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26th Conference of European Comparative Endocrinologists (CECE). University of Zurich, Irchel Campus 21–25 August 2012 — with Special Section on Fish Model system edited by Oliana Carnevali.-- 11 pages, 3 figures, 3 tables, It is well established that Kisspeptin regulates the onset of puberty in vertebrates through stimulation of the secretion of gonadotropin-releasing hormones. However, the function of kisspeptin in peripheral tissues and in other functions is still poorly understood. Recently, the evolution and distribution of kisspeptin genes in vertebrates has been clarified. In contrast to placental mammals, which have a single gene for the ligand (Kiss) and for the receptor (Kissr), fish may have up to three Kiss genes and up to four Kissr genes because of genome duplications. However, information on the genomic structure of the piscine kiss and kissr genes is still scarce. Furthermore, when data from several species is taken together, interspecific differences in the expression of kiss and kissr during the reproductive cycle are found. Here, we discuss data gathered from several fish species, but mainly from two flatfishes, the Senegalese sole and the Atlantic halibut, to address general questions on kiss gene structure, regulation and function. Flatfish are among the most derived fish species and the two species referred to above have only one ligand and one receptor, probably because of the genome reduction observed in Pleuronectiformes. However, gene analysis shows that both species have an alternative splicing mechanism based on intron retention, but the functions of the alternative isoforms are unclear. In the Senegalese sole, sex-related differences in the temporal and spatial expression of kiss and kissr were observed during a whole reproductive cycle. In addition, recent studies suggested that kisspeptin system gene expression is correlated to energy balance and reproduction. This suggests that kisspeptin signaling may involve different sources of information to synchronize important biological functions in vertebrates, including reproduction. We propose a set of criteria to facilitate the comparison of kiss and kissr gene expression data across species, A.S.M. was supported by a predoctoral scholarship from the Spanish Ministry of Education and Science (MEC) and J.V. by a postdoctoral contract from Genoma España through the “Pleurogene” project. This study was carried out with the financial help of projects “Pleurogene”. Research at the F.P. lab is partially supported by project “Aquagenomics” (CDS-2007-0002) funded by the Spanish Government

Memoria de tesis doctoral presentada por Alejandro S. Mechaly para optar al grado de Doctor por la Universitat Pompeu Fabra (UPF), realizada bajo la dirección del Dr. Francesc Piferrer Circuns del Institut de Ciències del Mar (ICM-CSIC) y del Dr. Jordi Viñas de Puig de la Universitat de Girona (UdG).-- 164 pages, [EN] The recently discovered decapeptide kisspeptin and its G-protein coupled receptor form a signaling system expressed ubiquitously and are implicated in a variety of still poorly characterized functions. In the brain, kisspeptin is secreted by specific neurons and its receptor is localized in GnRH neurons. Kisspeptin signaling has been fully established in the control of the onset of puberty in vertebrates, from fish to mammals. In this study, we characterized the kisspeptin gene in the Senegalese sole and characterized the kisspeptin receptor genes in both the Senegalese sole and in the Atlantic halibut. In contrast to other fish species, the two species analyzed here showed only the presence of one ligand and one receptor, probably as a consequence of the genome reduction characteristic of Pleuronectiformes. However, in both cases we found an alternative splicing mechanism based on intron retention that produces also non-functional isoforms, but whether this is part of a mechanism to control abundance of the active gene product is still not known. We document spatial and temporal changes of expression of kisspeptin and its receptor in the brain, pituitary and gonads related to the annual reproductive cycle. Finally, we present the first evidence of a possible link between energy balance and reproduction mediated by kisspeptin signaling in a non-mammalian vertebrate, [CAT] El recentment descobert decapèptid kisspeptina i el seu receptor associat a una proteïna G formen un sistema que s’expressa ubiqüitament i que està implicat en diverses funcions, moltes de les quals encara no estan ben caracteritzades. En el cervell, la kisspeptina és secretada per neurones específiques, mentre que el seu receptor es troba a les neurones GnRH. Aquest sistema s’ha relacionat amb el control de l’inici de la pubertat en diferents vertebrats, des de peixos fins a mamífers. En aquest estudi, hem caracteritzat el gen de la kisspeptina en el llenguado senegalès, i els gens del receptor de la kisspeptina tant a llenguado senegalès com en l’Halibut de l’Atlàntic. Al contrari del que ocorre en moltes altres espècies de peixos, aquestes dues espècies només presenten un gen pel lligand i un gen pel receptor. Aquest fet és probable que estigui relacionat amb la reducció de la mida del genoma que han sofert els Pleuronectiformes. Tot i així, en les dues espècies s’hi troba un mecanisme d’empalmament alternatiu conseqüència d’una retenció intrónica que produeix una isoforma no funcional. Ara bé, si aquest mecanisme està relacionat amb el control de l’abundància dels trànscrits de la isoforma funcional encara està per esbrinar. Per altra banda, hem trobat canvis en l’expressió gènica tant en l’espai com en el temps durant un cicle reproductiu dels gens de la kisspeptina i el seu receptor en el cervell, pituïtària i gònades. Finalment, també presentem la primera evidència, en un vertebrat no mamífer, d’una possible relació entre el balanç energètic i la reproducció controlada pel sistema kisspeptina

