Effects of Water Temperature on the Body Color and Expression of the Genes Related to Body Color Regulation in the Goldfish.

Melanin-concentrating hormone (MCH), melanocyte-stimulating hormone (MSH), and somatolactin (SL) in the hypothalamus-pituitary axis are associated with body color regulation in teleost fish. Although these hormones' production and secretion respond well to light environments, such as background color, little is known about the effects of different water temperatures. We investigated the effects of water temperature, 10°C, 20°C, and 30°C, on body color and the expression of these genes and corresponding receptor genes in goldfish. The body color in white background (WBG) becomes paler at the higher water temperature, although no difference was observed in black background (BBG). Brain mRNA contents of proMCH genes (pmch1 and pmch2) increased at 30°C and 20°C compared to 10°C in WBG, respectively. Apparent effects of background color and temperature on the pituitary mRNA contents of a POMC gene (pomc) were not observed. The pituitary mRNA contents of the SLα gene were almost double those on a WBG at any temperature, while those of the SLß gene (slb) at 30°C tended to be higher than those at 10°C and 20°C on WBG and BBG. The scale mRNA contents of the MCH receptor gene (mchr2) in WBG were higher than those in BBG at 30°C. The highest scale mRNA contents of MSH receptor (mc1r and mc5r) on BBG were observed at 20°C, while the lowest respective mRNA levels were observed at 30°C on WBG. These results highlight the importance of temperature for the endocrinological regulation of body color, and darker background color may stabilize those endocrine functions.

Effects of background color and feeding status on the expression of genes associated with body color regulation in the goldfish Carassius auratus.

Alpha-melanocyte-stimulating hormone (α-MSH), a peptide derived from proopiomelanocortin (POMC), and melanin-concentrating hormone (MCH), act as neuromodulators and regulate food intake in vertebrates. In teleosts, these peptides are also involved competitively in body color regulation; α-MSH induces a dark body color, while MCH induces a pale body color. Similarly, members of the growth hormone (GH) family, somatolactin (SL) and prolactin (PRL), which are involved in the regulation of energy metabolism, are also associated with body color regulation in teleosts. Since these hormones are involved in both body color regulation and energy metabolism, it is possible that feeding status can affect body color. Here, we examined the effects of fasting on the response of goldfish body coloration to changes in background color. Goldfish were acclimated for one week in tanks with a white or black background under conditions of periodic feeding or fasting. The results showed that body color and expression levels of pmch1 and pomc were affected by background color, irrespective of feeding status. Expression levels of sla were higher in fish maintained in tanks with a black background than in tanks with a white background, and higher in the fasted fish compared to the fed fish. However, the pattern of slb expression was almost the opposite of that observed in sla expression. The expression levels of gh and prl in the pituitary, and pmch2a and pmch2b in the brain, were not affected by background color. These results suggest that MCH, α-MSH, SLα, and SLß might be involved in body color regulation and that they are affected by background color in goldfish. The results also suggest that feeding status may affect body color regulation via SLα and SLß, although these effects might be limited compared to the effect of background color.

The effects of chromatic lights on body color and gene expressions of melanin-concentrating hormone and proopiomelanocortin in goldfish (Carassius auratus).

In the present study, the effects of photic environments, such as background color (white and black) and chromatic lights (blue, green, and red), on body color and gene expressions of melanin-concentrating hormone (mch) in the brain and proopiomelanocortin (pomc) in the pituitary, as well as the roles of the eyes and brain as mediators of ambient light to these genes, were examined in goldfish (Carassius auratus). Body color of goldfish exposed to fluorescent light (FL) under white background (WBG) was paler than those under black background (BBG). Gene expression levels for mch and pomc were reciprocally different depending on background color; under WBG, mRNA levels of mch and pomc were high and low, respectively, while under BBG, these levels were reversed. mch and pomc mRNA expressions of the fish exposed to chromatic light from LED were primarily similar to those exposed to FL, while blue light stimulated the expressions of mch and pomc. Ophthalmectomized goldfish exposed to FL or blue light showed minimum expression levels of mch gene, suggesting that eyes are the major mediator of ambient light for mch gene expression. Contrastingly, mRNA expressions of pomc in ophthalmectomized goldfish exposed to FL were different from those of intact goldfish. These results suggest that eyes play a functional role in mediating ambient light to regulate pomc gene expression. Since ophthalmectomy caused an increase in pomc mRNA contents in the fish exposed to blue light, we suggest that the brain is an additional mediator to regulate pomc gene expression.

Effects of tank color brightness on the body color, somatic growth, and endocrine systems of rainbow trout Oncorhynchus mykiss.

