm current dominated. In the 1994 transfer experiment, VT mRNA levels markedly increased in the SW-retained males, whereas the levels were decreased by FW-transfer in both sexes. Homing fish were not fully matured in 1993 and 1995 when the branch of cold current reached the Sanriku coast. In the 1995 transfer experiment, VT mRNA levels did not change in both FW-retained and FW-transferred fish. In conclusion, the warmer oceanographic conditions affected the maturity and salinity tolerance in homing salmon, which in turn altered the osmotically-induced expression pattern of VT gene.

DOI： 10.2108/zsj.18.389

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We examined the nucleotide sequences of 500 bp variable portion from the 5' end of mitochondrial (mt) DNA control region in about 500 individuals from 12 populations that were captured in 11 rivers, six in Hokkaido and five in Honshu, japan. Comparison of the sequences showed 10 variable sites, defining a total of 12 haplotypes in the examined individuals. All the 12 haplotypes occurred in seven Hokkaido populations, whereas only six haplotypes were found in the five Honshu populations. Among these haplotypes, two were common in all the Hokkaido and Honshu populations. The AMOVA analysis inferred a genetic differentiation among three geographic regions, i.e. Hokkaido, Pacific Ocean coast in Honshu, and Japan Sea coast in Honshu. Haplotype diversity was higher in the populations of Hokkaido than those of Honshu, indicating a greater genetic variation in the Hokkaido than the Honshu populations. The estimates of painwise population F-ST suggested that the regional differentiation was mostly ascribed to the divergence between populations in Hokkaido and the Pacific coast in Honshu.

DOI： 10.2108/zsj.18.99

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：MEDIMOND S R L  

Guanylyl cyclase (GC) mRNAs and nitric oxide synthase (NOS) activity were examined in the retina and pineal organ of immature rainbow trout by in situ hybridization (ISH) and NADPH-diaphorase (NADPHd) histochemistry. In the laminar retina, mRNAs encoding alpha and beta subunits of salmon soluble GC (ssGC-alpha and ssGC-beta) and salmon membrane GC (smGC) were mainly located in the outer and inner segments of photoreceptors. However, no hybridization signal for ssGC mRNAs was observed in the light and dark-adapted pineal organs, although strong signals for smGC mRNA were present in the pineal photoreceptors. NOS activity was widely distributed in the retinal layers and pinealocytes while with unevenly distributed levels. In conclusion, NOS activity and GC mRNAs were colocalized in the retinal and pineal photoreceptors, suggesting functional importance of the NO-cGMP system in trout phototransduction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Steroidogenic factor 1 (SF-1) or Ad4BP is a member of the fushi tarazu factor 1 (FTZ-F1) family and an orphan nuclear receptor that plays an important role in the hypothalamus-pituitary-gonadal axis and the adrenal cortex. Although its critical role in the differentiation of adrenals, gonads, and pituitary gonadotropes has been well demonstrated, regulatory function of SF-1 during sexual maturation is yet to be examined. To investigate the potential role of SF-1 in sexual maturation, expression of two salmon FTZ-F1 homolog genes, sFF1-I and sFF1-II, was examined in the pituitaries of chum and sockeye salmons, using specific and sensitive RNase protection assays. Only sFF1-I mRNA was found in the pituitary and other organs, such as the ovary, spleen, liver, brain, and skeletal muscle. In chum salmon during upstream migration from the bay to the hatchery, the level of sFF1-I mRNA in the male fish was increased on the midway in the river, where the levels of gonadotropin alpha- and II beta -subunit mRNAs were increased. In maturing sockeye salmon, the expression of the sFF1-I gene was elevated in the mature male fish, but the administration of CnRH analog did not further enhance the expression. These results indicate that sFF1-I gene expression in the pituitary is upregulated in maturing salmon, and this upregulation may not depend on GnRH.

