AIEC Posters Short ABS 090827 by TravisStanaway

VIEWS: 276 PAGES: 23

									                        7AIEC POSTER SESSION ABSTRACTS


LIST of POSTER PRESENTATIONS:
P1
OPTIMIZACIÓN DE UN MÉTODO DE ALTA SENSIBILIDAD PARA LA CUANTIFICACIÓN DE
MELATONINA MEDIANTE DERIVATIZACIÓN PRECOLUMNA Y DETECCIÓN POR FL-HPLC.
A. Macías, E. Velarde, C. Azpeleta, M.J. Delgado, A.L. Alonso Gómez y Ana Isabel Valenciano.

P2
PRESENCE OF GHRELIN AND ITS RECEPTOR IN A GONADOTROPIN-SECRETING PITUITARY
ADENOMA CAUSING OVARIAN HYPERSTIMULATION SYNDROME
Ana Quintero1, I Saigí2, AJ Martinez-Fuentes1, MJ Barahona2, RM Luque1, N Sucunza2, MM Malagón1, E.
Resmini2, SM Webb2, Justo P. Castaño1.

P3
CALCIOTROPIC EFFECT OF THE PTH FAMILY OF PEPTIDES IN THE CLAWED FROG
Ana S. Gomes, Juan Fuentes, João CR Cardoso, Deborah M Power & Adelino V M Canário.

P4
EVIDENCIAS SOBRE EL POSIBLE PAPEL DE LA LEPTINA COMO INTERMEDIADORA ENTRE
LA RESPUESTA GLUCOSENSORA Y EL CONTROL DE LA INGESTA EN LA TRUCHA ARCO
IRIS (Oncorhynchus mykiss)
Ariel Aguilar, M. Conde, J.L. Muñoz, S. Polakof, J.L. Soengas.

P5
VASOTOCIN AND ISOTOCIN DAILY CHANGES IN RAINBOW TROUT: EVIDENCE FOR A ROLE
IN THE REGULATION OF PINEAL MELATONIN PRODUCTION
Arnau Rodríguez-Illamola1,2, J.L.P. Muñoz2, J.M. Wilson1, J. Coimbra1, J.M. Míguez2

P6
HOW DOES AN ACUTE STRESS MODULATE THE INNATE IMMUNE SYSTEM OF
SENEGALESE SOLE (Solea senegalensis Kaup 1858)?
Benjamín Costas1,2, L. Conceição2, C. Aragão2, J.A. Martos3, I. Ruiz-Jarabo3, J.M. Mancera3, A. Afonso1.

P7
GENE EXPRESSION OF PARATHYROID HORMONE RECEPTOR 1 AND STRUCTURAL
EXTRACELLULAR MATRIX COMPONENTS DURING SEA BREAM FIN REGENERATION
Cristina Rocha, Redruello B., Canário A.VM. and Power, D.M.

P8
IN VITRO CHARACTERIZATION OF THE MELATONIN SYNTHESIS RATE-LIMITING ENZYME,
AANAT1, IN THE DIGESTIVE SYSTEM OF THE GOLDFISH, Carassius auratus.
Elena Velarde, Y. Vivas, E. Isorna, M.J. Delgado, A.I. Valenciano, A.L. Alonso-Gómez.

P9
THYROID HORMONE CELL TRANSPORTERS: DO THEY EXIST IN FISH?
Emanuel Esgaio, João CR Cardoso & Deborah M Power

P10
GENITAL PAPILLA MORPHOLOGY FOR SEXING Lipophrys pholis, A NEW SENTINEL SPECIES
FOR ESTROGENIC CHEMICAL POLLUTION.
F. Ferreira1, M.M. Santos1, M.A. Reis-Henriques1, N.M. Vieira1,2 & N.M. Monteiro 1,3

P11
MELATONIN RECEPTORS IN SEA BASS AND SOLE: DAILY AND SEASONAL EXPRESSION
ALONG THE RETINA-BRAIN-PITUITARY-GONAD AXIS.
Francesca Confente1, Patricia Herrera-Pérez 1, J.J Aarseth2, E.H.Jørgensen 2, J. Falcón3, JA. Muñoz-Cueto

P12
PRODUCCION DE FSHβ RECOMBINANTE DE LUBINA (Dicentrarchus labrax), OBTENCION DE
ANTICUERPOS Y ANALISIS DE PERFILES HIPOFISARIOS

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                        7AIEC POSTER SESSION ABSTRACTS


Gregorio Molés, M. Carrillo y S. Zanuy.

P13
THYROID AXIS mRNA EXPRESSION IN GILTHEAD SEABREAM (Sparus auratus) UNDER
DIFFERENT SALINITY CONDITIONS
Ignacio Ruiz-Jarabo1, F.J. Arjona1, L. Vargas-Chacoff1 , P. Pinto2, , M.P. Martín del Río1, D. Power2, J.M.
Mancera1.

P14
HORMONAL REGULATION OF BRANCHIAL RHESUS GLYCOPROTEIN AMMONIA
TRANSPORTER EXPRESSION IN ZEBRAFISH.
Inês Páscoa1,2; António Fontaínhas-Fernandes1; L. Filipe C. Castro2 & Jonathan M. Wilson2

P15
DAILY VARIATIOS IN CLOCK-GENE EXPRESSION IN RETINA AND HYPOTHALAMUS OF
RAINBOW TROUT.
Jesús M. Míguez, M. Conde-Sieira, A. Rodríguez-Illamola, J.L. Soengas, M.A. López Patiño.

P16
FUNCTIONAL CHARACTERISATION OF A NOVEL PTH/PTHRP RECEPTOR IN TETRAPODS
Jorge F Pascoal, Deborah M Power & João CR Cardoso

P17
AN IN VITRO STUDY OF THE EFFECTS OF L-TRYPTOPHAN AND DRUGS INTERACTING WITH
SEROTONINERGIC FUNCTION ON THE INTESTINAL MELATONIN PRODUCTION IN THE
RAINBOW TROUT
José L..P. Muñoz, M. Conde-Sieira, M. López-Patiño, J.L. Soengas, J.M. Míguez .

P18
CORTISOL INCREASED PLASMA MELATONIN LEVELS IN GILTHEAD SEA BREAM (Sparus
auratus)
Juan Miguel Mancera 1, L. Vargas-Chacoff 1, J.A. Martos 1, A. H. Kalamarz 2, G. Martínez-Rodríguez 3, E.
Kulczykowska 2.

P19
CHARACTERISATION OF GROWTH AND DIFFERENTIATION FACTOR- 9 IN Oreochromis
mossambicus OVARY AND GENE EXPRESSION DURING FOLLICLE GROWTH
Laurence Deloffre 1, J. Lesage 2, D.M. Power 1, A.V.M Canario.

P20
TEMPERATURE MODULATES PLASMA CORTISOL, MSH AND β-ENDORPHIN RESPONSE TO
HIGH DENSITY IN GILTHEAD SEABREAM Sparus auratus
Luis Vargas-Chacoff1,3, J. Metz2, F. J. Arjona1, P.H. M. Klaren2, G. Flik2 and J.M. Mancera1

P21
IDENTIFICATION OF A NOVEL HUMAN GHRELIN GENE TRANSCRIPT THAT CONTAINS
INTRON 2 AND IS UP-REGULATED, TOGETHER WITH GHRELIN O-ACYL TRANSFERASE
(GOAT) ENZYME BUT NOT WITH NATIVE-GHRELIN, IN BREAST CANCER TUMOURS.
Manuel David Gahete-Ortiz1, J. Cordoba-Chacón1, M. Hergueta2, F. Gracia-Navarro1, RD Kineman3, G.
Moreno-Bueno2, Raul M Luque1, Justo P Castaño1.

P22
CHANGES ON THE REPRODUCTIVE STAGE OF IMPUBER MALES EUROPEAN SEA BASS
(Dicentrarchus labrax) AFTER ADMINISTRATION OF RECOMBINANT SINGLE-CHAIN FSH.
María José Mazón, A.Gómez, S. Zanuy,

P23
MODULATION OF STEROIDOGENIC AND IMMUNE FUNCTIONS IN HEAD KIDNEY CELLS OF
GILTHEAD SEABREAM STIMULATED WITH A CHEMICAL STRESSOR
Mariana Teles1,2, Juan Castillo1 and Lluís Tort1.


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                       7AIEC POSTER SESSION ABSTRACTS



P24
HIGH-STOCKING DENSITY STRESS INTERACTS WITH PERIPHERAL GLUCOSENSOR
SYSTEM AND FOOD INTAKE IN RAINBOW TROUT Oncorhynchus mykiss
Marta Conde Sieira, A. Aguilar, J.L.P. Muñoz, J.M. Míguez, J.L. Soengas.

P25
DIETARY CARBOHYDRATE LEVEL AND EXERCISE EFFECT ON EXPRESSION OF
MOLECULES RELATED WITH MYOGENESIS IN SEA BREAM (Sparus aurata) WHITE MUSCLE
Mònica Rius, Daniel García de la serrana, Encarnación Capilla, Isabel Navarro, Joaquim Gutiérrez.

P26
HYDROSTATIC PRESSURE INCREASES HEPATIC ARYLALKYLAMINE N-
ACETYLTRANSFERASE (AANAT) TRANSCRIPT EXPRESSION IN RAINBOW TROUT (O. mykiss)
Odete Marinho-Gonçalves1; A.F. Gonçalves1,2; J. Ings3; A. Damasceno-Oliveira1; N. Aluru3; M.M. Vijayan3; J.
Coimbra1,2; J.M. Wilson1

P27
EL SISTEMA ENDOCANNABINOIDE EN ÁREAS CIRCUNVENTRICULARES DE LA RATA
Pedro Fernández-Llebrez1, J. Suárez2, F.J. Bermúdez-Silva2, J. Pérez1, F. Rodríguez de Fonseca2.

P28
PHOTOPERIODIC CONTROL OF THE EARLY PUBERTY IN MALE SEA BASS (Dicentrarchus
labrax)
Rafael Rodríguez, E. Hala, A. Felip, S. Zanuy, M. Carrillo.

P29
KISSPEPTIN SIGNALS THROUGH MITOGEN-ACTIVATED PROTEIN KINASE, PROTEIN
KINASE C AND INTRACELLULAR CALCIUM STORES TO DIRECTLY INCREASE GH RELEASE
IN PRIMARY PITUITARY CELL CULTURES FROM A NON-HUMAN PRIMATE (Papio anubis)
Raúl M. Luque1, J Cordoba-Chacon1, E Gutierrez-Pascual1, MD Gahete1, A. Pozo1, MM Malagón1, AJ
Martinez-Fuentes1, M Tena-Sempere1, RD Kineman2, Justo P. Castaño1.

P30
MOLECULAR CLONING AND EXPRESSION OF HYDROXYINDOLE-O-METHYLTRANFERASE
IN SENEGALESE SOLE (Solea senegalensis)
Rosa María Martínez-Álvarez1, F. Confente1, E. Isorna2, A.J. Martín-Robles1, J.A. Muñoz-Cueto1.

P31
TROUT AND CHICKEN GLUCOKINASE IS DIFFERENTIALLY REGULATED BY A HEPATIC
PROTEIN
Sergio Polakof A.Aguilar, J.M. Míguez, J.L. Soengas.

P32
MOLECULAR CLONING OF A cDNA FRAGMENT ENCODING A PUTATIVE NEUROPEPTIDE Y
FROM THE HYPOTHALAMUS OF RAINBOW TROUT Oncorhynchus mykiss
Vanesa Lozano1, M. Mancebo2, B. Fondevila2, J.J. Pérez2, M. Aldegunde2.

P33
DEPENDENCIA CIRCADIANA DE LAS ACCIONES ANORÉTICAS DE LA LEPTINA EN EL
CARPÍN (Carassius auratus).
Yurena Vivas, C. Azpeleta, A. Feliciano, E. Velarde, A. Macias, E. Isorna, M.J. Delgado, N. De Pedro.

P34
THYROID HORMONE FEEDBACK ON PERIPHERAL DEIODINATION IN THE SENEGALESE
SOLE (SOLEA SENEGALENSIS)
Yvette Wunderink1, Felipe Espigares1, Gert Flik2, Peter Klaren2 and Juan M. Mancera1




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                        7AIEC POSTER SESSION ABSTRACTS




P1
OPTIMIZACIÓN DE UN MÉTODO DE ALTA SENSIBILIDAD PARA LA CUANTIFICACIÓN DE
MELATONINA MEDIANTE DERIVATIZACIÓN PRECOLUMNA Y DETECCIÓN POR FL-HPLC.

