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Sponsors
GENERALITAT VALENCIANA (GV)
PROGRAMA PROMETEO
(Prometeo II 2015/085 y II 2015/051)
CONSEJO SUPERIOR DE INVESTIGACIONES
CIENTÍFICAS (CSIC)
SOCIEDAD IBÉRICA DE ENDOCRINOLOGÍA
COMPARADA (AIEC)
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Local Organizing
Committee Members
(in alphabetical order)
Josep Calduch-Giner
José Miguel Cerdá-Reverter
Alicia Felip Edo
Ana Gómez Peris
Félix Hernández
Evaristo Mañanós Sánchez
Jaime Nácher-Mestre
Jaume Pérez-Sánchez (Chair)
Society Council Members
(in alphabetical order)
Pedro Guerreiro
Esther Isorna
Juan Miguel Mancera
José Antonio Muñoz-Cueto
Isabel Navarro (Vicepresident)
Jaume Pérez-Sánchez
Deborah Power (President)
José Luis Soengas
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WEDNESDAY (23rd
September 2015)
OPENING LECTURE
OL - ON THE SHOULDERS OF GH-IANTS: A SOMATOTROPE JOURNEY
FROM COMPARATIVE TO TRANSLATIONAL ENDOCRINOLOGY. J.P.
Castaño, F. Gracia-Navarro, S. Pedraza-Agüera, A. Sarmento-Cabral, M.C.
Vázquez-Borrego, F. López-López, D. Hormaechea-Agulla, A. Villa-Osaba, E.
Rivero-Cortés, A.J. Martínez-Fuentes, A. Ibáñez-Costa, M.D. Gahete, R.M.
Luque.
SESSION 1: REPRODUCTION
Chairperson: Dr. Fransesc Piferrer
O1 - SPECIFIC RECOMBINANT GONADOTROPINS INDUCE
SPERMATOGENESIS AND SPERMIATION BY FIRST TIME IN A TELEOST
FISH, THE EUROPEAN EEL. David S. Peñaranda, V. Gallego, M.C. Vílchez,
L. Pérez, A. Gómez, I. Giménez, J.F. Asturiano
O2 - CHARACTERIZATION OF NUCLEAR AND MEMBRANE
PROGESTIN RECEPTORS IN EUROPEAN EEL, AND THEIR
EXPRESSION IN VIVO THROUGHOUT SPERMATOGENESIS. Marina
Morini, D.S. Peñaranda, M.C. Vílchez, R. Nourizadeh-Lillabadi, J.F.
Asturiano, F.A. Weltzien, L. Pérez
O3 - ASSESSMENT OF THE RA-SIGNALLING PATHWAY DURING
GONADAL DEVELOPMENT AND MATURATION IN THE EUROPEAN
SEA BASS (Dicentrarchus labrax). Paula Medina, A. Gómez, S. Zanuy, M.
Blázquez
O4 - DIFFERENTIAL ACTIVATION OF KISS RECEPTORS BY Kiss1 and
Kiss2 PEPTIDES IN THE SEA BASS. Alicia Felip, F. Espigares, S. Zanuy, A.
Gómez
O5 – EFFECT OF HIGH REARING DENSITY ON SEX DIFFERENTIATION
IN ZEBRAFISH (Danio rerio): SEX RATIO AND GENE EXPRESSION
ANALYSIS. Alejandro Valdivieso, L. Ribas, F. Piferrer
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THURSDAY (24th
September 2015)
JOSEP PLANAS' CONFERENCE
JP'C - FROM INSULIN TO IGFS IN FISH: A SCIENTIFIC JOURNEY.
Gutiérrez, J., Navarro, I., Fernández-Borràs, J., Blasco, J., Planas, J.V., Vélez,
E.J., Lutfi, E. and Capilla, E. E-mail: [email protected]
SESSION 2: ENERGY METABOLISM
Chairperson: Dr. José Luis Soengas
O6 - DAILY VARIATIONS OF CARBOHYDRATE AND LIPID
METABOLISM-RELATED PARAMETERS IN LIVER OF RAINBOW
TROUT. INFLUENCE OF LIGHT AND FOOD AVAILABILITY. Juan
Hernández-Pérez, J.M. Míguez, J.L. Muñoz, F. Naderi, M. Librán-Pérez, R.
Ceinos, M.A. López-Patiño
O7 - EFFECTS OF GHRELIN ON THE RESPONSE TO OLEATE OF
HYPOTHALAMIC FATTY ACID SENSORS IN RAINBOW TROUT
(Oncorhynchus mykiss): INVOLVEMENT IN THE CONTROL OF FOOD
INTAKE. Cristina Velasco, M. Librán-Pérez, C. Otero-Rodiño, F. Naderi, R.
Álvarez-Otero, M.A. López-Patiño, J.L. Soengas
O8 - FEEDING-INDUCED CHANGES IN THE GHRELINERGIC SYSTEM
IN THE PLASMA, BRAIN AND INTESTINAL BULB OF GOLDFISH
(Carassius auratus). Ayelén M. Blanco, M. Gómez-Boronat, I. Redondo, A.I. ,
M.J. Delgado
O9 - SERUM METABOLOME OF FASTED GILTHEAD SEA BREAM. A
NON-TARGETED APPROACH FOR THE IDENTIFICATION OF ROBUST
BIOMARKERS OF MALNUTRITION IN FISH. Rubén Gil Solsona, J.
Nácher-Mestre, L. Lacalle, J.A. Calduch-Giner, J.V. Sancho, F. Hernández, J.
Pérez-Sánchez
O10 - ENERGY SENSING IN GILTHEAD SEA BREAM. MOLECULAR
AND FUNCTIONAL CHARACTERIZATION OF SIRTUINS. Paula Simó,
J.A. Calduch-Giner, J. Pérez-Sánchez
O11 - METFORMIN EXERTS DIRECT ANTI-PROLIFERATIVE EFFECTS
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IN PROSTATE CANCER CELLS IN VITRO AND INHIBITS PROSTATE
TUMOR GROWTH IN VIVO UNDER NORMAL-FED AND, SPECIALLY,
UNDER HIGH FAT FED CONDITIONS. André Sarmento-Cabral, F. López-
López, M.D. Gahete, J.P. Castaño, R.M. Luque
SESSION 3: WIDE FUNCTIONAL ANALYSIS
Chairperson: Dr. Deborah Power
O12 - WIDE-TRANSCRIPTOMIC ANALYSIS OF INTESTINE IN
MEDITERRANEAN FISH. Jaume Pérez-Sánchez, J.A. Calduch-Giner, A.
Sitjà-Bobadilla
O13 - TRANSCRIPTOMICS PROVIDE INSIGHT INTO MUSSEL (Mytilus
galloprovincialis) MANTLE FUNCTION. Nadège Zaghdoudi-Allan, T. Yarra,
A. Churcher, R. Felix, M. Clark, D.M. Power
O14 - THE SKIN AS AN ENDOCRINE ORGAN: INSIGHTS FROM THE
SEA BASS SKIN TRANSCRIPTOME. Patrícia I. S. Pinto, M.A.S. Thorne, D.
M. Power
O15 - A MICROARRAY STUDY OF SKIN AND SCALE REGENERATION
IN THE SEA BREAM (Sparus aurata). Rita Alves Costa, F. Vieira, D.M.
Power
O16 - MULTI-CLASS SCREENING OF FEEDSTUFF CONTAMINANTS
AND POTENTIAL ENDOCRINE DISRUPTORS IN FARMED FISH. Jaime
Nácher-Mestre, R. Serrano, M. Ibáñez, T, Portolés, M.H.G. Berntssen, F.
Hernández, J. Pérez-Sánchez
SESSION 4: STRESS AND IMMUNE RESPONSE
Chairperson: Dr. Juan Miguel Mancera
O17 - ACUTE STRESS REGULATES VASOTOCINERGIC AND
ISOTOCINERGIC SYSTEM IN THE GILTHEAD SEA BREAM (Sparus
aurata). Arleta Krystyna Skrzyńska, E. Maiorano, M.Bastaroli, G. Martínez-
Rodríguez, J.A. Martos-Sitcha, J.M. Mancera
O18 - BRAIN MONOAMINERGIC NEUROTRANSMITTERS DURING
CHRONIC STRESS IN RAINBOW TROUT (Oncorhynchus mykiss). Fatemeh
Naderi, M.A. López-Patiño, M. Gesto, J.M. Cerdá-Reverter, C. Otero-Rodiño,
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C. Velasco, J.M. Míguez
O19 - PLASTIC PHYSIOLOGY, SOCIAL BEHAVIOUR AND ENDOCRINE
PROFILES OF AN INVASIVE CICHLID IN SOUTHERN PORTUGAL. Flávia
B. Silva, J.L. Saraiva, M. Vargas, M. Silva, J. Soares, A.V.M. Canário, P.M.
Guerreiro
O20 - TEMPERATURE IMPRINTING DURING EARLY DEVELOPMENT
AND THE ADULT STRESS AND IMMUNE RESPONSE. Ana Patrícia
Mateus, R. Costa, J.R. Cardoso, E. Gisbert, D.M. Power
FRIDAY (25th
September 2015)
SESSION 5: GROWTH FACTORS AND MOLECULAR EVOLUTION
Chairperson: Dr. Isabel Navarro
O21 - ACTIVATION OF SOMATOSTATIN RECEPTOR 3 REDUCES CELL
VIABILITY AND HORMONAL SECRETION IN NON-FUNCTIONING
PITUITARY ADENOMAS THROUGH MAPK SIGNALING. Mª Carmen
Vázquez-Borrego, A. Ibáñez-Costa, E. Venegas-Moreno, A. Toledano-Delgado,
A. Soto-Moreno, M.A. Gálvez, MD. Gahete, J.P. Castaño, R.M. Luque
O22 - LACK OF CORTISTATIN OR SOMATOSTATIN PLAYS
DIFFERENTIAL ROLES IN THE CONTROL OF MAMMARY GLAND
TUMORIGENESIS IN LEAN AND OBESE MICE. Alicia Villa-Osaba, M.D.
Gahete, F López-López, A.I. Pozo-Salas, R. Sánchez-Sánchez, R. Ortega-Salas,
M. Álvarez-Benito, J. López-Miranda, R.M. Luque, J.P. Castaño
O23 – THE TRUNCATED SOMATOSTATIN RECEPTOR sst5TMD4 IS
OVEREXPRESSED IN PROSTATE CANCER AND INCREASES
AGGRESSIVENESS FEATURES THROUGH REGULATION OF
ANGIOGENIC FACTORS AND Wnt/B-CATENIN PATHWAY. Daniel
Hormaechea-Agulla, M.D. Gahete, A. Ibáñez-Costa, E. Gómez-Gómez, J.A.
Ramos-Fernández, J. Valero-Rosa, J. Carrasco-Valiente, M.M. Moreno, M.D.
Culler, M.J. Requena, J.P. Castaño, R.M. Luque
O24 - IN1-GHRELIN INCREASES MALIGNANCY FEATURES OF BREAST
CANCER CELL LINES. David Rincón-Fernández, M.D. Gahete, V. Ruiz-
Murillo, R. Santamaría, R.M. Luque, J.P. Castaño
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O25 - EXPLORING ALLATOSTATIN-A, KISS AND GALANIN GPCRS IN
LOPHOTROCOZOANS. João C.R. Cardoso, N. Zaghdoudi-Allan, R.C. Félix,
D.M. Power
SESSION 6: MINERALIZATION AND PIGMENTATION
Chairperson: Dr. Pedro Guerreiro
O26 - RESPONSIVENESS OF SEA BASS SCALES TO ESTRADIOL AND
GENISTEIN. M. Dulce Estêvão, P. I. S. Pinto, A. Andrade, S. Santos, D. M.
Power O27 - AN APPROACH TO STANNIOCALCIN SECRETION IN GILTHEAD
SEABREAM (Sparus aurata). Ignacio Ruiz-Jarabo, S.F. Gregório, J. Fuentes
O28 - TARGETED PTH4 CELL ABLATION LEADS TO AN UNCOUPLING
OF BONE FORMATION AND MINERALIZATION. Paula Suarez-Bregua, A.
Saxena, M.E. Bronner, P. Moran, J. Rotllant
O29 - THE GENE REGULATORY NETWORK UNDERPINNING DORSAL–
VENTRAL PIGMENTATION PATTERNING IN FISH. ANALYSIS OF asip1
CIS-REGULATORY LANDSCAPE. Laura Cal, I. Braasch, J.L. Gómez-
Skarmeta, R. Kelsh, J.M. Cerdá-Reverter, J. Rotllant
CLOSING LECTURE
CL - 40 YEARS OF RESEARCH IN COMPARATIVE ENDOCRINOLOGY.
Olivier Kah
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Opening Lecture (OL)
ON THE SHOULDERS OF GH-IANTS: A SOMATOTROPE JOURNEY
FROM COMPARATIVE TO TRANSLATIONAL ENDOCRINOLOGY
J.P. Castaño, F. Gracia-Navarro, S. Pedraza-Agüera, A. Sarmento-Cabral,
M.C. Vázquez-Borrego, F. López-López, D. Hormaechea-Agulla, A. Villa-
Osaba, E. Rivero-Cortés, A.J. Martínez-Fuentes, A. Ibáñez-Costa, M.D. Gahete,
R.M. Luque. Email: [email protected]
1Department of Cell Biology, Physiology and Immunology, University of
Córdoba, Hospital Universitario Reina Sofía (HURS), Instituto Maimónides de
Investigación Biomédica de Córdoba (IMIBIC), CIBERobn and, ceiA3;
Córdoba, Spain.
Pituitary somatotropes provide a unique cell model to investigate the regulation
of hormone secretion from an integrative viewpoint. Over the last thirty years,
our group has investigated multiple aspects of the biology of somatotropes, in a
journey encompassing different animal species, organizational levels, and
functional research targets. A retrospective view of the steps covered reveals the
genuine value of comparative endocrinology, and its potential to develop novel
scientific knowledge. Our initial research, focused on frog pituitary, aimed at
understanding the role of hypothalamic hormones on the regulation of
somatotrope function. Combining in vivo and in vitro studies, EM-
immunocytochemistry and quantitative image analysis, we demonstrated for the
first time that TRH is a stimulatory factor for frog somatotropes. This discovery
was later extended by studies on cells from porcine and rat pituitary, and human
pituitary tumors, which unveiled a multifactorial regulation of somatotropes
across species by multiple neuropeptides, including GHRH, ghrelin, PACAP,
TRH, somatostatin, cortistatin, kisspeptin, etc, and helped to define the common
and unique signaling pathways and receptors mediating such regulation.
Ultrastructural analysis of somatotropes led us to the notion of cell
heterogeneity, and prompted us to investigate its biological meaning by
characterizing the different subpopulations of somatotropes composing the
pituitary during lifespan. Morphological and functional in vitro studies in pig
and rat somatotropes unveiled a unique dynamic secretory cell cycle that
includes not only hormone storage and secretion, but also the distinct ability of
somatotropes to respond to regulatory cues through an integrated control of
their molecular and cellular identity. The knowledge gathered in this travel led
us to explore the pathological side of somatotropes and GH. First, by studying
the cell biology of somatotropes from human pituitary tumors, we discovered
and characterized the presence and pathological role of aberrantly spliced
receptors for somatostatin (sst5TMD4) and of the In1-ghrelin variant in
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acromegaly patients, and examined their potential as therapeutic targets. A
second area of study aims at understanding the pathophysiological importance,
beyond somatic growth, of GH and somatotropes, which function as a true
endo-metabolic hub integrating, in a balanced manner, multiple components of
the homeostatic regulatory machinery of the glucose/insulin/IGF-I axis, whose
deregulation critically influences pathologies like diabetes, obesity and cancer.
Thus, departing from a comparative endocrinology onset, our journey provided
us a unique opportunity to achieve scientific knowledge, enjoy discoveries,
traverse fascinating translational research avenues, and treasure the privilege of
receiving the wisdom from GH-iant mentors.
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Josep Planas' Conference (JP'C)
FROM INSULIN TO IGFS IN FISH: A SCIENTIFIC JOURNEY
Gutiérrez, J., Navarro, I., Fernández-Borràs, J., Blasco, J., Planas, J.V., Vélez,
E.J., Lutfi, E. and Capilla, E. E-mail: [email protected]
Department of Physiology and Immunology, School of Biology, University of
Barcelona, Barcelona, Spain
A lecture in honor of Professor José Planas has been created in the 10th
congress of AIEC and we believe that it is fair to dedicate it to who can be
considered one of the first comparative endocrinologists in Spain. Dr. Planas
presented his thesis at the University of Barcelona (UB) in 1955, directed by
Prof. F. Ponz entitled “Insulin extraction from tuna (Thunnus thynnus, L.)” that
demonstrated his early interest in fish hormones. Later he also studied tuna
glucagon showing its effects and all these findings were reported in
international publications.
After a period at the University of Valladolid Dr. Planas returned to UB and
recovered the line of research in fish, directing the thesis of S. Zanuy and M.
Carrillo (1975) and later those of J. Fernández-Borràs and J. Gutiérrez (1985).
In 1984, in collaboration with A. Thorpe and IATS-CSIC, the first paper on sea
bass insulin was published, which was followed by a series of papers on insulin
function in different fish species. In 1986 the first international report on fish
plasma glucagon levels was published. Insulin receptor research was initiated
with E. Plisetskaya (1988/89) and was followed by the first characterization of
IGF-I receptors in fish (1993). During these years J. Planas and the group was
very active publishing an interesting series of papers on phylogenetic aspects of
insulin and IGF-I receptors and their role in fish glucose intolerance. After Dr.
Planas dead the group followed this research and E. Capilla and J.V. Planas
published the first report of a fish Glut4 (2000) and in 2001 the group published
for the first time the existence of IGF-II receptors in ectotherms.
In the last years, I. Navarro and J. Gutiérrez have dedicated a big effort to study
the regulation, mainly by IGFs and GH, of adipose tissue and muscle
development and physiology, generating numerous thesis and publications.
However, all this research has the origin in the pioneer work of Dr. Planas, who
was able to start it in a very difficult time for science in Spain, being the
predecessor of different teams that have contributed importantly to develop this
scientific area in our country.
(Supported by funds from MINECO, EU-FPs and SGR).
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Closing Lecture (CL)
40 YEARS OF RESEARCH IN COMPARATIVE ENDOCRINOLOGY
Olivier Kah. E-mail: [email protected]
Research Institute in Health, Environment and Occupation (INSERM U1085),
University of Rennes 1, Rennes, France
Comparative endocrinology played an important role in understanding how our
hormones and their receptors are inherited from our ancestors, diversify through
genome duplications and gain new functions. By taking the example of GnRH,
this lecture intends to cover about 40 years of research in comparative
neuroendocrinology. The primary sequence of GnRH was published in 1971
and through the evolution of the field, we shall see how ideas evolved, dogma
collapsed and new concepts appear that, in some cases, totally changed the way
we have to consider our past and future research.
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O1
SPECIFIC RECOMBINANT GONADOTROPINS INDUCE
SPERMATOGENESIS AND SPERMIATION BY FIRST TIME IN A
TELEOST FISH, THE EUROPEAN EEL
David S. Peñaranda1, V. Gallego
1, M.C. Vílchez
1, L. Pérez
1, A. Gómez
2, I.
Giménez3, J.F. Asturiano
1. E-mail: [email protected]
1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología
Animal. Universitat Politècnica de València. Spain. 2Instituto de Acuicultura de
Torre la Sal (CSIC). Castellón, Spain. 3Rara Avis Biotec S.L., Valencia, Spain.
Eel recombinant gonadotropins have induced spermatogenesis in vitro, but had
limited effect in vivo. New specific single-chain recombinant gonadotropins
(Rara Avis Biotec S.L.) were tested treating European eel males with rFSH and
rLH. Males received rFSH in three doses (2.8, 1.4 and 0.7 µg/fish; high,
medium and low treatments) during 3 weeks. Later, an increasing rLH dose
(every 3 weeks; 1, 2, 6 µg/fish) was combined with rFSH. Three
males/treatment were sacrificed every 3 weeks. Blood and testis samples were
taken for steroids and histological analyses and GSI was calculated. Sperm
quality was determined by CASA software.
rFSH by itself was able to induce the spermatogenesis (week 3), causing higher
androgen levels and development until the spermatogonia 2 stage in the high
rFSH group. The rLH (week 6) promoted further maturation to spermatocyte 2
stage in the medium and high groups, but no gonadal progression was observed
in the low group. Higher T levels were reached in all the groups, and the biggest
11KT values in the rFSH high group. A second increase of rLH (week 9)
induced further maturation to spermatid-spermatozoa 1 and spermatocyte 1
stages in the high and low groups, respectively. The medium group showed no
further maturation (spermatocyte 1-2 stages). The highest dose of rLH (week
12) induced the most advanced stage (spermatozoa 2), and significant GSI
increase in all the groups. A progressive 11KT (high group) and T (all
treatments) decrease was observed from week 9 to 12. All treatments induced
spermiating males, however, the best sperm quality with ?50% motile cells and
volumes ~0.4 ml was observed in males treated with the highest rFSH dose and
a progressive increase of rLH treatment. On the other hand, a 20% of non-
responders were observed and the sperm quality was variable. So, further
experiments are required to improve these treatments.
Funded by IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement nº:
642893) and MINECO (REPRO-TEMP; AGL2013-41646-R).
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O2
CHARACTERIZATION OF NUCLEAR AND MEMBRANE
PROGESTIN RECEPTORS IN EUROPEAN EEL, AND THEIR
EXPRESSION IN VIVO THROUGHOUT SPERMATOGENESIS
Marina Morini1, D.S. Peñaranda
1, M.C. Vílchez
1, R. Nourizadeh-Lillabadi
2,
J.F. Asturiano1, F.A. Weltzien
2, L. Pérez*
1. E-mail: [email protected]
1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología
Animal. Universitat Politècnica de València, Valencia, Spain. 2Department of
Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences.
Campus Adamstuen, Oslo, Norway.
In male teleost, the role of progestins as 17α,20β-dihydroxy-4-pregnen-3-one
(DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20βS), have been reported
in the regulation of spermatogenesis. Two mechanisms of action can mediate
the progestins action: the classic genomic mechanism of steroid action relatively
slow involving nuclear receptors (nPRs or Pgrs), members of the nuclear steroid
receptor superfamily; and the membrane progestin receptors (mPRs), members
of the progestin and adipoQ receptor (PAQR) family, which exert an often non
genomic, rapid activation of intracellular signal transduction pathways.
We characterized and studied the mRNA expression of two nuclear progestin
receptors (pgr1 and pgr2) and for the first time in a teleost of five membrane
progestin receptors (mPRα, mPRγ, mPRδ, mPRAL1, mPRAL2) in brain,
pituitary and gonads, in the European eel. mPR phylogeny placed three eel
mPRs together with vertebrate mPRα, and were called mPRα, mPRAL1 (alpha-
like1) and mPRAL2 (alpha-like2). The two other eel mPRs (i.e.: mPRγ, mPRδ)
clustered together with their respective mPR types amongst vertebrate
representatives.
Eel tissue distributions reveal that nPR and mPR transcripts were expressed in
neuroendocrine and non-reproductive tissues, which suggest the involvement of
these receptors not only in reproduction, but also in other physiological
functions. Furthermore, in vivo studies of mRNA expression throughout
spermatogenesis suggest that progestins exert their actions in the eel brain and
pituitary by both nPRs and mPRs. In the testis, the nuclear pgr2 and membrane
mPRγ and mPRδ seem to be involved on the induction of meiosis, whereas
mPRa, mPRAL1 and mPRAL2 seems to be involved on the process of final
sperm maturation.
The emergence of both mPRs and nPRs in early vertebrates and the
colocalisation of both mPRs and nPRs mRNA suggest the possibility that they
may act in concert to regulate the progestin-mediated functions.
18
03
ASSESSMENT OF THE RA-SIGNALLING PATHWAY DURING
GONADAL DEVELOPMENT AND MATURATION IN THE EUROPEAN
SEA BASS (Dicentrarchus labrax)
Paula Medinaac
, A. Gómezb, S. Zanuy
b, M. Blázquez
a. E-mail:
aInstitut de Ciències del Mar (ICM-CSIC), Barcelona, Spain.
bInstituto de Acuicultura de Torre la Sal (IATS-CSIC), Castellón, Spain.
cUniversidad de Antofagasta, Antofagasta, Chile.
