Fisiología de la reproducción del lenguado senegalés (Solea senegalensis): mecanismos endocrinos y aplicaciones en acuicultura

Memoria de tesis doctoral presentada por Maria Josep Agulleiro Gozalbo para obtener el título de Doctora en Ciencias Biológicas por la Universitat de València (UV), realizada bajo la dirección del Dr. Joan Cerdà Luque del Institut de Ciències del Mar (ICM-CSIC) y el Institut de Recerca i Tecnologia Agroalimentaria (IRTA)El presente trabajo se puede dividir en tres partes. En la primera parte, o Introducción General, se resume el estado actual de conocimientos sobre los mecanismos endocrinos implicados en la reproducción de teleósteos, y se introduce al lector en los beneficios y problemas de la acuicultura de peces planos, especialmente del lenguado senegalés (Solea senegalensis). En la segunda parte, Capítulos 2 y 3, se describen los cambios estacionales de las principales hormonas, moléculas y receptores que intervienen en el proceso de crecimiento ovárico en el lenguado. Y en la tercera parte, Capítulos 4 y 5, se diseñan y aplican métodos hormonales para controlar la reproducción del lenguado en condiciones de cautividad. El objetivo principal de la presente tesis ha sido profundizar en el conocimiento de la endocrinología de la reproducción en teleósteos, y particularmente en el lenguado senegalés. Esta especie posee actualmente un alto interés comercial para la acuicultura marina mediterránea, pero su fisiología reproductiva es todavía muy desconocida, lo que imposibilita mejorar su producción y calidad a nivel industrial. Por ello, el objetivo final de este trabajo ha sido desarrollar métodos para controlar la reproducción del lenguado en condiciones de cautividad los cuales puedan ser transferidos a la industriaPeer Reviewe

Fisiología de la reproducción del lenguado senegalés (Solea senegalensis): mecanismos endocrinos y aplicaciones en acuicultura.

