Takeshi Yamanome

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.

Three GnRH systems in the brain and pituitary of a pleuronectiform fish, the barfin flounder Verasper moseri.

Abstract. To clarify the possible function of gonadotropin-releasing hormone (GnRH) in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, the distribution of three forms of GnRH in various areas of the brain was examined by radioimmunoassay, and the localization of GnRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary was determined by immunocytochemistry. The dominant form in the pituitary was seabream GnRH (sbGnRH), levels of which were much higher than those of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In contrast, sbGnRH levels were extremely low in all other brain areas examined. Levels of sGnRH and cGnRH-II were high in the anterior and posterior part of the brain, respectively. sbGnRH-ir cell bodies were located in the preoptic area, whereas sbGnRH-ir fibers were localized mainly in the preoptic area-hypothalamus-pituitary and formed a distinctive bundle of axons projecting to the pituitary. sGnRH-ir cell bodies were located in the ventromedial part of the rostral olfactory bulbs and in the terminal nerve ganglion (the transitional area between the olfactory bulb and the telencephalon). cGnRH-II-ir cell bodies were localized to the midbrain tegmentum. sGnRH-ir and cGnRH-II-ir fibers were observed throughout the brain except in the pituitary gland. These results indicate that sbGnRH is responsible for the neural control of the reproductive endocrinology of the barfin flounder (hypothalamo-hypophysial system), and that sGnRH and cGnRH-II function as neurotransmitters or neuromodulators in the brain.

Green light stimulates somatic growth in the barfin flounder Verasper moseri.

We examined the effects of different light wavelengths-blue, green, and red-on the somatic growth of the barfin flounder Verasper moseri, a flatfish. The light sources used were fluorescent lamps and a combination of daylight and fluorescent lamps that produced ambient light. These light sources were filtered using blue, green, or red filters. During the experiments, the fish were reared in indoor tanks with running seawater of natural temperature and fed with commercial pellets twice daily until satiety. The tanks were white in color. Fish were exposed to constant light emitted from the fluorescent lamps (9:15, light:dark; 08:00-17:00, light) for 14 weeks from October or September to January or to ambient light with a 14-week natural photoperiod from September to December. The wavelengths that were filtered from the fluorescent lamp light modified the growth of the fish, i.e., fish reared under green or blue light exhibited a greater total length (TL; P<0.01) and body weight (BW; P<0.01) than those reared under red light. In contrast, in the case of fish exposed to filtered ambient light, fish reared under green light exhibited a greater TL (P<0.01) and BW (P<0.01) than fish exposed to other wavelengths-blue-, red-, and nonfiltered ambient light. Our results indicate that flounder growth was modified by certain wavelengths, namely, green and red light, which had growth-stimulating and growth-inhibiting effects, respectively.

Possible paracrine function of α-melanocyte-stimulating hormone and inhibition of its melanin-dispersing activity by N-terminal acetylation in the skin of the barfin flounder, Verasper moseri

Melanocyte-stimulating hormone (MSH) is generated from a precursor protein, proopiomelanocortin (POMC), mainly in the pituitary. The barfin flounder, Verasper moseri, expresses three different POMC genes (Pomc), among which Pomc-c is also expressed in the skin. Herein, we characterized the biological significance of POMC and MSH produced in barfin flounder skin. The reverse transcription polymerase chain reaction showed the expression of Pomc-c in isolated non-chromatophoric dermal cells. Mass spectrometry analyses of fractions of skin extract separated by high-performance liquid chromatography revealed the presence of a peptide with a molecular mass corresponding to Des-acetyl (Ac)-alpha-MSH-C derived from POMC-C. These results indicate that, in addition to endocrine functions, MSH in barfin flounder is associated with skin pigmentation via paracrine mechanisms. On the other hand, in vitro studies showed that Des-Ac-alpha-MSH-C dispersed pigments in both melanophores and xanthophores. These functions are similar to those of Des-Ac-alpha-MSH, which differs from Des-Ac-alpha-MSH-C only at the C-terminus, generated from POMC-A and -B. Alpha-MSH, which has an acetyl group at the N-terminus, led to pigment dispersion in xanthophores, but showed no effect in melanophores. A series of bioassays indicated that acetylation enhances MSH activity in xanthophores, but inhibits it in melanophores, suggesting that receptors for MSHs expressed in xanthophores and melanophores are different from each other.