In higher vertebrates, the retinoic acid (RA) signalling pathway stimulates
meiosis by regulating the transcription of several genes that result in a spatio-
temporal balance between the synthesis and degradation of RA. The goal of this
research is to gain a better understanding of the role of the RA-signalling
pathway in fish meiosis using the European sea bass as a model. The study is
focussed on the expression of nine RA-related genes by the time of sex
differentiation and the onset of meiosis in males and females. Moreover, in
females it was also extended to the first vitellogenesis and final maturation. The
selected genes include a retinol receptor (stra6), oxidizing proteins that
synthesize RA (aldh1a2, aldh1a3), binding proteins (crabp, crbp4), nuclear
receptors (rarα, rxrα, pparγ), and the RA catabolizing enzyme (cyp26a1). The
results show that in males, stra6, crbp4, rarα, rxrα, cyp26a1, and crabp reached
lowest levels by the onset of puberty. The highest expression of aldh1a2,
aldh1a3, and pparγ was attained after sex differentiation, decreasing to their
lowest levels by the time of puberty. In females, a steady increase of stra6, rarα,
rxrα, and cyp26a1 up to the start of vitellogenesis, was followed by a
progressive decrease during maturation. A similar scenario applied for aldh1a2,
aldh1a3, pparγ and crabp, although in this case, a final increase occurred by the
time of maturation. The decrease in the expression of cyp26a1 in both sexes
marked the onset of meiosis, indicating that an increase in the availability of RA
is required to trigger the process. In addition, in females, an increase in the
levels of both aldhs and thus of RA was also observed by the time of final
oocyte maturation, coinciding with the resumption of meiosis, further
supporting the need of high RA levels for the process. Regardless of these
evidences, more studies are required to elucidate the influence of RA in fish
meiosis and the network of genes that might be involved.
Funded by projects Reprosex (AGL2011-28890) and Reprobass
(PROMETEOII/2014/051). PM was sponsored by a grant MECE2 (ANT0806).
19
04
DIFFERENTIAL ACTIVATION OF KISS RECEPTORS BY KISS1 AND
KISS2 PEPTIDES IN THE SEA BASS
Alicia Felip, F. Espigares, S. Zanuy, A. Gómez. E-mail: [email protected]
Instituto de acuicultura de Torre de la Sal (CSIC), Ribera de Cabanes,12595
Castellón, Spain
Two forms of kiss gene (kiss1and kiss2) have been described in the teleost sea
bass. This study assesses the cloning and characterization of two Kiss receptor
genes, namely kissr2 and kissr3 (known as gpr54-1b and gpr54-2b,
respectively), and their signal transduction pathways in response to Kiss1 and
Kiss2 peptides. Phylogenetic and synteny analyses indicate that these paralogs
originated by duplication of an ancestral gene before teleost specific
duplication. The kissr2 and kissr3 mRNAs encode proteins of 368 and 378
amino acids, respectively, and share 53.1% similarity in amino acid sequences.
In silico analysis of the putative promoter regions of the sea bass Kiss receptor
genes revealed conserved flanking regulatory sequences among teleosts.
Both kissr2 and kissr3 are predominantly expressed in brain and gonads of sea
bass, medaka and zebrafish. In the testis, the expression levels of sea bass
kisspeptins and Kiss receptors point to a significant variation during the
reproductive cycle.
In vitro functional analyses revealed that sea bass Kiss receptor signals are
transduced both via the protein kinase C (PKC) and protein kinase A (PKA)
pathway. Synthetic sea bass Kiss1-15 and Kiss2-12 peptides activated Kiss
receptors with different potencies, indicating differential ligand selectivity. Our
data suggest that Kissr2 and Kissr3 have a preference for Kiss1 and Kiss2
peptides, respectively, thus providing the basis for future studies aimed at
establishing their physiologic roles in sea bass.
Supported by GV (GV06/268) and (ACOMP2010), Programa PROMETEO
(Prometeo/2010/003; PrometeoII/2014/051) and EU grant 655 FP7222719
(LIFECYCLE). A.F was supported by a Ramón y Cajal and F.E by a JAE-
Predoc CSIC.
20
05
EFFECT OF HIGH REARING DENSITY ON SEX DIFFERENTIATION
IN ZEBRAFISH (Danio rerio): SEX RATIO AND GENE EXPRESSION
ANALYSIS
Alejandro Valdivieso, L. Ribas, F. Piferrer. E-mail:[email protected]
Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas.
Passeig Marítim, 37–49. 08003 Barcelona
Fish manifest all kind of reproduction systems known in vertebrates in which
sex determination and sex differentiation establish the final gender. Rearing
density is one of the most studied environmental factors in fish and it has been
shown that some species placed at high density can exhibit altered sex ratios,
usually in favor of males. In addition, it has been shown that some fish
subjected to high density exhibit increased cortisol plasma levels, which can
have masculinizing effects. Here, we used a domesticated zebrafish strain,
which behaves as having a polygenic system of sex determination, being more
susceptible to the environmental factors than the wild type strains. To
understand the effect of density and cortisol on sex determination and
differentiation in zebrafish we examined population sex ratios and gene
expression in ovaries and testes of juveniles by RT-PCR. We were interested in
canonical reproduction-related genes, genes involved in the glucocorticoid
pathway and genes related to epigenetic regulatory mechanisms. To better
determine the role of density and final sex of the individual we set up two
experiments. In experiment one fish were subjected to different densities during
6–90 days post fertilization (dpf) while in experiment two fish were fed using
different cortisol treatments between 10–45 dpf. Elevated densities resulted in
an increase in the number of males while cortisol treatment resulted in complete
masculinization of the population. Moreover, elevated density downregulated
nr3c1, cy19a1a and dnmt1 in both ovaries and testes. However, despite the
observed masculinization, cortisol treatment did not result in significant
differences in gene expression. Taken together, these results show that density
affects sex ratios in zebrafish, inducing masculinization. Results also show that
high density downregulated the expression of gene related to the female
differentiation pathway. Nevertheless, the underlying mechanisms related to the
masculinizing effects of cortisol still remain unclear.
Supported by MINECO grant AGL2013-41047-R “Epifarm” to FP.
21
06
DAILY VARIATIONS OF CARBOHYDRATE AND LIPID
METABOLISM-RELATED PARAMETERS IN LIVER OF RAINBOW
TROUT. INFLUENCE OF LIGHT AND FOOD AVAILABILITY.
Juan Hernández-Pérez*, Jesús M. Míguez, J.L. Muñoz
#, Fatemeh Naderi, Marta
Librán-Pérez, Rosa Ceinos, Marcos A. López-Patiño. E-mail:
Laboratorio de Fisioloxía Animal. Dept. Bioloxía Funcional e Ciencias da
Saúde, Facultade de Bioloxía, Universidade de Vigo; and ECIMAT. #Current
address: Centro I-Mar. Universidad de Los Lagos (Chile).
Most physiological processes, such as feeding behaviour and energy
metabolism, exhibit circadian rhythmicity, thus playing a main role in the
maintenance of the functional homeostasis. Light-dark and feeding-time cycles
are mainly entraining such processes in order to adjust them to the
chronological arrangement of the external world. The aim of the current study
was to investigate in rainbow trout the existence of daily changes in activity and
mRNA abundance of proteins involved in major pathways of carbohydrate and
lipid metabolism in liver. The influence exerted by both the light-dark cycle and
food availability in synchronizing such rhythms was also evaluated. For this
purpose, four cohorts of animals previously adapted to 12L:12D (lights on at
ZT0; feeding time at ZT2) were subjected to: normal conditions (LD); 48-h
light isolation (DD); 96-h food deprivation (LD + Fasting); or food deprivation
and light isolation (DD + Fasting) respectively. After such time periods, fish
were sacrificed and sampled every 4-h on the following 24-h period (ZT/CT0,
4, 8, 12, 16, 20 and 0´). Our results reveal gk, pepck, g6pase, pk, glut2, hoad
and fas rhythms of mRNA abundance to persist in the absence of light and/or
food as synchronizers. Enzyme activities did not display significant daily
variations although these parameters mostly fluctuated in parallel to the
respective gene expression. Rhythms of mRNA abundance did mostly depend
on the presence of food, but the influence of the light/dark cycle could not be
discarded. The present results indicate that liver metabolism in trout is linked to
a circadian system which may play a main role in generating rhythms of
enzymes expression, but also influences enzyme activities. Further research
must be carried out in order to understand such interaction.
Acknowledgements: Funded by Ministerio de Economía y Competitividad and
European Fund for Regional Development (AGL2013-46448-3-1-R and
FEDER) and AEO-ECIMAT.
22
O7
EFFECTS OF GHRELIN ON THE RESPONSE TO OLEATE OF
HYPOTHALAMIC FATTY ACID SENSORS IN RAINBOW TROUT
(Oncorhynchus mykiss): INVOLVEMENT IN THE CONTROL OF FOOD
INTAKE
Cristina Velasco, M. Librán-Pérez, C. Otero-Rodiño, F. Naderi, R. Álvarez-
Otero, M.A. López-Patiño, J.L. Soengas. E-mail: [email protected]
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e
Ciencias da Saúde. Facultade de Bioloxía, Universidade de Vigo and ECIMAT.
We have previously demonstrated the existence in hypothalamus of rainbow
trout of fatty acid sensing systems responding to changes in the levels of LCFA
like oleate or MCFA like octanoate. The activation of these systems induces an
anorectic effect through changes in the expression of hypothalamic
neuropeptides like NPY/AgRP and POMC/CART. The hypothalamic neurons
possessing these sensors are known in mammals to integrate information from
levels of circulating hormones like insulin, leptin or ghrelin. Ghrelin is a
gastrointestinal hormone that plays an important role in the regulation of
hormone release, energy balance, and control of food intake. We hypothesize
that ghrelin counteracts the response to increased fatty acid levels of
hypothalamic fatty acid sensing systems. Thus, we administered ICV to 100g
rainbow trout 1µl of saline alone (control) or containing 2 ng ghrelin, 1 µmol
oleate or both ghrelin+oleate. After oleate treatment we observed an activation
of fatty acid sensor systems, increase of anorectic potential and inhibition of
food intake in agreement with our previous studies thus validating the
experimental design. After ghrelin treatment, we observed a significant
increased production of orexigenic factors (NPY, AgRP) and a significant
decrease in the production of anorexigenic factors (POMC and CART) whose
overall balance would be an increased orexigenic potential also in agreement
with the finding of a raised food intake. The administration of oleate+ghrelin
resulted in a response of fatty acid sensing systems, expression of
neuropeptides, and food intake similar to control group, thus suggesting that
ghrelin is effectively counteracting the effects induced by raised oleate levels.
We therefore demonstrate, for the first time in fish, the modulatory action of
ghrelin of the metabolic hypothalamic integration related to the control of food
intake.
Acknowledgements: Funded by Ministerio de Economía y Competitividad and
European Fund for Regional Development (AGL2013-46448-3-1-R and
FEDER) and AEO-ECIMAT.
23
O8
FEEDING-INDUCED CHANGES IN THE GHRELINERGIC SYSTEM
IN THE PLASMA, BRAIN AND INTESTINAL BULB OF GOLDFISH
(Carassius auratus)
Ayelén M. Blanco, M. Gómez-Boronat, I. Redondo, A.I. Valenciano, M.J.
Delgado. E-mail: [email protected]
Department of Physiology (Animal Physiology II), Faculty of Biology,
University Complutense of Madrid, Spain
Ghrelin is a gut-derived peptide hormone with a key role in food intake
regulation and energy balance. In mammals, ghrelin secretion and expression is
known to increase prior to a meal, suggesting a role for this hormone in hunger
and meal initiation. However, results regarding this aspect in fish are
controversial. Therefore, the aim of this study was to characterize the pre- and
postprandial pattern of plasma acyl-ghrelin (AG), desacyl-ghrelin (DAG) and
ghrelin O-acyl transferase (GOAT) in goldfish (Carassius auratus) that were
either fed or unfed the day of the experiment. Moreover, the expression of
preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was
quantified in brain and peripheral tissues. Circulating hormone levels were
measured by immunoassays, and gene expression was determined by real-time
RT-PCR. Results show a significant preprandial rise in circulating levels of AG,
but not DAG, and GOAT, supporting the role of AG as a meal initiator in this
teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1-h
before scheduled feeding time in intestinal bulb, suggesting that this receptor
subtype might be involved in the preprandial action of ghrelin in this tissue.
Neither preproghrelin nor ghs-r1a1 are observed to be modified postprandially
in intestinal bulb; by contrary, goat and ghs-r1a2 mRNA levels are significantly
higher in unfed fish after subjective feeding time. In brain, none of the studied
genes are modified preprandially, except for goat in the hypothalamus, whose
expression is reduced 1-h before scheduled feeding time. Postprandially, a
significant increase in mRNA levels of unfed fish is detected for preproghrelin
in telencephalon, goat in both telencephalon and hypothalamus, ghs-r1a1 in
vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus, and ghs-r2a2 in
telencephalon and vagal lobe. Collectively, the periprandial variations observed
in this study support the role of ghrelin as an orexigenic factor in fish, and show
for the first time a tissue-dependent implication of GOAT and the four subtypes
of GHS-R in the responses of the ghrelinergic system to feeding.
Acknowledgements. Supported by the Spanish MINECO (AGL 2013-46448-C3-
2-R). A.M. Blanco and M. Gómez-Boronat are predoctoral fellows from the
Spanish MEC and MINECO, respectively.
24
O9
SERUM METABOLOME OF FASTED GILTHEAD SEA BREAM. A
NON-TARGETED APPROACH FOR THE IDENTIFICATION OF
ROBUST BIOMARKERS OF MALNUTRITION IN FISH
Rubén Gil Solsona1, J. Nácher-Mestre
1, L. Lacalle
1, J.A. Calduch-Giner
2, J.V.
Sancho1, F. Hernández
1, J. Pérez-Sánchez
2. E-mail: [email protected]
1Research Institute for Pesticides and Water (IUPA), University Jaume I,
Castellón, Spain.
2Nutrigenomics and Fish Growth Endocrinology Group, Institute of
Aquaculture Torre de la Sal (CSIC), Castellón, Spain.
Up-stream regulators of the endocrine cascade are largely altered by
malnutrition due to feed deprivation or unbalanced diet composition. This
includes many changes in circulating catabolic and growth-promoting factors,
also related to plasma metabolite levels. This complex trade-off has been
addressed in both fish and higher vertebrates, although modern omic
approaches are required to identify and validate the most robust biomarkers
with either diagnostic and/or prognostic value. For this goal, a non-targeted
metabolomics approach is developed.
Nuclear magnetic resonance has been often used for routine metabolomic
profiling in a vast array of species due to its high elucidation power and
universality. However, liquid chromatography-high resolution mass
spectrometry (LC-HRMS) has emerged as a reliable alternative technique with
greater sensitivity and selectivity. This methodological advantage has been
exploited to assess the serum metabolome of two-year old gilthead sea bream
(Sparus aurata) in continuously fed fish and 10-days fasted fish.
Feed deprivation resulted in a significant reduction of condition factor (2.91 vs
2.63), hepatosomatic index (1.32 vs 0.90) and mesenteric fat index (1.90 vs
1.64). Deproteinized serum samples were analysed by LC-HRMS for individual
fish (n=10) and data was peak picked, aligned and normalized with XCMS.
Partial Least Squares–Discriminant Analysis (PLS-DA) clearly separated the
two experimental groups, explaining the first component more than 90% of
total variance. Orthogonal PLS-DA highlighted more than 850 differentiation
ions (P>0.80), which primarily yielded 46 compounds that were classified into
different physiological pathways (phospholipid metabolism, lysophosphatidic
acid metabolism, fatty acid oxidation, bile salt metabolism, methionine-cysteine
metabolism, amino acid catabolism, biotin metabolism). A targeted refined
search in combination with transcriptional validation procedures were
conducted to go further into fish biomarker research.
The study was partially funded by the ARRAINA EU Project.
25
O10
ENERGY SENSING IN GILTHEAD SEA BREAM. MOLECULAR AND
FUNCTIONAL CHARACTERIZATION OF SIRTUINS
Paula Simó, J.A Calduch-Giner, J. Pérez-Sánchez. E-mail: [email protected]
Nutrigenomics and Fish Growth Endocrinology, Institute of Aquaculture Torre
de la Sal, CSIC, Spain.
Sirtuins (SIRTs) use nicotinamide adenine dinucleotide (NAD+) as co-substrate
in their acetylated reactions, sensing specific metabolic states that intimately
link the intracellular NAD+/NADH ratio with post-translational modifications
of histones and metabolic enzymes. Several examples in mammals agree with
this, and the increase of the intracellular NAD+/NADH ratio with dietary
restriction and exercise leads to increase SIRT activity and mRNA expression.
Thus, SIRTs are master regulators of energy metabolism and nearly all enzymes
involved in glycolysis, gluconeogenesis, FA oxidation, AA catabolism and
OXPHOS have been identified as acetylated proteins in the proteome of
mammalian cells. The SIRT family is virtually ubiquitous throughout all
kingdoms of life with a number of SIRTs that ranges from one in bacteria to
seven in vertebrates, but few studies have addressed the molecular
characterization and functional analysis of SIRTs in livestock animals and fish
in particular. To solve this gap of knowledge, the molecular characterization and
gene expression regulation of SIRTs was assessed in gilthead sea bream.
Searches in the Nutrigroup transcriptomic database (www.nutrigroup-
iats.org/seabreamdb) yielded seven full nucleotide sequences with a conserved
enzymatic core domain (SIRT1-7) and mitochondrial targeting sequences
(SIRT3-5). Phylogenetic analysis identified three major clades corresponding to
SIRT1-3, SIRT4-5 and SIRT6-7 that reflected the present hierarchy of
vertebrates. Transcriptional studies corroborated a ubiquitous gene expression
that was tissue-specific for each SIRT isoform. This was evidenced by PCA and
hierarchical clustering which identified two main clusters corresponding to
SIRT isoforms with relatively high (SIRT1, 2 and 5) and low (SIRT3, 4, 6 and
7) expression levels. A nutritional regulation was also evidenced in 10-days
fasted fish, and SIRT2, 3 and 4 exhibited an overall down-regulated expression.
In contrast, the overall response of SIRT 1, 5, 6 and 7 was the up-regulation,
which is indicative of the different adaptive responses to cope with the
changing tissue-energy demands.
The study was funded by EU (ARRAINA) and Spanish (Mi2-Fish,
PrometeoII/2014/85) Projects.
26
O11
METFORMIN EXERTS DIRECT ANTI-PROLIFERATIVE EFFECTS IN
PROSTATE CANCER CELLS IN VITRO AND INHIBITS PROSTATE
TUMOR GROWTH IN VIVO UNDER NORMAL-FED AND,
SPECIALLY, UNDER HIGH FAT FED CONDITIONS.
André Sarmento-Cabral, F. López-López, M.D. Gahete, J.P. Castaño, R.M.
Luque. E-mail: [email protected]
Department of Cell Biology, Physiology and Immunology, University of
Córdoba; Hospital Universitario Reina Sofía, Instituto Maimónides de
Investigación Biomédica de Córdoba (IMIBIC); CIBER Fisiopatología de la
Obesidad y Nutrición (CIBERobn); and, Campus de Excelencia Internacional
Agroalimentario (ceiA3), Córdoba, Spain.
Obesity (Ob) is a chronic endocrine-metabolic disease and one of the most
serious and complex threats for the human health, which is associated with an
increased incidence of some types of cancers such as prostate cancer (PC), the
second most common cancer in men worldwide. Interestingly, metformin (Met),
an antidiabetic drug, might represents a very promising opportunity to treat Ob
and PC as some retrospective clinical studies have shown that the incidence of
PC is lower in patients treated with Met. However, the endocrine-metabolic,
cellular and molecular mechanisms underlying the association between Ob and
higher incidence/aggressiveness of PC and the putative pharmacological
effectiveness of Met in PC are still unknown. We used primary normal prostate
(NP) cell cultures from mice and human PC cell lines (PC3, 22Rv1 and LNCaP)
as well as, immuno-suppressed mice inoculated with PC3 cells, fed a high-fat
diet (HFD) or low-fat diet (LFD), as models to test the beneficial effect of Met
(in vitro: 10µM-10mM; in vivo: 250mg/Kg). Our results indicate that Met
modulates key metabolic, endocrine and pathologic components (e.g.
expression of components of insulin/IGF-I/somatostatin/ghrelin systems) in NP
cell cultures. Interestingly, Met had no evident effect on 22Rv1 cells
proliferation but significantly reduced PC3 and LNCaP cells proliferation
and/or migration. Remarkably, we found that Met also have a significant
beneficial in vivo effect as it reduced tumor volume and weight in mice fed a
LFD and, specially, on those fed a HFD compared to their vehicle-treated
control mice. Altogether, our data suggest that Met modulates NP cell function
and exerts beneficial effects in the inhibition of PC cells growth in vitro and in
vivo, specially, under HF-conditions.
Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-
2012, and CIBERobn.
27
O12
WIDE-TRANSCRIPTOMIC ANALYSIS OF INTESTINE IN
MEDITERRANEAN FISH
J. Pérez-Sánchez1, Josep A. Calduch-Giner
1, A. Sitjà-Bobadilla
2. E-mail:
1Nutrigenomics and Fish Growth Endocrinology, Institute of Aquaculture Torre
de la Sal, CSIC, Spain.
2Fish Pathology, Institute of Aquaculture Torre de la Sal, CSIC, Spain.
The intestinal tract of teleosts is involved in water and electrolyte balance,
immunity, and endocrine regulation of nutrient availability and metabolism.
Hence, gastrointestinal syndromes due to nutritional deficiencies and infectious
agents are important causes of low growth performance, poor health or serious
mortalities in farmed fish. We presented the current research conducted in our
laboratory with gilthead sea bream and European sea bass. Attention is mostly
focused on the plasticity at the molecular level of the intestine tract in order to
identify/validate new intestine health biomarkers of diagnostic and/or
prognostic value.
In a first stage, microarray expression profiling of sea bass intestine highlighted
almost a constant gene expression profile of middle (MID) and anterior (ANT)
intestine segments. Conversely, more than 1,800 genes were differently
expressed between posterior (POS) and MID-ANT intestine. Thus, POS
intestine emerged as a highly immune-regulated tissue, with also relevance on
vitamin B12 and bile acid metabolism (entero-hepatic pathway). The
differential expression of different chemosensors along the intestine tract was
also evidenced. This issue was further verified in sea bream and more than
5,000 unique genes were differentially expressed across the intestine, although
this different expression pattern is especially evident in active feeding periods
(summer). Microarray and RNA-seq only revealed minor gene expression
changes in fish fed balanced low FM/FO diets and interestingly the use of feed
additives is able to reverse most of the observed changes towards the wild
phenotype. When season was considered, the number of differentially expressed
genes (summer vs winter) was higher than 6,500 genes. The magnitude of the
change is even higher than that reported after massive destruction of intestine
epithelium in chronically parasitized fish. Likewise, previous studies reflected
that the plasticity of the transcriptome of anaerobic and highly oxidative muscle
tissues is quite lower in comparison to that of the intestine tract.
The study was funded by EU (ARRAINA) and Spanish (Mi2-Fish,
PrometeoII/2014/85) Projects.
28
O13
TRANSCRIPTOMICS PROVIDE INSIGHT INTO MUSSEL (Mytilus
galloprovincialis) MANTLE FUNCTION
Nadège Zaghdoudi-Allan1, T. Yarra
2, A. Churcher
1, R. Felix
1, M. Clark
2 and
D.M. Power1. E-mail: [email protected]
1Comparative Endocrinology and Integrative Biology, Centre of Marine
Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,
Portugal. 2British Antarctic Survey, Natural Environment Research Council,
High Cross, Madingley Road, Cambridge, CB3 0ET, UK.
