Search

The aim of the present thesis is directed towards the comprehension of the mechanisms involved in the regulation of lipid and carbohydrate metabolism in relevant species in European aquaculture. Special attention has been placed on adipose tissue due to its key role in the control of the overall energy homeostasis and its endocrine functions. Six diets were formulated to determine the feasibility of replacing dietary lipids (from 23 to 17%) by starch (from 12 to 28%) and subsequent replacements in starch by fibre up to levels of 18% in gilthead sea bream. The dietary treatments did not affect fish growth nor feed utilization efficiency and triggered only moderate changes in lipid and carbohydrate metabolism. Gilthead sea bream adipose tissue exhibited an activation of the lipogenic pathway and a transcriptional regulation of relevant transcription factors directed to modulate the preferential use of lipids or carbohydrates according to availability. Despite of a lack of hepatic regulation of gluconeogenesis, an efficient metabolic hepatic adaptation to dietary starch was found through the modulation of enzymes directed to increase glucose metabolization and balance fatty acid uptake and lipolysis. These metabolic adaptations encourage the formulation of diets with a relatively high content of starch or fibre. In addition, the capacity of Atlantic salmon adipocytes to synthesise fatty acids from glucose has been demonstrated for the first time. However, the capacity of the pathway is relatively low, which might be contributing to the previously reported glucose intolerance in salmonids. Atlantic salmon adipocytes mainly transformed glucose to glycerol and Krebs-cycle intermediates. The effect of glucose on the transcriptional regulation of enzymes involved in glycolysis, the pentose phosphate pathway and glyceroneogenesis, highlights the relevance of carbohydrate metabolism in fish adipocytes as well as the metabolic adaptation of these cells to ensure the availability of necessary factors and other substrates for lipid synthesis and storage. The implication of adiponectin and TNFα in the regulation of glucose uptake in rainbow trout adipocytes has been explored, showing that both adipokines stimulated glucose uptake without modifying AKT or TOR phosphorylation. However, adiponectin failed to stimulate glucose uptake in insulin-treated adipocytes. Insulin regulated the transcription of the adiponectin system enhancing the mRNA levels of the peptide and reducing those of adiponectin receptors, following the mammalian model. However, neither adiponectin nor TNFα mRNA levels were affected by each other, concluding that the reciprocal suppressive effects reported in mammals are not conserved in fish. TNFα supressed PPARγ at a protein level, corroborating its anti-adipogenic role. However, TNFα enhanced glucose uptake in insulin-stimulated adipocytes, suggesting that the interactions between this cytokine and insulin are not so well defined in fish. Finally, the transcriptomic profile along primary culture of rainbow trout preadipocytes has been characterized revealing that adipogenesis is a complex process involving the coordinated activation or repression of genes associated to the different stages of cell development. It presented high similarities to the mammalian model, being characterized by two main phases: proliferation and differentiation. Proliferation phase was enriched with genes involved in cellular remodelling, autophagy and angiogenesis. In addition, the implication of the eicosanoid signalling pathway was highlighted during this phase. Differentiation phase was enriched with genes involved in energy production, lipid and carbohydrate metabolism. During this phase, the activation of the TR/RXR and the PPAR signalling pathways were revealed. The whole process was driven by a coordinated activation of transcription factors and epigenetic modulators. Overall, these studies have contributed to better understand the mechanisms regulating lipid and carbohydrate metabolisms in teleost fish, as well as adipose tissue growth and development. All this information might be applied to modulate and optimize farming practices.