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The profile of homing behavior in chum salmon (Oncorhynchus keta) that migrate from coastal sea to their natal river was not known well. We thus investigated temporal behavioral profiles of pre-spawning chum salmon in terms of water depth and temperature in Ishikari Bay using a micro data logger in 1997 and 1998. Fish were caught by a set net, tagged and attached with a data logger under MS222 anesthesia, and were released at the points 5 and 3 km off from the mouth of the Ishikari River in 1997 and 1998, respectively. A temporal profile of water depth and ambient temperature along the migratory pathway of recaptured salmon indicated that they usually stayed near the surface where water temperature was relatively low. Conductivity-temperature-depth recorder (CTD) data and the sea surface temperature estimated with National Oceanic and Atmospheric Administration (NOAA) Advanced Very High Resolution Radiometer (AVHRR) indicated that the influent of river water formed a low-temperature brackish subsurface layer near the mouth of the Ishikari River. These results indicate that chum salmon homing to the Ishikari River prefer low water temperature, and wander in the subsurface layer of nearshore area close to the mouth of the Ishikari River until they are motivated to migrate upstream. The main factor that regulates behavioral pattern of returned chum salmon in coastal sea should be distribution of water temperature.

DOI： 10.2108/zsj.17.1247

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Pituitary hormones regulate various physiological functions during spawning migration in salmonid. Cytological features of pituitary cells were therefore immunocytochemically examined by use of antisera against homologous hormones in pre-spawning chum salmon (Oncorhynchus keta) caught from Ishikari Bay and the Chitose River in October, 1996. Immunoreactivity and sizes of pituitary cells were determined by a computer-aided image analyzing technique. Immunoreactivity in growth hormone (GH) cells was stronger with enlarged cell sizes in freshwater (FW) fish than in seawater (SW) ones of both sexes. Majority of prolactin (PRL) cells also had significantly stronger immunoreactivity with enlarged cell sizes in FW fish than in SW animals of both sexes. Immunoreactivity in somatolactin (SL) cells was markedly stronger with enlarged cell sizes in FW fish than in SW ones of both males and females. In addition, greater portions of SL cells were strongly stained in FW animals than in SW ones. A greater portion of gonadotropin (GTH) I cells had stronger immunoreactivity with reduced cell sizes in FW fish of both sexes, when compared with SW fish. Conversely, GTH II cells had significantly stronger immunoreactivity with enlarged cell sizes in FW ones of both sexes. In proopiomelanocortin (POMC)-derived hormone producing cells, adrenocorticotropin (ACTH) cells had stronger immunoreactivity in FW animals of both sexes, while cell sizes did not change. In melanotropes, the cells immunoreactive to alpha-melanophore stimulating hormone (alpha-MSH) antiserum had stronger immunoreactivity with reduced cell sizes only in FW males, while the cells immunoreactive to beta-endorphin antiserum had stronger immunoreactivity with reduced cell sizes in FW fish than in SW animals of both sexes. Implication of these results was discussed along with previous reports on gene expression of pituitary hormone precursors.

DOI： 10.2108/zsj.17.805

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Gonadotropin-releasing hormone (GnRH) is a possible secretagogue of growth hormone (GH) and somatolactin (SL) in teleosts. Effects of GnRH on the levels of pituitary mRNAs encoding GH, prolactin (PRL), and SL were therefore examined in prespawning sockeye salmon (Oncorhynchus nerka). A capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon for 3 weeks. The levels of hormonal mRNAs were then determined by a quantitative dot blot analysis using single-stranded sense DNA of the same sequence of mRNA as the standard. Further, we analyzed effects of GnRHa on expression of the genes encoding pituitary-specific transcription factor (Pit-1/GHF-1). Relative levels of Pit-1/GHF-1 mRNAs were estimated by Northern blot analysis, which showed specific 2- and 3-kb bands of mRNAs. GnRHa significantly increased the level of SL mRNA in the males, but not in the females, compared to the control fish. It did not induce significant increases in the levels of GH and PRL mRNAs in both the males and the females. The levels of Pit-1/GHF-1 mRNAs in the control males tended to be higher than those in the initial controls, so that GnRHa might not be effective in enhancing expression of Pit-1/GHF-1 gene, except for the level of 3-kb Pit-1/GHF-1 mRNA in the females treated with 150 lug GnRHa. The pattern of changes in the levels of Pit-1/ GHF-1 mRNAs were similar to those of GH and PRL mRNAs in both the males and the females and to that of SL mRNA in the females. These results indicate that, in prespawning sockeye salmon, GnRH can stimulate SL gene expression, but probably not through the Pit-1/ GHF-1-dependent system. (C) 2000 Academic Press.