With over 90,000 extant species, phylum Mollusca is the most successful group
of animals on Earth. Common to all molluscs, the mantle is a multi-functional
highly muscular tissue that contacts the shell and envelops vital organs. In
bivalves, the epithelial cells of the mantle secrete the external shell by a
complex network of mechanisms that remain poorly understood. We
hypothesize that the mantle edge in Mytilus species is heterogenous in cellular
structure and function and use next generation sequencing to test this
hypothesis. The mantle edge of the Mediterranean mussel (Mytilus
galloprovincialis) was sectioned into three parts and sequenced using Illumina
(HiSeq 1500). The transcriptome sequences generated assembled into 18440
genes with an N50 of 2055. The transcriptome was annotated and GO
(geneontology.org) and String analysis (http://www.string-db.org) used for a
preliminary characterisation of biological processes. To test our hypothesis, we
compared the transcripts from the 3 mantle segments and the expression levels
of 8 candidate genes in the sectioned mantle of 6 individuals using qPCR.
Candidates were chosen based on their regulatory function and potential
involvement in shell formation. Candidates such as Dermatopontin and SMAD
are known to regulate cell proliferation and growth. While the Buccalin-related
neuropeptide mediates neuromuscular transmission in molluscs. Our results
show significant differences in transcript abundance and cellular function
amongst the three mantle sections. Combining our transcriptomic study with
histological studies of the mantle tissue, we present evidence of both molecular
and structural heterogeneity of the mussel mantle and identify several putative
regulatory networks.
Acknowledgements: This study was financed by the European Regional
Development Fund (ERDF) COMPETE and Portuguese funds through
Foundation for Science and Technology (FCT, project UID/Multi/04326/2013)
and CACHE a Marie Curie ITN (PITN-GA-2013-605051).
29
O14
THE SKIN AS AN ENDOCRINE ORGAN: INSIGHTS FROM THE SEA
BASS SKIN TRANSCRIPTOME
Patrícia I. S. Pinto1, M. A. S. Thorne
2 and D. M. Power
1. E-mail:
1 CCMAR - Centre of Marine Sciences, Universidade do Algarve, Faro,
Portugal. 2 British Antarctic Survey (BAS), Cambridge, UK
The skin is an important organ with a wide range of roles including mechanical,
thermal and immune barrier functions and its sensory and communication
functions. Significant attention has been paid to understanding skin homeostasis
and regeneration in mammals but much less is known about this organ in other
vertebrates. The skin is a target for several hormones that regulate its multitude
of functions including pigmentation and appendage growth. Fish skin has a
similar structural organization to mammals with the exception of the calcified
scale and the outer non-keratinized epithelia and it has specialized roles in
osmoregulation and mineral homeostasis. The endocrine regulation of the
function of skin in fish is not very well explored and the emerging concept of
skin as an endocrine organ is unstudied in fish. The present study uses next-
generation sequencing to explore the potential endocrine function of skin, by
generating the transcriptome of sea bass (Dicentrarchus labrax) dorsal skin.
The sea bass skin transcriptome contained 31,856 unique transcripts with
minimum 500 and average 1070 base pairs of length, of which approx. 97%
were assigned to the sea bass genome and 94% to existing proteins or cDNAs in
databases. A focused analysis identified several hormones and receptors from
different systems (including steroid or thyroid hormones and calcemic
hormones) which are expressed in the skin. The results from in silico and
targeted quantitative PCR support the role of fish skin as both a target and a
source of hormones. They will be an important resource for future research on
the impact of the environment on skin functions as well as the recognition of
the skins contribution to whole organism physiology, with potential relevance to
aquaculture production, fish welfare and conservation.
Acknowledgments: Funded by FCT – Portuguese Science and Technology
Foundation through projects UID/Multi/04326/2013 and PTDC/AAG-
GLO/4003/2012 and grant FCT SFRH/BPD/25247/2005 to PISP.
30
O15
A MICROARRAY STUDY OF SKIN AND SCALE REGENERATION IN
THE SEA BREAM (Sparus aurata)
Rita Alves Costa, F. Vieira and D.M. Power. Email: [email protected]
Comparative Endocrinology and Integrative Biology, Centre of Marine
Sciences, University of Algarve, Campus of Gambelas, 8005-139 Faro,
Portugal
Wound healing in teleost fish is a multistage process that involves several
biological processes that overlap in time and space and re-establish tissue
integrity. We previously reported using a validated microarray that 3 days after
scale removal in sea bream significantly up-regulated genes were involved in
cell cycle regulation, cell proliferation and adhesion, immune response and
antioxidant activities and that by day 7 only 8 genes were up-regulated (Vieira
et al. 2011). In the present study we targeted down-regulated genes in the
microarray to identify regulatory factors and immune processes that are
suppressed during scale regeneration. The results revealed that 3 days after scale
removal 109 genes were down-regulated. The 72 annotated probes were
analysed using STRING and the most represented biological processes were
vascular endothelial growth factor receptor signalling (3.2e-4
), endothelial cell
proliferation (3.2e-4
), cell migration and motility (8.4e e-4
), hydrogen peroxide
metabolic process (6e-3
) and leukocyte migration (6.7 e-3
). Several genes
associated with innate and acquired immunity and the inflammatory response
(17/72) were identified amongst down-regulated transcripts, although in the
previous study up-regulated immunoendocrine genes were identified (10/53).
To carry out a detailed analysis of the neuroimmunoendocrine response during
regeneration a new experiment was performed in which scales were removed
from the left flank of adult sea bream and regenerating and intact skin and
scales were collected at 0 and 6 hours and 1, 2, 3 and 4 days after scale removal.
The results revealed that genes related with the cellular response of the innate
immunity, such as myeloperoxidase (mpo), cytochrome b-light chain (p22phox)
and macrophage colony stimulating factor 1 receptor (csf1r) were down-
regulated while the expression of galectin-3-binding protein precursor (gal3)
and receptor-transporting protein 2 (rtp2) varied during the time course studied.
This study demonstrated that during four days of skin and scale regeneration in
sea bream there is a balance between different regulatory processes that dictate
the stimulation and impairment of immune related genes.
Acknowledgements: RC is in receipt of FCT scholarship SFRH/BD/81625/2011
grant. Vieira et al. BMC Genomics 2011, 12:490
31
O16
MULTI-CLASS SCREENING OF FEEDSTUFF CONTAMINANTS AND
POTENTIAL ENDOCRINE DISRUPTORS IN FARMED FISH
Jaime Nácher-Mestre1, R. Serrano
1, M. Ibáñez
1, T, Portolés
1, M.H.G.
Berntssen2, F. Hernández
1, J. Pérez-Sánchez
3. E-mail: [email protected]
1Research Institute for Pesticides and Water (IUPA), University Jaume I.
2National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029,
Nordnes, N-5817 Bergen, Norway.
3Nutrigenomics and Fish Growth Endocrinology Group, Institute of
Aquaculture Torre de la Sal (CSIC).
Food and feedstuffs could seat undesirables associated with potential risk for
animal welfare and food safety. Pesticides, polycyclic aromatic hydrocarbons
(PAHs), classical halogenated organic contaminants (HOCs), mycotoxins,
emergent pharmaceuticals and personal care products (PPCPs), marker dyes
among others, may be an unintentionally contamination source. These
contaminants have several potential toxic outcomes by several modes of action,
including endocrine disruption and genotoxicity. To be aware of current and
recurrent disrupting affairs, an analytical strategy based on a generic extraction
followed by ultra-high performance liquid chromatography (LC) and gas
chromatography (GC) both coupled to high resolution mass spectrometry
(HRMS), using a quadrupole time-of-flight mass analyzer (QTOF MS), has
been developed and applied to real world samples.
Up to our knowledge, HOCs and mycotoxines do not represent a problem in
food safety although their study remains relevant. Only residue levels of
pirimiphos-methyl and chlorpyrifos have been mainly found in plant based feed.
These pesticides may disturb steroid production and biosynthesis, and
histopathological changes have been reported in animals. In addition, some light
PAHs have been found. These compounds may be related to steroid level
suppression and steroid synthesis inhibition as well as retinoid signaling
disruption. In some cases, the presence of unintended background levels of
illegal pharmaceutical dyes, unreported antibiotics, or unauthorized anti-
inflammatory agents might give cause to legal actions, making their monitoring
relevant. The potential applicability of this strategy helps to assure possible
emergent risk exposition of food and feed industry to more than 2500
compounds, including previous non-reported undesirables. The proposed
strategy is presented as a useful risk assessment tool in order to widen the
knowledge on novel ingredients and also traditional ingredients under use.
The study was funded by the ARRAINA EU Project.
32
O17
ACUTE STRESS REGULATES VASOTOCINERGIC AND
ISOTOCINERGIC SYSTEM IN THE GILTHEAD SEA BREAM (Sparus
aurata)
Arleta Krystyna Skrzyńska1, E. Maiorano
1, M.Bastaroli
1, G. Martínez-
Rodríguez2, J.A. Martos-Sitcha
2, J.M. Mancera
1. E-mail:
1Department of Biology, Faculty of Marine and Environmental Sciences,
Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cádiz,
11510 Puerto Real, Cádiz, Spain; 2Instituto de Ciencias Marinas de Andalucía,
Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto
Real, Cádiz.
The hypothalamus-pituitary-interrenal axis is involved in stress response.
Although corticotrophin releasing hormone (CRH) is considered the main
hormone that activates this axis, other hormones as CRH binding protein (CRH-
BP), arginine vasotocin (AVT) and isotocin (IT) are considered as important
players in stress regulation. This study assessed, using the gilthead sea bream
(Sparus aurata) as a biological model, hypothalamic mRNA expression changes
of different endocrine factors (CRH, CRH-BP), as well as different
neuropeptides (AVT e IT) and their receptors (AVTR V1-type, AVTR V2-type,
and ITR) after an acute stress situation. Specimens were exposed to air for 3
min by lifting the wire-net cage out of the tank and put back in their respective
tanks after that, being sampled in a time course response (15 min, 30 min, 1, 2,
4 and 8 hours post-stress). At hypothalamic level, acute stress affects mRNA
expression of all precursors (pro-VT and pro-IT) and receptors (AVTRs and
ITR), as well as hormonal factors (CRH and CRH-BP), by increasing and
modulating their mRNA expression levels from the first 15 min post-emersion.
Furthermore, our data provide evidence on existence of different variation
pattern in the mRNA expression of both AVT receptors suggesting that each of
them can be involved in different phases of stress response to cope with this
adverse situation. Thus, the AVT V1-type receptor can be responsible for
primary endocrine control while the AVTR V2-type can be involved in the
regulation of several metabolic pathways to support energy demand
enhancement required during stress processes. Our results demonstrate the
activation of both endocrine pathways in the regulation of hypothalamic-
pituitary-interrenal axis of S. aurata, which is stated, at least, through changes in
mRNA expression levels of these genes.
33
O18
BRAIN MONOAMINERGIC NEUROTRANSMITTERS DURING
CHRONIC STRESS IN RAINBOW TROUT (Oncorhynchus mykiss).
Fatemeh Naderi1*
, Marcos A. López-Patiño1, Manuel Gesto
$, José M. Cerdá-
Reverter2, Cristina Otero-Rodiño
1, Cristina Velasco
1, Jesús M. Míguez
1. E-mail:
1Lab. Fisioloxía Animal. Dept. Bioloxía Funcional e Ciencias da Saúde, Fac.
Bioloxía, Universidade de Vigo; and ECIMAT. 2Dept. Fish Physiology and
Biotechnology, Inst. Acuicultura de Torre de la Sal (IATS-CSIC). $: Current
address: Natl. Institute of Aquatic Resources, Technical University of Denmark.
Stress is negatively affecting animal welfare in such a way that most behavioral
and physiological functions, such as food intake, are jeopardized. In fish, the
stress response initiates with the activation of the hypothalamus-sympathetic-
cromaffin (HPC), and the hypothalamus-pituitary-interrenal cells (HPI) axes
leading to increased plasma catecholamines and cortisol levels. In addition, our
previous results point to the important role played by both dopaminergic and
serotonergic systems in initiating the response to acute stress. However, little is
known regarding the involvement of both systems under chronic stress, but also
their influence on food intake. To address that question an experiment was
performed consisting on stressing rainbow trout (Oncorhynchus mykiss) by high
stocking density for 3 and 10 days. Food intake was evaluated all over the
experiment in non-stressed and stressed fish. Plasma cortisol, glucose and
lactate levels, the content of DA and 5-HT, and their main metabolites (DOPAC
and 5-HIAA) in different brain regions, and hypothalamic TPH1 mRNA
abundance were assessed. Our results reveal that stressed animals showed
reduced food intake, increased plasma cortisol levels, and enhanced
dopaminergic and serotonergic activities in telencephalon, hypothalamus, optic
tectum and hindbrain, independently of the stress duration. TPH1 mRNA
abundance also increased in stressed trout at both 3 and 10 days. Our present
results support the hypothesis of a key role played by both dopaminergic and
serotonergic systems in initiating and maintaining the neuroendocrine response
to stress. In addition their involvement in mediating the stress-related food
intake inhibition might be taken in consideration.
Acknowledgements: Funded by Ministerio de Economía y Competitividad and
European Fund for Regional Development (AGL2013-46448-3-1-R and
FEDER) and AEO-ECIMAT.
34
O19
PLASTIC PHYSIOLOGY, SOCIAL BEHAVIOUR AND ENDOCRINE
PROFILES OF AN INVASIVE CICHLID IN SOUTHERN PORTUGAL
Flávia B. Silva, J.L. Saraiva, M. Vargas, M. Silva, J. Soares, A.V.M. Canário
and Pedro M. Guerreiro. Email:[email protected]
CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.
Invasive species are a growing concern for habitats worldwide. The chanchito,
Australoheros facetus, is a neotropical cichlid which displays high tolerance to
abiotic factors and marked social behaviours. The species is currently found in
the upper regions of the Guadiana, Sado and Odelouca basins in habitats with
striking seasonal variations in hydrological regimes. We studied its endocrine
physiology in the context of environmental acclimation, hierarchy formation
and reproductive behavior.
Fish were exposed to a range of salinities of upper estuaries during short and
long-term trials. A significant reduction in growth and in social behavior was
noticed at higher salinities (18 ppt), at which plasma osmolality, cortisol and
enzymatic activity were significantly elevated. Whether the rise in cortisol is
related to salinity acclimation mechanisms or a result of stress is still to be
determined.
Monogamous pairs establish and defend breeding territories. Social groups were
followed at different periods of the year. Dominant status was attributed to fish
that formed a territorial pair and a subsequent linear hierarchy ensued.
Dominance indexes and fish size are highly correlated (?: R=0.55; ?: R=0.84;
p<0.05) and even very small size differences account for social ranking. No
correlation to sex, GSI or HIS was found. Significant differences in initial and
final 11K-testosterone(11KT) levels occurred among dominant and subordinates
males but not in females of different status. Estradiol(E2) in non-territorial
males was slightly reduced after hierarchy formation. Females showed high but
variable E2 levels not related to status.
Size is key in hierarchy formation but 11KT may influence male status, while
the variance found in females E2 may reflect different maturation stages.
Members of the pair perform parental duties during offspring development but
males spend more time patrolling while females care for the young. Whether
such behaviors are under hormonal control is currently under investigation.
FBS has a Science Without Borders doctoral fellowship from the Brazilian
CNPq. Funded partially by FCT - Foundation for Science and Technology -
UID/Multi/04326/2013.
35
O20
TEMPERATURE IMPRINTING DURING EARLY DEVELOPMENT
AND THE ADULT STRESS AND IMMUNE RESPONSE
Ana Patrícia Mateus1,2
, R. Costa1, J.R. Cardoso
1, E. Gisbert
3, D.M. Power
1.
Email: [email protected]
1Comparative Endocrinology and Integrative Biology, Centre of Marine
Sciences, University of Algarve, Gambelas, 8005-139 Faro, Portugal, 2Health
Superior School, University of Algarve, Av. Dr. Adelino da Palma Carlos, 8000-
510 Faro, Portugal, 3IRTA, 43540 Sant Carles de la Ràpita, Spain
Despite the variations in the HPA axis organization in mammals and fish, the
regulatory loop is conserved. CRH is released in response to stress, stimulating
the production of POMC/ACTH in the pituitary and the subsequent release of
cortisol by modulation of StAR in the head kidney in fish. Since fish do not
thermoregulate, changes in early life temperatures are sub-lethal stressors which
have a strong impact on the development plasticity of fish.
The impact of thermal imprinting in early life on the plasticity of the HPI axis
and stress response in juvenile seabream (Sparus aurata) was assessed by
rearing fish under 4 thermal regimes: 2 constant (18-18ºC; 22-22ºC) and 2
variable temperatures between egg incubation and larval rearing (22-18ºC; 18-
22ºC); and exposing the resulting juvenile to acute confinement stress to
monitor plasma cortisol levels and genes of the HPI axis by qPCR. Changes in
the immune response, a common result of stress, was also evaluated using
histomorphometric measurements of MMCs in the head kidney and by
monitoring transcripts related with their immunological role.
Thermal imprinting caused a change in whole animal physiology and growth,
reflected by a significant increase in pituitary GH. Early thermal history
significantly modified HPI responsiveness as assessed by the amplitude of the
cortisol response, reflected by a significant shift in the relative abundance of
pituitary POMC and head kidney StAR. Furthermore, a modification in immune
responsiveness after an acute stress challenge was evident and there was a
decrease in the number of MMCs and a significant down-regulation of DCT
after acute stress. The results demonstrate the impact of early life history on
juveniles of the teleost sea bream.
Acknowledgements: This study was financed by the European Regional
Development Fund (ERDF) COMPETE and Portuguese funds through
Foundation for Science and Technology (FCT) under the project
UID/Multi/04326/2013 and the EC FP7 Program (2007-2013) grant agreement
nº 222719 – Lifecycle.
36
O21
ACTIVATION OF SOMATOSTATIN RECEPTOR 3 REDUCES CELL
VIABILITY AND HORMONAL SECRETION IN NON-FUNCTIONING
PITUITARY ADENOMAS THROUGH MAPK SIGNALING
Mª Carmen Vázquez-Borrego1, A. Ibáñez-Costa
1, E. Venegas-Moreno
2, A.
Toledano-Delgado3, A. Soto-Moreno
2, MA. Gálvez
4, MD. Gahete
1, JP. Castaño
1,
RM. Luque1. E-mail: [email protected]
1Department of Cell Biology, Physiology and Immunology, University of
Córdoba (UCO); Hospital Universitario Reina Sofía (HURS); Instituto
Maimónides de Investigación Biomédica de Córdoba; CIBERobn and ceiA3,
Córdoba, Spain; 2Metabolism and Nutrition Unit, Hospital Virgen del Rocío,
Sevilla; 3
Service of Neurosurgery, HURS; 4Service of Endocrinology and
Nutrition, HURS/IMIBIC.
Non-functioning pituitary adenomas (NFPA) are characterized by the lack of
hormone hypersecretion and are associated to severe comorbidities related to
mass effects. Surgical resection of NFPAs is generally needed because
pharmacological treatments using currently available somatostatin analogs (SA)
are ineffective and therefore, identification of novel therapies deem necessary to
manage this pathology. One of the reasons of this lack of response to SA might
be related to the fact that NFPAs express high levels of sst3 and low sst2 and
sst5 levels. Thus, the aim of this study was to determine the effect of an sst3-
specific non-peptidic analogue (L-796,778; Merck) on key functional
parameters (cell viability, apoptosis/necrosis, hormone secretion and
intracellular signaling) in primary human NFPA cell cultures. Firstly, we
analyzed the expression profile of a series of 39 NFPAs and 10 normal
pituitaries (NP) by quantitative real-time PCR. We found a clear alteration in the
expression of key hormones (increased FSH levels) and receptors (increased
sst3 and truncated sst5TMD4 levels) in NFPAs compared to NP. Remarkably, in
vitro treatment of NFPA cultures with L-796,778 significantly reduced cell
viability and chromogranin-A secretion and increased necrosis by a Ca2+
-
independent mechanism. These direct effects of L-796,778 might be mediated
by the inhibition of MAPK signalling (ERK1/2-phosphorylation). In
conclusion, our study provides new evidence regarding the role and potential
clinical implications of sst3 in the pathophysiology of NFPAs, suggesting that
pharmacological treatment specifically targeting sst3 could be a promising
therapeutic alternative for this pathology.
Funding: CTS-1406, PI-0639-2012, BIO-0139, PI13-00651, BFU2013-43282-
R, CIBERobn and Merck Serono.
37
O22
LACK OF CORTISTATIN OR SOMATOSTATIN PLAYS
DIFFERENTIAL ROLES IN THE CONTROL OF MAMMARY GLAND
TUMORIGENESIS IN LEAN AND OBESE MICE
Alicia Villa-Osaba1,2,3
, MD Gahete1,2,3
, F López-López1,2,3
, AI Pozo-Salas1,2
, R
Sánchez-Sánchez4, R Ortega-Salas
4, M Álvarez-Benito
2,5, J López-Miranda
2,3,6,
RM Luque1,2,3
, JP Castaño1,2,3
. Email: [email protected]
1Department of Cell Biology, Physiology, and Immunology, University of
Córdoba. 2Instituto Maimónides de Investigación Biomédica de Córdoba
(IMIBIC), Hospital Universitario Reina Sofía (HURS). 3CIBERobn.
4Anatomical Pathology Service, HURS.
5Mammary Gland Unit, HURS.
6Lipids
and Atherosclerosis Unit, HURS; Córdoba, Spain.
Somatostatin (SST) and cortistatin (CORT) are two peptides that exert common
biological actions, including the suppression of tumor cell proliferation,
offering a potential therapeutic target. However, attempts to apply SST analogs
in the treatment of breast cancer have been controversial and the specific role of
SST and/or CORT in mammary gland tumorigenesis remains uncertain,
especially in obesity. We studied the role of endogenous SST and CORT on
carcinogen-induced mammary gland tumorigenesis under normal-weight and
obesity conditions, by treating SST- and CORT-knockout (KO) and littermate-
controls mice, fed a low fat (LF) or a high fat (HF) diet, with 7, 12-
dimethylbenzaanthracene. Lack of SST did not impact DMBA-induced tumor
incidence under lean conditions, while lack of CORT aggravated DMBA-
induced tumorigenesis in LF-fed mice. These differences were not attributable
to altered mammary gland development or changes in relevant circulating
hormones. Interestingly, HF-diet increased the sensitivity to DBMA-induced
carcinogenesis in control and SST-KO mice while, tumor incidence was not
statistically altered in HF-diet compared with LF-diet CORT-KO; however,
tumor incidence was already significantly elevated in LF-fed CORT-KO, which
might preclude a further elevation in obesity. Therefore, our data indicate that
endogenous SST and CORT distinctly contribute to the control of DMBA-
induced tumorigenesis, which is strongly influenced by the metabolic/endocrine
milieu, suggesting that CORT, rather than SST, might represent a key inhibitor
factor of mammary tumorigenesis. Thus, these data invite to suggest that CORT-
like rather than SST-like molecules could be more promising tools for the
treatment of breast cancer tumors.
Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-
2012 and CIBERobn.
38
O23
THE TRUNCATED SOMATOSTATIN RECEPTOR SST5TMD4 IS
OVEREXPRESSED IN PROSTATE CANCER AND INCREASES
AGGRESSIVENESS FEATURES THROUGH REGULATION OF
ANGIOGENIC FACTORS AND WNT/B-CATENIN PATHWAY.
Daniel Hormaechea-Agulla1, MD Gahete
1, A Ibáñez-Costa
1, E Gómez-Gómez
2,
JA Ramos-Fernández1, J Valero-Rosa
2, J Carrasco-Valiente
2, MM Moreno
3, MD
Culler4, MJ Requena
2, JP Castaño
1, RM Luque
1. Email:
1Department of Cell Biology, Physiology and Immunology, University of
Córdoba, Hospital Universitario Reina Sofía (HURS), Instituto Maimónides de
Investigación Biomédica de Córdoba (IMIBIC), CIBERobn and, ceiA3;
Córdoba, Spain. 2Urology Service, HURS.
3Anatomical Pathology Service,
HURS; Cordoba, Spain. 4IPSEN Bioscience, Cambridge, MA, USA.
Somatostatin is a highly conserved, pleiotropic neuropeptide that binds to a
family of G protein-coupled receptors with seven transmembrane domains
(TMD), called somatostatin receptors (sst1-5), to exert its biological function.
Our group has identified and characterized in several species additional sst5
variants originated from alternative splicing that lack several TMDs.