A. Macías, E. Velarde, C. Azpeleta, M.J. Delgado, A.L. Alonso Gómez y Ana Isabel Valenciano.
E-mail: aivalenciano@bio.ucm.es
Departamento de Fisiología (Fisiología Animal II). Facultad de Biología. Universidad Complutense de Madrid.

La presencia de melatonina (MEL) en localizaciones periféricas se ha descrito recientemente en vertebrados, si
bien su reducido contenido dificulta su cuantificación. El objetivo de este estudio ha sido la optimización de un
método preciso y sensible que permita cuantificar niveles de femtomoles de MEL utilizando la capacidad
fluorescente de un derivado de esta indolamina. La técnica se basa en la oxidación y posterior condensación
aldólica intramolecular de la MEL en N-6-metoxi-4-oxo-1,4-dihidroquinolin-3-meltilacetamida (6-MOQMA) en
presencia de peróxido de hidrógeno y carbonato sódico, seguida de su cuantificación por HPLC acoplada a
detección fluorimétrica. A partir de los espectros de emisión y absorción para el 6-MOQMA se fijaron las
longitudes de onda de excitación y emisión en 245 y 380 nm, respectivamente. La emisión de fluorescencia
aumenta significativamente cuando se utiliza una fase móvil de pH ácido (3,5 respecto 6,5). El producto de la
reacción de derivatización, 6-MOQMA, muestra una fluorescencia 6 veces superior a la de la MEL. El límite de
sensibilidad de este método es de 7,8 fmoles. La linealidad de la reacción de derivatización respecto a la
concentración de sustrato se conserva hasta 1 nmol de MEL. Tiempos de reacción de 30-60 min no modifican
significativamente la cantidad de 6-MOQMA producido. La eficacia de la reacción de derivatización es
independiente de la presencia de acetonitrilo, sin embargo, se ve inhibida en presencia de etanol. El ácido 5-
metoxiindol-3-acético y la 5-metoxi-N-ciclopropanoiltriptamina se transforman tras la reacción de
derivatización en productos fluorescentes con longitudes de onda y tiempos de retención adecuados para su
valoración conjunta con 6-MOQMA, por tanto pueden utilizarse como estándares internos. La aplicación de esta
técnica ha permitido cuantificar niveles diurnos de MEL circulante y contenido de MEL en tejidos periféricos
del teleósteo Carassius auratus.




P2
PRESENCE OF GHRELIN AND ITS RECEPTOR IN A GONADOTROPIN-SECRETING PITUITARY
ADENOMA CAUSING OVARIAN HYPERSTIMULATION SYNDROME

Ana Quintero1, I Saigí2, AJ Martinez-Fuentes1, MJ Barahona2, RM Luque1, N Sucunza2, MM Malagón1, E.
Resmini2, SM Webb2, Justo P. Castaño1. E-mail: aquica@hotmail.com
(1) Dpt. of Cell Biology, Physiology & Immunology. Univ. of Córdoba, IMIBIC, and CIBERobn 06/03,
Córdoba, Spain.
(2) Hospital Sant Pau, Autonomous University of Barcelona, and CIBERER Unit 747, Barcelona, Spain.

Gonadotropin-synthesizing pituitary adenomas (GnPA) represent 25% of all pituitary tumors but rarely cause
any clinical syndrome as they usually secrete their hormone products inefficiently and in low concentrations.
Consequently, most GnPAs are diagnosed as a result of sellar mass effect. Here, we report a unique case of a 40-
yr-old woman diagnosed of a GnPA with a secondary mild ovarian hyperstimulation syndrome (OHS), which
disappeared after neurosurgical resection. OHS in premenopausal women with GnPA is a rare entity, as these
adenomas commonly secrete nonfunctional free α-subunit. We suspected the existence of a GnPA causing OHS
when we found an amenorreich woman with enlarged multicystic ovaries of long evolution, accompanied by
increased FSH levels, suppressed LH and highly elevated estradiol levels. To investigate the molecular features
associated with this GnPA, expression of genes related to gonadotrope regulation were evaluated by RT-PCR in
the tumor. This confirmed expression of β-FSH, β-LH, α-subunit, gonadotropin releasing hormone receptor
(GnRH-R), and estrogen receptors α-ER, β1-ER, and β2-ER. However, this expression pattern could not explain
the differential regulation and consequent imbalance of FSH/LH release. In search for other molecules
potentially related to this pathology, we tested the presence of the ghrelin system. Ghrelin was originally
identified as a growth hormone (GH) releasing peptide expressed in stomach and hypothalamus, but was
subsequently found to exert a wide array of patho-physiological actions, including modulation of gonadotropic

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                        7AIEC POSTER SESSION ABSTRACTS


axis, corticotrope tumors, etc. Ghrelin acts through its 7-transmembrane receptor, GHS-R1a, which has a
truncated variant GHS-R1b, which is related to some tumors. Uniquely, binding and activation of GHS-R1a
requires ghrelin to be octanolylated at its Ser-3 residue by the enzyme ghrelin O-acyltransferase (GOAT). In this
context, by applying quantitative real time PCR we discover that this tumor expresses unusually high levels of
ghrelin, GOAT, GHS-R1a and GHS-R1b. In cultured rat pituitary cells, ghrelin stimulates both LH and FSH
release, yet, interestingly in presence of GnRH, ghrelin reduces the LH response and enhances FSH response.
Moreover, in vivo, ghrelin decreases the LH response to GnRH at the pituitary level. Thus, our data demonstrate
the unusual presence of high levels of the complete ghrelin/GOAT/GHS-R system in a GnPA causing OHS,
suggesting a potential involvement of this system in the pathophysiology of this tumor.
Support: RYC-2007-00186, BIO-0139, CTS-01705, BFU2007-60180/BFI, BFU2008-01136/BFI.




P3
CALCIOTROPIC EFFECT OF THE PTH FAMILY OF PEPTIDES IN THE CLAWED FROG

Ana S. Gomes, Juan Fuentes, João CR Cardoso, Deborah M Power & Adelino V M Canário.
Email: acgomes@ualg.pt
Comparative and Molecular Endocrinology Group, Centre of Marine Sciences, Universidade do Algarve,
Campus de Gambelas, 8005-139 Faro, Portugal.

Parathyroid hormone (PTH) and Parathyroid hormone-related peptide (PTHrP) are endocrine factors important
for calcium homeostasis that share a highly conserved (1-34) N-terminal region and activate a common G
protein coupled receptor (PTHR1). A novel PTH-like peptide (PTH-L) was recently identified in teleosts which
shares conserved structure and function with other PTH family members, raising intriguing questions about the
origin and evolution of PTH/PTHrP systems and their role in calcium homeostasis during the vertebrate
radiation. Xenopus laevis which belongs to the first group of terrestrial vertebrates to possess a parathyroid
gland was the model organism used to obtain insight into the evolution of the PTH family. In the Xenopus
genome, three PTH family members (PTH, PTHrP and PTH-L), homologues of teleost members, were
identified. Expression studies using gene specific primers revealed that PTH family genes have a widespread
tissue expression with PTHrP as the most widely distributed transcript, in agreement with its pleiotropic
paracrine role in vertebrates. We characterised the calciotrophic activity of (1-34) N-terminal PTH family by
analysing their function in Xenopus abdominal skin using Ussing chambers. The homologous Xenopus PTH,
PTHrP and PTH-L N-terminal (1-34) peptides all stimulated calcium flux from the apical/basolateral membrane
site. The (1-34) PTHrP was the most potent peptide followed by (1-34) PTH-L and (1-34) PTH at a
concentration of 10 nM, and this action seems to be mediated by the receptor form PTHR1. The results confirm
that amphibian skin may plays a role in calcium homeostasis and provides a solid model for future studies of
calcitropic factors.
Acknowledgement: Work funded by the Fundação para a Ciência e a Tecnologia grant Nº PTDC/CVT/
66735/2006 PTHL and AG received a Bolsa de Técnico de Investigação (BI) in the project.




P4
EVIDENCIAS SOBRE EL POSIBLE PAPEL DE LA LEPTINA COMO INTERMEDIADORA ENTRE
LA RESPUESTA GLUCOSENSORA Y EL CONTROL DE LA INGESTA EN LA TRUCHA ARCO
IRIS (Oncorhynchus mykiss)

Ariel Aguilar, M. Conde, J.L. Muñoz, S. Polakof, J.L. Soengas. E-Mail: Ariel.aguilar@uvigo.es
Depto. Bioloxía Funcional e Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, España.

No se conoce cuales de los sistemas orexigénicos o anorexigénicos conocidos en peces pueden intermediar entre
la respuesta glucosensora y la ingesta de alimento. En este estudio presentamos evidencias sobre el posible papel
de la leptina. En un primer experimento se inyectaron intracerebroventricularmente (ICV) truchas arco iris con 1
µl de salino solo (control) o conteniendo dosis crecientes de leptina (0.3, 1, 3, 5, 10 y 30 µg/µl/100 g animal).
Transcurridas 6h se tomaron muestras de plasma así como de las áreas glucosensoras centrales (hipotálamo y
cerebro posterior) para evaluar cambios en los parámetros glucosensores (niveles de metabolitos, actividades

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                         7AIEC POSTER SESSION ABSTRACTS


enzimáticas y expresión génica). Los tratamientos con leptina produjeron cambios dosis-dependientes en los
parámetros evaluados (niveles de glucógeno, actividad GSase…) que en general coinciden con los descritos
previamente en truchas arco iris en situaciones de hiperglucemia. Dado que la hiperglucemia en trucha inhibe la
ingesta y que la leptina en peces es anorexigénica, ésta es la primera evidencia obtenida en peces sobre el
posible papel intermediador de la leptina entre los cambios en el sistema glucosensor a nivel central y la ingesta.
Para confirmar la especificidad de la respuesta de la leptina, en un segundo experimento se inyectaron ICV
truchas con 1 µl de salino solo (control) o conteniendo 5 µg/µl/100 g animal de leptina, o conteniendo 5
µg/µl/100 g animal de leptina más un fármaco antagonista de los mecanismos de acción conocidos de la leptina
en mamíferos (inhibidor de PI-3K: 0.01 nmol wortmannin, inhibidor de PDE: 12.5 µg milrinone, inhibidor de
K+ATP: 500 µmol diazoxide, inhibidor de Ca2+-PKC: 50 ng GF109203X, o inhibidor de la síntesis de NO: 100 µg
L-NAME). En general, el tratamiento con los fármacos revierte el efecto producido por el tratamiento con
leptina sola, lo cual demuestra la especificidad de la acción de la leptina y proporciona evidencias sobre los
mecanismos de acción implicados en su acción a nivel de las áreas glucosensoras.
Financiado por MEC (AGL2007-61211/ACU) y Universidade de Vigo (Contrato-Programa)




P5
VASOTOCIN AND ISOTOCIN DAILY CHANGES IN RAINBOW TROUT: EVIDENCE FOR A ROLE
IN THE REGULATION OF PINEAL MELATONIN PRODUCTION

Arnau Rodríguez-Illamola1,2, J.L.P. Muñoz2, J.M. Wilson1, J. Coimbra1, J.M. Míguez2
e-mail: arnau@uvigo.es
(1) Laboratory of Ecophysiology. Centro Interdisciplinar de Investigação Marinha e Ambiental. Porto.
(2) Lab. Fisiología Animal y Comparada. Departamento de Bioloxía Funcional e Ciencias da Saúde. Facultade
de Bioloxía, Universidade de Vigo.

Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptidic hormones involved in the
regulation of the ion and water balance in fish. Plasma AVT content has been shown to display a daily rhythm
with a peak during the day-night transition, suggesting a role of this neuropeptide as a time-keeping signal. In
mammals, several studies with pinealocyte cultures have also implicated vasopressin (the analogue hormone to
fish AVT) in the regulation of pineal melatonin production. Taking this in mind, the present work aimed, firstly,
to evaluate the existence of daily variations in trout pituitary and plasma AVT and IT contents, as well as their
dependence on the lighting cycle by testing the effect of constant darkness. Secondly, the effect of AVT on
melatonin secretion from trout pineal organs was assayed in vitro. The results showed a clear rhythm of AVT
and IT pituitary contents with a peak during the day-night transition. This rhythm was similar to that of
circulating AVT levels, suggesting a direct relationship between hypothalamic synthesis and pituitary secretion
of the peptide to the blood. After exposure of trout to constant darkness, the daily rhythms of pituitary and
plasma AVT but not those of IT, remained with similar characteristics than those found under exposure to the
light-dark cycle. By other hand, incubation of trout pineals with AVT (10-9 and 10-11 M) increased melatonin
synthesis under low lighting conditions. All these results suggest that synthesis and/or release of AVT might be
endogenous processes with potential involvement in the circadian time-keeping system in fish. The data also
suggest that AVT might have a role in modulating pineal melatonin production.




P6
HOW DOES AN ACUTE STRESS MODULATE THE INNATE IMMUNE SYSTEM OF
SENEGALESE SOLE (Solea senegalensis Kaup 1858)?

Benjamín Costas1,2, L. Conceição2, C. Aragão2, J.A. Martos3, I. Ruiz-Jarabo3, J.M. Mancera3, A. Afonso1. E-
mail: bcostas@ualg.pt
(1) CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental; and ICBAS – Instituto de
Ciências Biomédicas Abel Salazar, University of Porto, Porto, Portugal
(2) CIMAR/CCMAR – Centro de Ciências do Mar do Algarve, University of Algarve, Faro, Portugal
(3) Department of Biology, Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real,
Cadiz, Spain

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                         7AIEC POSTER SESSION ABSTRACTS



Stress-related physiological changes present an impact on metabolism and cell processes, including that of
immune cells, and it is generally considered that stress causes decreased immune function in fish. The objective
of this study was to investigate the subsequent effects of an acute stress on cellular and humoral non-specific
immune functions. Therefore, eight groups of six specimens of Senegalese sole (Solea senegalensis) (136.1 ±
58.4 g wet weight) were maintained undisturbed, while other eight groups of six specimens were used for acute
stress challenge (air exposed during 3 minutes). A group of six specimens was sampled for blood and head-
kidney collection immediately after air exposure (time 0), while the remaining groups were sampled at 5 and 30
minutes, 1, 2, 4, 6 and 24 hours. Undisturbed fish were sampled at the same times and used as control. Fish were
fasted for 24 hours prior to air exposure and sampling. Plasma cortisol level was used as stress parameter, while
superoxide anion (O2-) production from head-kidney leucocytes and plasma complement (ACH50) and
lysozyme activities were used as cellular and humoral innate immune parameters, respectively. Plasma cortisol
level significantly increased from time 0 until peak level at 1 hour in air exposed fish. Respiratory burst activity
of head-kidney leucocytes from stressed fish was significantly higher than that of the control groups at 2 and 6
hours, with the highest levels at 6 hours. Moreover, control groups showed different O2- production, with
significantly higher levels at 4 hours. Furthermore, ACH50 values followed an inverse linear relationship with
respect to cortisol levels, being the lowest values at 1 hour in air exposed fish. Although lysozyme activity also
decreased in stressed specimens, significant lower values were only found at 4 hours after air exposure. Results
suggest that S. senegalensis presents a stress response similar to that observed in other fish species. O2-
production observed suggests that an acute stress can initially stimulate the non-specific cellular immune
system, at least for O2- production under these experimental conditions. However, air exposure suppressed the
innate humoral response. The stress response observed in this study appears to modulate the innate immune
system by increasing the cellular response while humoral response is suppressed. These data provide further
information that the neuroendocrine responses in flatfish can affect the activity of the innate immune system.




P7
GENE EXPRESSION OF PARATHYROID HORMONE RECEPTOR 1 AND STRUCTURAL
EXTRACELLULAR MATRIX COMPONENTS DURING SEA BREAM FIN REGENERATION

Cristina Rocha, Redruello B., Canário A.VM. and Power, D.M. Email: dpower@ualg.pt
Centre of Marine Sciences, Comparative and Molecular Endocrinology, University of Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal.

The study of regeneration processes are important resources to understand the mechanisms involved in cell
growth and differentiation. Among vertebrates, amphibians and teleosts are the most popular biological models
and several studies have been carried out in zebrafish and medaka, in which important transcription factors and
parathyroid hormone (PTH) receptors were identified to be involved in this process. The PTH/PTHrP system is
a complex calcitropic system in vertebrates with multiple functions associated and potential role in cell
proliferation. For example, PTH receptor 1 (PTH1R) is highly expressed in pre-hypertrophic cells and PTHrP
down-regulates mRNA of the extracellular matrix protein osteonectin (OSN) in sea bream scales. In zebrafish
and medaka, OSN is up regulated during fin regeneration. In this work the potential involvement of PTH1R and
structural extracellular matrix components (Col1A1, Col5A2, LUM, FN and OSN) in fin regeneration was
studied by analysing their gene expression by semi-quantitative RT-PCR and in situ hybridazation, as well as of
some gene markers of cartilage and bone development (Cilp, Cbfa1 and TRACP) and the transcription factors c-
fos and Creb. Gene expression was monitored during the first 6 days sea bream of fin regeneration. Expression
for FN, Cbfa1, TRACP, c-fos and Creb was found as soon as 6 hours post-amputation. After 24 hours
extracellular matrix genes and PTH1R are expressed but strong upregulation was found at day 6. The results
suggest that the initial processes after injury involve the rearrangement of cells and tissues, a process that is
finished within the first 24 hours post amputation, after which blastem formation occurs. The strong expression
at day 6 may be related to ossification processes.
Acknowledgments: supported by the Ministry of Science and Higher Education and European Social Funds
through the Portuguese National Science Foundation: project POCI/MAR/61091/2004.




                                                         7
                         7AIEC POSTER SESSION ABSTRACTS




P8
IN VITRO CHARACTERIZATION OF THE MELATONIN SYNTHESIS RATE-LIMITING ENZYME,
AANAT1, IN THE DIGESTIVE SYSTEM OF THE GOLDFISH, Carassius auratus.

Elena Velarde, Y. Vivas, E. Isorna, M.J. Delgado, A.I. Valenciano, A.L. Alonso-Gómez. E-mail:
alalonso@bio.ucm.es
Departamento Fisiología (Fisiología Animal II), Facultad de Biología, Universidad Complutense de Madrid.

Since melatonin (N-acetyl-5-methoxytryptamine, MEL) was found in the gastrointestinal tract of vertebrates
including fish, it has been proposed as a peripheral site of MEL production. This indoleamine is synthesized
from the amino acid tryptophan in four steps, including the N-acetylation of serotonin by the arylalkylamine N-
acetyltransferase (AANAT), the rate-limiting enzyme of MEL synthesis. In teleost fish, several isoforms of the
enzyme are found with differences in their distribution pattern, enzyme kinetics and substrate preference. In the
present study, in vitro NAT enzymatic assays were performed on liver and gut from goldfish (Carassius
auratus) to demonstrate the possible MEL synthesis in such locations. Neural retina was used as a control tissue
for AANAT1 isoform. Enzymatically obtained products were analyzed by HPLC with fluorescence detection.
Tryptamine was used in the assay as reference acceptor amine, and different substrates and inhibitors were
added to the reaction in order to characterize the enzymatic activity in the studied organs. Either, the addition of
folic acid (inhibitor) or phenetidine (specific substrate of arylamine N-acetyltransferase activity, ANAT) did not
modify the reaction. These results refute the presence of ANAT in our assay conditions, and support that the
reaction was catalyzed by one or more AANAT isoforms. To identify the AANAT subtype involved in the
transferase reaction, the tyramine (specific substrate for AANAT1) was added together with tryptamine. Results
showed a significant decrease in the N-acetyltryptamine formation in the three tissues. This suggests that the
enzymatic activity in liver and gut can be attributed, at least in part, to AANAT1. The cloning of goldfish
AANAT1 is currently ongoing in our laboratory and the screening in peripheral tissues would confirm the
presence of this enzyme in goldfish liver and hindgut. Our present results concerning in vitro AANAT1 activity
reinforces the existence of a peripheral melatoninergic system in goldfish.




P9
THYROID HORMONE CELL TRANSPORTERS: DO THEY EXIST IN FISH?

Emanuel Esgaio, João CR Cardoso & Deborah M Power E-mail: esgaio.emanuel@gmail.com
Comparative Molecular Endocrinology, Centre of Marine Sciences, Universidade do Algarve, 8005-139 Faro

The thyroid hormones (THs), T3 (Triiodothyronine) and T4 (Thyroxine) are produced by the follicular cells of
thyroid tissue and regulate a diversity of physiological functions, such as growth, development and metabolism.
The THs bring about their action by binding to nuclear thyroid hormone receptors (TRs) and modulating gene
transcription. Until recently it was assumed that the lipophilic character of THs permitted their free diffusion
into cells, however the identification of highly specific membrane cell transporters has modified this paradigm.
In humans, three transporters: MCT8 and MCT10 (monocarboxylate transporters) and OATP1C1 (an organic
anion-transporting polypeptide) have been identified and mutations modifying their ability to transport THs
causes specific human syndromes. Despite the functional importance of TH cell transporters their
mechanism/regulation of action are poorly understood. The aim of the present study was to characterise thyroid
hormones transporters in a model organism, the zebrafish, using a series of in silico and molecular approaches in
order to establish an experimental model for functional studies. Teleost EST and genome databases were mined
with the sequence of human MCT8, MCT10 and OATP1C1 and single MCT8 and MCT10 genes retrieved. In
silico comparison of mammalian and zebrafish TH cell transporters revealed good conservation of sequence
(>55%) and gene organisation although the fish genes were at least 10 times smaller. The zebrafish transporters
have a similar tissue distribution to that in humans and were found in liver, brain and heart, raising intriguing
questions about potential conservation of function. In vitro cell transfection assays are under development to
permit pharmacological characterisation of the zebrafish cell transporters.



                                                         8
                         7AIEC POSTER SESSION ABSTRACTS


Acknowledgments: E.E. was funded by Bolsa de Integração na Investigação (BII) from Fundação para a Ciência
e a Tecnologia and a British Society of Endocrinology grant. The work was supported by a CCMAR Pluriannual
project and Fp7 EU project Lifecyle.




P10


GENITAL PAPILLA MORPHOLOGY FOR SEXING Lipophrys pholis, A NEW SENTINEL SPECIES
FOR ESTROGENIC CHEMICAL POLLUTION.

F. Ferreira1, M.M. Santos1, M.A. Reis-Henriques1, N.M. Vieira1,2 & N.M. Monteiro 1,3 E-mail:
nmonteir@fc.up.pt
(1) CIIMAR, Centre of Marine and Environmental Research, Rua dos Bragas, 289, 4050-124 Porto, Portugal
(2) Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, rua do
Campo Alegre, 4169-007 Porto, Portugal
(3) CEBIMED, Faculdade de Ciências da Saúde da Universidade Fernando Pessoa, Rua Carlos da Maia, 296,
4200-150 Porto, Portugal

Some aquatic species, such as the zebrafish, the Japanese medaka, the fathead minnow or even some bivalves,
have been increasingly used worldwide as sentinels in aquatic ecosystem bio-monitoring programmes. Although
useful, some of these species represent only a restricted group of habitats and life-strategies. Consequently,
questions relating the sensitivity to chemicals of species with very distinct life histories or the necessity of
applying less straightforward laboratorial exposure experiments, reintroduced the need for new candidate
sentinel species, especially those that inhabit marine coastal areas since pollution is not circumscribed to
freshwater or estuarine ecosystems. The shanny, Lipophrys pholis (Linnaeus, 1758), is a common inhabitant of
the northeastern Atlantic rocky intertidal, from Mauritania to Norway, usually found in rock pools from where it
emerges at high tide to feed. Recently, it has been demonstrated that this species is responsive both to estrogenic
chemicals as well as polycyclic aromatic hydrocarbons. These observations, if taken together with a wide
geographical distribution and a well documented life history, converted into an excellent choice as a sentinel
species, that could be used under field or laboratory conditions, and thus integrated in monitoring programs of
European marine ecosystems. Although a vast body of literature exists describing the basic biology of the most
common intertidal blenniid in the Portuguese rocky coast, on the shanny, there is no detailed information on the
morphology of its genital area. Taking into account the renewed interest on this species, that led to its inclusion
as a ‘sentinel’ given its response to environmental contaminants, a rapid and reliable method that enables the
distinction of both sexes, namely outside the breeding season where the amount of secondary sexual characters
is reduced, is now increasingly needed. In this work, detailed information on the external and internal
morphology of the genital area of males and females is provided. Moreover, given the impracticality of
distinguishing specific anatomical details, the distance of the anus to the genital papilla is proposed as a valid
proxy for gender distinction, since females always present larger distances, both within or outside the breeding
season, when compared to males.