International audience; Kisspeptin signaling in the brain is involved in the control of the onset of puberty in vertebrates. In this study, we present novel evidence indicating that kisspeptin may link energy balance and reproduction. For that purpose, we determined the complete gene structure of kisspeptin in a teleost fish, the Senegalese sole (Ss). In contrast to the situation evident in several fish, in this species only Kiss2 was found. Yet, two Ss isoforms generated by alternative splicing through intronic retention were detected: Ss , producing the functional protein, and Ss , coding for a truncated, non-functional protein. Specific qPCRs showed that the expression of these two isoforms varied differently in brain and gonads throughout maturation. In addition, and in contrast to what has been observed in mammals, fasting increased hypothalamic mRNA levels of Ss , which also caused a concomitant rise in pituitary Ss and Ss mRNA. Together, these data indicate the impact of the nutritional status on mRNA expression as a potential regulatory mechanism for the metabolic control of reproduction in non-mammalian species, albeit with some significant differences with respect to the situation described in mammals.

XII Jornada de Biologia de la Reproducció. Barcelona, 1 de Juny de 2011

Proceedings of 9th International Symposium on Reproductive Physiology of Fish, Cochin, India.-- 2 pages, 1 figure

International audience; Kisspeptin and its receptor, Kiss1r, play an essential role in the control of the onset of puberty in vertebrates. We characterized the cDNA and genomic DNA encoding Kiss1r in Atlantic halibut (). The 1,146bp open reading frame predicts a 381 amino acid protein with high homology to the Kiss1r-2 of other teleost fish. Phylogenetic analysis of Kiss1r sequences suggests that the mammalian Kiss1r-1 form arose by way of a gene duplication prior to the emergence of amphibians. Synteny analysis demonstrated the highly conserved nature of the region in teleosts, suggesting that flanking regulatory sequences are also likely to be conserved. Bioinformatic analysis identified six conserved regions in piscine upstream sequences, providing potential targets for future in-depth investigation of regulation. expression in the brain increased coinciding with the onset of puberty. Expression levels in the gonads were two orders of magnitude lower than those of the brain, a characteristic apparently conserved in other fishes, and expression in gonads was only detected in immature fish.

9th International Congress on the Biology of Fish. 5-9 Julio de 2010, Barcelona, The Kisspeptin system plays an important role in the onset of puberty in vertebrates including fishes. This system includes several orthologs and paralogs of the Kisspeptin genes (Kiss) and of its receptor (Kiss1r). Cloning of the full coding region of both genes in an aquacultured fish, the Senegalese sole (Solea senegalensis) (Ss), revealed unexpected features of these genes. For the first time in vertebrates two splice variants were detected, which originated by intron retention in both the Kiss and Kiss1r. However, no paralog genes were found in this species. Analysis of both retained introns showed several features characteristic of an alternative splicing mechanism. Interestingly, the two splice variants showed distinct expression patterns according to the different stages of sexual maturation. In conclusion, presence of alternative splice variants on the kisspeptin ligand and receptor in vertebrates may contribute to our understanding of the regulation kisspeptin- KISS1R signaling

7º Congreso de la Asociación Ibérica de Endocrinología Comparada AIEC, 6-9 Septiembre 2009, Porto, Portugal