Particularly, the human sst5TMD4 variant, a receptor with four TMDs, seems to
have pathological consequences since it is abundantly expressed in a subset of
pituitary tumors and breast cancer samples, wherein it could exacerbate the
malignant phenotype. Here, we analyzed the presence and role of sst5TMD4 in
human prostate cancer (PC). We found a clear overexpression of sst5TMD4 in
PC samples compared to control tissues and, its expression was higher in
tumors from patients with metastasis compared to those without metastasis.
Thus, the in vitro functional role of sst5TMD4 was explored by using androgen
dependent (VCaP) and independent (PC-3) PC cell lines. Specifically,
sst5TMD4 overexpression increased cell proliferation and migration in both cell
lines while, its silencing by siRNA decreased basal cell proliferation. Moreover,
several important pathways were deregulated after overexpression of
sst5TMD4, including a dramatic downregulation of two tumor suppressor
proteins from the Wnt/B-catenin pathway (APC and SFRP1) and an increase of
angiogenic factors such as VEGF and ANG1. Altogether, our results suggest a
relevant role of sst5TMD4 in PC where is associated to enhanced features of
aggressiveness through regulation of angiogenic factors and Wnt/B-catenin
pathway.
Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-
2012 and CIBERobn.
39
O24
IN1-GHRELIN INCREASES MALIGNANCY FEATURES OF BREAST
CANCER CELL LINES
David Rincón-Fernández, MD Gahete, V Ruiz-Murillo, R Santamaría, RM
Luque, JP Castaño. E-mail: [email protected]
Department of Cell Biology, Physiology and Immunology, University of
Córdoba, Maimonides Institute for Research in Biomedicine of Cordoba
(IMIBIC), Reina Sofia University Hospital and CIBER Fisiopatología de la
Obesidad y Nutrición (CIBERobn). 14014-Córdoba, Spain
Aberrant alternative splicing is a hallmark of tumoral process that can worsen
cancer outcome by inducing the appearance of alternative proteins, which can
increase malignancy features of the tumoral cells. Our group has recently
discovered a splicing variant of the ghrelin gene, named In1-ghrelin, which
results from the retention of intron 1. In1-ghrelin variant is overexpressed in
breast cancer, and its expression correlates with proliferation markers,
suggesting the involvement of In1-ghrelin in the development and/or
progression of this pathology. The aim of this study was to determine the
functional role of In1-ghrelin (and native-ghrelin) in two breast cancer derived
cell lines: MDA-MB-231 and MCF7. Particularly, treatment with In1-ghrelin
peptides or native-ghrelin stimulated proliferation and migration in both cell
lines. Similarly, overexpression of native-ghrelin and In1-ghrelin increased the
proliferation rate, while only In1-ghrelin overexpression was able to increase
migration ability in MDA-MB-231 and MCF7 cells. These results were further
corroborated by silencing endogenous In1-ghrelin in MDA-MB-231 cells. In
addition, In1-ghrelin, but not native-ghrelin, overexpression induced an increase
in pERK1/2 and a decreased in pAKT. We then analyzed dedifferentiation
processes in response to native-ghrelin and In1-ghrelin by studying the
percentage of cells with mesenchymal-like phenotype and found that In1-
ghrelin, but not ghrelin, increased epithelial to mesenchymal transition in
MDA-MB-231 cells. In addition, we studied the capacity to form
mammospheres and found that only In1-ghrelin overexpression and treatment
led to the formation of more and bigger mammospheres. In summary, our data
demonstrate that In1-ghrelin splicing variant increases malignancy features of
breast cancer cells from two different cell models, suggesting a relevant role of
In1-ghrelin in breast cancer development and/or progression.
Funding: BFU2013-43282-R, PI13-00651, BIO-0139, CTS-1406, PI-0639-
2012 and CIBERobn.
40
025
EXPLORING ALLATOSTATIN-A, KISS AND GALANIN GPCRS IN
LOPHOTROCOZOANS
João C.R. Cardoso, N. Zaghdoudi-Allan, R.C. Félix, D.M. Power. E-mail:
Comparative Endocrinology and Integrative Biology, Centre of Marine
Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,
Portugal
The Allatostatin type-A (AST-A), kisspeptin (KISS) and galanin (GAL) G-
protein coupled receptor (GPCRs) and their activating peptides play a major
role in the regulation of feeding, energy metabolism and reproduction.
Receptors and peptides of this family emerged early in the bilaterians however
relatively few studies of their evolution and function in invertebrates exist. In
nematodes and arthropods, only the AST-A system exists, suggesting that KISS
and GAL receptors and peptides were lost during the protostome radiation. The
occurrence of the AST-A, KISS and GAL system in Lophotrocozoans, a major
group of the animal kingdom remains largely unstudied. The aim of the present
study was to exploit available molecular data for the Lophotrocozoans to study
the evolution of the AST-A/KISS/GAL system in protostomes and to compare it
with the deuterostomes. The existence of the peptides and receptors in the
genomes and transcriptomes of representatives of several Lophotrocozoan
clades (annelids, gastropods, bivalves, bryozoans, rotifers and
plathyhelminthes) was established. The Lophotrocozoan AST-A and KISS
receptors were compared with those in vertebrates and arthropods and revealed
that orthologues existed in the genomes of all the species analysed but that GAL
receptors were only present in annelid genomes. AST-A and KISS receptor gene
expansion was observed in some species suggesting that receptor gene
evolution was affected by lineage-specific events. The results from
Lophotrocozoans supported the recent hypothesis that AST-A/KISS/GAL
GPCRs were under distinct evolutionary pressures in the protostomes and that
the AST-A and the KISS receptors are more closely related than the GAL
receptors (Felix et al., 2015).
Acknowledgements: Funded through Foundation for Science and Technology
(FCT) through project UID/Multi/04326/2013 and European Union Seventh
Framework Programme (grant agreement n° 605051, CACHE-ITN). JCRC is
funded by an auxiliary research contract (UID/Multi/04326/2013), NA by
CACHE-ITN and RCF by FCT SFRH/BPD/89811/2012 grant.
41
O26
RESPONSIVENESS OF SEA BASS SCALES TO ESTRADIOL AND
GENISTEIN
M. Dulce Estêvão 1,2
, P. I. S. Pinto 1, A. Andrade
1, S. Santos
1 and D. M. Power
1. E-mail: [email protected]
1 CCMAR - Centre of Marine Sciences, Universidade do Algarve, Faro,
Portugal. 2Escola Superior de Saúde, Universidade do Algarve, Faro, Portugal
Phytoestrogens are plant-derived compounds with estrogen-like activity, to
which fish may be exposed in the wild due to aquatic contamination or in
aquaculture through ingestion of fish feeds enriched in plant-based ingredients.
While several endocrine-disrupting effects have been described for
phytoestrogens in fish reproductive functions, little is known about their
impacts on mineralized tissues. The present study focused on a fish scale model
because of their well recognized responsiveness to estrogen (E2). European sea
bass were exposed to estradiol and the phytoestrogen genistein (Gen) and their
effects on calcium and phosphorus plasma levels and the activities of alkaline
phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers of
osteoblast (OSB) and osteoclast (OSC) activity respectively, were evaluated.
The repertoire of nuclear and membrane ERs was characterized, plasma
vitellogenin (Vtg) was quantified and candidate E2 responsive genes were
analyzed in scales and liver. Results show that Ca and P plasma content
significantly increased 5 days after E2 injection although Gen had no effect. In
contrast, TRAP and ALP activities significantly increased 1 d after Gen
injection although 5 d after injection no significant effect was detected and E2
failed to stimulate these enzymes. Both Gen and E2 induced an increase in
plasma Vtg levels 1 d after the injection but after 5 d this effect was only
induced by E2. Both E2 and Gen induced significant changes in gene expression
in scales and an overlapping but not identical response was found in the liver,
suggesting different mechanisms of action and responsiveness to estrogens in
these tissues. In line with this observation different patterns of ERs expression
occurred in scales and liver. Overall the results suggest that exposure to
estrogens and phytoestrogens impacts on both liver (a classical E2 responsive
tissue) and mineralized tissues modifying their homeostasis and ultimately their
function.
Acknowledgements: Funded by FCT – Portuguese Science and Technology
Foundation through projects UID/Multi/04326/2013 and PTDC/AAG-
GLO/4003/2012 and grant FCT SFRH/BPD/25247/2005.
42
027
AN APPROACH TO STANNIOCALCIN SECRETION IN GILTHEAD
SEABREAM (Sparus aurata)
Ignacio Ruiz-Jarabo, S.F. Gregório, J. Fuentes. E-mail: [email protected]
Centre of Marine Sciences (CCMar), Universidade do Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
Calcium balance is of vital importance for vertebrates. In teleost fish, the most
important endocrine modulators of calcium homeostasis are stanniocalcin
(STC) and the parathyroid hormone-related protein (PTHrP), which act as
antagonists. In turn, both hormones are apparently regulated by a calcium-
sensing receptor (CaSR), and no other regulators are yet described for either
hormone. In this sense, teleost fish provide an unique model to study STC
regulation, as they have a specialized STC-producing gland called the
corpuscles of Stannius (CS). Here, using the seabream as a model, we have
developed an ex vivo assay to culture CS of fish and a competitive ELISA
method to measure STC concentrations. STC release from the CS is mediated
by a CaSR, as indicated by the effects of incubation with different calcium
levels, and the use of calcimimetic and calcilytic compounds. Moreover, we
describe for the first time the presence of two PTHrP receptors in the seabream
CS i.e. PTHR1 and PTHR3. Thus, ex vivo incubations revealed a dose-response
inhibitory effect of PTHrP on STC secretion under basal Ca2+
concentrations.
This inhibition is carried out through specific and reversible second messenger
pathways i.e. transmembrane adenylyl cyclases and phospholipase C. Together,
these results revealed a fascinating novel process of endocrine modulation
between two antagonist hormones, involved in calcium regulation, that
resembles the terrestrial regulation of calcium balance in the parathyroid gland.
Supported by PTDC/MAR/104008/2008, FCT, Portugal
43
O28
TARGETED PTH4 CELL ABLATION LEADS TO AN UNCOUPLING
OF BONE FORMATION AND MINERALIZATION
Paula Suarez-Bregua1,2
, A. Saxena3, M.E. Bronner
3, P. Moran
2 and J. Rotllant .
E-mail: [email protected]
1Aquatic Molecular Pathobiology Lab, IIM- CSIC, Vigo, Pontevedra, Spain
.
2Dept. of Biochemistry, Genetics and Inmunology, University of Vigo.
Pontevedra, Spain. 3Division of Biology and Biological Engineering, California
Institute of Technology, Pasadena, United States.
Bone formation and mineralization are dynamic processes that occur during the
embryonic development but also along adult life through remodeling.
Mineralization is the most distinctive feature of the bone as it makes the
skeleton a great store of phosphate and calcium in order to contribute to the
mineral balance in vertebrates. Recent studies have provided evidence that the
central and peripheral nervous systems directly control bone homeostasis.
Therefore, the hypothalamus has been documented to be a potent regulator of
the skeletal mass through efferent neural signals and/or neuroendocrine signals.
Given its “sensing” properties, the hypothalamus is able to integrate
physiological information from the whole body and sends inputs to bone cells
that regulate skeletal mass accordingly. We have recently identified a new PTH
family member in zebrafish, Parathyroid hormone 4 (Pth4), produced by two
clusters of cells in the lateral hypothalamus. We found that Pth4 is involved in
bone mass accrual through phosphate homeostasis regulation in adult fish,
which indicates a new functional link between brain and bone. To better
understand Pth4 and its role during skeletal development, we have characterized
Pth4-expressing cells and performed single-cell resolution two-photon laser
ablation and confocal imaging in vivo. We found that hypothalamic cells
expressing pth4 are post-mitotic neurons. Targeting these neurons via two-
photon laser ablation resulted in impaired bone mineralization and altered
expression of mineralization and phosphate-regulating genes as well as
osteoblast differentiation markers. Collectively, these results suggest that Pth4-
expressing hypothalamic neurons may play a key in the regulation of bone
metabolism, possibly through regulating phosphate balance.
This work was funded by the Spanish Science and Innovation Ministry project
AGL2014-52473R to JR. Paula Suarez Bregua was supported by Campus Do
Mar-Xunta de Galicia PhD fellowship.
44
O29
THE GENE REGULATORY NETWORK UNDERPINNING DORSAL–
VENTRAL PIGMENTATION PATTERNING IN FISH. ANALYSIS OF
ASIP1 CIS-REGULATORY LANDSCAPE
Laura Cal1, I. Braasch
2, J.L.Gómez-Skarmeta
3, R. Kelsh
4, J.M. Cerdá-Reverter
5, J. Rotllant
1. E-mail: [email protected]
1Institute of Marine Research, CSIC. Vigo, Spain.;
2Institute of Neuroscience,
University of Oregon, EE.UU.; 3CABR-CSIC;
4University Of Bath, UK.,
5Instituto de Acuicultura de Torre de la Sal (IATS-CSIC). Castellón, Spain.
Dorso-ventral pigment pattern differences are the most widespread pigmentary
adaptations in vertebrates. In mammals, ASIP have been reported as the key
factor in this process by regulating melanin types and levels in melanocytes. In
fish, studies of pigment patterning have focused on stripe formation, identifying
a core striping mechanism dependent upon interactions between different
pigment cell types. We have recently demonstrated that, in fact, zebrafish utilize
two distinct adult pigment patterning mechanisms -an ancient dorso-ventral
patterning mechanism, and a more recent striping mechanism based on cell-cell
interactions; remarkably, the dorso-ventral patterning mechanism also utilizes
Asip. We hypothesize that agouti have a conserved role in the countershading
pigment pattern formation in vertebrates. Yet the genetic mechanisms
underlying this dorso-ventral asip1 expression gradient remain largely
unexplored. Thus, we report here the asip1 expression in the holostei spotted
gar (Lepisosteus oculatus), one of Darwin's defining examples of 'living fossils'
member of a lineage diverged from teleosts before to Teleost Genome
Duplication (TGD), and through phylogenetic footprinting and transgenesis in
zebrafish, we examined the cis-regulatory landscape of Asip1.
Funded by MINECO (ALG2011-23581) and Xunta de Galicia (INCITE-09 402
193)
46
P1
INFLUENCE OF AVT AND CORTISOL TREATMENT ON STRESS AND
THYROID PATHWAYS IN THE GILTHEAD SEA BREAM (Sparus
aurata)
Juan Antonio Martos-Sitcha1,2
, I. Jerez2, G. Martínez-Rodríguez
1, J.M.
Mancera2. E-mail: [email protected]
1Instituto de Ciencias Marinas de Andalucía, Consejo Superior de
Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto Real, Cádiz, Spain. 2Department of Biology, Faculty of Marine and Environmental Sciences,
University of Cádiz, Campus de Excelencia Internacional del Mar (CEI-MAR),
11510 Puerto Real, Cádiz, Spain.
The endocrine system could be considered as a complex net of pathways for
which an interconnection between some of them cannot be ruled out. Cortisol,
the main corticosteroid in teleosts, is the final step on the hypothalamic-
pituitary-interrenal axis, and its functions are related to intermediary
metabolism, osmotic and ionic regulation, growth, stress and immunity. In turn,
the vasotocinergic, homologous to the mammalian vasopressinergic pathways,
and thyroid systems have also an important role in several physiological
processes in which cortisol plays a role (e.g. osmoregulation, metabolism or
stress). The aim of this study was to evaluate the possible interaction between
different endocrine axes (vasotocinergic, stress and thyroid pathways) in the
gilthead sea bream (Sparus aurata), through assessing the effects of exogenous
arginine vasotocin (AVT) and cortisol administration. Specimens acclimated to
seawater (38 ‰ salinity) were intraperitoneally implanted with vegetable oil
(mixture 5:1 coconut and seeds oils) alone (control) or containing different
doses of AVT (0.5 or 1.0 ?µg/g body weight) or cortisol (50?µg/g body weight)
and sampled at 12 hours, and 1 and 3 days after injection. Thus, both hormones
produced a significant decrease of hypothalamic corticotrophin releasing
hormone (CRH) mRNA expression from 12/24 hours onwards, while CRH-
binding protein (CRH-BP) mRNA levels did not altered its values in any of the
intraperitoneally injected specimens during the time that the experiment lasted.
Moreover, AVT and cortisol administration independently produced a
significant decrease in thyrotropin-releasing hormone (TRH) gene expression
from 12 h post-injection, whereas an up-regulation in thyroid receptor β (TRβ)
mRNA expression was observed just 12 h after hormonal administration. Our
results clearly suggest that both AVT and cortisol hormones independently co-
regulate the functions of these endocrine systems.
47
P2
STUDY OF THE SOMATOTROPIC HORMONES EXPRESSION
DURING THE EARLY DEVELOPMENT OF THICK LIPPED GREY
MULLET (Chelon labrosus)
Neda Gilannejad, J.A. Martos-Sitcha, V. de las Heras, M. Yúfera, G. Martínez-
Rodríguez. E-mail: [email protected]
Instituto de Ciencias Marinas de Andalucía (ICMAN), CSIC, 11510 Puerto
Real, Cádiz.
Mullets are considered as potential species for sustainable aquaculture due to
their feeding characteristics. Among all the mullets found in the Spanish
territory, Chelon labrosus is having higher commercial interest because, in
comparison to the rest, it reaches larger sizes in a shorter period of time.
Recently hatched larvae go through a series of molecular, biochemical and
morphological changes during growth and development to juveniles. Some of
the processes involved in larval development, including the growth hormone
(GH) and insulin-like growth factor I (IGF-I), are subjected to endocrine
control. With the purpose of deepening the knowledge of the possible factors
involved in the early development and differentiation in this species, expression
of the two somatotropic genes, gh and igf1, was studied during the first 92 days
of life using real time PCR. These genes showed different expression patterns.
The expression of gh increased significantly, reaching to its maximum at 43
days post hatch (dph), and decreased drastically afterwards, maintaining
relatively low levels during the rest of the studied period. However, igf1 showed
a gradual raise, peaking at 50 dph, and maintaining thereafter relatively constant
levels. Moreover, the maximum growth rate, between 22 and 54 dph, coincides,
to a large extent, with elevated expression levels of gh and the beginning in the
increase of igf1 observed in this work. This can suggest that gh and igf1 are
expressed in an independent manner during the first 50 days of life, while the
GH/IGF-I axis starts to be functional afterwards.
Acknowledgments: This study has been partially funded by a grant for applied
innovation and transfer of knowledge in the professional formation of the
educative system from the Ministry of Education in Spain, entitled
“Optimización del Proceso de maduración y desove del mugílido Chelon
labrosus”, and co-financed by ESF. The authors acknowledge the IES Els
Alfacs (Sant Carles de la Rápita, Tarragona, Spain) for providing the eggs.
48
P3
LONG-TERM EFFECTS OF rBGH TREATMENT ON GH/IGFs AXIS IN
GILTHEAD SEA BREAM (Sparus aurata)
Emilio J. Vélez, M. Perelló, E. Lutfi, A. Moya, J. Fernández-Borràs, J. Blasco,
I. Navarro, E. Capilla, J. Gutiérrez. E-mail: [email protected]
Department of Physiology and Immunology, Faculty of Biology, University of
Barcelona, Barcelona 08028, Spain.
Fish growth is mainly regulated by the growth hormone (GH)/insulin-like
growth factors (IGFs) axis. GH is secreted by the pituitary and stimulates the
synthesis of IGF-I, which is the largest growth inducer in muscle and bone
tissues. IGF-I binds to membrane receptors activating different signalling
pathways to regulate metabolism and to promote growth. The aim of this work
was to study the long-term effects of sustained-release of recombinant bovine
GH (rBGH) in fingerlings of gilthead sea bream, in order to improve knowledge
on the role of GH/IGFs axis in this species. Six weeks post-injection, liver, bone
and muscle tissue samples were collected. mRNA levels of IGFs (IGF-I and II),
growth hormone receptors (GHR-I and II), IGF-I receptors (IGF-IRa and Rb), as
well as some IGF binding-proteins (IGFBP-2, 4 and 5) important for controlling
IGFs bioavailability, were measured by quantitative PCR. Body-weight results
showed that rBGH-injected fish grew significantly more than control fish. Gene
expression analyses indicated increased expression of several genes of the
GH/IGFs system in liver, whereas only minor differences were observed in bone
and muscle. Liver expression of GHR-II and both peptides, IGF-I and IGF-II,
were increased in rBGH-treated fish, whereas no differences were observed in
these molecules in the other tissues studied. Regarding IGF-IRs, in rBGH-
treated fish an increased expression of IGF-IRb was found in liver, whereas in
bone and muscle this receptor was down-regulated, as also IGF-IRa in bone.
With respect to IGFBPs, only IGFBP-2 showed an increase in the liver of
rBGH-treated fish, while IGFBPs 4 and 5 remained stable in muscle and bone
tissues. These findings reveal the main effects of long-term GH treatment in this
species through hepatic IGFs, and provide new insights to better understand the
endocrine regulation of fish somatic growth, which may contribute to improve
aquaculture production.
Thanks to Elanco Animal Health for kindly providing the rBGH. Supported by
MINECO (AGL2012-39768) and Catalonian Government (2014SGR-01371).
49
P4
HYPER- AND HYPO-OSMOTIC CHALLENGES MODIFY
HORMONAL PATHWAYS IN THE GILTHEAD SEA BREAM (Sparus
aurata): A MICROARRAY APPROACH
Juan Antonio Martos-Sitcha1,2
, J.M. Mancera2, J.A. Calduch-Giner
3, J. Pérez-
Sánchez3, M. Yúfera
1, G. Martínez-Rodríguez
1. E-mail:
1Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), E-11519 Puerto
Real, Cádiz, Spain. 2Department of Biology, Faculty of Marine and
Environmental Sciences, University of Cádiz, Campus de Excelencia
Internacional del Mar (CEI-MAR), E-11519 Puerto Real, Cádiz, Spain. 3Nutrigenomics and Fish Growth Endocrinology Group, Institute of
Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, E-12595
Castellón, Spain.
Euryhaline teleost can live in a wide range of environmental salinities. This
ability is carried out by different osmoregulatory and non-osmoregulatory
tissues, being gills, liver and hypothalamus very important due to their
implications in ion and water exchange, metabolism and endocrine regulation,
respectively. Transcriptomic analysis in these tissues by means of a specific
oligo-microarray revealed new aspects related to the osmoregulatory processes
mediated by different endocrine pathways after hypo- (38‰ ? 5‰) or hyper-
(38‰ ? 55‰) osmotic challenges to gilthead sea bream juveniles. In liver,
aldosterone-related signalling pathways, represented by 8 heat shock proteins
(DNAJC8, DNAJC11, DNAJC17, DNAJC30, HSP90AB1, HSPA14, HSPD1 and
HSPE1), showed an up-regulation in both salinity challenges, while other
enzymes related to cell growth (PRKCA), division (KRAS) and maturation
(PIK3C3) were down-regulated. In gills, serotonin, melatonin and thyroid
hormone pathways were affected by the differential up-regulation of genes
involved in xenobiotic and protective metabolism facing transfer to 5‰
(CYP1A1, SULT1A1 and UGT1A1) or 55‰ (CYP1B1, UGT2A3). A similar
pattern was also observed for several genes involved in the melatonin
metabolism pathway (CYP2J2, CYP1B1 and SULT1A1) in the hypothalamus of
specimens transferred to 5‰, whereas hyperosmotic challenge induced a clear
down-regulation in another gene (UGT1A9) involved in xenobiotic metabolism.
Taking together, these results point out the importance of liver, gills and
hypothalamus in the response to osmolality change, showing the differentially
regulated endocrine components and pathways that process the harmful
metabolites related to osmotic stress.
Acknowledgments: project Aquagenomics (MINECO; CSD2007-00002).
50
P5
CLONING, SEQUENCING AND EXPRESSION OF GENES BRDT AND
TERT IN SEABREAM (Sparus aurata): POTENTIAL NEW SEX
MARKERS IN FISH.
M. Úbeda-Manzanaro, J.B. Ortiz-Delgado, C. Sarasquete. E-mail:
Instituto de Ciencias Marinas de Andalucía ICMAN-CSIC
Campus Universitario Río San Pedro. CEIMAR. Apdo oficial, 11510, Puerto
Real, Cádiz
Applying molecular amplification and cloning techniques obtained cDNA
sequences of Brdt and Tert genes, which has allowed to analyze levels of
differential expression in gonads, brain and somatic tissues, by q-PCR
quantitative, comparatively, using two reference genes (β-actin and 18S rRNA)
At the same time, its cellular-tissue distribution will be analyzed, and
phylogenetic analyses are performed.