Molecular cloning of three cDNAs encoding different gnrhs in the brain of barfin flounder

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly-Lys-Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.

Melanocortin receptor subtypes in interrenal cells and corticotropic activity of α-melanocyte-stimulating hormones in barfin flounder, Verasper moseri

The aim of this study was to characterize the pituitary-interrenal axis in barfin flounder, a flatfish. Adrenocorticotropic hormone (ACTH) and melanocortin 2 receptor (MC2R) have been shown to be indispensable substances in pituitary and interrenal cells for cortisol release, respectively. We previously identified ACTH in the pars distalis of the barfin flounder pituitary gland, and detected transcripts of Mc1r, Mc4r, and Mc5r in the head kidney wherein interrenal cells are located. We have now demonstrated the presence of MC2R, which is a specific receptor for ACTH, in interrenal cells by molecular cloning of Mc2r cDNA and in situ hybridization, and confirmation of the in vitro cortisol-releasing activity of ACTH. These results show the presence of a classical pituitary-interrenal axis in this fish. We also evaluated the role of α-melanocyte-stimulating hormone (α-MSH) and its related peptides. In situ hybridization was used to demonstrate the expression of Mc5r in interrenal cells; both desacetyl-α-MSH and diacetyl-α-MSH showed in vitro cortisol-releasing activities, while the activity of α-MSH was negligible. These findings indicate the presence of an additional pituitary-interrenal axis consisting of α-MSH-like peptides secreted from the neurointermediate lobe of the pituitary and MC5R in the interrenal cells. The cortisol-releasing activity of desacetyl-α-MSH and diacetyl-α-MSH, compared with the low activity of α-MSH, suggest a unique and specific functional role of these forms of MSH peptides. The interrenal co-expression of two subtypes of Mcrs may play a role in this specialization.

Differential expressions of melanocortin receptor subtypes in melanophores and xanthophores of barfin flounder

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a member of the melanocortin (MC) family, and the MC receptor (MCR) is a member of the G protein-coupled receptor (GPCR) superfamily. We previously found that in barfin flounder, a flatfish, alpha-MSH with an acetyl group at the N-terminus stimulated pigment dispersion in xanthophores; however, this effect was not observed in melanophores. Therefore, the present study was undertaken to find which MCR subtypes are expressed in these pigment cells in order to elucidate how acetylation regulates activities of alpha-MSH-related peptides. Here, we also report the cloning of Mc1r and Mc5r from barfin flounder. Three types of cells-melanophores, xanthophores, and nonchromatophoric dermal cells-were isolated from the skin samples collected from the dorsal fin. These cells were then tested for the expression of Mc1r and Mc5r as well as Mc2r and Mc4r that we had previously cloned. Mc1r and Mc5r transcripts were detected in melanophores, and a sole Mc5r transcript was detected in xanthophores. We had previously found that the efficiency of alpha-MSH was higher than that of desacetyl-alpha-MSH for pigment dispersion in xanthophores. Acetylated MSH peptide may have increased binding affinity to MC5R, whereas alpha-MSH lacks melanin-dispersing activity. Increasing evidences indicate that many GPCRs form heterodimers, and this may affect the affinity of the ligand for the corresponding GPCR. Taken together, the expression of two different Mcr subtypes in melanophores may suggest that a heterodimer consisting of MC1R and MC5R may have a low binding affinity toward alpha-MSH. The present results clarify the types of MCRs that are expressed in melanophores and xanthophores of barfin flounder; furthermore, the results provide important clues about the functional regulation of alpha-MSH-related peptides.