Biologia de la reproducció.-- 13 pages, 4 figures, 1 table, [CA] El recent descobriment de la kisspeptina (producte del gen KISS1), conjuntament amb el seu receptor (producte del gen KISS1R), i de la seva relació amb el control de l'inici de la maduresa sexual en tots els vertebrats, ha estat descrit com una de les troballes més rellevants de la biologia reproductiva en els últims anys. La kisspeptina té un efecte directe sobre l'hormona alliberadora de gonadotrofines, la qual desencadena una cascada hormonal que finalment acaba a les gònades amb la secreció dels esteroides sexuals, els quals provoquen la consegüent maduració de l'individu. En aquest treball s'explica el descobriment dels gens KISS1 i KISS1R, l'estructura d'aquests gens i dels seus productes proteics, i es fa referència als mecanismes endocrins responsables tant de la regulació endògena com dels possibles factors externs que afecten el sistema kisspeptina-KISS1R. També, i amb especial èmfasi en els peixos teleostis, es discuteixen les possibles causes de les diferències i similituds trobades en l'expressió cerebral del sistema kisspeptina-KISS1R. Finalment, s'avaluen les perspectives futures en el camp del control de la reproducció mitjançant la manipulació del sistema kisspeptina-KISS1R, [EN] The discovery of kisspeptin (the product of the KISS1 gene) together with its receptor (product of the KISS1R gene), and the relationship with the control of the onset of puberty in all vertebrates has been described as one the most important findings in reproductive biology in the last years. Kisspeptin stimulates gonadotrophin-releasing hormone (GnRH), initiating the hormonal cascade which ends in the gonads with the secretion of the sex steroids which, in turn, drive sexual maturation. This paper describes the origin and discovery of the KISS1 and KISS1R genes, the structure of these genes as well as their protein products. The endocrine mechanisms and the exogenous factors affecting the kisspeptin- KISS1R complex are also discussed. Finally, we describe the multiple functions in relationship with this system in all vertebrates and discuss the differences and similarities regarding the neural expression of kisspeptin-KISS1R complex in several teleost fishes. Finally, the future perspectives in the field of the control reproduction by the manipulation of the kisspeptin-KISS1R complex are discussed

XI Jornada de Biologia de la Reproducció. Girona, 30 d'abril de 2009.-- 4 pages, 3 figures, Aquest treball se centra en la descripció del sistema de la kisspeptina i el seu receptor (KISSIR), ja que tenen una funció clau com a reguladors de l'inici de la pubertat en animals. Es descriu i es discuteix l'evolució deis gens i de les diferents isoformes del KISSIR trobades recentment. Aquests gens i isoformes són produtts per duplicacions geniques o per mecanismes d'empalmament altematiu dins d'un mateix gen. Finalment, es plantegen les possibles línies d'investigació sobre el sistema i es mencionen els possibles factors de transcripció que poden formar part del mecanisme regulador de KISSIR

1st World Conference on Kisspeptin Signaling in the Brain, 8-11 October 2008, Córdoba, The KISSPEPTIN-1 receptor (KISS1R) and its ligands (KISSPEPTINS) are implicated in the regulation of the onset of puberty. We report the coding region and genomic structure of the kiss1r gene of a modern teleost, the Senegalese sole (Ss). Ss kiss1r cDNA contained an opening frame of 1137 bp, which results in a predicted 378 amino acid protein. Searching genomic databases allowed the identification of kiss1r orthologues in six new species belonging to three vertebrate groups and established the evolutionary relationships of all KISS1R sequences available to date. Analysis of Ss kiss1r revealed for the first time in any vertebrate KISS1R gene the presence of features that are characteristic of a mechanism of alternative splicing. This was confirmed by the identification of two transcripts, Ss kiss1r_v1 and Ss kiss1r_v2. The latter, arising from intron III retention, contained a 27 codons insert in transmembrane region 4 with two stop codons, suggesting it may lead to a truncated protein. The mRNA of the two variants was differently expressed in several tissues. In the brain, levels of the Ss kiss1r_v1 were higher than those of Ss kiss1r_v2. In the gonads, the opposite was observed. Both isoforms exhibited changes depending on sex and maturity stage. The presence of two variants may help to explain some discrepancies observed in past studies regarding KISS1R expression during puberty. Thus, the existence of alternative splicing for the KISS1R gene may contribute to our understanding of the many physiological functions suspected to be mediated by KISSPEPTIN-KISS1R signaling

I Simposi d’Aqüicultura de Catalunya. Investigació, desenvolupament i transferència en aqüicultura des de Catalunya a Europa, 6-8 de febrer de 2008, Barcelona, El descobriment del neurotransmissor kisspeptina (Kiss1), del seu receptor (Kiss1r) i la seva relació en el control de la pubertat en vertebrats és un dels avanços més importats dels últims anys en reproducció. El complex Kiss1/Kiss1r actua directament a l'hipotàlem provocant l’alliberació de les GnRHs, les quals inicien la secreció dels esteroides sexuals a les gònades per finalment incitar la maduració gonadal i la pubertat. Aquests dos gens estan ben estudiats en mamífers, però en peixos existeix molt poca informació. Així, no hi ha cap seqüència del gen Kiss1 publicada per cap espècie. Pel que fa al Kiss1r, estan disponibles fins a cinc seqüències: peix zebra (Danio rerio), cobia (Rachycentron canadum), corball (Micropogonias undulatus), llissa gris (Mugil cephalus) i tilapia (Oreochromis niloticus). En aquest estudi presentem la seqüència del gen Kiss1r del llenguado senegalès. Aquest gen conté 1137 bp que codifiquen un pèptid de 378 aminoàcids. El cribratge de les bases de dades genòmiques ha permès la identificació de gens Kiss1r en altres teleostis: el tetraodon (Tetraodon nigroviridis), fugu (Takifugu rubripes) i l’espinós (Gasterosteus aculeatus). D'altra banda, l’anàlisi d'expressió gènica va mostrar que en cervell els nivells de mRNA no van ser diferents entre mascles i femelles púbers i madurs. En canvi, en gònades els nivells més baixos d'expressió van ser observats en individus madurs. El clonatge d’una nova seqüència del Kiss1r ha de contribuir a un millor coneixement dels mecanismes de control de la pubertat en el llenguado senegalès i, en un àmbit més general, a l’àrea de l’endocrinologia reproductiva