P11
MELATONIN RECEPTORS IN SEA BASS AND SOLE: DAILY AND SEASONAL EXPRESSION
ALONG THE RETINA-BRAIN-PITUITARY-GONAD AXIS.

Francesca Confente1, Patricia Herrera-Pérez 1, J.J Aarseth2, E.H.Jørgensen 2, J. Falcón3, JA. Muñoz-Cueto 1e-
mail: patricia.herrera@uca.es; francesca.confente@uca.es
(1) Departamento de Biología. Facultad de Ciencias del Mar y Ambientales. Universidad de Cádiz. E-11510.
Puerto Real. España.
(2) Department of Aquatic Biology, Norwegian College of Fishery Science, University of Tromsø, 9010 Tromsø,
Norway.
(3) Laboratoire Aragó - UMR7628 (CNRS et UPMC) et GDR2821 (CNRS/Ifremer) - F-6665.

Melatonin receptors (MelR) are expressed in neural and peripheral tissues and mediate melatonin actions on the
synchronization of circadian and circannual rhythms in vertebrates. Recently, we have cloned three different

                                                        9
                         7AIEC POSTER SESSION ABSTRACTS


MelR (MT1, MT2 and Mel1c) in sea bass and sole, two fish species with high relevance for aquaculture. To
better understand the physiological role of this hormone, we have analyzed daily and seasonal MelR expression
in the retina, brain and pituitary of sea bass and sole by quantitative real time PCR (qPCR). Furthermore, we
have analyzed MT1 expression in sea bass gonad by RT-PCR and in situ hybridization as well as daily and
annual ovarian MT1 and MT2 expression in sole by qPCR. Finally, a possible direct action of melatonin on in
vitro estradiol (E2) secretion was examined in sole. The results obtained reveal the existence of daily and
seasonal variations in MT1 mRNA expression in retina and optic tectum from both species. However, daily and
seasonal differences in MT2 mRNA levels were less conspicuous or absent. In sole pituitary, only seasonal
variation in MT1 and MT2 expression was detected, being mRNA levels higher in summer for both MelR. In
gonads, MT1 receptors were expressed both in ovary and testis of sea bass, being mRNAs detected in
spermatogonia. In sole ovary, there were no daily differences in MT1 and MT2 mRNA expression but both
MelR showed a higher expression in autumn and spring than in summer and winter. However, these MelR do
not seem to mediate melatonin effects on E2 secretion in sole ovaries. Our results suggest that not only daily and
seasonal variations in melatonin levels, but also in MelR expression (in particular MT1) could be mediating
circadian and circannual rhythms in sea bass and sole. Moreover, the expression of MelR along the brain-
pituitary-gonad axis suggests a role of melatonin in the control of reproduction in both species.
Grants: MCINN (AGL2001-0593-C03-02; AGL2004-07984-C02-02; HF2005-0047), Marie Curie Training
Program (EU) and CNRS/IFREMER (GDR2821).




P12
PRODUCCION DE FSHβ RECOMBINANTE DE LUBINA (Dicentrarchus labrax), OBTENCION DE
ANTICUERPOS Y ANALISIS DE PERFILES HIPOFISARIOS

Gregorio Molés, M. Carrillo y S. Zanuy. E-mail: zanuy@iats.csic.es
Departamento de Fisiología de Peces y Biotecnología. Instituto de Acuicultura de Torre la Sal (IATS), CSIC.
Torre de la Sal s/n, Ribera de Cabanes, 12590 Castellón, España.

La FSH es una hormona hipofisaria clave en la reproducción de vertebrados. Es una glicoproteina compuesta
por dos subunidades, la GP-α, común a la de LH y TSH, y la FSHβ que confiere especificidad de función a la
hormona. Desde la década de los 80 se han aislado y caracterizado en numerosas especies de peces, pero solo en
unas pocas se ha logrado desarrollar inmunoensayos específicos para la determinación de sus niveles en
hipófisis o plasma. La producción de FSHβ recombinante de lubina, mediante un sistema de expresión
baculovirus, nos ha permitido obtener cantidades suficientes de FSHβ para la inmunización de conejos y
obtención de anticuerpos policlonales anti-FSHβ. Los anticuerpos obtenidos reaccionaron específica y
eficientemente con la FSH cuando se usan condiciones reductoras. Sin embargo, se detecto un reconocimiento
escaso de la forma nativa de FSH, imprescindible para la puesta a punto de un inmunoensayo tipo ELISA. En su
lugar se desarrolló un inmuno-dot-blot, en el cual mediante desnaturalización e inmovilización de las proteínas
en membranas de PVDF se consiguió cuantificar FSH de diferentes orígenes. La inmunodetección se realizo por
quimioluminiscencia en un sistema de imagen Versadoc (Bio-Rad) y la intensidad de la señal fue determinada
por densitometría. La sensibilidad del ensayo fue de 100 ng/ml, con coeficientes intra- e inter-ensayo de 9,8 % y
11,5 % respectivamente. Mediante el inmuno-dot-blot desarrollado hemos determinado los perfiles anuales de
FSH en hipófisis de hembras de lubina adultas. Dichos perfiles muestran un aumento considerable de FSH
durante las fases de postvitelogenesis y maduración de los oocitos, correlacionándose con los perfiles de
expresión de receptor de FSH en gónada y los niveles plasmáticos de estradiol. Los anticuerpos obtenidos anti-
FSHβ nos permite disponer de herramientas para identificar y cuantificar FSH y abre la posibilidad de realizar
nuevos estudios inmunohistoquimicos en esta especie.




P13
THYROID AXIS mRNA EXPRESSION IN GILTHEAD SEABREAM (Sparus auratus) UNDER
DIFFERENT SALINITY CONDITIONS

Ignacio Ruiz-Jarabo1, F.J. Arjona1, L. Vargas-Chacoff1 , P. Pinto2, , M.P. Martín del Río1, D. Power2, J.M.
Mancera1. E-mail: ignacio.ruizjarabo@alum.uca.es

                                                       10
                         7AIEC POSTER SESSION ABSTRACTS


(1) Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Puerto
Real 11510, Cádiz, Spain
(2) Lab. of Molecular and Comparative Endocrinology. Centre of Marine Sciences (CCMAR), Universidade do
Algarve, Portugal

Information regarding the involvement of thyroid hormones (TH) in teleost osmoregulation and osmotic
acclimation is scarce and contradictory. Most experimental designs have been focused on plasma TH levels and
central interactions with other endocrine axes, i.e. stress axis, without assessing the main source of bioactive
TH: outer ring deiodination (ORD) pathway. This metabolic pathway, catalysed by deiodinases, involves the
removal of one iodine atom from the outer (phenolic) ring of thyroid hormone molecule. By ORD, the
prohormone 3,5,3’,5’-tetraiodothyronine (T4) yields the bioactive 3,5,3’-triiodothyronine (T3), which once
inside the cellular nucleus binds to its specific nuclear receptor, acting the T3-receptor complexes (despite of
non-genomic actions for TH have been described too) as transcription factor for plenty of genes controlling
multiple metabolic routes. In some teleost species, activation of several components of the thyroidal network,
i.e. deiodinase type II (DII), occurs at lower salinities. In this work, we aimed to provide more insight about the
involvement of the thyroid system in the osmotic acclimation of Sparus auratus . Modulation of ORD activities
and mRNA expression of those genes encoding for deiodinases, as well as the transcription of other relevant
indicators (TSH and TH receptors) of thyroidal status where also investigated. For this purpose we acclimated
juveniles of S. auratus to four different ambient salinities (5, 15, 40 and 55 ppt) (N=8 per group) for two weeks,
and according to previous studies with this species (Ruiz-Jarabo et al., 2007), we decided to extract mRNA from
the main osmoregulatory organs (gills and kidney). Real time quantitative PCR using Taqman technique was
used to quantify gene expression of the two deiodinases that seems to have ORD activity in this species (DI and
DII), in peripheral organs as gills and kidney. Branchial, renal and pituitary thyroid hormone receptor β (TRβ)
expression as well as thyroid stimulating hormone (TSH), only in the pituitary, were also assessed. With this set
of measurements, we expect to shed light on the acclimation mechanism to different salinities and its relation
with the thyroid axis and associated peripheral metabolism. Results indicate, from a molecular and biochemical
point of view, that the thyroid system is involved in the osmotic acclimation of S. auratus.




P14
HORMONAL REGULATION OF BRANCHIAL RHESUS GLYCOPROTEIN AMMONIA
TRANSPORTER EXPRESSION IN ZEBRAFISH.

Inês Páscoa1,2; António Fontaínhas-Fernandes1; L. Filipe C. Castro2 & Jonathan M. Wilson2
E-mail: mipascoa@ciimar.up.pt
1
  Escola Ciências da Vida e do Ambiente / Centro de Investigação e de Tecnologias Agroambientais e
Biológicas (CITAB), Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal
2
   Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto, Porto,
Portugal

Rhesus glycoproteins have recently been identified as ammonia transporters and hormonal mechanisms of
regulation have only begun to be studied. The pituitary hormone prolactin (PRL) has an important role in
freshwater adaptation, decreasing branchial permeability of osmoregulatory organs to water and ions. Yet it is
unknown if it has similar effects on ammonia excretion pathways. Cortisol is the main corticosteroid, that
increases in response to stress, enhancing the circulatory glucose through gluconeogenesis. Consequently,
cortisol can raise the production and excretion of ammonia. The aim of this study was to investigate the effects
of exogenous administration of these hormones on ammonia excretion and on transcript expression of branchial
genes involved in ammonia transport mainly, the Rhesus glycoproteins Rhbg, Rhcg1 and Rhcg2, as well as,
mineralocorticoids receptor (MR) and glucocorticoids receptor (GR) levels. The experiment was done in adult
zebrafish that were injected intraperitoneally with coconut oil (vehicle), cortisol (50ug/g) or ovine prolactin
(5ug/g) and a control group of fish. Ammonia flux rates were measure 48h after recovery and animals sacrificed
24h after the end of the experiment for tissue collection. Previously, the distribution of these genes was studied
in whole embryos and in different tissues of adults. All genes were found in the gill but showed different
distributions within other tissues: MR and GR were expressed in all samples; Rhbg and Rhcg1 were expressed
only in gill and embryos; and Rhcg2 in gill, intestine anterior and posterior, embryos, organs and body. The
ammonia excretion rates were not statistically different between the treatment groups. The main differences in
gene expression were observed in the gill of fish injected with PRL. In this group, a decrease of MR and Rhbg
and increase of Rhcg2 was observed. Cortisol induced an up-regulation in gene expression of Rhbg. GR

                                                       11
                        7AIEC POSTER SESSION ABSTRACTS


expression was not affected by either hormone. Coconut oil had subtle effects on gene expression mainly
decreasing MR and increasing Rhbg levels. This response might indicate that fish were responding to a stressor
(injection and/or handling). Our results show that PRL may play a role in ammonia transport, but it will be
necessary to continue this research in order to clarify this relationship.
This work was supported by FCT grant SFRH/BD/39378/2007.




P15
DAILY VARIATIOS IN CLOCK-GENE EXPRESSION IN RETINA AND HYPOTHALAMUS OF
RAINBOW TROUT.

Jesús M. Míguez, M. Conde-Sieira, A. Rodríguez-Illamola, J.L. Soengas, M.A. López Patiño. E-mail:
jmmiguez@uvigo.es
Departamento de Biología Funcional y CC Salud, Facultad de Biología, Universidad de Vigo, Spain.