Tert gene encodes a catalytic subunit of the enzyme telomerase reverse
transcriptase, which is which keeps the telomeric ends of chromosomes,
protecting them from abnormal folds and their degradation. In the majority of
cells, telomeres will shorten progressively as this divides, and after a certain
number of successive divisions, the telomeres are so short that stops cell
division or cell apoptosis is induced. In mammals, the activity of telomerase in
tissues of adults is limited to stem cells with high potential for proliferation,
finding expression in the cells of the germline, tumors and neoplastic cells.
However, in fish the Tert gene seems to express themselves, so ubiquitous, in
different gonad, brain and somatic tissues.
On the other hand, the Brdt gene encodes a protein regulating the transcription,
which in mammals is expressed almost exclusively in the testis, and is essential
for the proper sexual function of males. BRDT protein control reorganization
post-meiotic of the genome that occurs after the hiperacetilation of histones in
the phase of elongation of the spermatids, to be crucial in the development of
spermatogenesis and therefore, determinants of male fertility.
The results for both genes showed relative expression levels significantly higher
in testis, in relation to ovarian, and other tissues, corroborating their great
importance during the reproductive process in general, and in the
spermatogenesis in particular. It was shown expression in postmeiotic stages, as
in spermatocytes and spermatids, and negative expression in gametes.
51
P6
AVT AND IT SYSTEMS MEDIATED METABOLIC EFFECTS
INDUCED BY AIR EXPOSICION IN THE GILTHEAD SEA BREAM
(Sparus aurata)
Arleta Krystyna Skrzyńska1, M. Bastaroli
1, E. Maiorano
1, G. Martínez-
Rodríguez2, J.M. Mancera
1, J.A. Martos-Sitcha
2. E-mail:
1Department of Biology, Faculty of Marine and Environmental Sciences,
Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cádiz,
11510 Puerto Real, Cádiz, Spain; 2Instituto de Ciencias Marinas de Andalucía,
Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto
Real, Cádiz.
Arginine vasotocin (AVT) and isotocin (IT) are considered as important players
in stress regulation. The present study assesses the response of both
vasotocinergic and isotocinergic systems in the gilthead sea bream (Sparus
aurata), through their specific receptors (AVTR V1a2-type, AVTR V2-type and
ITR), after an acute stress situation. Specimens were exposed to air for 3 min by
lifting the wire-net cage out of the tank and put back in their respective tanks
after that, being sampled in a time course response (15 min, 30 min, 1, 2, 4 and
8 hours post-stress). Plasma cortisol values increased after few minutes post-
exposure, decreasing during the experimental time while a metabolic
reorganization occurred in both plasmatic and hepatic levels. Thus, an
enhancement in hepatic glycogenolysis and lactate production, together with
higher plasma glucose and lactate levels, as well as a decrease in free plasma
proteins was observed. Acute stress response involves changes in gene
expression of both hypothalamic AVT and IT precursor levels as well as in their
specific hepatic receptors, which are differentially expressed depending on the
receptor type (AVTR V1-type, AVTR V2-type or ITR). This response suggests
that activation of both vasotocinergic and isotocinergic systems is important
during metabolic mobilization induced by an acute stress situation, which is
stated, at least, through changes in mRNA expression levels of these genes
analyzed.
52
P7
INFLUENCE OF GLUCOSE LEVELS ON LACTATE METABOLISM IN
BRAIN GLUCOSENSING AREAS OF RAINBOW TROUT
(Oncorhynchus mykiss)
Cristina Otero-Rodiño, M. Librán-Pérez, C. Velasco, J. Hernández-Pérez, R.
Álvarez-Otero, M. Conde-Sieira, J.L. Soengas. E-mail: [email protected]
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e
Ciencias da Saúde, Facultade de Bioloxía y ECIMAT, Universidade de Vigo
There is no direct evidence in fish brain demonstrating the existence of changes
in lactate metabolism in response to alterations in glucose levels to support the
existence of an astrocyte-neuron lactate shuttle (ANLS) in these areas.
Therefore, we induced in rainbow trout, through IP treatments, hypo-glycaemic
or hyper-glycaemic changes to assess the response of parameters involved in
putative ANLS in glucosensing areas like hypothalamus and hindbrain. To
distinguish those effects from those induced by peripheral changes in the levels
of metabolites or hormones, we also carried out ICV treatments with 2-deoxy-
D-glucose (2-DG, a non-metabolizable glucose analogue thus inducing local
glucopenia) or glucose. Finally, we also incubated hypothalamus and hindbrain
in vitro in the presence of increased glucose concentrations. As a whole, the
induction of changes in glucose levels in hypothalamus and hindbrain of
rainbow trout resulted in the activation of the glucosensing mechanism
dependent on glucokinase (GK) in these areas in agreement with similar
previous studies. However, when we assessed in these areas the response of
parameters related to lactate metabolism, the results obtained were
contradictory. The increase in glucose levels did not produce in general the
expected changes in those pathways with only a minor increase in their capacity
of lactate production. The decrease in glucose levels was however more clearly
related to a decreased capacity of the pathways involved in the production and
use of lactate, and this was especially evident after ICV treatment with 2-DG in
both areas. In conclusion, the present results only partially support the existence
of an ANLS in hypothalamus and hindbrain of rainbow trout relating glucose
availability to lactate production and use.
Acknowledgements. This study was supported by Ministerio de Economía y
Competitividad and European Fund for Regional Development (AGL2013-
46448-3-1-R and FEDER).
53
P8
ORGANOTIN ENDOCRINE DISRUPTORS ACT AS OBESOGENS IN
RAINBOW TROUT CULTURED ADIPOCYTES
Lutfi, E.1 Córdoba, M.
1 Nerín, G.
1 Porte, C.
2 Gutiérrez, J.
1 Capilla, E.
1
Navarro, I.1. E-mail: [email protected]
1Department of Physiology and Immunology, Faculty of Biology, University of
Barcelona, Barcelona 08028, Spain. 2Environmental Chemistry Department,
IDAEA-CSIC, 08034 Barcelona, Spain
In recent years, several environmental pollutants have been identified to act as
obesogenic compounds affecting endocrine signalling and lipid homeostasis.
Among them, organotins such as tributyltin (TBT) and triphenyltin (TPT) have
been the most widely studied in aquatic environments. In vitro and in vivo
studies have demonstrated that TBT and TPT promote adipogenesis trough
peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X
receptor (RXR) activation. The aim of the present study was to investigate in
vitro the effects of TBT and TPT on the development and lipid metabolism of
rainbow trout (Onchorynchus mykiss) primary cultured adipocytes. Results
obtained by oil red O staining indicated that TBT and TPT induce only slightly
lipid accumulation compared with the differentiation medium in the absence of
lipid mixture; but when present, lipid accumulation was significantly higher
with and without organotins. Moreover, triglyceride content measurement
revealed that TPT produces stronger lipid accumulation than TBT, although the
highest accumulation of triglycerides was observed when adding lipid mixture,
also in those cells exposed to TBT or TPT. Furthermore, protein expression of
two key regulators of adipogenesis was evaluated by immunofluorescence.
Results showed that TBT and TPT activate PPARγ and CEBPα protein
expression; however, the stronger effect was observed again when lipid mixture
was added to the differentiation medium. Overall, the activation of PPARγ and
CEBPα, together with the increase of triglyceride levels inside the cells
confirmed that TBT and TPT enhance adipocyte differentiation and act as
potential obesogens in rainbow trout. Moreover, our results highlighted that
lipid mixture is essential to achieve complete cell differentiation in cultured
adipocytes from this species. Additionally, quantitative real-time PCR analyses
are being performed to further clarify the effects of TBT and TPT on the gene
expression of lipid metabolism and cell proliferation/differentiation markers.
Supported by MINECO (AGL2011-24961) and DECO (2014SGR-01371).
54
P9
EFFECTS ON FEEDING RAINBOW TROUT WITH A LIPID-
ENRICHED DIET ON FATTY ACID SENSING, FOOD INTAKE
REGULATION AND CELLULAR SIGNALING PATHWAYS IN
HYPOTHALAMUS AND LIVER
Marta Librán-Pérez, C. Otero-Rodiño, C. Velasco, J. Hernández-Pérez, F.
Naderi, J.M. Míguez, J.L. Soengas. E-mail: [email protected]
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e
Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, E-36310
Vigo, Spain.
In previous studies carried out in rainbow trout, we have characterized in
hypothalamus and liver the presence and functioning of fatty acid-sensing
systems whose activation ultimately lead to decreased food intake. Since a
reduced food intake has been observed after feeding fish with lipid-enriched
diets, changes in fatty acid-sensing systems are expected in fish fed with diets
containing different lipid levels that have not been assessed yet. There is
evidence in rainbow trout for the presence and functioning of AMPK and
proteins involved in cellular signaling like mTOR and Akt but to date there is
no information in any fish tissue regarding the response of these sensors and
proteins to changes in the levels of nutrients like fatty acids. The aim of this
study in rainbow trout fed with low fat (LF) or high-fat (HF) diets was to
determine if the response of food intake, mRNA abundance of hypothalamic
neuropeptides, and fatty acid sensing systems in hypothalamus and liver is
similar to that previously observed when levels of specific fatty acid were raised
by injection and to determine if the phosphorylation state of AMPK, Akt and
mTOR display changes in hypothalamus and liver in response to changes in
dietary lipid levels. The increased levels of fatty acids in hypothalamus and liver
of rainbow trout fed the HF diet only partially activated fatty acid-sensing
systems with no changes in food intake allowing us to suggest that fatty acid-
sensing response in fish to increased levels of fatty acids is more dependent on
the presence of specific fatty acids rather than to the global increase in fatty
acids. In hypothalamus and liver, AMPK, Akt and mTOR are generally activated
in fish fed the HF diet, suggesting an enhanced response of the cellular
signaling pathways to the increased availability of fatty acids.
Funded by Ministerio de Economía y Competitividad and European Fund for
Regional Development (AGL2013-46448-3-1-R and FEDER).
55
P10
REGULATION OF ZEBRAFISH GAMETOGENESIS: A TRANSGENIC
APPROACH
Sandra Navarro1, R. Guillot
1, M. Mischitelli
2, E. Sánchez
1, R. Cortés
1, L.
Soletto1 & J.M. Cerdá-Reverter
1. E-mail: [email protected]
1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of
Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),
Castellón, Spain. 2
Department of Biological and Environmental Sciences,
University of Messina, S. Agata (ME), Italy.
Early puberty adversely affects growth, food efficiency conversion and market
values. Conversely, the delayed puberty can also be a problem for the
aquaculture which involves an increase in costs and health risk to maintain
stocks of fish.
In Poecilid fish, the first sexual maturation is regulated by a unique sex-linked
Mendilian locus named locus P. Recent experiments have demonstrated that
locus P is occupied by multiple copies of the melanocortin receptor 4 (MC4R).
Melanocortins are small peptides encoded by a complex precursor called
proopiomelanocortin. These peptides include the melanocyte-stimulating
hormones and the adrenocorticotropic hormone. Five different receptors
(MC1R-MC5R) mediate their physiological actions. Melanocortin signaling is
regulated also by endogenous antagonists, agouti-signaling protein (ASIP) and
agouti-related protein (AGRP) that compete with the melanocortin peptides by
binding to MCRs. AGRP is mainly expressed in the hypothalamus and works as
an inverse agonist and/or competitive antagonist at MC4R. ASIP is mainly
expressed in the ventral skin and binds MC1R to inhibit ventral melanogenesis.
Very recently, we have developed a new transgenic zebrafish line ovexpressing
ASIP. This fish line exhibits profuse alteration of the dorso-ventral pigment
pattern but also exhibits faster and bigger growth than wildtype fish both in
length and weight. We have carried out experiments to evaluate the puberty
onset in zebrafish using same-aged embryos from different ASIP and wildtype
breeding mates. Our results demonstrated that ASIP males reach puberty earlier
than wildtype fish (unpublished data) however studies on female transgenic fish
remains to be done. In addition, we demonstrate the presence of MC4R in ovary
and testis by ISH. The involvement of melanocortin system in the puberty onset
will be discussed.
Funded by Ministerio de Economía y Competitividad and European Fund for
Regional Development (AGL2013-46448-3-3-R and FEDER).
56
P11
MRAPS PARTICIPATION IN THE FISH MELANOCORTIN SYSTEM
Raúl Cortés1, M.J. Agulleiro
1, M. Michel
2, S. Navarro
1, E. Sánchez
1, L. Soletto
1,
R.D. Cone2, J.M. Cerdá-Reverter
1. E-mail: [email protected]
1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of
Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),
Castellón, Spain.
. 2Department of Molecular Physiology & Biophysics,
Vanderbilt University. Nashville, Tennessee (USA).
Melanocortin system is composed of hormones derived from
proopiomelanocortin precursor (POMC), including adrenocorticotropic
hormone (ACTH) and melanocyte-stimulating hormones (MSHs), melanocortin
receptors (MC1R-MC5R), and endogenous antagonists, i.e. agouti signaling
protein (ASIP) and agouti-related protein (AgRP). Recently, a new member of
melanocortin system has be added, the melanocortin receptor accessory protein
(MRAP). Our studies have demonstrated the presence of three MRAPs in the
zebrafish genome named as MRAP1, MRPA2a and MRAP2b. Similar to
mammalian system, MRAP1 is necessary for the ACTH signaling via MC2R
but MRAP2s were unknown.
In order to explore possible interactions with MCRs we studied the expression
profiles of all zebrafish MRAPs and MCRs by qPCR. Both MRAP2s were
highly expressed in the brain together with MC4R. Immunoprecipitation studies
showed physical interaction MRAP2s/MC4R. Double in situ hybridization
showed MRAP2 and MC4R co-localization in the neurons of the tuberal
hypothalamus meanwhile MRA2b/MC4R are co-localized within the preoptic
area. Pharmacological experiments demonstrated that co-transfection of
MRAP2a/MC4R, transform zebrafish MC4R into an ACTH receptor whereas
MRAP2b/MC4R results in a reduction of the constitutive activity of the
receptor. We have also demonstrated that MRAP2s interact with MC1R, but no
effects at signaling level were observed. We have further demonstrated that
ACTH-induced inhibition of food intake requires a functional MC4R, thus
providing a plausible physiological role to the MRAP2/MC4R interaction. We
are now using genome editing techniques like CRISPR/Cas9 system to
corroborate this result. We have successfully deleted MRAP2a from zebrafish
genome and we have discarded the involvement of MRAP2a in the larval
growth as previously suggested.
Funded by Ministerio de Economía y Competitividad and European Fund for
Regional Development (AGL2013-46448-3-3-R and FEDER).
57
P12
POSSIBLE ROLE OF MELANOCORTIN IN THE CONTROL OF
CIRCADIAN ACTIVITY RHYTMS
Lucia Soletto1, S. Puchol
1, S. Navarro
1, R.. Cortés
1, E. Sánchez
1, J.M. Míguez
JM2, J.F. Rosel
3, J. Rotllant
4 , J.M. Cerdá-Reverter
1 E-mail:
[email protected] 1Department of Fish Physiology and Biotechnology, Institute of Aquaculture of
Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC),
Castellón, Spain. 2Lab. Fisiología animal, Depto. Biol. Funcional y CC Salud,
Facultad de Biología, Universidad de Vigo, Spain. 3Departamento de
Psicología Evolutiva, Educativa, Social y Metodología, Universidad Jaume I,
Castellon, Spain. 4 Aquatic Molecular Pathobiology Lab, IIM- CSIC, Vigo,
Pontevedra, Spain.
Melanocortin system is formed by peptides derived from proopiomelanocortin
(POMC). Signaling is mediated by five different melanocortin receptors
(MC1R-MC5R). In addition, two different endogenous antagonists of MCRs
have been characterized: the agouti-signaling protein (ASIP) and agouti-related
protein (AGRP). ASIP is expressed in the ventral skin where it antagonizes the
effects of MSH on MC1R and is responsible for the dorso-ventral pigment
pattern in fish. AGRP is made in the hypothalamus and is involved in the
control of food intake by antagonizing central MC4R function. Zebrafish
genome exhibits an extra copy of AGRP named AGRP2. AGRP1 is expressed in
the hypothalamus whereas AGRP2 is mainly made in the pineal complex.
Previous studies have shown that ASIP functions as an antagonist for zebrafish
MC4R and ASIP over-expression in transgenic systems results in increased
growth and food intake. Our brain-pituitary transcriptomic analysis have
revealed two important sets of evidence concerning the involvement of the
circadian timing system in melanocortin function. A comparison of
differentially expressed genes (DEG) in the brain between wild type (WT)
males and female fish shows an enrichment of GO terms related with circadian
rhythms (p<5.2x10-9
) suggesting that males and females may show fundamental
differences in circadian timing system function. However, our comparison of
the brain-pituitary transcriptome of males and females over-expressing ASIP
(ASIP zebrafish) revealed that these differences were mitigated. A comparison
between ASIP vs WT males or females, revealed GO enrichment only in males
(p<1.7x10-3
) but not when comparing females of different genotypes. Results
suggest that WT males and females seem to exhibit differences in their
circadian timing system but that ASIP is able to mitigate such differences.
Funded by Ministerio de Economía y Competitividad and European Fund for
Regional Development (AGL2013-46448-3-3-R and FEDER).
58
P13
THE INVOLVEMENT OF GALANIN IN THE REPRODUTIVE CYCLE
OF SEA BASS.
Zélia Velez1, P. Pinto
1, R.S. Martins
1, S. Santos
1, A. Andrade
1, A. Gómez
2, S.
Zanuy2, A. Canário
1 . E-mail: [email protected]
1Laboratory of Comparative Endocrinology and Integrative Biology, CCMAR –
Centre of Marine Sciences, University of Algarve; 2 Instituto de Acuicultura de
Torre de la Sal, Castellon, Espana.
In commercial aquaculture systems the sea bass (Dicentrarchus labrax)
consistently presents high rates of precocious puberty males, which is correlated
with negative aspects to aquaculture production. Preliminary data suggest that
galanin (GAL), a neuropeptide associated with increased appetite, is potentially
involved in the control of the reproductive cycle. Taking in account that the
identification and characterization of regulators of the reproductive cycle in sea
bass would be extremely beneficial to commercial aquaculture systems, the aim
of the present work was the evaluation of the putative involvement of GAL in
the reproductive cycle of sea bass. Dispersed pituitary cells collected from fish
in different stages of the reproductive cycle were incubated with GAL and the
amount of released gonadotropins luteinizing hormone (LH) and follicle
stimulating hormone (FSH) was quantified and compared with that of control
cells. In addition, the effects of GAL in cAMP production was also accessed.
Our results showed that there was a peak of FSH release in September in two
year-old males (pre-pubertal) in response to GAL, while there were no
differences relative to control cells in November nor in January (spermiating
males). No effects of GAL were induced on pituitary LH release in September
and November, while in January there was a significant increase of LH release
in all the concentrations tested. Galanin alone had no effect on pituitary cells
cAMP production, however in cells previously stimulated with forskolin (an
activator of adenylate cyclase) there was an inhibition of cAMP production by
GAL, in September and October but not in January. RT-PCR allowed to detect
the expression of several subtypes of galanin receptors in pituitaries collected at
the analysed time points of the year. These results suggest the involvement of
galanin in the reproductive cycle of sea bass.
Acknowledgements: Funded by FCT – Portuguese Science and Technology
Foundation through projects UID/Multi/04326/2013 and PTDC/MAR-
BIO/3890/2012 and grant FCT SFRH/BPD/25247/2005 to PISP.
59
P14
SEASONAL SYNCHRONIZATION TO ENVIRONMENTAL CUES IN
SALMONIDS: ARE PHOTOPERIOD AND TEMPERATURE ACTING
THROUGH A COMMON PATHWAY?
Laura Gabriela Nisembaum1, A. Bantz
1, E. Magnanou
1, M. Fuentes
1, P.
Martin2, L. Besseau
1, J. Falcón
1. E-mail: [email protected]
1Sorbonne Universités, UPMC Univ Paris 06, CNRS, Biologie Intégrative des
Organismes Marins (BIOM), Observatoire Océanologique, Banyuls/Mer,
France; 2Conservatoire National du Saumon Sauvage, Chanteuges, France.
The complex life cycle of the Atlantic salmon (Salmo salar) involves a
downstream migration from fresh to sea water. The timing of this physiological
and behavioral transformation (smoltification) is triggered by external cues such
as photoperiod and temperature. Their combination is currently changing with
global warming: ambient temperatures are rising while daily and seasonal
photoperiod variations remain the same. The secretion of the time-keeping
hormone, melatonin (MEL), is a nocturnal and Ca2+
dependent process that
takes place in the pineal and retinal photoreceptor cells. The duration of MEL
secretion reflects night length, while the amplitude of its surge is influenced by
ambient temperature. While the mechanisms of the light/dark regulation of
melatonin secretion are quite well understood, those of temperature remain
unelucidated. We hypothesize that thermoreception in salmonids might be
mediated by transient receptor potential channels (TRPs), that mediate
temperature sensing in mammals. TRPs are activated by different ranges of
temperature and are permeable to calcium. Therefore, light and temperature are
likely to act through the same intracellular messenger, Ca2+
in the
photoreceptors. In the present study, photoperiod and/or temperature were
manipulated during smoltification process in order to assess the effect of each
signal and/or mimic the predicted effect of global warming. Salmons were
sampled every two months from December to June of the following year, and
the gene expression of two receptors from the vanilloid subfamily (TRPV1 and
TRPV4) was quantified in the retina, pituitary gland and gills. A combined or
separated impact of temperature and photoperiod was observed on the
expression levels of TRPVs, in a tissue specific-way. These results support TRP
as good candidates for thermoreception in fish and provide clues to understand
temperature modulation of melatonin secretion and synchronization to seasonal
events.
60
P15
QUANTIFICATION OF PLASMA STEROIDS IN SOLE (Solea
senegalensis) BY ULTRA HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY COUPLED TO TANDEM MASS
SPECTROMETRY
Eduardo Beltrán1, V. Piquer
2, J.M. Guzmán
3, P. Swanson
3, E. Mañanós
2, R.
Serrano1. E-mail: [email protected]
1Instituto Universitario de Plaguicidas y Aguas (IUPA), Universidad Jaume I,
12071-Castellón, España. 2Instituto de Acuicultura de Torre la Sal (IATS),
CSIC, 12595-Castellón, España. 3Northwest Fisheries Science Center, NOAA,
Seattle, Washington 98112, USA
Steroidal hormones control the reproductive functions of vertebrates. Their
study is crucial to understand the different stages of fish development
(ontogeny, gonad differentiation and reproductive periods). Steroids are present
in biological matrices at low levels (in plasma at ng ml-1
to pg ml-1
). Therefore,
a robust and sensitive analytical method for multiple detection is required.
Immunoassays are the preferred technique for steroid determination due to its
high sensitivity; however, they require processing one sample for each analyte.
Liquid chromatography coupled to tandem mass spectrometry is the alternative
technique which allows a sensitive and reliable simultaneous determination of
several hormones. In this work, an ultra high performance liquid
chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS)
method was developed for the analysis of 11 steroids (17β-estradiol, estrone,
androstenedione, testosterone, progesterone, pregnenolone, 11ketotestosterone,
cortisol, 11-deoxycortisol, 17α,20β-dihydroxy-4-pregnen-3-one and
17α,20β,21,trihydroxy-4-pregnen-3-one) in plasma samples of sole. Sample
treatment was based on protein precipitation with acetonitrile and subsequent
clean-up with OASIS HLB SPE cartridge. Target compounds were determined
in selected reaction monitoring mode, using a triple quadrupole analyser (TQS,
Waters) with a rapid chromatographic separation (6 min), using a reversed
phase column (Acquity BEH 1.7µm, 50 x 2.1 mm). The method was validated
by means of recovery experiments, obtaining satisfactory results of accuracy
and precision. Lowest level validated was 0.1 ng ml-1
. Limits of detection
(LODs) were in the range of pg ml-1
. Finally, the method was applied to the
analysis of plasma samples. The acquisition of two MS/MS transitions for each
compound allowed the unequivocal confirmation of positive samples.
Acknowledgements: Funded by Ministerio de Economía y Competitividad
(“Solembryo” AGL2013-49027-C3-3-R).