DOI： 10.1006/gcen.2000.7480

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-hox was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the "dimerization" domain (I-box) is essential for the transcriptional activity of zFF1A Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAIA-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its Banking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain "cross-talk" may confer the full transcriptional activity of the protein.

DOI： 10.1074/jbc.M000121200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Juveniles of chum salmon (Oncorhynchus keta) held at high population density were apparently smaller than those held at medium and low population densities. The effects of high population density on pituitary growth hormone (GH) cells in juvenile chum salmon were examined using immunocytochemical and in situ hybridization techniques. The ratio of GH-immunoreactive (ir) area to the whole pituitary was almost constant in all of the high, medium and low population density groups, although the number and sizes of GH-ir cells were decreased in the high population density group. Image-analysis of GH-ir cells indicated the presence of a population of heterogenous cells, in which medium or rather strongly stained smaller cells and as extreme weakly stained larger cells. The medium or rather strongly stained smaller cells predominated in the high population density group, while weakly stained larger cells in the low population density group. In situ hybridization study showed somewhat different distributions and intensities of hybridization signals for mRNAs encoding GH I and II precursors. The area showing signals for GH II mRNA in the high population density group was significantly smaller than those in the medium and low population density groups. In contrast, the sizes of areas showing signals for GH I mRNA did not differ among the groups, although the intensity was slightly higher in the high population density group. These results indicate that high population density decreased the number of weakly immunoreactive larger GH cells, and also suppressed expression of the gene encoding GH II precursor, which may result in retarded somatic growth.

DOI： 10.2108/zsj.16.945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) production by activating GTH subunit gene transcription. In salmonid fish, the expression of the beta subunit gene of GTH II (sGTH II beta) is stimulated by GnRH at the final stages of reproduction. DNA elements required for the GnRH stimulation were examined by analyzing sGTH II beta promoter activity by transfection studies in a gonadotrope-derived cell line, alpha T3-1. A GnRH analog (GnRHa) specifically stimulated the sGTH II beta promoter (3358 bp) expression 3.6-fold, while phorbol myristate acid (PMA) stimulated it 6.2-9-fold. Analysis of a series of 5'-deletion mutants has revealed that a proximal region (-258 to -199) was important in GnRHa stimulation through protein kinase C (PKC)-independent signal transduction pathways, because an internal deletion mutant (Delta(246-217)/3358) showed a significant decrease in the level of GnRHa stimulation, but showed no change in stimulation by PMA. A large upstream region (-3358 to -1260) showed an enhancing activity of the GnRHa stimulation, and a far upstream 530 bp segment in this region (-3358 to -2829) may be responsible for this activity. The present results suggest that sGTH II beta gene may be controlled by GnRH through multiple DNA elements including those responsive to PKC-dependent and -independent signal transduction pathways. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0303-7207(99)00153-7

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In relevance to osmoregulatory and reproductive functions, activity of the hypothalamic magnocellular neurosecretory system may vary seasonally in teleosts. The changes in the expression of vasotocin (VT) and isotocin (IT) genes were thus studied by an in situ hybridization technique and an immunohistochemical avidin-biotin complex method in immature female masu salmon (Oncorhynchus masou). The plasma levels of testosterone and estradiol were also measured by enzyme immunoassay. Fish were sampled in March, May, August, and November 1994 and January 1995. The intensity of autoradiographic hybridization signals and immunoreactivity were determined in individual neurosecretory cells (NSC) in the rostroventral, middle, and dorsocaudal regions of the magnocellular part of the preoptic nucleus (PM). The VT hybridization signals and immunoreactivity were high in November, along with the elevation of plasma levels of testosterone and estradiol. These results suggest that sex steroid hormones are involved in seasonal regulation of VT gene expression. The hybridization signals for IT mRNA were increased in May and decreased in November, whereas IT immunoreactivity was low in March and high in November. NSCs thus showed seasonal variations in the intensity of hybridization signals for VT and IT mRNAs and immunoreactivity of VT and IT, although the patterns of changes were different between VT and IT. VT and IT genes may be seasonally expressed under different regulatory mechanisms. (C) 1999 Academic Press.