Living organisms show daily rhythms in physiology, behavior, and gene expression, which are due to the
presence of endogenous clocks that synchronize biological processes to the 24-h light/dark cycle. In metazoans,
the generation of circadian rhythmicity is consequence of specialized tissues known as “master clocks”, showing
different locations among species. The pineal gland and retina appear to be the location of the master clock in
teleosts. To date only a few studies describe clock-gene expression in fish brain but do not discriminate between
different brain regions. In this way the present study was designed to test the presence/absence of circadian
clock-gene expression in teleost hypothalamic region (which contains the master clock in mammals) and retina
(which appears to contain the master clock in fish). The comparison between both tissues would help in
localizing a “master clock” in teleosts hypothalamic region. Immature rainbow trout (Oncorhynchus mykiss)
were kept in 120L water tanks (13ºC water temperature), under a 12L:12D light-dark cycle for two weeks prior
to any experimental procedure. Animals were sacrificed by decapitation every 3 h in a 24-h cycle, and the
hypothalamus and neural retina were immediately removed, frozen on dry ice and stored at -80ºC. Real-time
quantitative RT-PCR (QPCR) assays were performed to quantify expression of core circadian genes clock1,
bmal1 and per1 as representative members of the positive (clock1 and bmal1) and negative (per1) regulatory
loops that involve the core oscilator of the circadian clock. All clock genes analyzed in the retina and
hypothalamus showed circadian rhythmicity. However, gene expression peaked in rainbow trout hypothalamus
with a 3-h (clock1 and bmal1) or 6-h (per1) delay relative to that observed in neural retina, the latest showing
the higest expression levels at ZT9 for clock1 and bmal1, and at ZT21 for per1. This suggests that the 6-h delay
observed in hypothalamic per1 expression could be responsible of the 3-h delay on clock1 and bmal1 expression
in this tissue, relative to gene oscillation in neural retina. The present study documents the presence of a core
oscillator in rainbow trout central nervous tissue (hypothalamus), in consistency with that observed in mammals.
The results also suggest that regulation of daily rhythms in physiology and behaviour could be due to an
interaction between both retina and hypothalamic circadian clocks.




P16
FUNCTIONAL CHARACTERISATION OF A NOVEL PTH/PTHRP RECEPTOR IN TETRAPODS

Jorge F Pascoal, Deborah M Power & João CR Cardoso
E-mail: jfpascoal@gmail.com
Comparative Molecular Endocrinology, Centre of Marine Sciences, Universidade do Algarve, 8005-139 Faro,
Portugal

Parathyroid hormone (PTH)/PTH related Peptide (PTHrP) family is part of a complex endocrine system
involved in calcium regulation. They are major calcitropic factors and mobilize calcium from bone. In
mammals, two proteins PTH and PTHrP which activate two specific family 2 GPCRs (PTHR1 and 2) have been
characterised and in teleosts which possess duplicate PTH and PTHrP hormones and a novel calcitropic factor,
PTH-like peptide (PTH-L), a further receptor (PTHR3) an orthologue of teleost PTHR1 has been identified.
Recently homologues of PTH-L have been characterised in Xenopus and chicken along with a potential PTHR3
receptor raising questions about the evolution and function of this system. In this study we aimed to

                                                       12
                         7AIEC POSTER SESSION ABSTRACTS


characterise Xenopus and chicken PTHR3 which share 70% of amino acid sequence similarity using an in vitro
cell transfection assay and compare it to its teleost homologue. Stable lines of mammalian HEK293 cells
expressing Xenopus and chicken PTHR3 were established and receptor stimulation assays were performed with
100 nM and 10 nM of species homologous N-terminal peptides. All peptides tested provoked receptor activation
as assessed by cAMP levels. Xenopus PTHR3 was strongly stimulated by PTH and less by PTH-L which
contrasts with zebrafish PTH3R in which PTHrP is the most potent whilst PTH-L only provoked weak
activation. The chicken PTHR3 is still being characterised. The results suggest that evolutionary pressure has
led to modification in ligand preference of PTH3R in Xenopus, chicken and zebrafish but the relative
importance of ligand and receptor remains to be established. It will be important in the future to characterise the
biological processes in which PTHR3 is involved.
Acknowledgments: The authors would like to acknowledge to Pedro Pinheiro and Ana Gomes for providing the
chicken and Xenopus PTH3R expression vectors and peptides. JFP was funded by Bolsa de Integração na
Investigação (BII) from Fundação para a Ciência e a Tecnologia. This work was supported by CCMAR
Pluriannual project.




P17
AN IN VITRO STUDY OF THE EFFECTS OF L-TRYPTOPHAN AND DRUGS INTERACTING WITH
SEROTONINERGIC FUNCTION ON THE INTESTINAL MELATONIN PRODUCTION IN THE
RAINBOW TROUT

José L..P. Muñoz, M. Conde-Sieira, M. López-Patiño, J.L. Soengas, J.M. Míguez .
E-mail: jmunoz@uvigo.es
Dept. Functional Biology and Health Sciences, Faculty of Biology, University of Vigo. Spain.

The hormone melatonin intermediates between the environment and rhythmic physiological processes in the
body. The pineal organ is believed as the main source of blood melatonin in vertebrates. However, there is
evidence that the gastrointestinal tract (GIT) is capable to synthesize and secrete melatonin independently on the
pineal organ. The synthesis of melatonin begins from the uptake of the amino acid L-tryptophan (L-Trp) which
is hydroxylated to 5-HT, and then N-acetylated and 5’-methylated to give melatonin. Although the regulation of
melatonin synthesis is well known in the pineal organ, little is known as it occurs in GIT, particularly in fish.
Taking this into account, the present study aimed to investigate the relationships between 5-HT availability and
melatonin production in fish GIT by using in vitro tissue preparations from trout intestine. Measurements of 5-
HT, 5-HIAA (the main 5-HT metabolite), and melatonin contents in tissues and the incubation media were done
by HPLC. In a first experiment, the role of 5-HT synthesis on GIT melatonin production was evaluated by
incubating intestinal tissues with five concentrations L-Trp (from 0.1 to 2.0 mM) for 18 h. In a second
experiment, the effect of L-Trp was evaluated in the presence of L-Ala in the incubation media, in order to study
the role of the neutral L-aminoacid transport in melatonin production. In a third experiment, the effect of drugs
that interact with 5-HT catabolism (pargyline, MAO inhibitor) and release (fenfluramine) was evaluated by
incubating the GIT tissues for 3 hours with these drugs. The results showed that L-Trp induces a concentration-
dependent increase in melatonin synthesis which is mediated by increased 5-HT content in GIT cells. The effect
of L-Trp on melatonin synthesis was abolished by the presence of L-Ala in the incubation medium. By other
hand, the MAO inhibitor pargyline was followed by an increased melatonin synthesis associated with higher 5-
HT content and lower 5-HIAA content. Fenfluramine, a 5-HT releaser/uptake inhibitor, was without effect on
GIT melatonin production. These data suggest that melatonin synthesis in fish is affected by processes (5-HT
synthesis, degradation) involved in the regulation of free cytoplasmatic 5-HT available in the GIT cells. The
results also suggest that the presence in the diet of L-Trp, and its relation with other neutral L-aminoacids, are
important to modulate melatonin synthesis in the GIT of fish.




                                                       13
                         7AIEC POSTER SESSION ABSTRACTS




P18
CORTISOL INCREASED PLASMA MELATONIN LEVELS IN GILTHEAD SEA BREAM (Sparus
auratus)

Juan Miguel Mancera 1, L. Vargas-Chacoff 1, J.A. Martos 1, A. H. Kalamarz 2, G. Martínez-Rodríguez 3, E.
Kulczykowska 2. E-mail: juanmiguel.mancera@uca.es
(1) Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, E-11510
Puerto Real (Cádiz), Spain.
(2) Department of Genetics and Marine Biotechnology, Institute of Oceanology of Polish Academy of Sciences,
Sopot, Poland.
(3) Instituto Ciencias Marinas de Andalucía y RIAC, Consejo Superior Investigaciones Científicas, E-11510
Puerto Real (Cádiz), Spain.

The main pineal indole melatonin (N-acetyl-5-methoxytryptamine; MEL) is a pleiotropic hormone which can
influence several physiological processes in vertebrates, including fish. However, the evidence for a potential
role of MEL in stress response is very scarce and sometimes results are contradictory and depend on the species
and kind of stress. The aim of the present study was to determine the response of MEL in S. auratus to
exogenous cortisol. Gilthead seabream Sparus auratus were injected intraperitoneally with slow-release
implants of coconut oil alone or with cortisol (50 µg.g-1 body weight), and sampled after 3, 7 and 14 days. No
differences in plasma cortisol levels between untreated fish (time 0 days) and implanted fish with coconut oil
alone (control) were observed. Cortisol implant induced a chronic increase in plasma cortisol during two
experimental weeks. Moreover, the cortisol levels were similar to those reported for the same species during the
first three days of confinement under high density. Therefore, cortisol treatment used in this study may simulate
the chronic stress, with persistent high cortisol levels. Plasma MEL levels in cortisol-treated fish were also
significantly increased. These results are in agreement with plasma cortisol and MEL levels reported earlier in S.
auratus while kept under high density for two weeks. We suggest that MEL may play a role during chronic
stress in this species.




P19
CHARACTERISATION OF GROWTH AND DIFFERENTIATION FACTOR- 9 IN Oreochromis
mossambicus OVARY AND GENE EXPRESSION DURING FOLLICLE GROWTH

Laurence Deloffre 1, J. Lesage 2, D.M. Power 1, A.V.M Canario. 1 Email: deloffre@ualg.pt
(1) Comparative and Molecular Endocrinology Group, Centre of Marine Sciences, Universidade do Algarve,
8005-139 Faro, Portugal.
(2) Neurosciences et Neurophysiologie Adaptatives, Equipe Stress Périnatal, Université de Lille I, Villeneuve
d'Ascq, France.

The production of high quality eggs is essential to ensure successful fertilization and reproduction [Duranthon &
Renard. Theriogenology 2001, 55:1277]. It has become apparent recently that oocytes regulate folliculogenesis
by regulating their microenvironment by secretion of soluble growth factors, called oocyte-secreted factors
(OSFs). In mammals, several essential OSFs of the transforming growth factors beta (TGF-β) superfamily have
been identified, among them growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-
15, also known as GDF9-b) [Knight & Glister. Reproduction 2006; 132: 191]. In contrast, less is known about
the BMP system in the ovary of lower vertebrates such as fish. BMP has been reported in zebrafish during the
early development but only BMP-15 has been associated with ovarian maturation [Clelland et al. Endocrinology
2007; 148: 5451].
To study the role of BMPs in ovarian maturation Oreochromis mossambicus GDF-9 was isolated and its
spatiotemporal expression analysed during oogenesis. A single transcript of 2.1 kb was detected by Northern
blot in ovary. Semi-quantitative RT-PCR confirmed the ovarian specific expression of GDF-9. To investigate its
expression during the ovarian cycle, ovarian follicles were dissected and separated according to their
size/maturation stage. The highest level of GDF-9 expression was present in oogonia and primary oocytes, and


                                                       14
                         7AIEC POSTER SESSION ABSTRACTS


decreased significantly in the vitellogenic stages. In situ hybridization confirmed the data obtained by semi-
quantitative RT-PCR suggesting that GDF-9 is probably involved in teleost ovarian maturation.
Acknowledgements: This research has been carried out with the financial support of European Social Fund and
the Portuguese National Science Foundation (FCT; project POCTI/CVT/47124/2002). LD was supported by a
Post-doc FCT fellowship (SFRH/BPD /31339 /2006).




P20
TEMPERATURE MODULATES PLASMA CORTISOL, MSH AND β-ENDORPHIN RESPONSE TO
HIGH DENSITY IN GILTHEAD SEABREAM Sparus auratus

Luis Vargas-Chacoff1,3, J. Metz2, F. J. Arjona1, P.H. M. Klaren2, G. Flik2 and J.M. Mancera1
E-mail: luis.vargas@uca.es
(1) Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510
Puerto Real, Cádiz, Spain. (2) Department of Animal Physiology, Institute for Water and Wetland Research,
Faculty of Science, Radboud University Nijmegen, The Netherlands
(3) Centro Andaluz de Ciencia y Tecnologías Marinas, Instituto de Investigación de la Universidad de Cádiz,
11510 Puerto Real, Cádiz, Spain.