61
P16
THE ROLE OF LIPIDS AND FATTY ACIDS THROUGHOUT
SPERMATOGENESIS OF EUROPEAN EEL (Anguilla anguilla) AND
EFFECT OF DIETS ON REPRODUCTIVE PERFORMANCE
Rosa Baeza1, I.A.E. Butts
2, M.C. Vílchez
1, V. Gallego
1, D.S. Peñaranda
1, H.
Tveiten3, L. Pérez
1, J.F. Asturiano
1 . E-mail: [email protected]
1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología
Animal. Universitat Politècnica de València, 46022 Valencia, Spain. 2Technical
University of Denmark, National Institute of Aquatic Resources. Kavalergården
6, 2920 Charlottenlund, Denmark. 3Norwegian Institute of Fisheries and
Aquaculture, Muninbakken 9-13 Breivika, 6122, Tromsø, Norway
During the last decades, European eel populations have declined considerably
and this species is considered outside its safe biological limits. The life cycle of
the eel is quite complex including transoceanic migration where native
conditions are still unknown and the natural spawning process has never been
observed. Eels cease feeding during migration and that is why their energy
reserves, such as lipids, play a crucial role in obtaining good quality gametes
(eggs and sperm). In the last years, efforts have been directed at studying the
influence of fatty acids on the reproductive performance of females, while less
effort has been devoted to males.
The present study describes the results obtained in two experiments on
European eel males. The first experiment focused on the effect of fatty acids
throughout sexual maturation, their relationship with steroid hormones, and
their effect on sperm quality parameters. The second experiment involved the
comparison of different dietary regimes on sperm quality traits.
Results of the first experiment showed ARA, EPA and DHA maintenance in the
testis during eel spermatogenesis, while all other fatty acids decreased. ARA
and EPA maintenance may have a physiological function, i.e. as prostaglandins
precursors, while the maintenance of DHA levels may have a structural role for
spermatozoa membrane formation. On the other hand, EPA and DHA acted as
modulators of androgens synthesis particularly during the final phase of the
sperm maturation, and ARA had an effect on sperm velocity. With the acquired
knowledge, a second experiment was carried out in which we designed feeds
with different fatty acid percentages in order to evaluate the influence of these
diets on sperm quality. Together, this work demonstrated higher sperm motility
and increased milt volume from eels fed balanced diets.
62
P17
CHARACTERIZATION OF ALDEHYDE DEHYDROGENASES aldh1a2
AND aldh1a3 IN THE EUROPEAN SEA BASS
Paula Medinaac
, A. Gómezb, S. Zanuy
b, M. Blázquez
a. E-mail:
aInstitut de Ciències del Mar (ICM-CSIC), Barcelona, Spain.
bInstituto de
Acuicultura de Torre la Sal (IATS-CSIC), Castellón, Spain. c
Universidad de
Antofagasta, Antofagasta, Chile
The aldh1a family is involved in the oxidation of aldehydes in different tissues.
Several members of this family are responsible for the synthesis of retinoic acid
(RA), known to act as a meiotic stimulating factor in higher vertebrates, but in
fish this is yet to be confirmed. The main goal of the study was the molecular
characterization of the aldh1a family in the European sea bass, Dicentrarchus
labrax. The study reports the isolation and characterization of aldh1a2 and
aldh1a3, as well as the identification of both genes in other teleost and non-
teleost genomes. Their tissue distribution in males and in females was also
assessed. In addition, testicular cultures were used to study the effect of
exogenous RA and FSH, an endocrine key regulator of germ cell development,
on the expression of aldh1a2 and aldh1a3. The study shows that both proteins
exhibit the main structural amino acid motifs that define them as active
NAD(P)-dependent catalytic enzymes. The phylogenetic and synteny analyses
of the proteins confirm that they are different orthologs further supporting that
the aldh1 family appeared before the divergence of teleosts from the
evolutionary lineage. Tissue-specific mRNA expression in juvenile sea bass
showed that aldh1a2 levels were higher than those of aldh1a3. Both mRNAs
were ubiquitously expressed in several tissues including testis and ovaries, with
a clear preference and female-specific expression for aldh1a3 in gonads and
brain. This sex-differential expression in gonads suggests a tight regulation on
the oxidation of aldehydes and the subsequent synthesis of RA, pointing at their
role in cell proliferation and differentiation during gonadal development.
Moreover, the expression in testicular cultures of both aldhs during early stages
of maturation was sensitive to exogenous RA and FSH, further suggesting an
important role of these enzymes in gonadal maturation and reproduction.
Funded by projects Reprosex (AGL2011-28890) and Reprobass
(PROMETEOII/2014/051). PM was sponsored by a grant MECE2 (ANT0806).
63
P18
EXPOSURE OF SOLE (Solea senegalensis) LARVAE TO DIFFERENT
ENVIRONMENTAL CONDITIONS AFFECT THE SYNTHESIS OF
REPRODUCTIVE HORMONES
Vanesa Piquer1, M. Aliaga
2, J.A. Paullada
2, J. Ramos
1, J.A. Muñoz-Cueto
2, E.
Mañanós1. E-mail: [email protected]
1Instituto de Acuicultura de Torre la Sal (IATS-CSIC), Castellón, España.
2Departamento de Biología, Facultad de Ciencias del Mar y Ambientales,
Universidad de Cádiz, Puerto Real, España
In fish, environmental factors have profound effects on larval development. In
sole, we have recently shown that daily thermocycles, compared to constant
temperatures, affect gonad differentiation (GD) and change sex ratios. Also,
blue light affects positively several metabolic and reproductive parameters,
compared to other light spectra. The mechanisms involved in the integration of
environmental signals in fish larvae and the early establishment of the
reproductive axis are largely unknown. This study investigated the combined
effects of light and temperature during early development. Sole larvae were
exposed to, 1) daily thermocycle and blue light (18-21ºC; TCblue) or, 2)
constant temperature and white light (19.5ºC; cteW; routine aquaculture
conditions). In addition, and to determine a potential sensitive period, the
TCblue conditions were applied in different time frames, 1) until
metamorphosis (0-12 days post hatching (dph)), 2) post-metamorphosis (0-30
dph) or, 3) gonad differentiation (0-100 dph). Effects were studied by analyzing
steroid levels (ELISA) and gonadotropin gene expression (qPCR). Body weight
was similar in all TCblue groups and, from 30 dph onwards, higher than in
cteW larva. Metamorphosis happened at 13-20 dph in all groups. Expression of
follicle-stimulating hormone beta (fshb) and luteinizing hormone beta (lhb))
subunits increased at metamorphosis, but only under TCblue; they peaked again
at GD and, in most sampling points, expression was higher in TCblue groups
than cteW. Levels of estradiol, testosterone and 11-ketotestosterone increased at
metamorphosis and GD, whereas the progestrogen 17,20β-P increased after
metamorphosis and was maintained high until 138 dph; no clear effect of
treatments was evident. The results showed a significant synthesis of
reproductive hormones during larval ontogeny and GD which was influenced by
light and temperature regimes.
Work funded by the Spanish Ministry of Economy and Competitiveness
(AGL2010-22139-C03-02 and AGL2013-49027-C3-3-R).
64
P19
IN VIVO EXPRESSION OF NUCLEAR AND MEMBRANE ESTROGEN
RECEPTORS IN EUROPEAN EEL THROUGH SPERMATOGENESIS
Marina Morini1, D.S. Peñaranda
1, M.C. Vílchez
1, A.G. Lafont
2, L. Pérez
1, S.
Dufour2, J.F. Asturiano*
1. E-mail: [email protected]
1Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología
Animal. Universitat Politècnica de València, Valencia, Spain. 2UMR BOREA
"Biology of Aquatic Organisms and Ecosystems", MNHN, CNRS, IRD, UPMC,
Paris, France
The sex steroid estrogens can bind nuclear receptors (ER) or membrane
receptors (GPR30 or GPER) to elicit a slow genomic action by modulating the
gene transcription and translation activity, or a rapid, non-genomic activation of
intracellular signal transduction pathways. The nuclear ER include ERα and
ERβ, with at least three distinct subtypes, including ERα, ERβ1 and ERβ2 in the
Actinopterigii species. The GPER (G-protein coupled ER) is recognized as a
membrane-bound ER binding estradiol that initiate subsequent signalling
cascade and present at least one copy in all the vertebrates.
In this study, we measured for the first time the expression in vivo of three
nuclear (ERα, ERβ1 and ERβ2) and two membrane (GPER1 and GPER2)
estrogen receptors simultaneously in the tissues of the brain-pituitary-gonad
axis, through the spermatogenesis, in an early teleost, the European eel
(Anguilla anguilla).
Male eel ER mRNA expression in the brain, pituitary and gonads, receptors
increased with the change from freshwater to sea water, suggesting that nuclear
ERs in the tissues of the gonadotropic axis play a critical part in osmoregulatory
adaptations. Gonad nuclear ERs, which were more expressed at spermatogonia
stage, are likely to play a role in the spermatogonia renewal; whereas both
membrane receptors (GPERs), which are more expressed at spermatozoa stage,
seems to be involved in the final spermatogenesis. Furthermore, brain and
pituitary nuclear ERs and pituitary GPERs seems to mediate autocrine or
paracrine communication of local estrogen production, through the
spermatogenesis. Finally, the expression of both nuclear ERs and GPERs in the
brain-pituitary-gonad axis indicates possible cooperation between genomic and
non-genomic estrogen actions in controlling reproduction.
Funded by IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement nº:
642893) and MINECO (REPRO-TEMP; AGL2013-41646-R).
65
P20
INFLUENCE OF EXOGENOUS MELATONIN ON GROWTH,
GONADAL MATURITY AND kiss/gnrh GENE EXPRESSION
PATTERNS IN THE BRAIN OF MALE SEA BASS (Dicentrarchus labrax)
M. Victoria Alvarado, M. Carrillo, A. Felip. E-mail: [email protected]
1Instituto de Acuicultura Torre la Sal, Consejo Superior de Investigaciones
Científicas (CSIC), Castellón, Spain.
The influence of long-term melatonin administration, as an implant, on growth
performance and reproduction of adult male sea bass was studied in vivo. Two
years old male sea bass were organized into two groups: in the first, which acted
as a control group, the animals were administered empty implants, whereas in
the second, the animals were administered melatonin implants. Our results
provided that this hormone had an anorexic action, thus affecting the
performance of fish. Melatonin reduced the fish weight and condition factor,
with the mean weight of melatonin-treated fish amounting to only 77.7% as
compared to controls. Melatonin also affected gonadogenesis following
testicular maturation in adult sea bass when administered over an extended
period of time throughout their entire reproductive cycle. A decrease in the
gonadosomatic index after 150 days of treatment (29% lower than in controls)
and a lower percentage of running males during the spermatogenesis and full
spermiation stages (January-March) was observed in this species. Exogenous
melatonin also resulted in lower plasma levels of testosterone and 11-
ketotestosterone during the reproductive period, and showed a significant
decrease of circulating Lh and Fsh levels after 30 and 60 days of treatment,
respectively. Furthermore, the hypothalamic expression of kiss1 was
significantly higher in melatonin-treated fish than in controls after 30 days of
treatment, while a significant increase in kiss2 expression was detected on day
90 of treatment. By contrast, melatonin showed a significant decrease in
kisspeptin expression in the dorsal brain on day 150 of treatment and also
affected the expression of gnrh-1 and gnrh-3 as well as gnrhr-II-1a and 2b and
the fshβ gene in the pituitary. Taken together, these findings reveal that
melatonin evokes seasonal changes in key reproductive hormones that affect
testicular maturity. In addition, melatonin might presumably induce the
downregulation of kisspeptin-gnrh members on the dorsal brain thus affecting
fsh-beta ?transcription during early gametogenesis that appear to mirror
disturbances in spermatogenesis.
66
P21
REGULATORY RESPONSES TO SALINITY IN SEA BASS
Pedro S. Palma1, P. M. Guerreiro
1, D. M. Power
1, A. V. M. Canário
1. E-mail:
1CCMAR - Centre of Marine Sciences, University of Algarve, Portugal.
Changes in salinity modifies the extracellular ionic environment which needs to
be regulated by multiple hormones. Calcium and sodium are key ions that need
to be regulated to maintain homeostasis as they are toxic and the two main
glands known to influence their extracellular levels are the pituitary and the
corpuscles of Stannius (CS). Calcium homeostasis is maintained by the
antagonistic actions of the parathyroid hormone-related protein (PTHrP) and
Stanniocalcin (STC). STC is a hypocalcemic hormone and is secreted by the
Corpuscles of Stannius (CS) which undergo changes in size/activity as water
calcium levels increase, and by the other hand PTHrP has hypercalcemic actions
promoting calcium uptake via gills and intestine. Prolactin (PRL) produced in
the pituitary is known to regulate sodium. Here we have analysed changes in
expression of genes that encode for these hormones as well as other genes
known or suspected to be involved in ion homeostasis during exposure of sea
bass, Dicentrarchus labrax, to salinity changes (transfer from 36 ppt to 3.6 ppt
starting at 2 hours after transfer and up to 30 days). Significant changes in gene
expression compared to control maintained at 36 ppt were found for PTHrPA
and STC1 in pituitary and CS, for PRL in the pituitary and for fibroblast growth
factor 23 (FGF23) in CS. There were no significant changes in calcium sensing
receptor and PTH receptors, with the exception of PTH3R in the pituitary.
Overall our results reflect coordinated and tightly controlled mechanisms of
gene expression underlying ion regulation during brackish water adaption at the
level of the pituitary and CS.
Acknowledgements: Funded by Portuguese Foundation for Science and
Technology projects PTDC/MAR/121279/2010 to AVMC and PDCT/BIA-
ANM/4225/2012 to PMG.
67
P22
VASOTOCINERGIC AND ISOTOCINERGIC CO-REGULATION IN
STRESS RESPONSE OF COMMON CARP (Cyprinus carpio L.)
Ismael Jerez1, J. M. Mancera
1, G. Flik
2 & M. Gorissen
2. E-mail:
1Department of Biology, Faculty of Marine and Environmental Sciences,
University of Cadiz, Spain. 2Department of Animal Physiology, Institute for
Water and Wetland Research, Radboud University, Nijmegen, The Netherlands.
Under stress conditions, the teleostean hypothalamic-pituitary-interrenal (HPI)
axis is stimulated in order to cope with a disturbance of the homeostasis.
Endocrine regulation of this axis is controlled at different levels, involving
several pathways that activate the synthesis and release of cortisol. This axis
starts with the integration of the perception and appraisal of the stressor(s) by
the brain and the release of the corticotropin-releasing factor (CRF) from the
hypothalamic nucleus preopticus (NPO). CRF stimulates mainly the synthesis
of adrenocorticotropic hormone (ACTH) from the pituitary pars distalis, which
in turn stimulates the production and release of cortisol by the interrenal cells in
the head kidney. Furthermore, the NPO innervates pars intermedia cells from
the pituitary gland to produce acetylated endorphins and α-MSH (α-melanocyte-
stimulating hormone). The role of α-MSH in the stress response is not yet clear,
being either associated with pigmentation and food intake, and depending on
the species in the regulation of cortisol levels under chronic stress situations.
The picture becomes even more complex as arginine vasotocin (AVT) and
isotocin (IT), homologous to mammalian vasopressin and oxytocin, have been
demonstrated to be associated with both the central and peripheral regulation of
the stress response. In this study, we aim to assess the effect of AVT and IT on
the release of α-MSH and cortisol in common carp (Cyprinus carpio L.). We
performed an in vitro superfusion experiment using pituitary glands and head
kidneys, and incubated them with different combinations of either CRF or
ACTH, with / without AVT and / or IT. Our results suggest a potentiation of the
stress response by AVT and IT, by stimulation of α-MSH and cortisol release in
co-operation with CRF and ACTH, respectively.
68
P23
CHANGES IN PLASMA AND BRAIN STRESS-RELATED
PARAMETERS IN RAINBOW TROUT EXPOSED TO CONSPECIFIC
CHEMICAL SIGNALS
Jésus M. Míguez, F. Naderi, M.A. López-Patiño, J.L. Muñoz*, J. Hernández-
Pérez, R. Álvarez-Otero, M. Gesto¥. Email: [email protected]
Lab. Fisiología animal, Depto. Biol. Funcional y CC Salud, Facultad de
Biología, Universidad de Vigo, Spain. *,¥Current address: *Centro i?mar, Univ.
Los Lagos, Puerto Montt, Chile; ¥Section for Aquaculture, DTU Aqua, Tech.
University of Denmark, Hirtshals, Denmark.
Many fish species use chemical cues for predator recognition and defensive
behavior. These chemical signals are generally produced, or contained, within
the skin or in body fluids, and are only released through mechanical damage or
injuries, eliciting alarm reactions on conspecifics that comprise behavioral and
physiological changes. It has been demonstrated that olfaction is mainly
involved on the alarm response but it is still unclear whether an activation of the
neuroendocrine stress pathways participate in this reaction in fish. In the present
study we evaluated the physiological response of rainbow trout (Oncorhynchus
mykiss) to the presence in water of blood and skin extracts coming from
conspecifics. We exposed trout for 3 min and 15 min to 4 different scents:
“stressed blood”, “unstressed blood” (obtained from stressed and unstressed
trout, respectively), skin extract or distilled water (control). Trout from all
groups were sacrificed avoiding handling stress, and blood and brain samples
were obtained and assayed for plasma levels of stress-related hormones
(catecholamines, cortisol) and metabolites (glucose, lactate), as well as for brain
monoaminergic neurotransmitters (serotonin, dopamine). Trout exposed to
blood from stressed fish or to skin extract showed increased plasma
catecholamines and cortisol levels. At the brain level, we found decreased
dopaminergic and serotoninergic activity in telencephalon and hypothalamus of
trout exposed to unstressed blood, whereas increases were noted after exposure
to blood of previously stressed trout or to skin extract. Interestingly, the levels
of dopaminergic activity in the olfactory bulb were increased in all exposed
groups. Altogether, these data indicate that different chemical cues from
conspecifics can induce a mild but detectable stress response in trout, thus
demonstrating that these cues, which were already known to induce behavioral
alterations in fish, could also have a metabolic cost. The relevance this kind of
stressor may have in fish farms, where fish often suffer skin lesions, is yet to be
determined.
Funded by MEC AGL2013-46448-3-1-R and FEDER
69
P24
GHRELIN MODIFIES CLOCK GENES EXPRESSION IN THE LIVER
OF GOLDFISH VIA PLC-PKC PATHWAY.
Aida Sánchez-Bretaño, A.M. Blanco, A.L. Alonso-Gómez, M.J. Delgado, E.
Isorna. E-mail: [email protected].
Department of Animal Physiology, Faculty of Biology, University Complutense
of Madrid.
The liver has been proposed in mammals as a main oscillator which links the
circadian system with metabolism. Ghrelin plays a key role in energy balance
regulation, promoting food intake, carbohydrate utilization and adiposity, and
other physiological processes. In goldfish, an intraperitoneal injection of ghrelin
modifies clock genes expression in several structures including the liver,
suggesting that it could act as a metabolic/food intake signal. The objective of
this work was to investigate if ghrelin acts directly on the liver to modify clock
genes expression and which is the intracellular pathway employed. To this aim,
we studied ghrelin effects on clock gene expression using in vitro static cultures
of goldfish liver. Livers (n=6) were sampled and preincubated in 1 ml DMEM
(15 mg liver/well) during 2 h. Then, the samples were divided into 10
experimental groups: 1 control, 3 ghrelin-treated groups (0.1, 1, 10 nM), 3
groups with a ghrelin-antagonist ([D-Lys-3]-GPRH; 1, 10, 100 nM), and 3
groups with a ghrelin antagonist (1, 10, 100 nM) plus ghrelin (10 nM). Cultures
were maintained for 1 or 5 h. In a second experiment to study if PLC-PKC
pathway is the intracellular pathway used by ghrelin, cultured liver were
preincubated with a PLC inhibitor (U73122; 1, 10 µM) and incubated with or
without ghrelin (10 nM) for 1 and 5 h. Ghrelin induced (2-6 fold) per1a, per1b,
per2a, per3, bmal1a, clock1a and rev-erbα expression after 1 h of treatment,
and this effect was decreased or completely abolished after 5 h. In all the cases,
ghrelin effects at 1 h were partially or completely eliminated by the antagonist
of the ghrelin receptors and with U73122. Present results show for the first time
in teleosts the direct action of ghrelin on the hepatic oscillator by modulating
clock genes expression. Moreover, our data indicate that ghrelin acts via PLC-
PKC pathway. Taken together, these results evidence that ghrelin is one of the
direct links between the food intake and energy balance with the circadian
system in teleosts.
Acknowledgements: Supported by Spanish MINECO (AGL 2013-46448-C3-2-
R). A. Sánchez-Bretaño and A.M. Blanco are predoctoral fellowship from
Spanish MINECO (FPI) and MEC (FPU) respectively.
70
P25
PROLACTIN IN THE MEAGRE (Argyrosomus regius): MOLECULAR
CLONING AND EXPRESSION ANALYSIS UNDER DIFFERENT
ENVIRONMENTAL SALINITIES
Rubén Ayala Suárez1, A. Astola
1, J.A. Martos-Sitcha
2, J. M. Mancera
3. E-mail:
1Dpto. de Biomedicina, Biotecnología y Salud Pública. Facultad de Ciencias.
Universidad de Cádiz.2Instituto de Ciencias Marinas de Andalucía (ICMAN-
CSIC). 11510 Puerto Real, Cádiz.3
Dpto. de Biología. Facultad de Ciencias del
Mar y Ambientales. Campus de Excelencia Internacional del Mar (CEI-MAR),
11510 Puerto Real, Cádiz. Universidad de Cádiz.
The meagre Argyrosomus regius has gained good popularity for both
aquaculture producers and consumers due to its rapid growth rates, ease in
processing, low fat content and somewhat rigid texture. Recent studies have
shown the good euryhalinity capacity of this species. The role of prolactin (prl)
is well understood in many perciformes species as a fresh-water adapting
hormone, preventing ion loss and promoting water removal, but its role in A.
regius has not been inquired yet. Thus, understanding of this role will provide
knowledge assisting in the development of molecular strategies to identify
saline stress and optimal environmental salinity to improve the culture of this
species. Degenerated primers were designed from published cDNA prl
sequences in perciformes. cDNA synthesis was carried out using total RNA
extracted from pituitary gland, and a prl cDNA was amplified, cloned and
sequenced. prl sequence belongs to partial CDS (GenBank acc. no.:
KP984534), and it shared high sequence identity with its counterparts in other
teleosts. Finally, prl expression level was tested in juveniles specimens
acclimated for 21 days to 4 different environmental salinities: 5 ppt
(hyposmotic, 140 mOsm kg?1
H2O), 12 ppt (isosmotic, 364 mOsm kg?1
H2O), 38
ppt (hyperosmotic; normal seawater, 1090 mOsm kg?1
H2O) and 55 ppt
(extremely hyperosmotic, 1546 mOsm kg?1
H2O). This analysis was
accomplished by quantitative polymerase chain reaction. Generated data was
statistically analysed using one-way analysis of variance (ANOVA) and Tukey-
HSD Post-Hoc test. Significant values were considered when P < 0.01. Results
showed that prl mRNA levels decreased as salinity increased, showing a three-
fold gain at 5 ‰ related to seawater group. According to the hyperosmotic role
of PRL, our results indicated that prl seem to be important under hyposaline
environments, supporting the high euryhaline capacity of A. regius.