DOI： 10.1006/gcen.1999.7343

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Our previous study showed the seasonal variations in expression of vasotocin (VT) and isotocin (IT) genes in preoptic magnocellular neurons of female masu salmon (Oncorhynchus masou). The changes in the level of VT mRNA were coincident with those in plasma testosterone and estradiol levels. In the present study, generality of this phenomenon in salmonid was verified in males. We examined changes in expression of VT and IT genes by an in situ hybridization technique and an immunohistochemical avidin-biotin complex method in the preoptic nuclei of ordinary and precocious male masu salmon. Plasma levels of testosterone and estradiol were measured by enzyme immunoassay. Fish were sampled in March, May, August, and November 1994 and January 1995. The intensities of hybridization signals for VT and IT mRNAs, as well as immunoreactivity of VT and IT, showed seasonal variations, although the profiles were different between the ordinary and precocious males. In the ordinary males, the intensities of hybridization signals for VT and IT mRNAs were high in January. These strong hybridization signals, representing elevation of VT and IT gene expression, were accompanied by increases in plasma levels of testosterone and estradiol. However, in precocious males, changes in VT and IT mRNA levels were not coincident with variation of plasma levels of sex steroid hormones. The sensitivity to sex steroid hormones of VT and IT gene expression may be different between the ordinary and precocious male masu salmon. (C) 1999 Academic Press.

DOI： 10.1006/gcen.1999.7344

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC ENDOCRINOLOGY  

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.

DOI： 10.1677/jme.0.0230189

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The behavior of genes encoding the corticotropin-releasing hormone (CRH) precursor in response to stress has not been extensively studied in teleosts. To clarify this problem, CRH cDNAs were isolated from a hypothalamic cDNA library of sockeye salmon, Oncorhynchus nerka, by screening with PCR products amplified from the hypothalamic mRNA with primers deduced from the sequence of the sucker CRH precursor. Two types of PCR products with a high degree of sequence homology were identified (CRH-I and CRH-II). A cDNA encompassing the entire ceding sequence of the salmon CRH-I precursor was isolated. The salmon CRH-I cDNA encodes a 167-amino-acid precursor, which consists of a signal sequence, a cryptic peptide, and the carboxyl terminal 41-amino-acid sequence of CRH. The deduced amino acid sequence of salmon CRH peptide exhibits 66 to 80% homology with mammalian, Xenopus, and sucker CRHs, whereas it shows about 50% homology with sucker, carp, or role urotensin I, a CRH-related neuropeptide in teleost fish. In situ hybridization histochemistry demonstrated CRH mRNA-positive perikarya in the preoptic nucleus in rainbow trout, Oncorhynchus mykiss, when the fish were stressed by confinement. Adjacent sections hybridized with probes for salmon vasotocin (VT) precursor showed many VT mRNA-positive neurons also in the preoptic nucleus, suggesting a colocalization of CRH and VT mRNAs in the same magnocellular neurons in the rainbow trout brain. The present results suggest that CRH may have important roles in the control of stress responses in salmonid fish. (C) 1999 Academic Press.

DOI： 10.1006/gcen.1998.7182

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：HORMONE RESEARCH CENTER  

Aiming to realize a genetic program of stereotyped behavior as the final goal, we have examined molecular basis of neuroendocrine mechanisms for salmon spawning migration. Since spawning migration is a kind of reproductive behavior, we presumed that preoptic neurons producing gonadotropin-releasing hormone (GnRH) have a crucial role in coordination of the neuroendocrine function of brain-pituitary-gonadal axis and the neural control of migratory behavior. Actually, the level of salmon (s) GnRH mRNA in the ventrocaudal telencephalon and the preoptic area in pre-spawning fresh-water chum salmon was higher than that in seawater fish. The amounts of mRNAs for gonadotropin (GTH) II, that is GTH alpha and II beta subunit mRNAs, but not GTH I alpha mRNA, in the pituitaries of fresh-water fish were higher than those in the seawater ones. In both male and female lacustrine sockeye salmon, GnRH analogue elevated the pituitary levels of mRNAs for GTH II subunits, and further shortened homing duration, concomitantly with elevation of plasma level of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. These findings indicate that expression of GnRH gene is involved in the genetic program of salmon spawning migration.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

