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The melanocortin system is one of the most complex of the hormonal systems. It involves different agonists encoded in the multiplex precursor proopiomelanocortin (POMC) or in different genes as β-defensins, endogenous antagonist, like agouti-signalling protein (ASIP) or agouti-related protein (AGRP), and five different melanocortin receptors (MCRs). Rounds of whole genome duplication events have preceded the functional and molecular diversification of the family in addition some co-evolutionary and tandem duplication processes have been proposed. The evolutionary patterns of the different partners are controversial and different hypotheses have emerged from a study of the sequenced genomes. In this review, we summarize the different evolutionary hypotheses proposed for the different melanocortin partners. © 2014 Elsevier Inc., This work was supported by Grants AGL2010-22247-C03-01 and CSD 2007-00002 from Spanish Science and Education Ministry (MEC) to J.M.C.-R. Additional funding was obtained from the “Generalitat Valenciana” (PROMETEO 2010/006). M.J.A. and R.C. are recipient of a “Juan de la Cierva” research contract (2009) and FPI fellow from the Spanish Science and Innovation Ministry, respectively.

The melanocortin system is one of the most complex hormonal systems in vertebrates. Atypically, the signaling of melanocortin receptors is regulated by the binding of endogenous antagonists, named agouti-signaling protein (ASIP) and agouti-related protein (AGRP). Teleost specific genome duplication (TSGD) rendered new gene copies in teleost fish and up to four different genes of the agouti family of peptides have been characterized. In this paper, molecular cloning was used to characterize mRNA of the agouti family of peptides in sea bass. Four different genes were identified: AGRP1, ASIP1, AGRP2 and ASIP2. The AGRP1 gene is mainly expressed in the brain whereas ASIP1 is mainly expressed in the ventral skin. Both ASIP2 and AGRP2 are expressed in the brain and the pineal gland but also in some peripheral tissues. Immunocytochemical studies demonstrated that AGRP1 is exclusively expressed within the lateral tuberal nucleus, the homologue of the mammalian arcuate nucleus in fish. Long-term fasting (8-29. days) increased the hypothalamic expression of AGRP1 but depressed AGRP2 expression (15-29. days). In contrast, the hypothalamic expression of ASIP2 was upregulated during short-term fasting suggesting that this peptide could be involved in the short term regulation of food intake in the sea bass. © 2014 Elsevier Inc., This work was supported by Grants AGL2010-22247-C03-01 and CSD 2007-00002 from the Spanish Science and Education Ministry (MEC) to J.M.C.-R. Additional funding was obtained from the “Generalitat Valenciana” (PROMETEO 2010/006). M.J.A. and R.C. are recipients of a “Juan de la Cierva” research contract (2009) and an FPI fellowship, respectively, from the Spanish Science and Innovation Ministry.

The melanocortin system integrates different agonists, competitive or inverse agonists, and receptors. Recent investigations have also discovered a specific system of melanocortin receptor accessory proteins (MRAPs) that are involved in the regulation of the functional expression of these receptors. MRAP1 mutations are responsible for type 2 familial glucocorticoid deficiency (FGD2), a rare autosomal disorder characterized by high plasma adrenocorticotropin hormone (ACTH) levels but severe cortisol deficiency. ACTH binds melanocortin 2 receptor (MC2R), a G protein-coupled receptor, in the adrenal gland to promote corticosteroid synthesis. In the absence of MRAP1, MC2R cannot translocate from the endoplasmic reticulum to the plasma membrane and ACTH-induced signaling is extinguished. A second MRAP protein, called MRAP2, also modulates MC2R activity. MRAPs also interact with the other melanocortin receptors, adjusting their pharmacological properties. In this paper, we briefly review the MRAP system and its interaction with melanocortin receptors. © 2013 Elsevier Inc., Grants AGL2010-22247-C03-01 and CSD 2007-00002 from Spanish Science and Education Ministry (MEC) to JMC-R. Additional funding was obtained from the >Generalitat Valenciana> (research grant PROMETEO 2010/006). MJA and RC are recipient of a >Juan de la Cierva> research contract and FPI fellowship, respectively, from MINECO.