71
P26
CLONING, SEQUENCING, AND mRNA EXPRESSION PATTERNS OF
GH AND IGF-I GENES IN MEAGRE (Argyrosomus regius) JUVENILES
ACCLIMATED TO DIFFERENT ENVIRONMENTAL SALINITIES
Khaled Mohammed-Geba1,2,3
, J.A. Martos-Sitcha2, G. Martínez-Rodríguez
2,
J.M. Mancera1. E-mail: [email protected]
1Genetic Engineering and Molecular Biology Division, Department of Zoology,
Faculty of Science, Menoufia University, Shebin El- Kom, Menoufia,
Egypt.2Instituto de Ciencias Marinas de Andalucía, Consejo Superior de
Investigaciones Científicas (ICMAN-CSIC), 11510 Puerto Real (Cádiz),
Spain.3Department of Biology, Faculty of Marine and Environmental Sciences,
Campus de Excelencia Internacional del Mar (CEI-MAR), University of Cadiz,
11510 Puerto Real, Cadiz, Spain.
The Mediterranean meagre Argyrosomus regius exhibits a growing interest for
aquaculture, presenting a good euryhaline capacity as well as enhanced growth
under isosmotic environments. However, the role of growth hormone
(GH)/insulin-like growth factor-I (IGF-I) axis mediating this euryhalinity is still
completely unveiled. Therefore, and for the first time in this species, cDNAs of
meagre’s pituitary growth hormone (gh), hepatic insulin-like growth factor (igf-
1), and β-actin (actb) were cloned. Their mRNA expression patterns were tested
in juveniles acclimated to 4 different environmental salinities: i) 5 ppt
(hyposmotic, 140 mOsm kg?1
H2O), ii) 12 ppt (isosmotic, 364 mOsm kg?1
H2O),
iii) 38 ppt (hyperosmotic; normal seawater, 1090 mOsm kg?1
H2O), and iv) 55
ppt (extremely hyperosmotic, 1546 mOsm kg?1
H2O). All investigated
transcripts shared high sequence identities with their counterparts in other
perciformes. gh mRNA enhanced significantly in both 12 and 55 ppt salinity
groups in comparison to control group. However, igf-1 showed its maximum
expression levels under isosmotic salinity. The results indicated that growth
enhancement observed in A. regius specimens under isosmotic environment is
supported by the stimulation of GH/IGF-I axis.
Acknowledgments: This work was carried out as a part of the Spanish-Egyptian
joint project AP/039755/11 (Agencía Española de Cooperación Internacional
para el Desarrollo, AECID)
72
P27
PHYSIOLOGICAL CHANGES IN SILVER CATFISH (Rhamdia quelen)
TRANSPORTED WITH ESSENTIAL OIL OF Myrcia sylvatica
E.M.H. Saccol1, J.A. Martos-Sitcha
2, I. Jerez
3, T.S. Pês
1, R.H.V. Mourão
4, B.
Baldisserotto1, M.A. Pavanato
1, G. Martínez-Rodríguez
2, J.M. Mancera
3. E-
mail: [email protected]
1Departmento de Fisiologia e Farmacologia, Universidade Federal de Santa
Maria (UFSM), Santa Maria, Rio Grande do Sul, Brazil. 2Instituto de Ciencias
Marinas de Andalucía, Consejo Superior de Investigaciones Científicas
(ICMAN-CSIC), Puerto Real, Cádiz. 3
Departamento de Biología, Facultad de
Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar
(CEI-MAR), Universidad de Cádiz, Puerto Real, Cádiz, Spain. 4Instituto de
Biodiversidade e Florestas, Universidade Federal do Oeste do Pará (UFOPA),
Santarém, Pará, Brazil.
Aquaculture practices include several procedures as capture, handling and
transport, which cause stress in fish. Anesthetics or sedative substances have
been used to reduce this stress. The aim of this study was to evaluate the effects
of essential oil of Myrcia sylvatica (EOMS) in the water on stress system
activation in the silver catfish Rhamdia quelen summited to transport. Fish were
captured in the production ponds and transferred to a 250-L tanks (density of 54
kg/m3). After 24 hours, 10 fish were caught, euthanized by section of the spinal
cord and sampled (basal group). The remaining fish were placed in plastic bags
containing 5 L of water (density of 150 kg/m3) with different doses of EOMS
(0, 25 or 35 μL/L diluted in 315 μL/L ethanol), in triplicate, and transported for
6 h. After transportation, 10 animals of each group were captured, euthanized
and sampled. Cortisol and glucose levels in plasma, as well as hypothalamic
corticotrophin-releasing hormone (CRH) and pituitary proopiomelanocortin
(POMC) “a” and “b” mRNA expressions were determined. Cortisol levels and
CRH expression enhanced after 24h of handling, decreasing during the
transport with addition of EOMS. Expression of POMCa was higher in fish
transported with 25 μL/L respect to the rest of groups. Therefore, it is suggested
the use of EOMS for transporting fish in order to avoid the stress associated
with this procedure.
73
P28
CLOCK GENES AS TARGET FOR FEEDING IN THE FOOD
ENTRAINABLE OSCILLATOR OF LIVER IN GOLDFISH
Miguel Gómez-Boronat, A. Sánchez-Bretaño, A.M. Blanco, I. Redondo, M.J.
Delgado, N. De Pedro, E. Isorna. E-mail: [email protected]
Department of Animal Physiology, Faculty of Biology, University Complutense
of Madrid.
The circadian system consists of a net of oscillators whose molecular function is
based on circadian regulatory genes named clock genes. Although daily
photoperiod is the most well-known synchronizer of circadian oscillators,
scheduled feeding can also drive circadian rhythms, especially in peripheral
tissues, as the liver. However, very little is known about how food intake is
detected by the hepatic clock in mammals, and nothing in fish. The objective of
this study was to identify the clock genes that could be acting as possible targets
for feeding signalling in the liver clock of goldfish (Carassius auratus). To this
aim, the 24-h expression pattern of several clock genes was analyzed in the liver
of goldfish maintained under a 12L:12D photoperiod and scheduled feeding at
ZT 2. Secondly, we studied the periprandial fluctuations of hepatic clock genes
in fed and unfed fish. Finally, clock genes expression in liver was analyzed in
three fish groups: daily fed (1% bw), fasted (48 h), and fasted plus refed. In all
the experiments, liver samples were collected and clock genes (per1a, per1b,
per3, bmal1a, rev-erbα and pparα) expression was determined using real-time
RT-PCR. Significant daily rhythms were found for all the studied clock genes in
the liver. Results of the periprandial experiment showed that expression of
per1a, per1b and rev-erbα decreases 3 h after food intake in both experimental
groups (fed and unfed fish). Thus, these changes would be a consequence of
their daily rhythmic profiles, regardless of feeding. However, expression of
bmal1a increased only in fed fish, suggesting that the daily profile observed in
this gene is mainly due to food intake. In the last experiment, 48-h fasting
decreased per1a and bmal1a expression, and this effect was reverted by
refeeding, in agreement with the feeding bmal1a induction observed in the
periprandial experiment. Taken all together, present results suggest that bmal1a
could be a target in the food entrainment of hepatic oscillator in fish.
Acknowledgements. Supported by the Spanish MINECO (AGL 2013-46448-C3-
2-R). A.M. Blanco and M. Gómez-Boronat are predoctoral fellows from the
Spanish MEC and MINECO, respectively.
74
P29
INSULIN AND IGF-I PLAY A RELEVANT ROLE IN THE
REGULATION OF NORMAL AND TUMORAL PROSTATE CELL
FUNCTION
F. López-López, A. Sarmento-Cabral, M.D. Gahete, J.P. Castaño, R.M. Luque.
E-mail: [email protected]
Department of CellBiology, Physiology and Immunology, University of
Córdoba; Hospital Universitario Reina Sofía, Instituto Maimónides de
Investigación Biomédica de Córdoba; CIBER Fisiopatología de la Obesidad y
Nutrición;and, Campus de Excelencia Internacional Agroalimentario,
Córdoba, Spain.
Obesity (OB), a multifactorial chronic endocrine-metabolic disease, represents
one of the most serious globalhealth threats. OB is associated with an increase
in the incidence of certain cancertypes, such as prostate cancer (PC), a
hormone-related, heterogeneous, and complex cancer with a high incidence.
Unfortunately, the precise molecular/cellular and endocrine-metabolic
mechanisms underlyingthepathophysiological association between OB and the
higher incidence/aggressiveness of PC are stillnot well understood.Indeed,
although androgens play a primary, crucial role in PC development/progression,
there is growing evidence that other hormones and growth factors that are
significantly altered in OB, such asinsulin (INS) and IGFI, might also play a key
role in the development and progression of PC. However, the direct and relative
effectsof INS and IGFI or the combination of both (I+I) in controlling prostate
cell function has not been completely elucidated. Here, we used normal prostate
(NP) tissues and primary NP cell cultures from mice,as well as two human PC
cell lines (PC3 and LNCaP),as models to evaluate, in parallel, the presence of
INS/IGFI-axes and the direct role of INS and IGF-I alone or in combination. We
found that IGFIR was consistently more expressed than Ins-R in all
experimental models analyzed. Remarkably, both INS and IGFI treatment
increased IGFI/IGFI-R, but not Ins-Rexpression in prostate cells, whichcould be
considered as an indication of PC progression. In fact, IGFI, but not INS or I+I,
treatmentwas able to increase cell proliferation in PC3 and LNCap; whereas,
INS, IGFI and I+I treatments increased migration capacity in PC3. Interestingly,
INS and IGFI alonedecreased PSA secretion in LNCaP cells.Altogether, our
data indicate that INS and IGFI could contribute to the regulation of malignancy
featuresin normal and tumoral prostate cells.
Funding: PI13-00651, BFU2013-43282-R, BIO-0139, CTS-1406, PI-0639-
2012, and CIBERobn
75
P30
LONG-TERM CROWDING STRESS INCREASED CORTISOL ON
SERUM AND SKIN MUCUS OF THE GILTHEAD SEABREAM (Sparus
aurata L.)
Francisco A. Guardiola1, A. Cuesta
1, J.Meseguer
1, M.A. Esteban
1. E-mail:
1Fish Innate Immune System Group, Department of Cell Biology and Histology,
Faculty of Biology, Campus Regional de Excelencia Internacional “Campus
Mare Nostrum”, University of Murcia, 30100 Murcia, Spain.
One of the primary responses to stress is mediated by the hypothalamus-
pituitary-interrenal (HPI) axis, which releases cortisol, the dominant steroid in
stress physiology, into the circulatory system. Plasma cortisol levels have been
used to evaluate the stress response in a variety of cultured fish species.
Nonetheless, little is known about the cortisol skin mucus levels as stress
response of fish. To date, this is the first report about the cortisol level in fish
skin mucus. Thus, the aim of this study was to determine the gilthead seabream
(Sparus aurata) cortisol response in serum and skin mucus after long-term
crowding stress. Fish were subjected to 10 kg m-3
(low density, control group)
and 50 kg m-3
(high density as crowding group) during 2, 24 and 48 h. Then, the
cortisol level was evaluated in serum and skin mucus samples using commercial
kits (Calbiotech, USA) following the manufacturer's procedures. Ours results
demonstrated that the cortisol level in serum and skin mucus of gilthead
seabream showed a statistically significant increase in fish that were exposed to
crowding stress after all experimental times. In addition, the cortisol levels in
both samples (serum and skin mucus) followed the same pattern to each point
of sampling, being observed a higher increase in fish exposed to high density
for 24 hours. This study represents an advance on our knowledge about the
interaction between stress and cortisol response in fish skin mucus. Further
investigations are needed in the detection of this stress hormone in skin mucus
of fish subject to other stressor situations and their possible use as stress
bioindicator in intensive aquaculture systems.
Financial support by grants AGL2011-30381-C03-01 (Ministerio de Economía
y Competitividad) and 04538/GERM/06 (Fundación Séneca de la Región de
Murcia, Grupo de Excelencia) are gratefully acknowledged.
76
P31
17β-ESTRADIOL ALTERS THE SUSCEPTIBILITY TO NODAVIRUS
AND THE VIRAL IMMUNE RESPONSE OF EUROPEAN SEA BASS
Yulema Valero1, M. Arizcun
1, FA Guardiola
2, M.A. Esteban
2, A. Cuesta
2, E.
Chaves-Pozo1. E-mail: [email protected]
1Centro Oceanográfico de Murcia, Instituto Español de Oceanografía, 30860
Puerto de Mazarrón, Spain. 2Fish Innate Immune System Group, Department of
Cell Biology and Histology, Faculty of Biology, Campus Regional de
Excelencia Internacional “Campus Mare Nostrum”, University of Murcia,
30100 Murcia, Spain.
In addition to their role in fish reproduction, sex steroid hormones are known to
modulate the immune response in vertebrates, including fish. In teleost fish, the
increase on the serum level of 17β-estradiol (E2) has been correlated with
immunosuppression and increased disease susceptibility whilst some studies
have also demonstrated stimulation of some humoral activities. This fact
highlights the complexity of the role of E2 in the regulation of the immune
response and point to the necessity to make further studies to understand how
pathogens modulate E2 serum levels in order to modulate the immune system
and evade the immune response. Nodavirus (NNV) is a vertical transmitted
pathogen that is able to decrease or increase the E2 serum levels of infected
European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus
aurata), respectively, as revealed by some data obtained in our laboratory.
Taking into account that the European sea bass is very susceptible to NNV we
have experimentally altered the sex steroid serum levels of European sea bass
juveniles specimens by an intraperitoneal injection of E2 concomitantly with an
in vivo infection with in order to determine whether high levels of E2 in serum
might modify the kinetic of the infection by means of studying some humoral
activities and gene expressions relevant to the innate immune response.
Financial support by grants AGL2013-43588-P (MINECO and FEDER), RYC-
2009-05441 (MINECO), 04538/GERM/06 (Fundación Séneca, Región de
Murcia) and fellowship to Y. Valero (Instituto Español de Oceanografía) are
gratefully acknowledged.
77
P32
KISSPEPTINS PATHWAY IS ALTERED IN THE EUROPEAN SEA BASS
UPON NODAVIRUS INFECTION
Yulema Valero1, A. Cuesta
2, M. Arizcun
1, FA Guardiola
2, M.A. Esteban
2, J.
Meseguer2, E. Chaves-Pozo
1. E-mail: [email protected]
1Centro Oceanográfico de Murcia, Instituto Español de Oceanografía, 30860
Puerto de Mazarrón, Spain. 2Fish Innate Immune System Group, Department of
Cell Biology and Histology, Faculty of Biology, Campus Regional de
Excelencia Internacional “Campus Mare Nostrum”, University of Murcia,
30100 Murcia, Spain.
In vertebrates, the kisspeptins pathway in the brain is an essential upstream
regulatory element of the hypothalamus - pituitary - gonad (HPG) axis which
regulates reproduction. In European sea bass it has been described two forms of
kisspeptin genes, kisspeptin 1 (kiss1) and kisspeptin 2 (kiss2), and two forms of
G-coupled protein receptor for kisspeptins, gpr54-1b and gpr54-2b. The
kisspeptins pathway activation is involved in the release of GnRH in the brain,
which subsequently, is involved in the release of the gonadotropin hormones
(LH and FSH) in the pituitary, and in turn acts in the regulation of the
steroidogenesis and gametogenesis in the gonad. One of the pathogens that
greatly affects the brain is nodavirus (NNV), a known vertically transmitted
virus, which causes the viral encephalopathy and retinopathy (VER) disease.
Previous studies showed that, in addition to infect the brain and retina and
cause a considerable fish death in European sea bass specimens, NNV is able to
colonize and replicate into the gonad, altering the sex steroid hormone levels
and triggering a high immune response into the tissue. In this study, we have
determined the alteration on the expression levels of kiss1 and 2, gpr54-1b and -
2b, gnrh1, 2 and 3 genes and also the GnRH receptor (gnrhr2a) gene in the
brain and retina of European seabass males upon NNV intramuscular or
intravitreal infection. This study represents an advance on our knowledge about
the interaction between host and NNV, needed to understand how this virus is
able to avoid the immune response of adult fish to spread to the progeny.
Financial support by grants AGL2013-43588-P (MINECO and FEDER), RYC-
2009-05441 (MINECO), 04538/GERM/06 (Fundación Séneca, Región de
Murcia) and fellowship to Y. Valero (Instituto Español de Oceanografía) are
gratefully acknowledged.
78
P33
DIETARY Pi RESTRICTION IN SEA BASS AND HORMONAL
MODULATION OF PHOSPHORUS AND CALCIUM INTESTINAL
ABSORPTION
Alexandra Alves, S.C. Silva, A.V.M. Canario, D.M. Power, P.M. Guerreiro.
Email:[email protected]
CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.
The endocrine factors regulating inorganic phosphorus (Pi) homeostasis in
vertebrates are not completely understood and our knowledge comes from its
association with calcium (Ca) where parathyroid hormone (PTH) or 1,25-
dihydroxyvitamin D3 (VitD) play relevant roles. A novel factor, fibroblast
growth factor-23 (FGF23) was described as a key Pi regulator, with actions on
urinary Pi excretion but also on the expression of PTH and VitD. There is no
information on how these factors may interplay in fish but our studies show that
the Corpuscles of Stannius, the source of the anti-hypercalcemic stanniocalcin
also express PTHrP and FGF23. Does Pi availability determine hormonal
profiles and the response of absorption/excretion mechanisms?
We measured the rates of intestinal Pi and Ca absorption in sea bass,
Dicentrarchus labrax, fed with different Pi diets, and under several hormonal
treatments ex vivo. Fish (~70g) were allocated in 5 groups in tall 500L open
system tanks and fed a commercial control diet (1.1%P) for at least two weeks.
For the feeding trial four experimental diets containing 0.05, 0.3, 1.1 and 3.0
%P, plus the control diet, were used. Fish were fed (2-3% w/w) twice a day and
the remaining pellets collected 30 minutes after feeding to establish Pi
consumption and growth and feeding parameters. At 10 and 30 days into the
trial blood samples were collected and analyzed for electrolytes and energetic
substrates. Intestinal sections from fish in different conditions were dissected
and mounted in Ussing chambers and uni-direccional fluxes were measured in
symmetric and asymmetric conditions with or without hormonal exposure using
radioactive tracers.
Different dietary Pi had a marked effect (P<0.001) on circulating Pi but not on
Ca (P=0.176), and quantification of circulating hormone levels is underway.
Regional differentiation in intestinal absorption was observed for both Pi and
Ca, and this was further exacerbated by dietary conditions. Preliminary data
shows effects of VitD and PTHrP on absorption but it still uncertain whether
their actions or FGF23 were modulated by available Pi.
Funded by FCT - Foundation for Science and Technology through project
PTDC/BIA-ANM/4225/2012.
79
P34
DOES THE URINARY BLADDER PLAY A ROLE IN FISH RENAL
FUNCTION? SOME HINTS FROM PHOSPHATE AND CALCIUM
TRANSPORT STUDIES
Pedro M. Guerreiro, A. Alves, S.C. Silva, A.G. Becker, A.V.M. Canario,
Jonathan M. Wilson1 and J. Fuentes. Email:[email protected]
CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal and 1CIIMAR, Portugal and Wilfrid Laurier University, Canada.
In contrast to the ectodermal amphibian bladder or the reptilian cloaca, and the
largely endodermal mammalian bladder, the teleost urinary bladder derives as
an expansion of mesonephric duct which gives origin to the kidney and it is
thus possible that it possesses cellular and functional properties of the distal
renal tubule. It has been shown that the bladder permeability to water can be
modulated in different environmental conditions by hormones such as prolactin
and cortisol. Additionally, teleosts show considerable diversity in the size, shape
and even localization of the bladder, which can correlate to important adaptive
functions. Here we aimed to evaluate a possible role in Ca/Pi balance during
urine formation.
We have used the Lusitanian toadfish Halobatrachus didactylus bi-lobulated
urinary bladder as an ex vivo model using several approaches. Histological
sections of the bladder show differential distribution of distinct cell types, some
consistent with high transepithelial transport activity. Gene expression studies
indicate that the receptors for relevant calciotropric or phosphatropic factors,
such as Parathyroid hormone related-protein (PTHrP) and fibroblast growth
factor 23 (FGF23) are present in the urinary bladder. Transepithelial transport
using unidirectional 33
P or 45
Ca fluxes in bladder sac preparations or in Ussing
chambers under symmetrical and asymmetrical clamped conditions indicate
relevant transport rates occur and are modified by the addition of intracellular or
membrane transport modulators such as Forskolin, Amiloride or Ouabain.
Although our current data is not sufficient to clearly establish a role in Ca/Pi
balance, the relatively high variability observed in transport amplitude among
individuals suggests this tissue may be regulated and such differences may be
related to nutritional status, as relevant differences were observed in plasma and
urine phosphate levels. Further experiments in different environmental
conditions are being carried out.
Funded by FCT - Foundation for Science and Technology through project
PTDC/BIA-ANM/4225/2012.
80
P35
EXPRESSION OF FGF23/KLOTHO SYSTEM IN THE SEA BASS:
TISSUE DISTRIBUTION AND REGULATION BY DIETARY Pi
Sandra C. Silva, A. Alves, A.V.M. Canario, D.M. Power, P.M. Guerreiro. E-
mail:[email protected]
CEIB - Centro de Ciências do Mar, Universidade do Algarve, Portugal.
Fibroblast growth factor-23 (FGF23) is a key regulator of phosphorus (Pi)
homeostasis, secreted in response to increased serum Pi. FGF23 binds to FGF
receptor 1 (FGFR1) when coupled to the transmembrane protein αKLOTHO,
decreasing PTH secretion and increasing urinary phosphorus excretion. Given
its crucial physiological role, abundant amounts of Pi are provided in fish feeds,
with deleterious effects to either fish or the environment. Here we aimed to
characterize the presence and regulation of the genes encoding the main players
in Pi uptake, regulation and excretion in Dicentrarchus labrax.
Tissue distribution was performed in 3 male and 3 female sea bass (~250g).
RNA extraction and cDNA synthesis were performed using the E.Z.N.A. Total
RNA kit from Omega Biotek and Ribolock/RevertAid from Life Technologies.
Fish were pooled by gender and expression evaluated by PCR using primers
designed upon the sea bass genome. For the diet experiment, fish (~70g)
allocated in 5 groups fed commercial control diet: 1.1%P, and four
experimental diets: 0.05, 0.3, 1.1 and 3.0 %P. At 10 and 30 days into the
feeding trial tissues were collected in RNA later and processed as above. Gene
expression was performed by QPCR using EvaGreen® Supermix and a iCycler
thermocycler (Bio-Rad).
FGF23 and FGFR1 are expressed in all the tissues analyzed but αKLOTHO
expression occurs only in liver, kidney, CS, pyloric ceca and pituitary, indicating
these may be targeted as a FGFR1/ αKLOTHO complex is required for
transactivation. The sodium-phosphate cotransporter NaPi-IIa was abundantly
expressed in kidney while intestinal expression of the different NaPi forms is
inconclusive at this point. The evaluation of dietary effects on gene expression
is on-going. We expect high dietary P to reduce renal reabsorption while
increasing Pi excretion, probably by up-regulation of the FGF23 system, and
dietary P restriction to increase expression of NaPis both in intestine and kidney.
The elevated expression of FGF23 and its receptor complex in the CS, which
secretes Stanniocalcin and expresses PTHrP suggests it may be a central player
in Pi regulation.
Funded by FCT - Foundation for Science and Technology through project
PTDC/BIA-ANM/4225/2012.
81
P36
CAN SALINITY MODULATE AN INTERPLAY BETWEEN PTHRP AND
STANNIOCALCIN THROUGH CALCIUM AND THE CALCIUM
SENSING RECEPTOR IN THE SEA BASS (Dicentrarchus labrax)?
Alexssandro G. Becker, P.M. Guerreiro, P.F. Palma and A.V.M.Canário. E-
mail:[email protected]
CEIB – Center of Marine Sciences, University of Algarve, Portugal.
Stanniocalcin 1 (STC) and Parathyroid Hormone related Protein A (PTHrP) are
endocrine factors that regulate calcium in fish, the first being anti-
hypercalcemic and the latter hypercalcemic and their expression coincides in
pituitary (Pit) and Corpuscles of Stannius (CS). Previous studies indicated that
both STC and PTHrP secretion may be regulated by environmental salinity but
the onset mechanism is not yet elucidated. We aimed 1) to characterize the
effects of salinity in plasma calcium, STC secretion by CS and on the
expression of the STC, PTHrP and CaSR at different salinities (in vivo) and 2)
to determine STC and PTHrP secretion in CS and Pit incubated in vitro at
various Ca2+
or calcimimetics conditions.
For Task 1, fish (~200g) were acclimated to different water salinities (0.4 (FW),
12.8 (BW) and 35 (SW)) to determine physiological, biochemical and molecular
responses. Blood and tissues (CS, Pit, gill and kidney) were collected. Plasma
ion levels and osmolality were determined by colorimetric kits and a Vapour
pressure osmometer. Na+/K+-ATPase activity was evaluated on an enzymatic
assay and STC and PTHrP expression via QPCR (ongoing). Branchial activity
was higher in FW and SW conditions and reduced in BW. Plasma Na+ was
reduced in lower salinities but no evident changes occurred in Ca2+
indicating
this ion was tightly regulated.