The distribution of mRNA encoding thyrotropin-releasing hormone (TRH) precursor in the brain of sockeye salmon was studied by in situ hybridization histochemistry using digoxigenin-labeled riboprobes as a basis to investigate its physiological functions in the salmon brain. Since seasonal variation in TRH gene expression was expected in relation to smelting or maturation, fish were sampled in February and October. In both groups, TRH mRNA was widely distributed in discrete brain regions including the internal cellular layer (ICL) of the olfactory bulb, postcommissural nucleus of area ventralis telencephali (Vp), nucleus preopticus parvocellularis anterioris (PPa), nucleus preopticus magnocellularis, dorsal zone of periventricular hypothalamus (Hd), torus semicircularis, and also the motor nucleus of vagus nerve in the medulla oblongata. TRH neurons in ICL and Hd are round and small with diameters of 5-10 mu m. In contrast, TRH neurons in the ventral telencephalon and the preoptic area are medium-sized (10-20 mu m), and appear to have multiple processes. Most of these cells are restrictively localized along the lateral margin of the preoptic nuclei. The number of TRH neurons in Vp and PPa was smaller in February than in October, suggesting a seasonal change of TRH neurons in the preoptic area. In the medulla oblongata, a cluster of large oval-shaped cells (20-30 mu m) showed signals for TRH mRNA. The present results suggest that TRH may function as a neurotransmitter or neuromodulator involved in olfactory activity and also autonomic motor integration, in addition to neurohormonal control of secretion of pituitary hormones.

DOI： 10.2108/zsj.15.945

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC SCI FISHERIES SCI  

The effects of a three week intramuscular implantation of gonadotropin-releasing hormone analog (GnRHa) on gonadal maturation and changes in serum gonadal steroid hormone levels were examined in anadromous, maturing sockeye salmon Oncorhynchus nerka during the pre-spawning season. Spermiation occurred in both GnRHa-implanted and control males, but serum 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) levels in the implanted males were higher than in controls. No differences were found in serum testosterone (T) levels between GnRHa-implanted and control males. Serum 11-ketotestosterone (11KT) levels in control males increased for three weeks, but those in GnRHa-implanted males did not change. All GnRHa-implanted females ovulated completely for three weeks, while 25% of control females ovulated partially and the rest of females ovulated 17-24 days after sampling. The number of ovulated eggs of GnRHa-implanted females was greater than that of control females, and percentages of eyed eggs were not different between GnRHa-implanted and control females. Serum levels of DHP increased dramatically in GnRHa-implanted females, but those of T and estradiol-17 beta (E-2) was similar to control females. These results confirm that GnRHa accelerates gonadal maturation of both sexes through elevation of serum DHP levels in maturing sockeye salmon.

DOI： 10.2331/fishsci.64.948

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Our previous study suggested that, in the pituitaries of pre-spawning chum salmon, salmon gonadotropin-releasing hormone (sGnRH) stimulates expression of genes for gonadotropin (GTH) II beta but not for I beta, since the levels of mRNAs encoding sGnRH and GTH II but not I were increased during the final stages of spawning migration. In the present study, a capsule of GnRH analog (GnRHa) was implanted into the dorsal muscle of maturing sockeye salmon to clarify function of GnRH on expression of GTH subunit genes in pre-spawning homing salmonids. The amounts of GTH subunit mRNAs in the individual pituitaries were analyzed by a quantitative dot blot analysis using single-stranded sense DNA as the standard. The levels of GTH alpha and II beta mRNAs in the GnRHa-implanted fish were significantly higher than those in the control fish in both the males and females, whereas the levels of GTH I beta mRNA did not show any significant differences in both sexes. These results indicate that GnRH elevates expression of GTH subunit genes which encode the components of GTH II, alpha and II beta chains, in the pituitary of maturing sockeye salmon, and then accelerates final maturation.