Structural and functional diversity of proopiomelanocortin in fish with special reference to barfin flounder

Proopiomelanocortin (POMC) is a precursor of adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), and endorphin (END). We have characterized POMC systems in barfin flounder. The results revealed unique aspects of POMC systems. Notable features in terms of pituitary functions are the occurrence of three functional POMC genes, the mutation of an essential sequence in the beta-END in one of the genes, occurrence of alpha-MSH in addition to ACTH in the pars distalis of the pituitary, and expression of the three genes in a single cell. While MSHs stimulate pigment dispersion, expression of the POMC gene and plasma levels of MSH do not always respond to background color changes between black and white. The functions of MSHs in skin pigmentation are very unique, because acetylation at the N-terminal of alpha-MSH inhibits its pigment dispersing activity. This is in contrast to results from other teleosts and amphibians, in which acetylation increases the activity. In the skin, the POMC gene is expressed in the non-chromatophoric dermal cells, indicating that MSH produced in the skin de novo has a paracrine function. The detection of MSH peptides in skin extracts seems to show that the control of skin pigmentation by MSHs is twofold-endocrine control by the pituitary, and paracrine control by the skin itself. Thus, fish provide an interesting model to help understand the structural and functional diversity of POMC systems. In this review, we provide an overview of our recent studies on the characterization of molecules and biological significance of POMC systems in barfin flounder.

Effects of thyroid hormones on phototaxis of chum and coho salmon juveniles

Abstract Changes in phototaxis of chum ( Oncorhynchus keta ) and coho ( O. kisutch ) salmon were examined after treatment with thyroid hormones, using troughs (0.25 × 2 m long, 20 cm deep) with open (1200–2400 lux, 1 m long) and shaded (400–680 lux, 1 m long) water. In some experiments, half of the trough was covered to provide shelter ( 4 , 30 mg 150 l , immersion) or thiourea ( 15 g 150 l , immersion) for 56 days from early April. Coho smolts were treated with triiodothyronine (T 3 , 15 mg 150 l , immersion) for 4 days in July–August. T 4 -treated chum preferred open water by 80–90%. More than 78% of fry treated with thiourea and the control preferred to be in shade or to be in shelter. T 3 -treated coho preferred open water after 3 and 4 days, whereas the control fish stayed in shaded water. The preference of T 3 -treated fish for open water was observed 1 week after the end of treatment and the preference disappeared during the following week. These findings suggest that thyroid hormones play an important role in the phototaxis of salmonids in freshwater.

Food Deprivation Increases the Expression of the Prepro-Orexin Gene in the Hypothalamus of the Barfin Flounder, Verasper moseri

Orexins (orexin-A and -B) are involved in the regulation of food intake in mammals. In the barfin flounder, Verasper moseri, we previously reported that orexin-A-like-immunoreactive (ir) cell bodies are localized in the hypothalamus, which is a possible orexigenic center in fish. However, the physiological roles of orexin in the barfin flounder remain unclear. Here, we cloned prepro-orexin cDNA and examined the effects of feeding status on orexin gene expression in the barfin flounder to obtain a better insight into the roles of orexins in feeding regulation. A molecular cloning study showed that barfin flounder prepro-orexin cDNA encodes a 145 amino acid (aa) polypeptide containing orexin-A (43 aa) and orexin-B (28 aa). Prepro-orexin gene transcripts were detected in the hypothalamus, pituitary, and several peripheral organs such as the eyeball, gills, head kidney, body kidney, spleen, testis, and the skin on the eye-side of the flounders body. Furthermore, the mean prepro-orexin mRNA expression level in the hypothalamus was significantly higher in fasted than in fed fish. These results show that fasting regulates orexin mRNA in the hypothalamus and suggest that orexin is involved in feeding regulation in barfin flounder.