In Task 2, fish (~700g) maintained in seawater conditions were sacrificed and
CS and Pit carefully removed to a perifusion setup. In dynamic conditions, CS
were perifused with 0.1, 0.25, 0.5, 1.25, 3.0, 5.0, 8.0 mM Ca2+
or 10, 50 and
500nM; NPS2143 in oxygenated Hank’s medium and 20 min perifusate
fractions collected for ~3 hours. In the static system, CS and Pit were incubated
as above in 48-well plates and the medium containing putative secreted proteins
was collected and substituted for fresh medium every 60 min also up to 3 hours.
Fractions and tissues were collected to determine hormone levels and gene
expression (ongoing analyses).
AGB has a postdoctoral grant from Portuguese Foundation for Science and
Technology SFRH/BPD/96187/2013. Funded by FCT projects
PTDC/MAR/121279/2010 and PTDC/BIA-ANM/4225/2012.
82
P37
GONADOTROPIN-INHIBITORY HORMONE IN THE FLATFISH Solea
senegalensis: MOLECULAR CLONING, BRAIN LOCALIZATION AND
PHYSIOLOGICAL EFFECTS.
María Aliaga-Guerrero1, P. De Terry-Castro
1, V. Gallego-Recio
1, J.A. Paullada-
Salmerón1, E. Mañanós
2, J.A. Muñoz-Cueto
1. E-mail: [email protected]
1 Department of Biology. Faculty of Marine and Environmental Sciences.
University of Cádiz. 11510-Puerto Real, Spain
2 Institute of Aquaculture of Torre de la Sal. CSIC. 12595-Ribera de Cabanes,
Spain
Gonadotropin-inhibitory hormone (GnIH) is a recently discovered neuropeptide
that plays a very important role in the regulation of reproduction in birds and
mammals. This neuropeptide from the RFamide family has neuromodulatory
functions and controls the synthesis and/or release of brain gonadotropin-
releasing hormone (GnRH) and pituitary gonadotropins. Scientific and
technological advances on the GnIH system in fish are, up to date, scarce,
contradictory and inconclusive. For this reason, research on fish GnIH system
appears necessary to better clarify its role in the neuroendocrine and
environmental control of reproduction in this important group of vertebrates. In
this context, we have cloned a full-length sequence for the GnIH precursor of
Senegal sole (ssGnIH), which codifies for three putative GnIH peptides. We
have also generated specific antisera against ssGnIH peptides, and used them to
elucidate the localization of GnIH cells and their projections in the brain and
pituitary of sole. ssGnIH-immunoreactive cell bodies were present in the
olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum
and rostral rhombencephalon. These GnIH cells innervated profusely the brain
and pituitary of sole. Intramuscular injection of ssGnIH-3 provokes a significant
reduction in GnRH-3 (dose of 1.0μg/g) and LH (doses of 0.1μg/g and 1.0μg/g)
expression at 4 hours post-injection. In contrast, no effect of ssGnIH-2 on the
transcript levels of the main reproductive genes was evident. Our
immunohistochemical and physiological results reveal that GnIH could
represent an important actor in the neuroendocrine control of reproduction of
sole. Further studies are being directed to elucidate if this inhibitory
neurohormone is involved in the reproductive dysfunctions that occur in the F1
generation of aquacultured sole.
Funded by MINECO (AGL2010-22139-C03-03 and AGL2013-49027-C3-2-R
grants to JAM-C and BES-2011-047276 Fellow to MA-G).
83
P38
THE ONTOGENY OF SOLE (Solea senegalensis) NEUROENDOCRINE
SYSTEMS IS AFFECTED BY ENVIRONMENTAL AND CULTURE
CONDITIONS.
María Aliaga-Guerrero1, V. Piquer
2, E. Mañanos
2, J.A. Muñoz-Cueto
1. E-mail:
1 Department of Biology. Faculty of Marine and Environmental Sciences.
University of Cádiz. 11510-Puerto Real, Spain. 2 Institute of Aquaculture of
Torre de la Sal. CSIC. 12595-Ribera de Cabanes, Spain
Aquaculture of sole is seen as an excellent choice for diversification and
economic expansion of the European aquaculture. Reproduction of wild sole
breeders in captivity is successful whilst sole specimens from F1 generation
present reproductive dysfunctions. Fish farming in captivity introduces
variations in environmental conditions compared to natural ecosystems, which
could affect the ontogeny of the brain and neuroendocrine systems and,
therefore, the reproductive performance and behavior. However, information on
how environmental and culture conditions affect the establishment of fish
reproductive systems is rather scarce. For this purpose, sole larvae were exposed
to: 1) daily thermocycle and blue light (18-21ºC; TCblue) or, 2) constant
temperature and white light (19.5ºC; cteW; routine aquaculture conditions). In
addition, and to determine a potential sensitive period, the TCblue conditions
were applied in different time frames: 1) until metamorphosis (0-12 days post-
hatching or dph), 2) post-metamorphosis (0-30 dph) or, 3) gonad differentiation
(0-100 dph). In other experiment, development of sole embryo and larvae under
routine aquaculture conditions and mesocosm conditions was compared. Effects
were determined by analyzing gonadotropin-releasing hormone (GnRH),
kisspeptin and gonadotropin-inhibitory hormone (GnIH) gene expression by
qPCR. The results obtained demonstrated that under routine aquaculture
conditions, developing sole exhibited higher GnIH transcript levels in critical
ontogenetic periods (sex differentiation) compared to specimens reared under
natural thermocycles and blue lights. Mesocosm culture conditions also affected
GnIH, sGnRH and kisspeptin ontogenetic expression patterns. Our results
evidence that routine conditions used in aquaculture practice can disturb the
natural organization of sole neuroendocrine systems, reinforcing the importance
of mimicking natural environmental conditions during fish rearing.
Funded by MINECO (AGL2010-22139-C03-03 and AGL2013-49027-C3-2-R
grants to JAM-C and BES-2011-047276 Fellow to MA-G).
84
P39
ONTOGENY OF LIGHT-SENSOR SYSTEMS OF SENEGAL SOLE
EXPOSED TO DIFFERENT LIGHT PHOTOPERIOD AND SPECTRA
Isabella Paradiso, S. Frau, J.A. Muñoz-Cueto, Á.J. Martín Robles. E-mail:
Department of Biology. Faculty of Marine and Environmental Sciences.
University of Cádiz. 11510-Puerto Real, Spain
Light represents the main environmental factor synchronizing biological
rhythms and entraining melatonin production, which is dependent on light
intensity, spectra (wavelength) and duration (photoperiod). The light that
penetrates into the seawater column changes in intensity and wavelength due to
selective absorption. As a result, most of the wavelengths are absorbed in the
upper waters, while the blue light reaches greater depths. The Senegal sole
undergoes a real metamorphosis between days 12 and 19 after hatching and
during this process it changes its life habits from pelagic to benthonic and from
diurnal to nocturnal. However, it is unknown if this metamorphic process is
accompanied by a differential expression pattern of opsins with different
photosensitivity. In the present study we investigated the expression of four
classes of opsins (UV opsin 5, green sensitive pigment, red opsin and teleost
multiple tissue opsin or TMT) during the development of this flatfish species by
using quantitative real time PCR. The eggs were collected immediately after
spawning during the night and exposed to light-dark (LD) 12L:12D cycles of
white (LDW), blue (LDB, λ peak = 463 nm), red (LDR, λ peak = 685 nm),
continuous white light (LL) and continuous darkness (DD). Our results show a
peak of expression of green sensitive pigment between day 6 and 12 in all the
conditions, decreasing thereafter. Red opsin expression shows a peak at D12 in
all the conditions and decreases during metamorphosis, but in LL, LDW and
LDB it increases again in post-metamorphosis (D25). UV opsin expression
presents a first peak at day 1 in all the conditions, increasing during
metamorphosis (D19) in LDB, LDW and LL and decreasing in post-
metamorphic animals (D25). Finally, TMT opsin transcript levels peaked at D12
in all conditions. Day-night difference in opsin expression was only evident
under blue lights. These results show a differential pattern of expression of
different opsins before, during and after the completion of metamorphosis in
Solea senegalensis and suggest that its bottom settlement is accompanied by
changes in photopigments.
Funded by a grant from MINECO (AGL2013-49027-C3-2-R) to JAM-C.
85
P40
PHOTOPERIODIC SIGNALLING MEDIATES THE CONTROL OF
KISS1 AND GNRH2 NEURONAL POPULATIONS ON THE
REPRODUCTIVE AXIS IN A TELEOST FISH, EUROPEAN SEA BASS
(Dicentrarchus labrax)
Felipe Espigares, A. Rocha, A. Gómez, M. Carrillo, S. Zanuy. E-mail:
Department of Fish Physiology and Biotechnology, Institute of Aquaculture of
Torre de la Sal (IATS), Spanish National Research Council (CSIC), Ribera de
Cabanes s/n, 12595, Castellón. Spain.
There are a wide variety of environmental factors that change seasonally and
that may modulate the activity of the reproductive axis. Of all, photoperiod is
the main environmental signal that most temperate fish species use to predict
the changing seasons and therefore anticipate spawning time through activation
of neuroendocrine pathways which, in turn, stimulate gonadotropic axis and
gonadal growth. However, many molecular and endocrine mechanism involved
in the onset of puberty and the influence of environmental conditions, such as
photoperiod signalling, are not well understood in fish. In this study, 1 year-old
male European sea bass (Dicentrarchus labrax) were exposed to three different
photoperiod conditions, natural photoperiod (NP), advanced photoperiod (AP)
and continuous light (LL) in combination with size-sorting and studied from
August to February. The obtained results clearly indicated that AP and LL were
able to train the rhythms of both kiss1 and gnrh2 mRNA levels in the forebrain-
midbrain (FB-MB), suggesting that the photoperiodic signalling may modulate
the activity of the habenular Kiss1 and the synencephalic Gnrh2 neurons to
drive reproductive axis. Also AP and LL regimes affected both plasma Fsh and
11-KT profiles, which in turn would explain the phase shift and reduction of
gonadal recrudescence under AP and LL, respectively. In conclusion, the
present study reveals a strong relation between environmental control of puberty
and the Kiss1 and Gnrh2 systems in agreement with previous ?ndings observed
in other teleost species, including European sea bass. We also hypothesize that
very likely, as a result of the modulatory role of the photoperiod on the Kiss1
and Gnrh2 activity, this is able to shift the hormone profiles and hence advance
the onset and progression of gametogenesis under AP, whereas under LL, the
unbalanced production of 11-KT, regulated by an altered Fsh profile, could be
limiting the stimulation of germ cell proliferation at the testicular level and thus
prevent the normal progression of the spermatogenesis.
Supported by GV (PROMETEO/2010/003 to S.Z.), EU (LIFECYCLE,
FP7222719 to S.Z.). F.E. was supported by a JAE Predoctoral fellowship
(CSIC).
86
P41
SPECIATION IN THE EVOLUTION OF ALLATOSTATIN-TYPE A
RECEPTORS (AST-AR) IN DIPTERA
Rute C Félix, Joao CR Cardoso and Deborah Power. Email. [email protected]
Comparative Endocrinology and Integrative Biology, Centre of Marine
Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro,
Portugal
The Allatostatins type-A (AST-A) receptors are a group of invertebrate G-
protein coupled receptors (GPCRs) that share ancestral origin with the
vertebrate receptors for the peptides Kiss and Galanin. In arthropods, the
evolution of AST-AR was recently resolved and in insects, a single gene was
found but in dipterans two AST-ARs (AST-AR1 and AST-AR2) with distinct
functions co-exist as the result of gene duplication (Felix et al., 2015).
Anopheles mosquitoes have a rapid rate of speciation and a rapidly evolving
genome and in A. gambiae (PEST strain) both AST-ARs map to a highly
modified chromosome but the impact of this on receptor gene structure and
function has not been determined. The aim of this study was to characterize the
evolution of AST-A receptor genes in Anopheles mosquitos and to determine
how adaptive evolutionary pressure has contributed to receptor gene divergence
in diverse Anopheles species. AST-AR genes were retrieved from 17 Anopheles
species genomes and receptor gene structure and sequence was compared
between species and with the orthologues isolated from Drosophila genomes.
Analysis revealed that in Anopheles, orthologue receptor gene structure was
different and exon tandem duplication and exon inversions modified the AST-
AR2 gene in some species. The potential effect on AST-AR gene evolution of
geographic localization of Anopheles, its efficiency as a malaria vector, host
feeding preference and taxonomic characterization will be analysed and
discussed.
Acknowledgements: This study was financed by the European Regional
Development Fund (ERDF) COMPETE and Portuguese funds through the
Foundation for Science and Technology (FCT) under the project
UID/Multi/04326/2013. RCF is in receipt of FCT SFRH/BPD/89811/2012 grant
and JCRC by an auxiliary research contract under the project
UID/Multi/04326/2013.Felix et al, 2015, PLoS One. 10, e0130347.
87
P42
SOMATOLACTIN α AND β SPECIFIC REGULATORY ELEMENTS
AND DIFFERENTIAL SPACIAL EXPRESSION IN PITUITARY OF
Cyprinus carpio
Valenzuela-Nieto, G.E., Stolzenbach, M.F., Henriquez, N., Vega, M., Romero, A.,
Figueroa, J., Kausel, G. E-mail:[email protected]
Instituto de Bioquímica y Microbiología, Universidad Austral de Chile,
Valdivia, Región de Los Ríos, Chile.
Environmental changes affect gene expression that we addressed in the
pituitary, a central regulatory organ at the interface between the central nervous
system and the endocrine system where endocrine disruptor effects are still
poorly understood.
Somatolactin (SL), a fish specific pituitary hormone belonging to growth
hormone (GH) and prolactin (PRL) superfamily, is involved in background
adaptation, osmoregulation, reproduction and fatty acid metabolism. In carp,
transcripts of two SL genes (α and β) were detected in pituitary gland and when
treated with potential endocrine disruptor compounds differential response was
detected by RT-qPCR. Adult male carp responded with decrease of expression
of both SL genes to ZnCl2 treatment. However, expression of SLα and SLβ was
modulated differentially in pituitary of male adult carp in response to estrogen,
which might imply differential binding of transcription factors to the promoters
of these duplicated genes. With the aim to characterize in detail the divergent
regulation of the two SL genes, promoters of both genes were analyzed.
Therefore, the proximal promoter region of SLα and β were cloned applying
inverse PCR, transcription start site was determined with 5’RACE for 5´-UTR
and sequences complemented by in silico cloning. In the proximal promoter of
both genes several putative binding sites for Pit-1 were identified and confirmed
in vitro by electro mobility shift assays suggesting regulation of SL by this
pituitary specific transcription factor. In addition, clearly, specific binding to an
estrogen response element that was identified only in SLβ promoter, might
explain differential regulation of both SL in response to estrogen. Comparison
of the complete coding sequences of slα and slβ revealed 61.6% identity at the
nucleotide level and 46.2% between the derived aminoacid sequences. We
prepared antibodies against a carp SLα- specific oligopeptide and double
immunostaining with an anti-chum salmon SL and the specific anti-carp SLα
antibody revealed spacially specific expression patterns in pars intermedia of
carp pituitary sagittal sections.
Taken together, here we show for the first time a cis-regulatory element related
to estrogen response in SL which is absent in SLα promoter and a clear
spacially different expression pattern in pituitary of male carp. These data
88
highlight the importance to study regulation of duplicated genes in carp for
evaluation of environmental changes including effects of endocrine disrupters
of estrogenic potential in the aquatic environment.
Supported by: DAAD 50750108, DAE-UACh, Mecesup AUS1203, UACh-DID-
SE2015-02 (GK), CONICYT 21999 (GV), Fermelo S.A
89
PRESENTING AUTHORS
Oral Communications
Alves Costa R O15
Blanco AM O8
Cal L O29
Cardoso JCR O25
Castaño JP OL
Dulce Estêvão MD O26
Felip A O4
Gil Solsona R O9
Guerreiro PM O19
Gutiérrez J JP'C
Hernández-Pérez J O6
Hormaechea-Agulla D O23
Kah O CL
Mateus AP O20
Medina P O3
Morini M O2
Nácher-Mestre J O16
Naderi F O18
Peñaranda DS O1
Pérez-Sánchez J O12
Pinto PIS O14
Rincón-Fernández D O24
Ruiz-Jarabo I O27
Sarmento-Cabral A O11
Simó P O10
Skrzyńska AK O17
Suarez-Bregua P O28
Valdivieso A O5
Vázquez-Borrego MC O21
Velasco C O7
Villa-Osaba A O22
Zaghdoudi-Allan N O13
90
Poster
Aliaga-Guerrero M P37, P38
Alves A P33
Asturiano JF P16
Becker AG P36
Cortés R P11
Felip A P20
Félix RC P41
Frau S P39
Gilannejad N P2
Gómez-Boronat M P28
Guardiola FA P30, P31, P32
Guerreiro PM P34
Isorna E P24
Jerez I P22
Librán-Pérez M P9
López-López F P29
Mancera JM P25, P26, P27
Mañanós E P15
Martos-Sitcha JA P1, P4
Medina P P17
Míguez JM P23
Morini M P19
Navarro I P8
Navarro S P10
Nisembaum LG P14
Otero-Rodiño C P7
Palma PS P21
Pinto P P13
Piquer V P18
Sarasquete C P5
Silva SC P35
Skrzyńska AK P6
Soletto L P12
Valenzuela-Nieto GE P42
Vélez EJ P3
Zanuy S P40
91
AUTHOR INDEX
Agulleiro MJ P11
Aliaga M P18
Aliaga-Guerrero M P37, P38
Alonso-Gómez AL P24
Alvarado MV P20
Alvarez-BenitoM O22
Álvarez-Otero R O7, P7, P23
Alves Costa RA O15, O20, P33, P34, P35
Andrade A O26, P13
Arizcun M P31, P32
Astola A P25
Asturiano JF O1, O2, P16, P19
Ayala Suárez R P25
Baeza R P16
Baldisserotto B P27
Bantz A P14
Bastaroli M O17, P6
Becker AG P34, P36
Beltrán E P15
Berntssen MHG O16
Besseau L P14
Blanco AM O8, P24, P28
Blasco J JP'C,P3
Blázquez M O3, P17
Braasch I O28
Bronner ME O29
Butts IAE P16
Cal L O29
Calduch-Giner JA O9, O10, O12, P4
Canário AVM O19, P13, P21, P33, P34, P35, P36,
Capilla E JP'C,P3, P8
Cardoso JCR O20, O25, P41
Carrasco-Valiente J O23
Carrillo M P20, P40
Castaño JP OL, O11, O21, O22, O23, O24, P29
Ceinos R O6
Cerdá-Reverter JM O6, O28, P10, P11, P12
Chaves-Pozo E P31, P32
Churcher A O13
Clark M O13
92
Conde-Sieira M P7
Cone RD P11
Córdoba M P8
Cortés R P10, P11, P12
Cuesta A P30, P31, P32
Culler MD O23
de las Heras V P2
De Pedro N P28
de Terry-Castro P P37
Delgado MJ O8, P24, P28
Dufour S P19
Espigares F O4, P40
Esteban MA P30, P31, P32
Estêvão MD O26
Falcón J P14
Felip A O4, P20
Félix RC O13, O25, P41
Fernández-Borràs J JP'C,P3
Figueroa J P42
Flik G P22
Frau S P39
Fuentes J O27, P34
Fuentes M P14
Gahete MD OL, O11, O21, O22, O23, O24, P29
Gallego V O1, P16
Gallego-Recio V P37
Gálvez MA O21
Gesto M O6, P23
Gil Solsona R O9
Gilannejad N P2
Giménez I O1
Gisbert E O20
Gómez A O4, O3, O4, P13, P17, P40
Gómez-Boronat M O8, P28
Gómez-Gómez E O23
Gómez-Skarmeta JL O29
Gorissen M P22
Gracia-Navarro S OL
Gregório, SF O17
Guardiola FA P30, P31, P32
Guerreiro PM O19, P21, P33, P34, P35, P36
Guillot R P10
Gutiérrez J JP'C, P3, P8
93
Guzmán JM P15
Henriquez N P42
Hernández F O9, O16
Hernández-Pérez J O6, P7, P9, P23
Hormaechea-Agulla D OL, O23
Ibáñez M O16
Ibáñez-Costa A OL, O21, O23
Isorna E P24, P28
Jerez I P1
Jerez I P27
Jerez I P22
Kah O CL
Kausel G P42
Kelsh R O28
Lacalle L O9
Lafont AG P19
Librán-Pérez M O6, O7, P7, P9
López-López F OL, O11, O22, P29
López-Miranda J O21
López-Patiño MA O6, O7, O18, P23
Luque RM OL, O11, O21, O22, O23, O24, P29
Lutfi E JP'C,P3, P8
Magnanou E P14
Maiorano E O17, P6
Mancera JM O17, P1, P4, P6, P22, P25, P26 P27
Mañanos E P15, P18, P37, P38
Martin P P14
Martín Robles AJ P39
Martínez-Fuentes, A OL
Martínez-Rodríguez G O17, P1, P2, P4, P6, P26, P27
Martins RS P13
Martos-Sitcha JA O17, P1, P2, P4, P6, P25, P26, P27
Mateus AP O20
Medina P O3, P17
Meseguer J P30, P32
Michel M P11
Míguez JM O6, O17, P9, P12, P23
Mischitelli M P10
Mohammed-Geba E P26
Moran P O28
Moreno MM O23
Morini M O2, P19
Mourão RHV P27
94
Moya A P3
Muñoz-Cueto JA P18, P37, P38, P39
Muñoz JL O6, P23
Nácher-Mestre J O9, O16
Naderi F O6, O7, O18, P9, P23
Navarro I P3, P8
Navarro S JP'C,P10, P11, P12
Nerín G P8
Nisembaum LG P14
Nourizadeh-Lillabadi R O2
Ortega-Salas R O22
Ortiz-Delgado JB P5
Otero-Rodiño C O7, O18, P7, P9
Palma PF P36
Palma PS P21
Paradiso I P39
Paullada-Salmerón JA P18, P37
Pavanato MA P27
Pedraza-Agüera, A OL
Peñaranda DS O1, O2, P16, P19
Perelló M P3
Pérez L O1, O2, P16, P19
Pérez-Sánchez J O9, O10, O12, O16, P4
Pês TS P27
Piferrer F O5
Pinto PIS O14, O26, P13
Piquer V P15, P18, P38
Planas JV JP'C
Porte C P8
Portolés T O16
Power DM O13, O14, O15, O11, O20, O25, O26,
P21, P33, P35, P41
Pozo-Salas AI O22
Puchol S P12
Ramos J P18
Ramos-Fernández JA O23
Redondo I O12, P28
Requena MJ O23
Ribas L O5
Rincón-Fernández D O24
Rivero-Cortés, A.J OL
Rocha A P40
Romero A P42
95
Rosel JF P12
Rotllant J O28, O29, P12
Ruiz-Jarabo I O27
Ruiz-Murillo V O24
Saccol EMH P27
Sánchez E P10, P11, P12
Sánchez-Bretaño A P24, P28
Sánchez-Sánchez R O22
Sancho JV O9
Santamaría R O24
Santos S O16, P13
Saraiva JL O19
Sarasquete C P5
Sarmento-Cabral A OL, O11, P29
Saxena A O28
Serrano R O16, P15
Silva FB O19
Silva M O19
Silva SC P33, P35, P35
Simó P O10
Sitjà-Bobadilla A. O12
Skrzyńska AK O17, P6
Soares J O19
Soengas JL O19, P7, P9
Soletto L P10, P11, P12
Soto-Moreno A O17
Stolzenbach MF P42
Suarez-Bregua P O28
Swanson P P15
Thorne MAS O14
Toledano-Delgado A O21
Tveiten H P16
Úbeda-Manzanaro M P5
Valdivieso A O5
Valenciano, AI O8
Valenzuela-Nieto GE P42
Valero Y P31, P32
Valero-Rosa J O23
Vargas M O19
Vázquez-Borrego MC OL, O21
Vega M P42
Velasco C O7, O18, P7, P9
Vélez EJ JP'C,P3