DOI： 10.2108/zsj.15.761

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

We only have little information on expression of gonadotropin (GTH) subunit genes during spawning migration in salmonids. Changes in the levels of mRNAs for GTH subunits (GTH alpha 2, I beta and II beta) were therefore analyzed in the pituitaries of chum salmon (Oncorhynchus keta) during the final stages of spawning migration to the Ishikari river. The fish were caught at Atsuta, a fisherman's village facing the Ishikari bay, and at Chitose, a tributary of the Ishikari river, in 1993 and 1994. The former is referred to as seawater (SW) fish, and the latter as freshwater (FW) fish. The levels of GTH subunit mRNAs in the pituitaries were determined by a quantitative dot blot analysis, using single-stranded sense DNA as the standard. The sense DNAs have the same nucleic acid sequences of mRNAs. The level of GTH alpha 2 mRNA in the FW males was higher than that in the SW ones. Similar tendency was seen in the females. No significant changes were observed in the levels of GTH I beta mRNA in both the males and females. Whereas, the level of GTH II beta mRNA in the FW fish was higher than that in the SW fish regardless of sexes in 1994. Although not statistically significant in the males, similar tendency was seen in the 1993 fish. The present study thus showed that the level of GTH II beta mRNA was increased concomitantly with that of GTH alpha 2 mRNA during the final stages of spawning migration.

DOI： 10.2108/zsj.15.753

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

The Hsc70t gene is a Hsp70 homolog gene expressed constitutively in spermatids in mice. This gene is linked to two heat-inducible Hsp 70 genes, Hsp 70.1 and Hsp 70.3, located in the MHC class III region. The syntenic region of human chromosome 6 contains the HSPA1B, HSPA1A, and HSPA1L genes. Here, we have isolated a HSPA1L cDNA clone from human testicular cells. The HSPA1L gene contained an intron 13 bp upstream of the initiating ATG, A similar genomic structure was found in the Hsc70t gene. The transcription initiation site of the Hsc70t gene was located at ca. 600 bp upstream of the heat-inducible Hsp70.3 gene, linked head-to-head. Sequence alignment of the mouse and human genes revealed that the human HSPA1L and HSPA1A genes were orthologous to the mouse Hsc70t and Hsp70.3 genes, respectively. Conserved sequence stretches observed in the 5' flanking region and the first exon of the spermatid-specific Hsp70 gene may be involved in regulation of the specific gene expression.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：J ENDOCRINOLOGY LTD  

Two types of genes encode salmon gonadotropin-releasing hormone (sGnRH), which is thought to act on both sexual maturation and reproductive behavior, in salmonids. We characterized the two sGnRH genes (sGnRH-I and -II) and their upstream regions in masu salmon, Oncorhynchus masou, since such information is a prerequisite for molecular approaches to salmon reproduction. The two genes have similar exon-intron structures composed of four exons and three introns. Sequence analyses of the two genes showed that coding regions are highly conserved, but upstream regions are distinctively divergent. In the upstream regions, only the sGnRH-II gene has a large palindromic sequence, which has been proposed to be involved in control of transcription via estrogen receptors. In contrast, the sGnRH-I gene is missing the large palindromic sequence, but has three distinct palindromes in the upstream region. These results may suggest divergent transcription regulatory mechanisms between the two sGnRH genes in masu salmon. The differences in the upstream regions of sGnRH genes in Atlantic salmon (Salmo salar), sockeye salmon (Oncorhynchus nerka) and masu salmon are discussed with respect to the evolution of sGnRH genes in salmonid fish.

DOI： 10.1677/jme.0.0190149

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：J ENDOCRINOLOGY LTD  

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 IT-I and IT-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.