ACTH binding to the human melanocortin-2 receptor (MC2R) requires the presence of the MC2R accessory protein1 isoforms, MRAPα or MRAPβ. This study evaluated the role of the isoform-specific C-terminal domains of MRAP with regard to their cellular localization, topology, interaction with MRAP2 and cAMP production. When stably expressed in HEK293/FRT cells or in B16-G4F mouse melanoma cells (an MSH receptor-deficient cell clone), MRAPα and MRAPdCT (truncated MRAP1, N-terminal only) localized mainly around the nuclear envelope and within dense intracellular endosomes, while MRAPβ exhibited a strong localization at the plasma membrane, and partially with rapid recycling endosomes. MRAPβ and MRAPdCT both exhibited dual-topology (N cyto/C exo and N exo/C cyto) at the plasma membrane whereas MRAPα exhibited only N cyto/C exo topology at the plasma membrane while adopting dual-topology in intracellular compartments. Both MRAPα and MRAP2 colocalized in intracellular compartments, as opposed to weak colocalization between MRAPβ and MRAP2. MRAP2 and MC2R enhanced the expression of MRAP1 isoforms and vice versa. Moreover, in both HEK293/FRT and B16-G4F cells, ACTH failed to activate MC2R unless MRAP1 was present. MRAP1 expression enhanced MC2R cell-surface expression as well as concentration-dependent cAMP accumulation. In the presence of human or zebrafish MC2R, MRAPβ induced the highest cAMP accumulation while MRAPdCT induced the lowest. Together, the present findings indicate that the C-terminal domains of MRAP dictate their intracellular localization in addition to regulating ACTH-induced cAMP production. These preferential localizations suggest that MRAPα is involved in MC2R targeting to the plasma membrane, while MRAPβ may enhance ACTH-MC2R coupling to cAMP production. © 2012 Elsevier Inc., This work was supported by Grants from the Canadian Institutes of Health Research to N.G.-P. (MOP-82819) and to J.L.P. (MOP-69085) and by the Canada Research Chairs Program. N.G.-P. is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland; J.L.P. is a recipient of a Chercheur-boursier senior scholarship from the Fonds de la Recherche en Santé du Québec. N.G.-P. and J.-L.P. are members of the FRSQ-funded Centre de Recherche Clinique Étienne-le Bel. J.M.C-R is a recipient of research funds from the Spanish Science and Innovation Ministry (AGL2010-22247-C03-01, CSD 2007-00002). S.R. is a recipient of a studentship from the Fonds de la Recherche en Santé du Québec. MJA is a recipient of a ‘‘Juan de la Cierva’’ research contract (2009) from the Spanish Science and Innovation Ministry.

31 p. Review, Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC1-MC5) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC5 receptor, while pufferfish have 4 receptors with no melanocortin MC3 receptor and one copy of melanocortin MC5 receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC2 receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC 1 receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC1 receptor are responsible for reduced melanization. Both melanocortin MC4 and MC5 receptor are expressed in the hypothalamus, and central melanocortin MC4 receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC2 and MC5 receptors. Both sea bass melanocortin MC1 and MC 4 receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively., This work was partially supported by grants from the Ministry of Science and Innovation (MICINN) AGL2007-65744-C03-02, CSD 2007-00002 and AGL2010-22247-C03-01 to JM C-R. MJA is recipient of a “Juan de la Cierva” research contract (2009) from the Spanish Science and Innovation Ministry.

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.

The melanocortin system is probably one of the most complex hormonal systems since it integrates agonist, encoded in the proopiomelanocortin precursor, endogenous antagonist, agouti signaling protein and agouti-related protein, five different G-protein coupled receptors and two accessory proteins. These accessory proteins interact with melanocortin receptors to allow traffic to the plasma membrane or to regulate the pharmacological profile. The MC1R fill the extension locus, which is primarily responsible for the regulation of pigmentation. In zebrafish, both MC1R and MRAP2 system are expressed in the skin. We demonstrate that zebrafish MC1R physically, or closely, interacts with the MRAP2 system, although this interaction did not result in modification of the studied pharmacological profile. However, progressive fasting induced skin darkening but also an upregulation of the MRAP2 expression in the skin, suggesting an unknown role for MRAP2a that could involve receptor desensitization processes. We also demonstrate that crowding stress induces skin darkening and a downregulation of MC1R expression in the skin. © 2014 Elsevier Inc., This work was supported by Grants AGL2010-22247-C03-01 and CSD2007-00002 from Spanish Science and Education Ministry (MEC) to JMC-R. Additional funding was obtained from the “Generalitat Valenciana” (PROMETEO 2010/006). MJA and RC are recipient of a “Juan de la Cierva” research contract (2009) and FPI fellow from the Spanish Science and Innovation Ministry, respectively.