Temperature is considered as the master abiotic factor that controls several physiological responses in teleostean
fishes. High stocking density effectively activates the stress system and is a common stressor in aquaculture
activity. In south-western Spain, gilthead sea bream is cultured mainly in earthen ponds with scarce deep water
and natural seasonal changes dominate. Water temperature range from 24º C in summer to 12º C in winter. The
aim of this study was to evaluate plasma cortisol, MSH and β -Endorphin levels in specimens acclimated to
three different temperatures (12º, 19º and 26º C) and submitted to two different stocking densities, viz. normal
density 2 kg/m3; and high density, HD, 60 kg/m3. Plasma cortisol levels are directly and linearly related to
temperature (lowest level at 12º C and highest at 26º C) and HD increased cortisol levels twofold at 12º and 19º
C, but not at 26º C. Plasma MSH values showed high values at 19º C compared to other environmental
temperature, while HD significantly decreased these values at this temperature. Plasma β-endorphin levels
decreased in specimens maintained at the lowest temperature and HD had no effect on these values. Our study
indicates that temperature modulates plasma cortisol and β -endorphin responses to HD in gilthead seabream.




P21
IDENTIFICATION OF A NOVEL HUMAN GHRELIN GENE TRANSCRIPT THAT CONTAINS
INTRON 2 AND IS UP-REGULATED, TOGETHER WITH GHRELIN O-ACYL TRANSFERASE
(GOAT) ENZYME BUT NOT WITH NATIVE-GHRELIN, IN BREAST CANCER TUMOURS.

Manuel David Gahete-Ortiz1, J. Cordoba-Chacón1, M. Hergueta2, F. Gracia-Navarro1, RD Kineman3, G.
Moreno-Bueno2, Raul M Luque1, Justo P Castaño1. E-mail: bc2gaorm@uco.es
(1) Dpt. of Cell Biology, Physiology & Immunology. Univ. of Córdoba, IMIBIC, and CIBERobn 06/03,
Córdoba, Spain.
(2) Institute of Biomedical Research Alberto Sols, Madrid, Spain.
(3) Dpt. of Medicine, Section of Endocrinology, Diabetes and Metabolism. Univ. of Illinois at Chicago;
Research & Development Division, Jesse Brown Veterans Affairs Medical Center, Chicago, IL, USA.

Ghrelin is a multifunctional hormone predominantly produced by stomach, which is also widely expressed in
several tissues, where it acts as a paracrine or autocrine factor. The fine molecular mechanisms of ghrelin
biosynthesis show that this peptide is but one piece of a puzzle comprising distinct additional peptides,
generated by alternative splicing of the ghrelin gene or from post-translational modifications (e.g. obestatin, des-
Gln14-ghrelin, exon3-deleted-ghrelin…). Our laboratory has recently identified a spliced mRNA ghrelin variant
in the mouse containing exon(Ex)2, intron(In)2 and Ex3, but lacking Ex1, 4 and 5 (In2-ghrelin variant), which
may be of biological relevance because its expression is tissue-dependent and is regulated in extreme metabolic
states (fasting and obesity). The aim of this work was to investigate the existence of a homologous In2-ghrelin
variant in humans. Using molecular and cellular techniques, we identified the existence of an alternative In2-

                                                        15
                         7AIEC POSTER SESSION ABSTRACTS


ghrelin variant transcript in humans containing Ex0, Ex1, In2, and Ex2 but lacking Ex3 and 4. This novel In2-
ghrelin variant was thoroughly expressed in human tissues, including stomach and pituitary. Translation of this
splice variant would originate a new prepropeptide of 117 amino acids that conserves only the signal peptide
and the first 12 amino acids of native ghrelin, but retains the acylation site on Ser3. In fact, expression of In2-
ghrelin variant in the 22 human tissues analyzed parallels the expression of the enzyme responsible for ghrelin
acylation (GOAT; R2=0,921) but not that of native-ghrelin (R2=-0,025) suggesting that In2-ghrelin variant could
be a primary substrate for GOAT in human tissues. Interestingly, In2-ghrelin variant was strongly up-regulated
in a series of breast cancer samples as compared with normal mammary gland (P<0.01). Moreover, In2-ghrelin
variant expression in breast cancer was highly correlated with GOAT (R2=0,655) and ghrelin receptor type 1b
(GHSR-1b) but not with native ghrelin or GHSR1a. Taken together, our results suggest that human In2-ghrelin
mRNA is a novel variant of the ghrelin gene that could give rise to novel peptide(s) within this family.
Furthermore, its overexpression in breast tumor samples coupled to that of GOAT and GHSR1b suggests that
the novel peptide(s) may play a relevant role in breast cancer.
Support: RYC-2007-00186, BIO-0139, CTS-01705, BFU2007-60180/BFI, BFU2008-01136/BFI, NIDDK
30677, VA Merit Award



P22
CHANGES ON THE REPRODUCTIVE STAGE OF IMPUBER MALES EUROPEAN SEA BASS
(Dicentrarchus labrax) AFTER ADMINISTRATION OF RECOMBINANT SINGLE-CHAIN FSH.

María José Mazón, A.Gómez, S. Zanuy, E-mail: zanuy@iats.csic.es
Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre la Sal (IATS), CSIC,
Torre la Sal s/n, Ribera de Cabanes, 12595Castellón, Spain

Follicle-stimulating hormone is the main gonadotropin stimulating the early stages of gametogenesis. FSH key
role is supposed to be the activation of Sertoli cells proliferation as well as the proliferation of germ cells
associated to them. It has been suggested that FSH acts on spermatogenesis alone or by its effects on the
production and secretion of androgens. Due to the different results obtained in diverse species, its definitive
function has not been yet established. In this study we evaluate the effect of a recombinant single-chain FSH on
gonad recrudescence of impuber sea bass. Fish were injected at day 0 and 3 with 0.5µg of scFSH produced by
CHO cell line; animals injected with culture medium were used as control. FSH effects were analyzed by
monitoring changes of plasma 11-ketotestosterone (11-kt) and gonadal morphology. Changes in gene
expression of LH receptor, FSH receptor and Anti-Müllerian Hormone were also evaluated by real-time PCR.
Three days after the first injection an increase of plasma 11-kt levels was detected being higher than in control
groups until day 15. Changes of gonad structure were observed at day 7, where a strong increase of
proliferation was detected. As a result, an increment in the number of cyst of spermatocytes and spermatids was
observed, as compared to control groups, which remained immature, showing only spermatogonia type A and
B. The increase of plasma FSH provoked suppression on Anti-Müllerian hormone encoding transcript and an
increase in LHR mRNA. This findings support the probable role of FSH as the initial signal of the
spermatogenesis process enhancing cell proliferation via Sertoli cells activation and as androgen inductor via
Leydig cells.




P23
MODULATION OF STEROIDOGENIC AND IMMUNE FUNCTIONS IN HEAD KIDNEY CELLS OF
GILTHEAD SEABREAM STIMULATED WITH A CHEMICAL STRESSOR

Mariana Teles1,2, Juan Castillo1 and Lluís Tort1. E-mail: mteles@ua.pt
(1)Department de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, 08193
Bellaterra, Barcelona, Spain.
(2)CESAM & Biology Department, Aveiro University, 3810-193 Aveiro, Portugal.

Changes in the transcription of relevant genes in fish can be used as indicators of the sublethal impact of
chemical stressors present in the aquatic environment (e.g. heavy metals). In the present study, head kidney cells
of Sparus aurata were stimulated with a heavy metal, namely copper, to evaluate steroidogenic and immune

                                                       16
                         7AIEC POSTER SESSION ABSTRACTS


functions by measuring changes in gene expression of steroidogenic acute regulatory protein (StAR) and in
cytokines, respectively. Cortisol levels were measured in the incubation medium. As a preliminary step, a cell
viability experiment was performed with head kidney cells and LC50-1h for copper was estimated at 3.55 mM.
After incubating the head kidney cells for 1 h with 1 or 100 µM of copper, the previous parameters were
assessed. Exposure to the highest copper concentration caused an up-regulation on StAR gene expression,
whereas the cortisol levels in the incubation medium remained unaltered. Copper also caused significant
changes in immune related genes, as the expression of interleukin-1β (IL-1β) and transforming growth factor-β1
(TGF-β1) was increased at 100 µM and 1 µM of copper, respectively. However, the expression of interleukin-6
(IL-6) significantly decreased for both copper concentrations. The present results demonstrate that copper has
the ability to induce StAR gene up-regulation in gilthead seabream head kidney cells. However, this effect was
not accompanied by a cortisol increase in the culture medium, indicating that the linkage mechanism between
StAR and cortisol secretion is complex and it requires factors that were absent in the present cell culture. Copper
induced changes in the expression of cytokines and revealed a linkage between metal exposure and an altered
immune function, giving new insights onto the mechanisms involved. The balance between pro-inflammatory or
anti-inflammatory cytokines seems to depend on copper concentration, being determined by the antiparallel
actions of IL-1β and TGF-1β. The immunosuppressive potential of copper can also be extended to the lower
concentration (1 µM). Concerning the interactions between steroidogenic and the immune functions, the results
suggest that IL-1β can be implicated on StAR induction. Globally, the results indicate that changes in the
transcription of selected genes can be useful indicators for the detection of toxic effects at sublethal level, and
that ecotoxicogenomics provides innovative approaches to investigate the mechanisms of action of toxicants.
However, the consequences of the changes that occur at the gene transcription level and their linkage to
proteomics and organismic effects are difficult to establish. The connection between molecular responses and
ecologically relevant parameters remains a challenging task and it implies future in vivo studies that will
validate present in vitro works.




P24
HIGH-STOCKING DENSITY STRESS INTERACTS WITH PERIPHERAL GLUCOSENSOR
SYSTEM AND FOOD INTAKE IN RAINBOW TROUT Oncorhynchus mykiss

Marta Conde Sieira, A. Aguilar, J.L.P. Muñoz, J.M. Míguez, J.L. Soengas. E-mail: mconde@uvigo.es
Laboratorio de Fisioloxía Animal. Depto. Bioloxía Funcional e Ciencias da Saúde. Facultade de Bioloxía.
Universidade de Vigo.

We have recently demonstrated the existence of central and peripheral glucosensing mechanisms in rainbow
trout. A common stress response in teleost fish is the food intake reduction. We aimed to evaluate the possible
interaction of stress on the glucosensor capacity and its role in the food intake regulation in rainbow trout. A
non-stressed group of fishes was submitted to three different glycemic conditions such as control,
hyperglycaemic or hypoglycaemic conditions for 5 days by intraperitoneal injections (10 µl·g-1 body mass) of
coconut oil alone (control), or containing D-glucose (500 mg kg -1 body mass) or bovine insulin (4 mg kg -1
body mass), respectively. Similar treatments were carried out in additional three groups but subjected to chronic
stress by high stocking density (70 kg m-3). The experimental design was appropriate since hypoglycaemia and
hyperglycaemia were obtained in unstressed fish. Food intake displayed lower levels in stressed fishes than in
controls. The activity of several enzymes (HK-GK, PK, GSase, GPase, FBPase) and metabolites (glucose,
glycogen, DHAP, GAP) involved in the glucosensing pathway were assessed in two brain regions
(hypothalamus and hindbrain), as well as in liver and Brockmann bodies (BB). Plasma cortisol levels in stressed
fish increased only under normoglycemic and hypoglycaemic but not hyperglycemic conditions. In central
glucosensing areas few interactions of stress with glucosensing mechanisms were noticed. In contrast, several
parameters related to glucosensing in liver (glycogen and DHAP levels, and GSase and GPase activities) and
BB (glucose, glycogen, DHAP, and GAP levels, and PK activity) displayed in stressed fish changes compared
with those of unstressed fish. In general, the glucosensing capacity of those peripheral tissues was altered in
stressed fish. Therefore, we may suggest that the reduced food intake of stressed fish could be related, at least in
part, with the alteration of the peripheral glucosensing mechanism involved in the control of food intake.
Funded by Ministerio de Educación y Ciencia (AGL2007-65744-C03-01/ACU) and Universidade de Vigo




                                                        17
                         7AIEC POSTER SESSION ABSTRACTS




P25
DIETARY CARBOHYDRATE LEVEL AND EXERCISE EFFECT ON EXPRESSION OF
MOLECULES RELATED WITH MYOGENESIS IN SEA BREAM (Sparus aurata) WHITE MUSCLE

Mònica Rius, Daniel García de la serrana, Encarnación Capilla, Isabel Navarro, Joaquim Gutiérrez.
e-mail: mriusfra7@alumnes.ub.edu
Departament de Fisiologia, Facultat de Biologia. Universitat de Barcelona, Spain.