We investigated the effects of tank brightness on body color, growth, and endocrine systems of rainbow trout (Oncorhynchus mykiss). Five different tank colors that produce varying levels of brightness were used, including black, dark gray [DG], light gray [LG], white, and blue. The fish were reared in these tanks for 59 days under natural photoperiod and water temperature. The body color was affected by tank brightness, such that body color brightness was correlated with tank brightness (white-housed ≥ LG-housed ≥ DG-housed ≥ blue-housed ≥ black-housed). No difference in somatic growth was observed among the fish reared in the five tanks. The mRNA levels of melanin-concentrating hormone (mch1) was higher in white-housed fish than those in the other tanks, and the mRNA levels of proopiomelanocortins (pomc-a and pomc-b) were higher in fish housed in a black tank than those in other tanks. mRNA level of somatolactin, a member of growth hormone family, was higher in black-housed fish than those in white-housed fish. The mRNA levels of mch1 and mch2 in blue-housed fish were similar to those in black-housed fish, while the mRNA levels of pomc-a and pomc-b in blue-housed fish were similar to those in white-housed fish. The current results suggest that tank color is not related to fish growth, therefore any color of conventional rearing tank can be used to grow fish. Moreover, the association between somatolactin with body color changes is suggested in addition to the role of classical MCH and melanophore stimulating hormone derived from POMC.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures.

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

Melanin-concentrating hormone is a major substance mediating light wavelength-dependent skin color change in larval zebrafish.

Melanosome dispersion is important for protecting the internal organs of fish against ultraviolet light, especially in transparent larvae with underdeveloped skin. Melanosome dispersion leads to dark skin color in dim light. Melanosome aggregation, on the other hand, leads to pale skin color in bright light. Both of these mechanisms are therefore useful for camouflage. In this study, we investigated a hormone thought to be responsible for the light wavelength-dependent response of melanophores in zebrafish larvae. We irradiated larvae using light-emitting diode (LED) lights with peak wavelengths (λmax) of 355, 400, 476, 530, and 590 nm or fluorescent light (FL) 1-4 days post fertilization (dpf). Melanosomes in skin melanophores were more dispersed under short wavelength light (λmax ≤ 400 nm) than under FL. Conversely, melanosomes were more aggregated under mid-long wavelength light (λmax ≥ 476 nm) than under FL. In addition, long-term (1-12 dpf) irradiation of 400 nm light increased melanophores in the skin, whereas that of 530 nm light decreased them. In teleosts, melanin-concentrating hormone (MCH) aggregates melanosomes within chromatophores, whereas melanocyte-stimulating hormone, derived from proopiomelanocortin (POMC), disperses melanosomes. The expression of a gene for MCH was down-regulated by short wavelength light but up-regulated by mid-long wavelength light, whereas a gene for POMC was up-regulated under short wavelength light. Melanosomes in larvae (4 dpf) exposed to a black background aggregated when immersing the larvae in MCH solution. Yohimbine, an α2-adrenergic receptor antagonist, attenuated adrenaline-dependent aggregation in larvae exposed to a black background but did not induce melanosome dispersion in larvae exposed to a white background. These results suggest that MCH plays a key role in the light wavelength-dependent response of melanophores, flexibly mediating the transmission of light wavelength information between photoreceptors and melanophores.

Effects of different green light intensities on the growth performance and endocrine properties of barfin flounder Verasper moseri.

We previously reported that the somatic growth of barfin flounder, Verasper moseri, was effectively stimulated by the green light compared to the blue and red lights. Herein, we report the effects of different green light intensities on the growth and endocrine system of the fish. Fish were reared in a dark room with light from a light-emitting diode (LED) at a peak wavelength of 518nm under controlled photoperiod (10.5:13.5h, light:dark cycle; 06:00-16:30, light) with three levels of photon flux density (PFD)-2 (low), 7 (medium), or 21 (high) µmol·m-2·s-1 at the water surface. The average water temperature was 10.2°C, and the fish were fed until satiety. The fish reared under high PFD of green light showed the highest specific growth rates, followed by the medium PFD group. Under high PFD, the fish showed the highest amount of melanin-concentrating hormone mRNA in their brains and insulin in plasma, while the lowest amount of growth hormone was observed in their pituitary glands. These results suggest that the green light stimulated the growth of barfin flounders in a light intensity-dependent manner in association with their central and peripheral endocrine systems. However, when the fish were reared in an ordinary room where they received both ambient and green LED lights, the fish under LED and ambient light grew faster than those under ambient light only (control). Moreover, no difference was observed in the specific growth rate of the fish reared under the three different green LED light intensities, suggesting that the growth was equally stimulated by the green light within a certain range of intensities under ambient light.

Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus.