Melanocortin 2 receptor (MC2R) is the only canonical ACTH receptor. Its functional expression requires the presence of an accessory protein, known as melanocortin receptor 2 accessory protein 1 (MRAP1). The vertebrate genome exhibits a paralogue gene called MRAP2, which is duplicated in zebrafish (MRAP2a and MRAP2b), although its function remains unknown. In this paper, we demonstrate that MRAP2a enables MC4R, a canonical MSH receptor, to be activated by ACTH with a similar sensitivity to that exhibited by MC2R. Both proteins physically interact and are coexpressed in the neurons of the preoptic area, a key region in the control of the energy balance and hypophyseal secretion in fish. ACTH injections inhibit food intake in wild-type zebrafish but not in fish lacking functional MC4R. Both MRAP1 and MRAP2a are hormonally regulated, suggesting that these proteins are substrates for feed-back regulatory pathways of melanocortin signaling. Fasting has no effect on the central expression of MRAP2a but stimulates MRAP2b expression. This protein interacts and is colocalized with MC4R in the tuberal hypothalamic neurons but has no effect on the pharmacologic profile of MC4R. However, MRPA2b is able to decrease basal reporter activity in cell lines expressing MC4R. It is plausible that MRAP2b decreases the constitutive activity of the MC4R during fasting periods, driving the animal toward a positive energy balance. Our data indicate that MRAP2s control the activity of MC4R, opening up new pathways for the regulation of melanocortin signaling and, by extension, for the regulation of the energy balance and obesity. © 2013 by The Endocrine Society.

Dopamine is synthesized from l-dopa and subsequently processed into norepinephrine and epinephrine. Any excess neurotransmitter can be taken up again by the neurons to be broken down enzymatically into DOPAC. The effect of dopamine on mammalian food intake is controversial. Mice unable to synthesize central dopamine die of starvation. However, studies have also shown that central injection of dopamine inhibits food intake. The effect of dopaminergic system in the fish feeding behavior has been scarcely explored. We report that the inclusion of l-dopa in the diets results in the activation of sea bass central dopaminergic system but also in the significant increase of the hypothalamic serotonin levels. Dietary l-dopa induces a decrease of food intake and feed conversion efficiency that drives a decline of all growth parameters tested. No behavioral effects were observed after l-dopa treatment. l-dopa treatment stimulated central expression of NPY and CRF. It suggests that CRF might mediate l-dopa effects on food intake but also that CRF neurons lie downstream of NPY neurons in the hierarchical forebrain system, thus controlling energy balance. Unexpectedly, dietary administration of haloperidol, a D2-receptor antagonist, cannot block dopamine effects but also induces a decline of the food intake. This decrease seems to be a side effect of haloperidol treatment since fish exhibited a decreased locomotor activity. We conclude that oral l-dopa inhibits sea bass food intake and growth. Mechanism could also involve an increase of hypothalamic serotoninergic tone. © 2013 Elsevier Inc., This work was supported by grants AGL2010-22247-C03-01 and CSD 2007-00002 from the Spanish Science and Education Ministry (MEC) to JMC-R, AGL2010-22247-C03-03 to JMM. Additional funding was obtained from the >Generalitat Valenciana> (research grant PROMETEO 2010/006). EL was recipient of a Geronimo Forteza fellowship from the Generalitat Valenciana (GV). MJA is recipient of a >Juan de la Cierva> research contract (2009) from the Spanish Science and Innovation Ministry. MC-S was recipient of a FPI fellow from the Spanish Science and Innovation Ministry.

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release. © 2013 Agulleiro et al., This work was supported by Spanish Science and Innovation Ministry Grants: Life Sciences and Agrofeeding Research Program (AGL2010-22247-C03-01) and Consolider Program (CSD 2007-00002) Generalitat Valenciana Grant: PROMETEO program (2010/006) to JMC-R. MJA is recipient of a >Juan de la Cierva> research contract (2009) from the Spanish Science and Innovation Ministry. EL was recipient of a Geronimo Forteza fellowship from the Generalitat Valenciana (GV).