RESUMEN En el presente trabajo investigamos algunos de los mecanismos endocrinos implicados en el crecimiento oocitario del lenguado senegalés (Solea senegalensis), así como el desarrollo de métodos para el control de su reproducción en cautividad. La tesis se dividió en tres partes diferentes: (i) Capítulo 1: Introducción General donde se resume el estado actual de conocimientos sobre los mecanismos endocrinos implicados en la reproducción de teleósteos, y se introduce al lector en los beneficios y problemas actuales de la acuicultura de peces planos, especialmente del lenguado senegalés (Solea senegalensis); (ii) Capítulos 2 y 3, donde se investiga el proceso de vitelogénesis en el lenguado senegalés. En el Capítulo 2, se caracteriza el ADN complementario de ambas isoformas del receptor de lipoproteínas de muy baja densidad/vitelogenina (Vtg), vtgr, y de una nueva proteína de unión a ácidos grasos (FABP), FABP11, encontrada exclusivamente en peces teleósteos. El análisis de expresión de estos genes en ovario de lenguado senegalés, sugiere que el nivel de los transcritos de vtgr puede ser utilizado como marcador molecular para determinar el número de oocitos reclutados en vitelogénesis, mientras que los niveles de expresión de fabp11 pueden funcionar como un marcador molecular muy útil para determinar los procesos celulares y factores ambientales que regulan el proceso de la atresia folicular. En el Capítulo 3, se valida un ELISA homologo para determinar la concentración de Vtg en plasma. Mediante este análisis se correlacionan las variaciones anuales de Vtg con los niveles de 17-estradiol, índice gonadosomático y porcentaje de oocitos en diferente estadio de desarrollo oocitario; y (iii) Capítulos 4 y 5, donde se investiga la aplicación de diferentes métodos hormonales para inducir a la ovulación y a la espermiación en lenguado senegalés en condiciones de cautividad. En el Capítulo 4, se tratan machos y hembras con un agonista de la hormona liberadora de gonadotropinas (GnRHa), tanto mediante inyecciones sucesivas como mediante implantes de liberación sostenida. El tratamiento con GnRHa resulta eficaz para inducir la ovulación y la puesta en hembras F1 incapaces de reproducirse en cautividad. En cambio, el mismo tratamiento con GnRHa en machos es aparentemente ineficaz para aumentar la producción de esperma y/o estimular la espermatogénesis. En el Capítulo 5, el tratamiento de machos adultos con GnRHa en combinación con 11-ketoandrostenediona (OA), precursor de 11-ketotestosterona (11-KT), acelera la espermatogénesis y aumenta los niveles plasmáticos de los metabolitos 5β-reducidos de 17α,20β-dihidroxi-4-pregnan-3-ona (17,20βP), esteroide implicado en la maduración del esperma en salmónidos. Además, la motilidad del esperma producido por los machos tratados con GnRHa+OA, se ve duplicada respecto al esperma producido por el grupo control o el grupo tratado sólo con GnRHa. Por tanto, el tratamiento de machos de lenguado senegalés con GnRHa+OA puede ser empleado como potencial terapia hormonal para mejorar disfunciones reproductivas de machos de lenguado senegalés en condiciones de cautividad. En resumen, la presente tesis ha contribuido a profundizar en el conocimiento de los mecanismos endocrinos implicados en la gametogénesis del lenguado senegalés, y ha establecido algunos protocolos para la inducción de la ovulación y la espermiogénesis a través de terapias hormonales en esta especie de elevado interés comercial. __________________________________________________________________________________________________The present work was aimed at the investigation of some endocrine mechanisms involved in oocyte growth in the Senegalese sole (Solea senegalensis), as well as to develop methods for the control of reproduction of this species in captivity. The thesis was divided into three separate parts: (i) Chapter 1, a General Introduction where it was reviewed the endocrine regulation of reproduction in teleosts, and it introduced the reader into the significance and current problems in the culture of flatfish, particularly the Senegalese sole; (ii) Chapters 2 and 3, in which it was investigated the vitellogenic process of Senegalese sole. In chapter 2, it is described the isolation of complementary DNAs encoding two isoforms of very low-density lipoprotein/vitellogenin receptor (VtgR) and a novel fatty acid-binding protein (FABP), FAB11. The analysis of expression of these genes suggested that the the level of vtgr transcripts in the ovary may be used as a functional marker to quantify the number of oocytes recruited for vitellogenesis, while that of fabp11 may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia. In chapter 3, an homologous EIA to quantify plasma vitellogenin was developed, and annual plasma levels of vitellogenin were correlated with changes in plasma 17-estradiol, gonadosomatic index, and percentage of oocyte at different developmental stage in the ovary; and (iii) Chapters 4 and 5, in which it was investigated the induction of ovulation and spermiation in Senegalese sole. In chapter 4, males and females were treated with gonadotropin-releasing hormone agonist (GnRHa). Treatment was able to induce ovulation and spawning of F1 females that often fail to reproduce. However, this treatment was ineffective in males. Further studies were performed in chapter 5, where GnRHa treatment in combination with a biosynthetic precursor of 11-ketotestosterone, stimulated spermatogenesis, increasing the production of conjugated forms of 17,20-dihydroxy-4-pregnen-3-one, enhancing the motility of spermatozoa by 2-fold. In conclusion, the present work has contributed with novel information on the endocrine mechanisms involved in gametogenesis in Senegalese sole, and has established some protocols for the induction of ovulation and spermiation in this species through hormonal therapies

Fish melanocortin system

31 p. ReviewMelanocortin 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.Peer reviewe

Role of melanocortin receptor accessory proteins in the function of zebrafish melanocortin receptor type 2

19 p., figuras, tablas y bibliografíaIn 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.Peer reviewe

Melanocortin system in fish: from feeding behavior to stress response

Ponencia presentada en el 17th International Congress of Comparative Endocrinology celebrado en Barcelona del 15 al 19 de julio de 2013Melanocortin system is one of the most complex hormonal systems in vertebrates. Agonists including melanocyte.stimulating hormones (MSH) and adrenocorticotropic hormone (ACTH) are encoded into a single precursor called proopiomelanocortin (POMC). The proteolytic cleavage of POMC by prohormone convertase 1 (PC1) generates ACTH and beta-lipotropin in the corticotrophs whereas cleavage by PC1 and PC2 produces alfa-MSH and beta-endorphin in the melanotrophs.Peer Reviewe

