[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 07/2014; · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: β-Endorphin (β-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 β-END have not been elucidated. In the present study, we evaluated the biological roles of β-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 β-END on cortisol release were examined in vitro. β-END alone suppressed the basal release of cortisol in a dose-dependent manner. Moreover, β-END inhibited the cortisol-releasing activity of ACTH1-24. Therefore, it is probable that the role of β-END in the interrenal cells is the suppression of cortisol release. Interestingly, the suppression of cortisol release was not observed with N-acetyl- β-END, indicating that acetylation decreases the activity of β-END in interrenal cells.
General and Comparative Endocrinology 05/2014; · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To better understand the physiological mechanisms underlying reproductive dysfunction, such as impaired vitellogenesis and final oocyte maturation, we assessed endocrinological differences between captive and wild female jack mackerel (Trachurus japonicus). The presence of three gonadotropin-releasing hormone (GnRH) peptides was immunologically evaluated in jack mackerel brain tissues. Full-length cDNAs encoding GnRHs (gnrh1, gnrh2, and gnrh3) and gonadotropin subunits (fshb, lhb, and gpa) were cloned, sequenced, and quantitatively assayed. Of the captive females, 60% failed to undergo vitellogenesis, displaying immature (IM) or atretic oocytes, whereas 80% of wild females were captured during late vitellogenesis (LV) or ovulation (OV). The gnrh1 expression was significantly lower in captive fish than in wild LV and OV fish, while there were no significant differences in the expression of gnrh2 or gnrh3. The expression of fshb was lower in captive IM fish than in wild LV fish, but no significant differences were observed between the captive IM and LV individuals. The lhb expression was elevated in the wild LV and OV fish, and gpa expression was greatest in the wild OV fish. Serum estradiol-17β levels were significantly lower in captive IM fish than in captive LV fish. The results indicate that captive-rearing stress may impair vitellogenesis and negatively influence the transcription of gnrh1 in the brain and GtH synthesis in the pituitary.
Aquaculture 05/2014; s 428–429:226–235. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cell and Tissue Research 01/2014; · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Barfin flounder larvae exhibit unique black coloration, as well as left-right asymmetry in juvenile stage as in other flatfish. In this study, we first assessed the changes in melanophores with development and then investigated their responsiveness to melanin-concentrating hormone (MCH) during metamorphosis. Larval-type melanophores appeared on both sides of the body before metamorphosis, whereas adult-type melanophores appeared only on the ocular side after metamorphosis. Even in the individuals of this species displaying black coloration, the density of larval-type melanophores was similar to that in transparent larvae of other species. However, unlike in transparent larvae, larval-type melanophores completely dispersed in the black larvae of this species. Therefore, the black coloration during larval stages was mainly due to dispersion, and not the density, of larval-type melanophores. In vitro MCH treatment revealed, for the first time, the responsiveness of melanophores in larval stages. On the ocular side, larval-type melanophores aggregated against MCH during larval stages, while, in the larvae at later metamorphic stages and in juveniles, larval-type melanophores did not aggregate, although aggregation of adult-type melanophores was noted. In contrast, on the blind side, the responsiveness of larval-type melanophores to MCH was consistently present from larval to juvenile stages. The metamorphic transition of MCH responsiveness from larval- to adult-type melanophores only on the ocular side suggests the larval (therefore, immature) nature of the blind side skin. We propose that the inhibited development, and thus the retention of the larval-type skin leads to the formation of the blind side characteristics and is the central mechanism for the flatfish asymmetry.
General and Comparative Endocrinology 09/2013; · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 03/2013; · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proopiomelanocortin (POMC) is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived α-melanocyte-stimulating hormone (α-MSH) having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, α-MSH, as well as desacetyl (Des-Ac)-α-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, α-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas Des-Ac-α-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of α-MSH as an important consequence of posttranslational modification.
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 11/2012; · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 08/2012; 179(1):78-87. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin family of adenohypophyseal hormones. In teleost fish, SL is encoded by one or two paralogous genes, namely SL-α and -β. Our previous studies have revealed that pituitary adenylate-cyclase-activating polypeptide stimulates SL release from cultured goldfish pituitary cells, whereas melanin-concentrating hormone suppresses this release. As in other fish, the goldfish possesses SL-α and -β. So far, however, no useful means of detecting the respective SLs immunologically in this species has been possible. In order to achieve this aim, we raised rabbit antisera against synthetic peptide fragments deduced from the goldfish SL-α and -β cDNA sequences. Using these antisera, we observed adenohypophyseal cells showing SL-α- and -β-like immunoreactivities in the goldfish pituitary, especially the pars intermedia (PI). Several cells in the PI showed the colocalization of SL-α- and -β-like immunoreactivities. Then, using single-cell polymerase chain reaction with laser microdissection, we examined SL-α and -β gene expression in adenohypophyseal cells showing SL-α- or -β-like immunoreactivity. Among cultured pituitary cells, we observed three types of cell: those that possess transcripts of SL-α, -β, or both. These results suggest a polymorphism of SL-producing cells in the goldfish pituitary.
Cell and Tissue Research 05/2012; 350(1):167-76. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: α-Melanocyte-stimulating hormone (α-MSH) is responsible for pigment dispersion in the chromatophores of fish and other tetrapods such as amphibians and reptiles. Recently, we discovered that α-MSH did not always stimulate pigment dispersion because this hormonal peptide exerted no effects on the melanophores of flounders. We assumed that the reduction of α-MSH activity was related to the co-expression of different α-MSH receptor subtypes - termed melanocortin receptors (MCR) - a member of G-protein-coupled receptors (GPCR) - based on several reports demonstrating that GPCR forms heterodimers with various properties that are distinct from those of the corresponding monomers. In this review, we summarize the relationships between the pigment-dispersing activity of α-MSH-related peptides, molecular forms of α-MSH-related peptides, and mcr subtypes expressed in fish chromatophores.
[Show abstract][Hide abstract] ABSTRACT: 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.
General and Comparative Endocrinology 12/2011; 176(1):9-17. · 2.82 Impact Factor