To evaluate the association of the melanotropic peptides and their receptors for morphological color change, we investigated the effects of changes in background color, between white and black, on xanthophore density in the scales and expression levels of genes for hormonal peptides and corresponding receptors (MCH-R2, MC1R, and MC5R) in goldfish (Carassius auratus). The xanthophore density in both dorsal and ventral scales increased after transfer from a white to black background. However, xanthophore density in dorsal scales increased after transfer from a black to white background, and that of ventral scales decreased after transfer from a black to black background, which served as the control. In the white-reared fish, melanin-concentrating hormone (mch) mRNA content in the brain was higher than that in black-reared fish, whereas proopiomelanocortin a (pomc-a) mRNA content in the pituitary was lower than that in the black-reared fish. Agouti-signaling protein (asp) mRNA was detected in the ventral skin but not in the dorsal skin. No difference was observed in the asp mRNA content between fish reared in white or black background, suggesting that ASP might not be associated with background color adaptation. In situ hybridization revealed that both mc1r and mc5r were expressed in the xanthophores in scales. The mRNA content of mc1r in scales did not always follow the background color change, whereas those of mc5r decreased in the white background and increased in the black background, suggesting that mc5r might be a major factor reinforcing the function of MSH in morphological color changes. White backgrounds increased mch mRNA content in the brain, but decreased mch-r2 mRNA content in the scales. These altered expression levels of melanotropin receptors might affect reactivity to melanotropins through long-term adaptation to background color.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish.

Melanocortin (MC) systems are composed of MC peptides such as adrenocorticotropic hormone (ACTH), several molecular forms of melanocyte-stimulating hormones (MSHs) and MC receptors (MCRs). Here we demonstrated that the cartilaginous fish, Dasyatis akajei (stingray) expresses five subtypes of MCR genes-mc1r to mc5r-as in the case of teleost and tetrapod species. This is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. Expression of respective stingray mcr cDNAs in Chinese hamster ovary cells revealed that Des-acetyl-α-MSH exhibited cAMP-producing activity indistinguishable to ACTH(1-24) on MC1R and MC2R, while the activity of Des-acetyl-α-MSH on MC3R, MC4R, and MC5R were similar to or slightly greater than that of ACTH(1-24). Notably, in contrast to the other vertebrates, MC2R did not require coexpression with a melanocortin receptor-2 accessory protein 1 (mrap1) cDNA for functional expression. One of the roles of MC system resides in regulation of the pituitary-interrenal (PI) axis-a homologue of tetrapod pituitary-adrenal axis. In stingray, interrenal tissues were shown to express mc2r and mc5r as major MCR genes. These results established the presence of functional PI axis in stingray at the level of receptor molecule. While MC2R participates in adrenal functions together with MRAP1 in tetrapod species, the fact that sensitivity of MC5R to Des-acetyl-α-MSH and ACTH(1-24) were two order of magnitude higher than MC2R without coexpression with MRAP1 suggested that MC5R could play a more important role than MC2R to transmit signals conveyed by ACTH and MSHs if MRAP1 is really absent in the stingray.

Chronic effects of light irradiated from LED on the growth performance and endocrine properties of barfin flounder Verasper moseri.

We investigated the effects of specific wavelengths of light on the growth of barfin flounder. The fish, reared in white tanks in a dark room, were irradiated with light from light-emitting diodes (LEDs) with peak wavelengths of 464nm (blue), 518nm (green), and 635nm (red) under a controlled photoperiod (10.5:13.5, light-dark cycle; 06:00-16:30, light). Fish were reared for four weeks in three independent experiments at three different water temperatures (averages of 14.9°C, 8.6°C, and 6.6°C). The fish irradiated with blue and green light had higher specific growth rates (% body weight⋅day(-1)) than fish irradiated with red light. Notably, green light had the greatest effect on growth among the three light wavelengths at 6.6°C. In the brains of fish reared at 6.6°C, the amounts of melanin-concentrating hormone 1 mRNA under green light were lower than those under red light, and amounts of proopiomelanocortin-C mRNA under blue and green light were higher than those under red light. No differences were observed for other neuropeptides tested. In the pituitary, no difference was observed in growth hormone mRNA content. In plasma, higher levels of insulin and insulin-like growth factor-I were observed in fish under green light than those of fish under red light. These results suggest that the endocrine systems of barfin flounder are modulated by a specific wavelength of light that stimulates somatic growth.

Involvement of melanin-concentrating hormone 2 in background color adaptation of barfin flounder Verasper moseri.