[spa] La hidratación de los oocitos de teleósteos marinos que producen huevos pelágicos es clave para la supervivencia de los embriones en el océano. Estudios previos han descu-bierto el papel de una acuaporina específica de teleósteos (Aqp1b) durante este proceso, pero se carece todavía de evidencias experimentales directas, así como información sobre la regulación molecular de la Aqp1ab. En la presente tesis, estudios iniciales con el fletan Atlántico (Hippoglossus hippoglossus) confirman que los parálogos aqp1aa y -1ab de teleósteos han surgido probablemente por duplicación génica en tándem, y han demostrado el papel esencial de la Aqp1ab durante la hidratación del oocito. Para investigar el control transcripcional del gen aqp1ab en los oocitos de la dorada (Sparus aurata) se ha aislado su región promotora, la cual contiene elementos cis-reguladores de unión al receptor nuclear de progestinas (Pgr), como la 17α,20β-dihidroxi-4-pregnen-3-ona (17,20β-P), y factores de transcripción Sox. Estudios de localización subcelular indican que el Pgr aparece en el citoplasma de las oogonias, así como en el núcleo de oocitos en crecimiento primario (pre-vitelogénicos) coincidiendo con la activación de la expresión de aqp1ab. En este estadio también se expresan cuatro isoformas diferentes del Pgr, dos de las cuales pueden inhibir la transcripción mediada por el Pgr de forma dominante negativa. Las oogonias también expresan sox3 y -8b, mientras que el sox9b aparece en el estadio de alveolo cortical, cuando se reduce la expresión de aqp1ab. Ex-perimentos de transactivación indican que el Pgr activa la transcripción de aqp1ab, con Sox3 y -8b actuando de forma sinérgica, mientras que el Sox9b reprime este mecanismo. El papel del Pgr se ha investigado sobre explantes ováricos pre-vitelogénicos incubados in vitro, lo cual ha demostrado que la 17,20β-P, producida en las células de la granulosa en respuesta a la hormona folículo estimulante, activa el promotor de aqp1ab en el oocito induciendo la síntesis de Aqp1ab. Los resultados de esta tesis revelan por primera vez una estricta regulación transcripcional del gen aqp1ab durante la oogénesis de teleósteos marinos, lo cual remarca la función fisiológica esencial de este canal de agua durante la formación de los huevos pelágicos, [eng] In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts. By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20β-P, followed by chromatin immunoprecipitation, confirmed that 17,20β-P-activated Pgr enhanced aqp1ab promoter activation. The production of 17,20β-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20β-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20β-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20β-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20β-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins. The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish.

En aquest treball de tesi doctoral es descriu l'estructura gonadal, gametogènesi i cicle anual de Scorpaena porcus, S. scrofa i S. elongata per tal d'aprofundir en el coneixement dels diferents graus d'especialització dins l'oviparisme en peixos. Es pretén fer èmfasi en aquells trets inusuals que difereixen del què s'ha descrit fins ara per la majoria de les espècies ovípares de teleostis, i relacionar-los amb l'estratègia reproductiva de les espècies que els presentenEl mostreig s'ha dut a terme de forma intensiva entre l'any 2000 i 2002, tot i que s'ha continuat recol·lectant mostres fins a l'agost de 2004, amb més o menys freqüència segons la necessitat. En total s'han obtingut 258 exemplars de S. porcus, 119 de S. scrofa i 46 de S. elongata.S'ha pogut veure com S. porcus, S. scrofa i S. elongata són espècies ovípares ovulípares especialitzades, ja que la seva estructura ovàrica presenta tot una sèrie de trets molt particulars i poc comuns a la resta de peixos ovípars estudiats fins ara: posició central del raquis musculo-connectiu que es troba envoltat completament pel lumen i la paret ovàrica; presència de peduncles en oòcits en fase de creixement secundari; escassetat i mida petita dels alvèols corticals; poc gruix de la zona radiata; i secreció d'una massa gelatinosa que engloba els ous en el moment de la posta. Totes aquestes característiques també s'han observat en Scorpaena notata, suggerint