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility

8 pages, 3 figures, 2 tablesThe effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 μg kg−1) in the presence or absence of OA (2 or 7 mg kg−1) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5β-reduced metabolite(s) of 17,20β-dihydroxy-pregn-4-en-3-one (17,20βP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5β-reduced metabolites of 17,20βP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20βP in the testis but further studies will be required to elucidate the specific mechanism involved.This work was supported by grants from the “Junta Asesora de Cultivos Marinos” (JACUMAR) of the Spanish Ministry for Agriculture and Fisheries (Spain), and from the Reference Center of Aquaculture, Generalitat de Catalunya(Spain) to JC. M.J.A. was recipient of a fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain).Peer reviewe

High Transcript Level of Fatty Acid-Binding Protein 11 but Not of Very Low-Density Lipoprotein Receptor Is Correlated to Ovarian Follicle Atresia in a Teleost Fish (Solea senegalensis)

13 pages, 8 figuresTranscripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly defined teleost fish FABP subfamily that is a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9. RT-PCR revealed high levels of vldlr transcript splicing variants in the ovaries and, to a lesser extent, in somatic tissues, whereas fabp11 was highly expressed in the ovaries, liver, and adipose tissue. In situ hybridization analysis showed vldlr and fabp11 mRNAs in previtellogenic oocytes, whereas no hybridization signals were detected in the larger vitellogenic oocytes. Transcript expression of fabp11 was strongly upregulated in somatic cells surrounding atretic follicles. Real-time quantitative RT-PCR demonstrated that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively. These results suggest that the expression level of vldlr transcripts may be used as a precocious functional marker to quantify the number of oocytes recruited for vitellogenesis and that fabp11 mRNA may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia in fishSupported by the French Ministry of Research and Education to P.J.B, and by the Junta Asesora de Cultivos Marinos (JACUMAR, Spain) and Reference Center in Aquaculture (Spain) to J.C. M.J.A. was supported by a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain). S.M. was supported by a postdoctoral fellowship from the French governmentPeer reviewe

Molecular cloning and functional chracterisation of two Elovl4 in Solea senegalensis

Trabajo presentado en Aquaculture Europe 2016: Food for Thought, celebrado en Edimburgo (Escocia) del 21 al 23 de septiembre de 2016Recent investigations in mammals have revealed that very long-chain (>C24) polyunsaturated fatty acids (VLC-PUFAs) play pivotal roles in physiological processes including phototransduction in retina, fertility and spermatogenesis in testis, and brain functioning. In fish, however, VLC-PUFAs and their biosynthesis have been barely studied despite many of the above biological processes have obvious implications in finfish aquaculture, where altered visual acuity (critical in visual predators such as most cultured fish species), fertility issues of broodstock, and disruptions of brain functioning can hinder normal development, and jeopardise the economical profit of the farm (Monroig et al., 2010). The biosynthesis of VLC-PUFAs in mammals has been proposed to proceed through consecutive elongation reactions that enzymes called elongases of very long-chain fatty acids-4 (Elovl4) catalyse from shorter chain (C20-22) PUFA substrates. Recent investigations demonstrated that the Senegalese sole Solea senegalensis has the ability to biosynthesise some precursors of VLC-PUFAs (Morais et al., 2015), although the actual Elovl4 complement has not been investigated. In the present study, we have undertaken the molecular cloning and functional characterisation of two Elovl4-like elongases from S. senegalensis.This work was funded by project AGL2013-40986-R LONGFAQUA and FP7-PEOPLE-2010-RG (Project No. 274184).Peer reviewe

The C-terminal domains of melanocortin-2 receptor (MC2R) accessory proteins (MRAP1) influence their localization and ACTH-induced cAMP production

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.Peer Reviewe

Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2

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.Peer Reviewe