In teleosts, melanin-concentrating hormone (MCH) plays a key role in skin color changes. MCH is released into general circulation from the neurohypophysis, which causes pigment aggregation in the skin chromatophores. Recently, a novel MCH (MCH2) precursor gene, which is orthologous to the mammalian MCH precursor gene, has been identified in some teleosts using genomic data mining. The physiological function of MCH2 remains unclear. In the present study, we cloned the cDNA for MCH2 from barfin flounder, Verasper moseri. The putative prepro-MCH2 contains 25 amino acids of MCH2 peptide region. Liquid chromatography-electrospray ionization mass spectrometry with a high resolution mass analyzer were used for confirming the amino acid sequences of MCH1 and MCH2 peptides from the pituitary extract. In vitro synthesized MCH1 and MCH2 induced pigment aggregation in a dose-dependent manner. A mammalian cell-based assay indicated that both MCH1 and MCH2 functionally interacted with both the MCH receptor types 1 and 2. Mch1 and mch2 are exclusively expressed in the brain and pituitary. The levels of brain mch2 transcript were three times higher in the fish that were chronically acclimated to a white background than those acclimated to a black background. These results suggest that in V. moseri, MCH1 and MCH2 are involved in the response to changes in background colors, during the process of chromatophore control.

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel, paying special attention to gonadotropin-releasing hormone.

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.

Inhibitory effects of ß-endorphin on cortisol release from goldfish (Carassius auratus) head kidney: an in vitro study.

ß-Endorphin (ß-END) is an endogenous opioid peptide derived from the common precursor proopiomelanocortin, together with adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). Although the roles of ACTH and MSH in fish are well known, the roles of circulating ß-END have not been elucidated. In the present study, we evaluated the biological roles of ß-END in the goldfish. First, we cloned the cDNAs of the delta opioid receptor (DOR), kappa opioid receptor (KOR), and mu opioid receptor (MOR) from the brain of the goldfish. Second, we analyzed the tissues that expressed these genes by using reverse transcription polymerase chain reaction. Among the several tissues that contained the opioid gene transcripts, the mRNAs of DOR, KOR, and MOR were detected in interrenal cells of the head kidney, which produce cortisol. On the basis of these results, the effects of ß-END on cortisol release were examined in vitro. ß-END alone suppressed the basal release of cortisol in a dose-dependent manner. Moreover, ß-END inhibited the cortisol-releasing activity of ACTH1-24. Therefore, it is probable that the role of ß-END in the interrenal cells is the suppression of cortisol release. Interestingly, the suppression of cortisol release was not observed with N-acetyl-ß-END, indicating that acetylation decreases the activity of ß-END in interrenal cells.

The pituitary-interrenal axis of fish: a review focusing on the lamprey and flounder.

In fish, the pituitary-interrenal axis is associated with stress response and a variety of biological processes such as metabolism, immune response, and growth. The major hormones involved in this axis are adrenocorticotropic hormone (ACTH), released from the pars distalis of the pituitary gland, and corticosteroid, released from the interrenal gland that is embedded in the head kidney in ray-finned fish. The ACTH signal, by which corticosteroid release is stimulated, is transmitted by melanocortin (MC) receptors on interrenal cells. Thus, the interaction of ACTH and MC receptors is the pivotal event for interrenal cells. Knowledge about ACTH and MC receptors in lamprey, cartilaginous fish, and ray-finned fish is available, and it suggests the pituitary-interrenal axis was established early in vertebrate evolution. Moreover, the data, including our recent results from flounders and lampreys, provide interesting features about ligand-receptor interactions. This review focuses on the characteristics of ACTH, the proopiomelanocortin gene encoding ACTH, and the MC receptor, and it is mostly based on the results of our investigations.

Interrelation between melanocyte-stimulating hormone and melanin-concentrating hormone in physiological body color change: roles emerging from barfin flounder Verasper moseri.

In teleosts, as their names suggest, the main target cells of melanocyte-stimulating hormone (MSH) and melanin-concentrating hormone (MCH) are the chromatophores in the skin, where these peptide hormones play opposing roles in regulating pigment migration. These effects are obvious especially when their activities are examined in vitro. On the contrary, while MCH also exhibits activity in vivo, MSH does not always stimulate pigment dispersion in vivo because of predominant sympathetic nervous system. A series of our investigations indicates that this is also the case in barfin flounder, Verasper moseri. Interestingly, we observed that mch expression and the tissue contents of MCH can be easily influenced by changes in environmental color conditions, while gene expression and tissue contents related to MSH scarcely respond to color changes. Transcripts of MSH and MCH receptor genes have been identified in a variety of tissues of this fish species, suggesting that these are multifunctional peptide hormones. Nevertheless, chromatophores in the skin still offer important clues in the efforts to elucidate the functions of melanotropic peptides. Herein, we review the most recent advancements of our studies on MSH and MCH and their receptors in the barfin flounder and discuss the interrelations between these peptides, focusing on their roles in influencing pigment migration in the skin.

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 flounder's 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.

Further evidence on acetylation-induced inhibition of the pigment-dispersing activity of α-melanocyte-stimulating hormone.

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.