que l'especialització de l'oviparisme ovulípar es dóna en tot el gènere Scorpaena.L'estructura testicular i l'espermatogènesi d'aquestes espècies també és força particular. Per una banda els testicles són del tipus lobular no-restringit, tot i que presenten algunes característiques com la ordenació parcial dels cists segons l'estadi de maduració de les cèl·lules germinals que contenen i l'absència de lumen central, que actualment només s'ha observat en testicles del tipus lobular restringit de les espècies de la sèrie Aterinomorpha. Per l'altre banda, l'espermatogènesi és del tipus semicística, és a dir, els cists s'obren abans d'acabar tot el procés i les cèl·lules germinals s'acaben de desenvolupar a la llum del lòbul fins a espermatozoides.La posta d'aquestes espècies és múltipla i constisteix en una massa gelatinosa d'ous pelàgica, que en el cas de S. porcus s'alliberen entre els mesos de juny i agost, i en S. scrofa, a partir del mes de juliol. S'ha relacionat la presència d'aquesta massa amb un aparellament mascle-femella, de manera que el mascle alliberaria l'esperma directament sobre la massa d'ous, assegurant així la fartilització total dels mateixos., The present work describes gonadal structure, gametogenesis and annual cycle of Scorpaena porcus, S. scrofa and S. elongate, to go more deeply into the knowledge of different degree of oviparity specialization in fishes. We pretend to emphasize unusual features that diverge from those that have been described until now for oviparous teleosts.Sampling was performed intensively between years 2000 and 2002. However, collection of specimens was continued until August 2004 sporadically according to experimental needs. We obtained a total of 258 specimens of S. porcus, 119 of S. scrofa and 46 of S. elongata.S. porcus, S. scrofa and S. elongata are specialized ovuliparous species. The ovary showed many characteristics that are not typical for oviparous fishes: central ovarian stroma, pedunculate oocytes, scarcity and small size of cortical alveoli, thinness of the zona radiata, and a gelatinous matrix secreted during the maturation. All these features also were observed in Scorpaena notata suggesting that specialized ovuliparity probably occurs in the entire genus Scorpaena.Testicular structure and spermatogenesis are unusual too. On one hand, these three species have an unrestricted lobular testis. However, they present a partial arrangement of cists according to the stage of development of germinal cells, and they don't have central lumen, two features that currently have only been described for the restricted lobular testes of aterinomorph fishes. On the other hand, spermatogenesis is semicystic: cysts open before the end of spermatogenesis, which is then completed in the lobule lumen.These species are multiple spawning fishes, and they release a pelagic gelatinous mass of eggs. Spawning in S. porcus occurs between June and August, and in S. scrofa begins in July. The presence of the gelatinous mass of eggs has been related to a mating behaviour between male and female that guarantees the fertilization of the whole pond.

[spa] A través de estudios de ayuno y realimentación, y de tipo y cantidad de dieta suministrada, hemos comprobado cómo afecta el estado nutricional a enzimas clave implicadas en glucólisis-gluconeogénesis, vía de las pentosas fosfato y metabolismo de aminoácidos en hígado del pez teleósteo Sparus aurata. La investigacion se ha centrado en el control de la expresión génica de la enzima bifuncional 6-PF 2-K/FRU 2,6-P2ASA, responsable de la sintesis y degradación de FRU 2,6-P2, principal activador de la glucólisis e inhibidor de la gluconeogénesis. Flujo glucolítico, ruta de las pentosas, así como actividad quinasa, cantidad de proteína y niveles de mRNA de 6-PF 2-K/FRU 2,6-P2ASA, parámetros todos disminuídos por el ayuno, correlacionaron con la cantidad de dieta suministrada y con el nivel de carbohidratos aportado por la dieta. Esta enzima ha mostrado ser regulada a corto plazo fundamentalmente por alosterismo y/o modificación covalente, y a largo plazo por el nivel de proteína y mensajero. El análisis de los niveles de mRNA por Northern Blot se ha realizado con una sonda homóloga obtenida por RT-PCR, cuya secuencia es la primera descrita para el mensajero de 6-PF 2-K/FRU, 2,6-P2ASA en peces.