The skeletal muscle development and growth are regulated by several molecules as the Myogenic Regulatory
Factors (MyoD, Myf5, myogenin and MRF4), Pax7, Sox8 and IGFs. Signal pathways, as PI3K/Akt and mTOR
system, could be also affected. Their function is not fully understood and their role in different fish conditions
remains unclear. The knowledge about the molecules and environmental conditions that can affect and control
muscle growth is essential. The present study was focused in how dietary carbohydrate levels and moderate
exercise could affect in fish the expression of these important factors that control muscle development. For
carbohydrate assay, triplicate groups of fish were assigned to each of three groups of isoenergetic and
isonitrogenous combination of experimental diets with different carbohydrate amount (0%, 15%, 25%). This
trial significantly decreased the white muscle expression of Myf5; myogenin expression slightly decreased and
MRF4 mRNA expression also show a tendency to reduce. Therefore, although not changes were found on
weight, it seems that dietary treatment affects the activation phase of MPCs. The moderate exercise trial forced
muscle to work but was not sufficient to affect significantly the expression of molecules related with
myogenesis in white muscle. However insulin-like growth factor I (IGF-I) tends to reduce it expression. Further
studies in red muscle will provide interesting information on the response to moderate exercise. The response of
MPCs suggests that dietary carbohydrate may affect the fillet quality and it should be further investigated. On
the other hand, the exercise should be tested for longer and more intensively treatments in order to observe
differences.
Supported by: AGL-MEC; LIFECYCLE EU FP7 22719; XRAq.




P26
HYDROSTATIC PRESSURE INCREASES HEPATIC ARYLALKYLAMINE N-
ACETYLTRANSFERASE (AANAT) TRANSCRIPT EXPRESSION IN RAINBOW TROUT (O. mykiss)

Odete Marinho-Gonçalves1; A.F. Gonçalves1,2; J. Ings3; A. Damasceno-Oliveira1; N. Aluru3; M.M. Vijayan3; J.
Coimbra1,2; J.M. Wilson1. E-mail: odete007@gmail.com
(1) Laboratório de Ecofisiologia, Centro Interdisciplinar Investigação Marinha e Ambiental, Porto, Portugal.
(2) Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal.
(3) Department of Biology, University of Waterloo, Ontario, Canadá

Hydrostatic pressure (HP) is an important abiotic factor in the aquatic environment, increasing 10 Pa per meter
depth. It is recognized to affect physiological processes at the organismal and cellular levels. In a preliminary
cDNA microarray study, rainbow trout (Oncorhynchus mykiss), a shallow water pressure-sensitive teleost fish,
subjected to increased HP showed increased hepatic expression of Arylalkylamine N-Acetyltransferase
(AANAT). AANAT is the key enzyme involved in synthesis of melatonin, a neurohormone responsible for the
regulation of the circadian clock and seasonal rhythms in vertebrates. Furthermore, melatonin is known to have
antioxidant properties important in the natural defense against free radicals. Unlike mammals, birds and anurans
that have a single representative of this gene family, the teleosts have two or three members, synthesizing
melatonin in retina (AANAT-1 gene) and pineal gland (AANAT-2). Some studies performed in rainbow trout,
have shown AANAT-2 gene expression in other tissues, including gastrointestinal related tissues, namely the
liver. In the present study with the objective of investigating the responsiveness of AANAT-2 gene expression
to HP, a group of trout was submitted to a HP of 5MPa (equivalent to 500 m depth) using a flow through
hyperbaric chamber for 24 hours and compared with a control group maintained at 0,01MPa (1 m depth). The
gene expression was studied in liver by PCR. The study demonstrated a statistically significant increase of the
hepatic AANAT-2 expression in the group subjected to high pressure. These results confirm previous reports of
extrapineal gene expression and our microarray results. Furthermore, the up-regulation of AANAT-2 expression

                                                       18
                         7AIEC POSTER SESSION ABSTRACTS


suggests a potential role of melatonin production (increase). Considering the role of AANAT in melatonin
synthesis, we put forward that the observed increase is part of an adaptation mechanism of the physiological
response to HP induced oxidative stress.
This work was supported by FCT grant PTDC/MAR/64016.




P27
EL SISTEMA ENDOCANNABINOIDE EN ÁREAS CIRCUNVENTRICULARES DE LA RATA

Pedro Fernández-Llebrez1, J. Suárez2, F.J. Bermúdez-Silva2, J. Pérez1, F. Rodríguez de Fonseca2. E-mail:
llebrez@uma.es
(1) Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga.
(2) Laboratorio de Medicina Regenerativa, Fundación IMABIS, Málaga.

Hemos investigado la distribución inmunocitoquímica de los receptors de endocannabinoides (ECs) CB1R y
CB2R, las enzimas de síntesis NAPE-PLD, DAGLa, y DAGLb, y las enzimas de degradación FAAH y MAGL
en el epéndimo, subepéndimo, órganos circunventriculares y glía marginal del cerebro de la rata adulta. El
receptor CB1R se localiza en fibras nerviosas que alcanzan la zona subventricular del estriado conocida como la
principal zona de neurogénesis de adultos. También están presentes en algunos órganos circunventriculares
como el órgano subcomisural, el subfornical y el area postrema y en la glía marginal y algunas zonas
subventriculares. Se observó una inmunoreactividad muy patente en las neuronas del locus ceruleus. Algunas
células pequeñas subependimarias con aspecto de microglía son reactivas a anti-CB2R. El epéndimo ciliado
expresa DAGLb y los astrocitos subepéndimarios y de la glía marginal, DAGLa. Los astrocitos de otras
regiones no expresan enzimas de síntesis de ECs lo cual establece una clara distinción entre estas dos
poblaciones de astrocitos. Las células ependimarias expresan también NAPE-PLD, MAGL y FAAH. Los
tanicitos del tercer ventrículo se comportan como los astrocitos subependimarios. Los plexos coroideos
muestran una inmunoreactividad muy fuerte a FAAH. El órgano subcomisural expresa muy fuertemente
DAGLa y DAGLb que también están presentes en la fibra de Reissner. El órgano subfornical y el area postrema
también sintetizan enzimas de síntesis y degradación de ECs. Todos estos resultados indican que los ECs deben
estar implicados en el control de la composición del líquido cefalorraquídeo (LCR), la función de los órganos
circunventriculares y el control de las barreras que separan el LCR ventricular, meníngeo, parenquimal y la
sangre.
GRANTS: MICINN, BFU 2006/11754, SAF 2004/07762; Junta de Andalucía PI-0220, P07-CVI-03079, SAS-
S0742, REDES RTA RD06/001; ISCiii: 07/1226, 07/0880; Plan Nacional Sobre Drogas (2006/142); 5th
Framework Programme TARGALC QLRT-2001-01048.




P28
PHOTOPERIODIC CONTROL OF THE EARLY PUBERTY IN MALE SEA BASS (Dicentrarchus
labrax)
Rafael Rodríguez, E. Hala, A. Felip, S. Zanuy, M. Carrillo. E-mail: carrillo@iats.csic.es
Instituto de Acuicultura de Torre de la Sal, Department of Fish Physiology and Biotechnology (IATS), CSIC,
Torre de la Sal s/n, Ribera de Cabanes, 12595, Castellón, Spain.

The anticipated puberty is a process in which the fish undergo precocious maturation which reduces its further
growth respect to non-precocious fish and affects commercialization. This has been observed in several cultured
species and is one of the main aquaculture research interests of the EU. Artificial light regimes treatments such
as continuous light (LL), applied during the entire first year of life, or shorter LL treatments, applied before or
after the supposed time of the onset of gametogenesis, have been highly effective to reduce male precocity.
However, a precise identification of a short photosensitive period and the understanding of the hormonal basis
controlling early puberty is still lacking in sea bass. The aim of the present study was to screen for a short
photosensitive period, placed between August-September, by LL exposures of one-month duration.
Accordingly, 6 photoperiod treatments were analyzed in duplicate: W1 (simulated natural photoperiod), W2 (LL
from August 1st to August 30th), W3 (LL from August 15th to September 16th), W4 (LL from September 1st to
September 30th), W5 (LL from August 1st to September 30th) and W6 (LL). In this vein, the effect of these

                                                       19
                         7AIEC POSTER SESSION ABSTRACTS


treatments on the maturation status was investigated including an evaluation of somatic growth, testicular
development, plasma levels of testosterone (T), 11-ketotestosterone (11-KT) and 17beta-estradiol (E2) and
content of sea bream gonadotropin-releasing hormone (sbGnRH) in the brain. Our results showed that growth in
all treatments was independent of photoperiod. On the other hand, all treatments were effective in reducing
significantly the rate of precocious male respect to control which were encompassed by the corresponding
altered hormonal profiles. A more detailed analysis indicated that the hormonal profiles of W3 group were
similar to W6 (LL all year), as well as the evolution of gonadal development. It is suggested that the period from
half August to half September could be the most sensitive to LL. Besides, these studies confirmed the
predominant role of 11-KT in the spermatogenesis and spermiogenesis of sea bass and the possible role of E2 in
the spermatogonial proliferation in this species.




P29
KISSPEPTIN SIGNALS THROUGH MITOGEN-ACTIVATED PROTEIN KINASE, PROTEIN
KINASE C AND INTRACELLULAR CALCIUM STORES TO DIRECTLY INCREASE GH RELEASE
IN PRIMARY PITUITARY CELL CULTURES FROM A NON-HUMAN PRIMATE (Papio anubis)

Raúl M. Luque1, J Cordoba-Chacon1, E Gutierrez-Pascual1, MD Gahete1, A. Pozo1, MM Malagón1, AJ
Martinez-Fuentes1, M Tena-Sempere1, RD Kineman2, Justo P. Castaño1. E-mail: raul.luque@uco.es
(1) Dpt. of Cell Biology, Physiology & Immunology. Univ. of Córdoba, IMIBIC, and CIBERobn 06/03,
Córdoba, Spain.
(2) Dpt. of Medicine, Section of Endocrinology, Diabetes and Metabolism. Univ. of Illinois at Chicago;
Research & Development Division, Jesse Brown Veterans Affairs Medical Center, Chicago, IL, USA.

Kisspeptins (kp), a family of peptides encoded by the Kiss1 gene, and their receptor Kiss1r were first identified
by their anti-metastatic actions, but have emerged as a key regulatory system for the reproductive axis, where it
mainly acts by controlling hypothalamic GnRH release. Recently, Kp has been proposed to exert additional
regulatory functions at the pituitary (PIT), since Kiss1r is expressed in the gland and Kp stimulate luteinizing
hormone (LH) secretion directly at the PIT level in several species (mouse, rat, pig, cow, goldfish). Moreover,
Kp has unexpectedly been found to directly stimulate growth hormone (GH) release from rat, cow, and goldfish
somatotropes. In contrast, the potential role of Kiss1/Kiss1r in regulating PIT cells from normal adult primates,
a model very close to humans at the physiologic and genomic levels, has not yet been addressed. Here, primary
PIT cell cultures from adult female baboons (Papio anubis), which expressed Kiss1r, were treated with
kisspeptin-10 (kp10) alone or in combination with GH-releasing hormone (GHRH), ghrelin and somatostatin
(SST) and GH release was assessed. In this model, kp10 increased GH release in a dose- and time-dependent
fashion and this action was fully blocked by SST (4h). Direct actions of Kp10 on primate somatotropes were not
restricted to stimulation of GH release but also included augmentation of GH expression after long-term (24h)
exposure. Combined treatment of kp10 with GHRH, but not with ghrelin, resulted in an additive effect on GH
release suggesting that each ligand activates distinct intracellular signaling pathways, as confirmed by use of
specific inhibitors. Of note, blocking MAPK and PKC activity as well as iCa2+ stores, but not adenylyl cyclase
activity or extracellular Ca2+ influx, completely abolished kp10-induced GH release. Further, since sex steroids
are key modulators for somatotrope function, we tested the influence of estradiol (E2) on the responsiveness of
this cell type to kp10. Although preincubation with E2 (36h) decreased baseline GH release by ~50%, it served
to enhance the relative GH-releasing effect of kp10 alone and in combination with GHRH, as compared to E2-
free control cultures. Taken together, our results provide the first evidence that kisspeptins may play a relevant
role in stimulating GH from primate somatotropes through a direct effect mediated by MAPK-, PKC- and iCa2+-
signaling, and that sex steroids may sensitize somatotrope responsiveness to the direct action of Kp.
Support: RYC-2007-00186, BIO-0139, CTS-01705, BFU2007-60180/BFI, BFU2008-01136/BFI, NIDDK
30677, VA Merit Award




P30
MOLECULAR CLONING AND EXPRESSION OF HYDROXYINDOLE-O-METHYLTRANFERASE
IN SENEGALESE SOLE (Solea senegalensis)


                                                       20
                        7AIEC POSTER SESSION ABSTRACTS


Rosa María Martínez-Álvarez1, F. Confente1, E. Isorna2, A.J. Martín-Robles1, J.A. Muñoz-Cueto1.
E-mail: rosa.martinez@uca.es

(1) Dpto. Biología, Facultad de Ciencias del Mar y Ambientales, Univ. Cádiz. España.
(2) Dpto. Fisiología (Fisiología Animal II), Facultad de Biología, Univ. Complutense de Madrid. España.