DOI： 10.1677/jme.0.0180049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Changes in expression of vasotocin (VT) and isotocin (IT) genes were analyzed in chum salmon during the last stages of spawning migration. Pre-spawning chum salmon were caught at following four locations in the Sanriku coast of the Pacific Ocean in Japan: 1) the off-coast area north to the Otsuchi Bay, 2) the mouth of the Otsuchi Bay, 3) inside of the Otsuchi Bay, and 4) the place 500 m upstream to the mouth of the Otsuchi River. In addition, effects of hypo-osmotic stimulation by transition from sea water (SW) to freshwater (FW) were examined in animals caught at the mouth of the Otsuchi Bay. The levels of VT and IT mRNAs in the forebrains were determined by Northern blot analysis. The plasma osmolality and the levels of Na+ and Cl- were also analyzed. Expression patterns of VT and IT genes were different between the males and the females. In the males, VT and IT gene expression were maintained essentially at the same levels from the off-coast area to the Otsuchi River. In contrast, in the females, the level of VT-I mRNA was significantly increased in the fish caught at the mouth of the bay. After entering the bay, the level of VT-I mRNA was decreased and maintained at a low level through the final stages of spawning migration. Such sexual difference in VT and IT gene expression found in the field fish was further analyzed by a SW to FW transition experiment, in which fish were divided into two groups, those retained in SW and others replaced with FW. In the FW-replaced fish, the levels of VT and IT mRNAs were decreased in both sexes, although much more conspicuous in the females. In the SW-retained animals, changes in the levels of VT and IT mRNAs were sexually different. The levels of VT and IT mRNAs were increased in the males, whereas they were decreased in the females, when compared to the initial levels just before the experimental treatments. These results suggest that regulation of VT and IT gene expression is sexually dimorphic in pre-spawning chum salmon.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Two types of cDNAs encoding thyrotropin-releasing hormone (TRH) precursors (TRH-A andTRH-B) were amplified from hypothalamic mRNA of sockeye salmon by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplification was achieved using two primers which correspond to TRH progenitor sequence (Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg). A full length cDNA encoding TRH-A was obtained by 5'- and 5'-RACE methods. It has a length of 1324 base pairs (bp) that contains sequences of 5' and 3' untranslated regions and an open reading frame of 259 codons. The sockeye salmon TRH-A deduced from the nucleotide sequence tandemly contains 8 copies of TRH progenitor sequences. Another cDNA which encodes a part of TRH-B consists of 242 bp, and the sequence homology between TRH-A and -B cDNAs is 90%. The result of Southern blot analysis of sockeye and masu salmon genomic DNAs supported the evidence that there are at least two TRH genes in the salmonid. A RT-PCR analysis of TRH gene expression in various tissues of sockeye salmon showed that strong expression was observed only in the brain. The primary structure of the sockeye salmon TRH-A shares low similarity to those of human, rat and Xenopus TRH precursors (35, 27 and 44%, respectively). However, their hydropathy profiles were almost the same with each other, The profile of sockeye salmon TRH-A showed the presence of two discrete hydrophobic regions, one in the N-terminal region which corresponds to the signal peptide and the other in the C-terminal region, All of the repetitive TRH progenitor sequences are included in three hydrophilic regions easily recognizable, The present results thus suggest that the three-dimensional structures of TRH precursors are highly conserved, although the primary structures of TRH precursors have diverged through the evolutionary pathway of vertebrates.

DOI： 10.1046/j.1365-2826.1996.05065.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Vasotocin (VT) and isotocin (IT) are teleost neurohypophysial hormones produced by neurosecretory neurons in the magnocellular part of preoptic nucleus (PM) of the hypothalamus. Several previous studies indicated that neurohypophysial hormones are involved in teleost reproductive behavior. The changes in the expression of VT and IT genes were thus studied by an in situ hybridization technique and an immunohistochemical avidin-biotin-complex method in pre-spawning chum salmon (Oncorhynchus keta). Male and female fish were caught at Atsuta, the mouth of the Ishikari Bay, and at Chitose, upstream to the Ishikari River in October, 1994. The former and the latter are referred to as bay fish and river fish, respectively. The intensity of autoradiographic hybridization signals were determined in individual neurosecretory neurons in the rostroventral, middle, and dorsocaudal parts of the PM. In the females, the levels of VT and IT mRNAs in the river fish were significantly lower than those in the bay fish in all the three loci in the PM, whereas VT and IT immunoreactivities in the river fish were higher than those in the bay fish. These results suggest that both the rates of transcription and release of VT and IT were decreased in pre-spawning female chum salmon. Contrary, in the males, the levels of IT mRNA and IT immunoreactivity in the river fish were greater than those in the bay fish particularly in the rostroventral part of the PM, whereas conspicuous changes were not seen in the levels of VT mRNA and VT immunoreactivity. The present results thus revealed sexually different expression of neurohypophysial hormone genes in the preoptic nucleus of pre-spawning chum salmon when compared between bay and river fish. The regulation of neurohypophysial hormone gene expression may be different between the male and the female during the last stages of spawning migration.