27 p, figuras, tablas y bibliografía, To assess mechanisms relating to food intake and glucosensing in fish, and their interaction with stress, we evaluated changes in the expression of orexigenic (NPY) and anorexigenic (POMC, CART and CRF) peptides in central glucosensing areas (hypothalamus and hindbrain) of rainbow trout subjected to normoglycaemic (control), hypoglycaemic (4 mg insulin kg(-1)) or hyperglycaemic (500 mg glucose kg(-1)) conditions for 6 h under normal stocking density (NSD; 10 kg fish mass m(-3)) or under stress conditions induced by high stocking density (HSD; 70 kg fish mass m(-3)). Hyperglycaemic NSD conditions resulted in decreased mRNA levels of NPY and increased levels of CART and POMC in the hypothalamus as well as increased mRNA levels of CART and CRF in the hindbrain compared with hypo- and normoglycaemic conditions. HSD conditions in normoglycaemic fish induced marked changes in the expression of all peptides assessed: mRNA levels of NPY and CRF increased and mRNA levels of POMC and CART decreased in the hypothalamus, whereas the expression of all four peptides (NPY, POMC, CART and CRF) decreased in the hindbrain. Furthermore, HSD conditions altered the response to changes in glycaemia of NPY and POMC expression in the hypothalamus and CART expression in the hypothalamus and the hindbrain. The results are discussed in the context of food intake regulation by glucosensor systems and their interaction with stress in fish., This study was supported by research grants from Ministerio de Educación y Ciencia (AGL2007-65744-C03-01/ACU and AGL2007-65744-C03-03/ACU), Xunta de Galicia (Consolidación e estructuración de unidades de investigacion competitivas, INCITE09ENA310002ES), Universidade de Vigo (Contrato-Programa con grupos de investigación consolidados) M.C-S. and A.J.A. were recipients of FPI and MAEC-AECI predoctoral fellowships, respectively. MJA is recipient of a “Juan de la Cierva” research contract from the Spanish Science and Innovation Ministry.

19 p., figuras, tablas y bibliografía, In this paper, we identify three different MRAPs in zebrafish, zfMRAP1, zfMRAP2a and zfMRAP2b, and demonstrate that zfMC2R is not functional in the absence of MRAP expression. ZfMRAP1 expression was restricted to adipose tissue and the anterior kidney whereas MRAP2a and MRAP2b were expressed in all the tissues tested. Quantification of surface receptor and immunofluorescence studies indicated that the receptor is unable to translocate to membrane in the absence of MRAP isoforms. MRAP1 and MRAP2b are localized in the plasma membrane in the absence of zfMC2R expression but MRAP2b is retained in perinuclear position. MRAP1 and MRAP2a displayed an equivalent translocation capacity to the membrane of zfMC2R but only zfMRAP1 expression led to intracellular cAMP increases after ACTH stimulation. ZfMRAP2b had no effect on zfMC2R activity but both zfMRAP2 isoforms enhanced the zfMRAP1-assited cAMP intracellular increase, suggesting an interaction between zfMRAP1 and zfMRAP2s when regulating zfMC2R activity., This work was supported by grants from Ministry of Science and Innovation (MICINN) AGL2007-65744-C03-02 and CSD 2007-00002 to JM C-R, Canadian Institutes for Health Research (MOP 10998) and Canada Chairs Program to NG-P. The authors thank Lucie Chouinard and the other members of the laboratory for their technical assistance in performing cAMP assays. MJA is recipient of a “Juan de la Cierva” research contract (2009) from the Spanish Science and Innovation Ministry. SR is a recipient of a Fonds de la Recherche en Santé du Québec studentship. NGP is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland.

11 pages, 4 figures, 1 table In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild-caught males. To gain insights into the molecular mechanismsthatmay be altered in the F1 testis,we used 2-DDIGE tocompare the protein profiling of the testis of wild-caught males at the spermiation stage with that of F1 males showing different stages of germcell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins byMS/MSrevealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germcell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males This work was supported by the PLEUROGENE project funded by a Genome Spain-Genome Canada joint program. The LP CSIC/UAB is a member of the National Institute for Proteomics (ProteoRed) funded in part by Genome Spain. I. F. and M. J. A. were supported by predoctoral fellowships from the Scientific Park of Barcelona (University of Barcelona) and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain), respectively

25 pages, 7 figures, 3 tables, Background: The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. Results: Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis (leukocyte cell-derived chemotaxin 2,), angiogenesis (thrombospondin), and prevention of apoptosis (S100a10 calcium binding protein). Conclusion: This study has identified a number of differentially expressed genes in the ovary that were not previously found to be regulated during ovarian development in marine fish. Specifically, we found evidence, for the first time in teleosts, of the activation of chemoattractant, angiogenic and antiapoptotic pathways in hypertrophied follicular cells at the onset of ovarian atresia, We thank staff of Oryzon Genomics (Dr. Elisabet Rosell, Dr. Tamara Maes, Olga Durany and Francesc Subirada) for their assistance during microarray hybridization and bioinformatic analysis. This work was supported by Genome Spain and Genome Canada within the framework of the international consortium Pleurogene™ coordinated by JC. MJA was supported by a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain), and by a postdoctoral fellowship from Juan de la Cierva Programme (Spanish Ministry of Education and Science)