Melatonin is a neurohormone that synchronizes many physiological and behavioural processes to the daily and
seasonal variations of photoperiod. In vertebrates, melatonin is synthesized and released rhythmically during the
dark phase by the pineal gland and retina. The pineal gland is the main source of plasma melatonin, whereas the
retinal melatonin seems to function as an autocrine/paracrine hormone. Melatonin biosynthesis from serotonin
requires the sequential activity of arylalkylamine N-acetyltransferase (AANAT), which converts serotonin to N-
acetylserotonin, and hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4), which transforms N-
acetylserotonin to melatonin. In this study we have elucidated the molecular structure of HIOMT in the
Senegalese sole, a flatfish species of growing interest in European aquaculture. We have cloned a full-length
sole HIOMT sequence by using reverse transcripction (RT) and rapid amplification of cDNA ends (RACE). We
have also examined its tissue distribution by RT-PCR. A 1687 bp cDNA sequence was cloned, which consist of
a 5’-untraslated region (302 bp), an open reading frame (1005 bp) encoding a putative sole HIOMT of 334 aa,
and a 3’ unstraslated region (380 bp), including a polyadenylation signal (AATAAA). The deduced protein
sequence contains a methyltransferase domain (residues 63-306) and eight highly conserved cysteine residues.
Sole HIOMT shares 68-90% identity with proteins from other fish species, and 46-61% with other HIOMT
vertebrate sequences. HIOMT mRNA expression was detected in retina and some peripheral tissues including
gill, heart, intestine, kidney and muscle. This peripheral distribution agrees with HIOMT expression reported in
mammals and AANAT expression in fish, supporting the notion that extrapineal and extraretinal melatonin
synthesis exits in fish, as reported in mammals. Within the brain, HIOMT expression was found in the
cerebellum, where melatonin receptors have also been detected recently in sole. Cloning of sole HIOMT will
provide a new tool to characterize the circadian system of sole and will contribute to improve our understanding
of the circadian rhythms in this species.
Supported by Grants from MEC (AGL2007-66507-C02-01), Junta de Andalucía (P06-AGR-01939), AECI
(A/7371/06 and A/011798/07) and CNRS/IFREMER (GDR2821).




P31
TROUT AND CHICKEN GLUCOKINASE IS DIFFERENTIALLY REGULATED BY A HEPATIC
PROTEIN

Sergio Polakof A.Aguilar, J.M. Míguez, J.L. Soengas. E-mail: spolakof@uvigo.es
Lab. Fisioloxía Animal, Depto. Bioloxía Funcional, Fac. Bioloxía, Universidade de Vigo.

Glucokinase (GCK) is a key enzyme involved in hepatic glucose metabolism and glucose homeostasis
regulation. In mammals, GCK is regulated in vivo by a regulatory protein (GCKR) through a nucleus-to-
cytoplasm translocation enhanced by fructose 1-phosphate and counteracted by fructose 6-phosphate.
There were no previous evidences in literature regarding the presence of GCKR in livers of other
vertebrates like fish and bird. Accordingly, in the present study we assessed GKCR presence in chicken,
and trout hepatic homogenates. The results obtained demonstrate for the first time the presence of a
GCKR-like protein in the liver of those species, with molecular weight (68kDa) and biochemical
properties similar to those described in mammals. Several of the biochemical properties of rainbow trout
GCKR were closer to the mammalian model whereas those of chicken protein were specific.
Furthermore, the response of GCKR-like in liver of rainbow trout fed with a diet rich in carbohydrates
was compared with the rat model under extreme glycemic conditions. We found that despite trout
GCKR-like was less active and expressed than in rat, the response against glycemic changes took place in
the same direction, and the ratio GCKR-like:GCK was affected in a similar way. As a whole, the results
obtained in the present study provide numerous and original data regarding the non-transcriptional
regulation of GCK in non-mammalian species of vertebrates. We provided, for the first time, evidence for
the presence of a GCK regulatory-like protein in liver extracts from chicken and rainbow trout with
biochemical characteristics similar to those reported for mammalian GCKR. The CGKR-like displayed a
differential capacity to regulate a third purified GCK, demonstrating that two glucose intolerant species
like chicken and rainbow trout possess opposite GCKR-like inhibitory properties (competitive in trout,


                                                       21
                        7AIEC POSTER SESSION ABSTRACTS


non-competitive in chicken), in accordance with the clear differences in GCK kinetics previously
demonstrated.
Research grants from Ministerio de Educación y Ciencia and European Fund for Regional Development
(AGL2007-65744-C03-01/ACU) and Universidade de Vigo.




P32
MOLECULAR CLONING OF A cDNA FRAGMENT ENCODING A PUTATIVE NEUROPEPTIDE Y
FROM THE HYPOTHALAMUS OF RAINBOW TROUT Oncorhynchus mykiss

Vanesa Lozano1, M. Mancebo2, B. Fondevila2, J.J. Pérez2, M. Aldegunde2.
E-mail: vanessa.lozano@usc.es.
(1) Departamento de Bioquímica y Biología Molecular, Instituto de Acuicultura, Universidad de Santiago de
Compostela.
(2) Laboratorio de Fisiología Animal, Departamento de Fisiología, Facultad de Biología, Universidad de
Santiago de Compostela.

In vertebrates, food intake and energy homeostasis are regulated by a complex system that involves both central
and peripheral signals. The hypothalamus plays a crucial role in the regulation via a range of neuropeptides
stimulating or inhibiting food intake. Neuropeptide Y (NPY) is one of the neuropeptides released within the
hypothalamus and it is considered the most potent orexigenic signal known to date in mammals. In fish the
presence of NPY in the nervous system suggests primarily neuronal roles including stimulation of food intake
and seems to be a regulator of feeding and fasting. In this study, we have cloned a cDNA fragment encoding a
putative NPY from Oncorhynchus mykiss. Total RNA was extracted from the hypothalamus of inmature
rainbow trout. A partial cDNA sequence was obtained by reverse transcription-PCR (RT-PCR) and cloned. The
O. mykiss prepro-NPY partial sequence was 208 bp in lenght, and contained a 114 bp 3’-untranslated region
(3’UTR) and a 94 bp open reading frame (ORF) encoding the carboxyl-terminal extension of the NPY (CPON)
with 31 amino acids. A homology search in the UNIPROT database (WU-BLAST2) showed 96 % identity with
Salmo salar, Oncorhynchus mykiss and Salvelinus namaycush NPY; 90 % identity with Paralichthys olivaceus;
87 % identity with Dicentrarchus labrax and Gadus morhua NPY; and 80% identity with Oryzias latipes NPY.
These results will be used to develop a quantitative RT-PCR and provide basis for further investigation into the
regulation of NPY expression in rainbow trout.




P33
DEPENDENCIA CIRCADIANA DE LAS ACCIONES ANORÉTICAS DE LA LEPTINA EN EL
CARPÍN (Carassius auratus).

Yurena Vivas, C. Azpeleta, A. Feliciano, E. Velarde, A. Macias, E. Isorna, M.J. Delgado, N. De Pedro.
E-mail: ndepedro@bio.ucm.es
Departamento de Fisiología (Fisiología Animal II), Facultad de Biología, Universidad Complutense de Madrid.

La leptina, hormona proteica implicada en la regulación de la homeostasis energética, se caracteriza por
presentar un efecto reductor de la ingesta y del peso corporal en la mayoría de vertebrados estudiados, incluido
el teleósteo Carassius auratus. Dichos efectos han sido descritos utilizando protocolos de diseño clásico en
estudios de regulación de la ingesta, en los que la administración de los factores reguladores de la ingesta se
realiza a la hora habitual de la alimentación. El objetivo del presente estudio es determinar si los efectos
anoréticos de la leptina previamente descritos en el carpín son dependientes de la hora del fotociclo diario a la
que se administra la hormona y/o del horario de alimentación. Se realizaron inyecciones intraperitoneales (IP)
de leptina humana (1 µg/g peso corporal) en peces sometidos a diferentes horarios de alimentación en dos
momentos diferentes del ciclo diario luz/oscuridad (12L:12D), durante la fotofase, a las 10:00 h, y en la
escotofase, a las 22:00 h. La ingesta, la actividad locomotora y los niveles plasmáticos de glucosa se
determinaron a las 8 h del tratamiento. La administración de leptina durante la fotofase (10:00 h) reduce la
ingesta y la actividad locomotora y aumenta la glucemia en el carpín, independientemente de si los peces son
alimentados durante el día (10:00 h) o durante la noche (22:00 h). Sin embargo, tales efectos no se observaron

                                                       22
                         7AIEC POSTER SESSION ABSTRACTS


en peces inyectados durante la escotofase (22:00 h), tanto si las inyecciones coinciden o no con el horario de
alimentación. Estos resultados muestran por primera vez en los peces que las respuestas anoréticas de la leptina,
así como sus efectos sobre la actividad locomotora y la glucemia, pueden ser dependientes del fotoperiodo,
sugiriendo que la magnitud de la respuesta hormonal depende fuertemente de la hora del día, como previamente
se ha indicado en los mamíferos. A partir de estos resultados proponemos que la variación diaria observada en
las acciones de la leptina en el carpín parece estar condicionada fundamentalmente por el fotoperido y no tanto
por el “zeitgeber” alimentación.




P34
THYROID HORMONE FEEDBACK ON PERIPHERAL DEIODINATION IN THE SENEGALESE
SOLE (SOLEA SENEGALENSIS)

Yvette Wunderink1, Felipe Espigares1, Gert Flik2, Peter Klaren2 and Juan M. Mancera1
E-mail: yvette.wunderink@uca.es
(1) Departamento Biología, Facultad Ciencias del Mar y Ambientales, Universidad Cádiz, 11510 Puerto Real,
Spain.
(2) Department of Animal Physiology, Institute for Water and Wetland Research, Faculty of Science, Radboud
University Nijmegen, Nijmegen, The Netherlands

The regulation of the thyroid system is generally modelled by the hypothalamus-pituitary-thyroid gland (HPT)
axis that describes the influence of the central nervous system on thyroid gland function. Stimulation of the
thyroid gland by TSH from the pituitary results in the release of thyroxine (T4), which is converted
extrathyroidally to the biologically active hormone triiodothyronine (T3). This process occurs through
peripheral outer ring deiodination (ORD) activities, catalysed by deiodinases, and is a key pathway in thyroid
hormone metabolism. Senegalese sole juveniles were intraperitoneally injected with slow-release coconut oil
implants with T4 (10 µg/g body weight) or T3 (1 µg/g body weight), and effects on plasma parameters and
peripheral thyroid hormone metabolism were assessed. T3 injection lowered plasma T4 levels; whereas T4
enhanced plasma T3 concentrations compared to control groups. T4 also stimulates liver T4-ORD activity at
day 7 after injection, indicating the peripheral conversion of T4 into T3. Plasma glucose and triglycerides levels
had decreased in T3 – as well as in T4 – treated animals, indicative of the stimulatory function of both hormones
on metabolism. Plasma T4 and T3 levels seems to be differentially regulated. As T3 is the end product of the
ORD pathway, it is plausible that deiodinase activity elevates in the presence of more substrate (T4), hence
enhancing the plasma T3 level. The decrease in plasma T4 levels following T3 treatment probably reflects the
negative feedback exerted by T3 on the pituitary and/or hypothalamus.




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