DOI： 10.2108/zsj.13.593

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

To investigate regulatory mechanisms of proopiomelanocortin (POMC) gene expression in sockeye salmon, we have isolated and characterized cDNAs encoding two types of sockeye salmon POMC, which are referred to as ssPOMC-A and -B. Two types of PCR products were amplified from total RNA of sockeye salmon pituitaries by use of rainbow trout sequences. Full length cDNA clones encoding ssPOMC-A and ssPOMC-B were obtained from a pituitary cDNA library of sockeye salmon using the PCR products as probes. The ssPOMC-A and -B cDNAs have a length of 1072 and 1709 bps, respectively. Northern blot analysis showed that both ssPOMC-A and -B mRNAs were expressed only in the pituitary, and their sizes were about 1.2 kb and 1.8 kb, respectively. The presence of two ssPOMC genes was confirmed by Southern blot analysis of genomic DNA obtained from a single sockeye salmon. The deduced amino acid sequences of the ssPOMC-A and -B contained 230 and 226 residues, respectively. The amino terminal of beta-endorphin in ssPOMC-B which corresponds to Met-enkephalin domain is YSGFM, which is different from YGGFM of Met-enkephalin found in many other vertebrate species. The homology of nucleotide sequences between ssPOMC-A and -B is 59% in the entire coding region, whereas alpha-MSH coding regions are highly homologous (91%). Although the deduced amino acid sequences of ssPOMCs show 43% overall similarity, their hydropathy profiles are coincident with those of several other vertebrate species, particularly the amino terminal of N-terminal peptide (NPP) shows almost the same pattern with other vertebrate NPPs.

DOI： 10.2108/zsj.13.421

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Complementary DNAs encoding precursor molecules of the pituitary glycoprotein hormone (PGH) alpha- and luteinizing hormone (LH) beta-subunits of Japanese quail were isolated from a quail adenohypophyseal cDNA library using corresponding chicken cDNAs as hybridization probes. The isolated cDNAs had a length of 695 and 724 bp, respectively, and contained sequences of 5' and 3' untranslated regions and an entire coding region of the precursor molecules. Two series of incompletely repeating and partially overlapping nucleotide sequences were observed over a part near the 3' end of the apoprotein coding region and the 3'-untranslated region in the beta-subunit cDNA. One of them consisted of two repeated sets of 71 bases and the other consisted of six repeated sets of 30 bases or less. The predicted amino acid sequence showed that signal peptide and apoprotein of the alpha-subunit consisted of 24 and 96 amino acid residues, respectively, and those of the beta-subunit consisted of 47 and 119 amino acid residues, respectively. Hybridization of the quail PGH alpha- and LH beta-subunit cDNAs to adenohypophyseal RNA showed that sizes of PGH alpha- and LH beta-subunit precursor mRNAs were about 1.1 and 0.9 kb, respectively. Amounts of both mRNAs were increased three to four times by castration in male quail. A comparison of the amino acid sequences of each of the PGH alpha- and LH beta-subunit precursors of the quail and other vertebrates indicated that the apoprotein, especially that of the alpha-subunit, was conserved (59 to 85% and 34 to 55% homology in the alpha- and beta-subunits, respectively), while the signal peptide was diversified (4 to 75% and 4 to 45% homology in the alpha- and beta-subunits, respectively). In the LH beta-subunit apoprotein, interclass homology values varied in a narrow range and did not show a clear relation with the phylogenic distance, while interclass homology values in the alpha-subunit apoprotein and both signal peptides reflected the phylogenic distance. This characteristic feature of the LH beta apoprotein can be explained by assuming that the variable portion of the LH beta apoprotein molecule evolved with a higher speed than that of the other peptides, and consequently no sequence common with other classes remained. This highly variable sequence may be responsible for the high animal-class specificity of the interaction between LH and its receptor. (C) 1994 Academic Press, Inc.

DOI： 10.1006/gcen.1994.1040

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Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Fisheries Science  

DOI： 10.2331/fishsci.60.791

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：J ENDOCRINOLOGY LTD  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Publisher：Japan Society for Comparative Endocrinology  

DOI： 10.5983/nl2008jsce.37.250

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Language：Japanese   Publisher：日本比較内分泌学会  

DOI： 10.5983/nl2008jsce.35.7

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：生物研究社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

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DOI： 10.3330/hikakuseiriseika.21.135

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Language：English   Publisher：Zoological Society of Japan  

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Other Link： http://id.nii.ac.jp/1141/00038233/

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Other Link： http://id.nii.ac.jp/1141/00037170/

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Other Link： http://id.nii.ac.jp/1141/00037194/

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Other Link： http://id.nii.ac.jp/1141/00037316/

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