I Simposi d’Aqüicultura de Catalunya. Investigació, desenvolupament i transferència en aqüicultura des de Catalunya a Europa, 6-8 de febrer de 2008, Barcelona

El objetivo del presente trabajo es estudiar condiciones físicas y ambientales en el Salitral de la Vidriera, Provincia de Buenos. Aires, localizado en la zona interna del estuario de Bahía Blanca y su relación con el principal componente del zooplancton, el crustáceo Artemia persimilis (Crustacea, Anostraca). El enfoque se centró en la colecta del material biológico durante los meses de mayo a agosto de 2001. Los muéstreos se realizaron en dos transectas con tres estaciones cada una. Se midió temperatura, salinidad, pH y profundidad. Adicionalmente se registraron datos sobre llora y fauna acompañante y se realizó el análisis del tipo de sustrato. El zooplancton del Salitral de la Vidriera estuvo representado casi en su totalidad por Artemia persimilis. Los resultados indicaron que los meses de mayo y junio no difirieron estadísticamente en el número de individuos (73.66 y 57.34 ind/1 Olitros), pero si respecto al mes de agosto que registró una menor abundancia de 24.17 ind/101itros. Esta última variación podría estar condicionada a los cambios de salinidad y temperatura que fluctuaron durante ese mes en el cuerpo de agua. La temperatura del agua siguió el ritmo de la temperatura del aire, no registrándose fluctuaciones de magnitud importante en las diferentes transectas y en los distintos meses, registrándose temperaturas entre 12 - 15,5 °C en ese período. La salinidad fue muy baja (23.73), si se lo compara con el valor de 95.5 obtenido para diciembre de 2001. Este último registro sería más representativo para un ambiente hipersalino como el Salitral de la Vidriera. Los bajos valores de este parámetro estuvieron relacionados con una abundante precipitación para esos meses. El pH del agua fluctuó entre 7-8 y la profundidad en las dos transectas entre 25 y 55 cm. El estudio de las poblaciones evidenció un comportamiento bisexual, no detectándose que la relación machos/hembras fuera 1:1, pero si observándose una ligera superioridad de las hembras. Tampoco se registraron interacciones entre fechas y sectores, hallándose ambos sexos, a diferentes profundidades. En cuanto a las tallas se obtuvo un rango de ejemplares que variaron entre 5,88 - 8,98 mm. El 31.7 % de los individuos correspondió a adultos (talla > 5,88 mm)., The objetive of this study was to analysed physical and environmental aspects of El Salitral de la Vidriera, Buenos Aires province and its relation with Artemia persimilis (Crustacea, Anostraca) one of the most important component of the zooplankton. Samples were taken during four months in winter time. Data of temperature, salinity, depth and pH were registered. Flora, fauna and substrate were analyzed. The results showed that the months of May and June did not differ statistically in the number of individuals, presenting values of 73.66 and 57.34 ind/1 Olitres but if differ from August, which registered an abundance of 24.17 ind/1 Olitres. This variation could be explain by changes in salinity and temperature, that fluctuated during that month. Water temperature varied 12 °C and 15,5 °C during the study period. Salinity was very low (23.73) comparig with the higher measurement (95.5) in december 2001. The last data is representative of the hypersaline environment of El Salitral de la Vidriera. The lowest values were related with the rainfall in preview months. The pH variation were 7-8 and depth between 25 to 55 cm. Populations showed a bisexual behavior, the relation male females not detected 1:1. A hight superiority of the females is observed. Interactions between dates and sectors were not registered. Both sexes, in different depths were found. Sizes of strain varied among 5,88 - 8,98 mm. The 31.7 % of the individuals were adults (size> 5,88 mm)., Asociación Argentina de Geofísicos y Geodestas