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The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of gametogenesis in vertebrates, and gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of their synthesis and secretion. Gonadal sex steroids have a feedback effect modulating the availability of gonadotropins. All these effects at the level of pituitary have a direct impact in gametogenesis progression. Previous in vivo studies in sea bass, during the sexual resting period, showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene. At the same time, different steroid implants repressed fsh-b expression, but activated the expression of lh-b in the pituitary. To elucidate how this system is organized in the pituitary of male sea bass, we analysed the annual expression profile of the three nuclear estrogen receptors (Esrs) in male pituitary and the circulating levels of estradiol in relation with the different stages of spermatogenesis. Also, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors and their relationship with gonadotrophs and GnRh1 fibers. Moreover, we have used an in vitro pituitary primary cell cultures stimulated with estradiol-17β (E2) to study its direct action on gonadotrophs, and E2 implants to study its effect in vivo on the pituitary and the gonad. We have concluded that E2 has an inhibitory effect on the gonadotropins, although with different specific actions, which is also reflected in the localization of the Esrs in the gonadotrophs, while Gnrh differentially regulates Lh and Fsh cells in male seabass., Funded by Spanish MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and CSIC (201640E073). PP was supported by the Portuguese FCT through project UID/Multi/04326/2019 and a researcher contract under “Norma Transitória”-DL57/2016/CP1361/CT0015.

Póster presentado en el XII Congress of Iberian Association for Comparative Endocrinology, celebrado en Faro (Portugal) del 26 al 28 de septiembre de 2019., The follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of gametogenesis in vertebrates, and gonadotropin-releasing hormones (Gnrh) have been postulated as the main regulators of their synthesis and secretion. In addition, gonadal sex steroids have a feedback effect modulating the availability of gonadotropins. All these effects at the level of pituitary have a direct impact in gametogenesis progression. Previous in vivo studies in sea bass, during the sexual resting period, showed that Gnrh injections stimulated Lh synthesis and release, but had no effect on the expression of the Fsh beta-subunit gene. At the same time, different steroid implants repressed fsh beta expression, but activated the expression of lh-beta in the pituitary. In this study we have focused on the action of estradiol in males in different moments of gametogenesis. To elucidate how this system is organized in the pituitary of sea bass, we analysed in a first step the annual profile of male pituitary expression of the three nuclear estrogen receptors, in relation with the different stages of spermatogenesis, the pituitary gonadotropin content and the circulating levels of estradiol. In addition, immunohistochemistry studies have been performed to identify the pituitary cells containing steroid receptors and their relation with gonadotrophs and GnRh1 fibers. Next, we have used an in vitro pituitary primary cell cultures stimulated with estradiol to study its direct action on gonadotrophs, and estradiol implants to study its in vivo effect on the pituitary and the gonad. We have concluded that estradiol has a general an inhibitory effect on the gonadotropins, although with different specific actions, which is also reflected in the distribution of the estradiol receptors in the gonadotrophs, while Gnrh differentially regulates Lh and Fsh cells in male seabass., Funded by MICINN (AGL2015-67477-C2-1-R, RTI2018-094667-B-C22) and CSIC (201640E073)

Poster.-- CIAC2022 Conference organized by the Cephalopod International Advisory Council, held from the 2nd to the 8th of April, 2022 in Sesimbra (Portugal)

Poster.-- Aquaculture Europe, Berlin, 7-10 October 2019

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017, Estrogens are involved in a wide range of processes in vertebrate reproduction, acting through ligand-activation of their specific cognate receptors. Nuclear estrogen receptors (Esr) are transcription factors that bind to estrogen response elements on gene promoters to regulate their expression. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a and Esr2b), probably involved in different physiological functions. Indeed, differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among Esr subtypes, suggest distinct roles during oogenesis, vitellogenesis and testicular development. In European sea bass, the complete cDNA sequence of esr1 and partial cDNA sequences of esr2a and esr2b had already been described, together with the brain distribution of their mRNAs and the transcriptional activity of Esr1. However, no information was available on the transcriptional activity of Esr2a and Esr2b, or on the significance of the Esr subtypes in other reproductive tissues. This study is focused on investigating the role of the Esr subtypes in European sea bass ovarian development and their endocrine regulation. We isolated the complete cDNAs of sea bass esr2a and esr2b and functionally characterized them using HEK293 cells. The coding genes of the three Esr subtypes are highly expressed in reproductive related tissues such as pituitary, gonad and liver. Quantification of esr1, esr2a and esr2b expression in the ovary during a whole reproductive cycle showed different expression patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis. Finally, for the first time in teleosts, localization of the different estrogen receptor subtypes in ovarian follicles and regulation by gonadotropins are described. Results showed differences among subtypes depending on the gonadal stage, reinforcing the hypothesis that each estrogen receptor has a specific role on ovarian development., Funded by MINECO (AGL2015-67477-C2-1-R) and GV (PROMETEOII-2014/051)

Trabajo presentado en el 8th International Symposium on Fish Endocrinology (ISFE), celebrado en Gotemburgo (Suecia), del 26 de junio al 2 de julio de 2016, Puberty is a developmental process in which the hypothalamus-pituitarygonadal axis is activated and animals become reproductively active. In European sea bass, Dicentrarchus labrax, precocious puberty among males results in smaller size at the time of marketing. Photoperiod manipulations are able to inhibit (continuous light - CC) or advance (shift from long to short days - AP) this process, although the underlying mechanisms are largely unknown. In this study, we explored the effects of CC and AP compared to natural photoperiod (NP) in the brain and adipose/pancreatic transcriptomes of pre-pubertal sea bass applied before the summer solstice (from April). We hypothesized that larger fish, who become reproductive during the first year, would have a different response to AP compared to non-reproductive smaller fish and other photoperiods. We constructed SuperSAGE libraries from brains and adipose/pancreatic tissue of immature fish of two sizes (Llarge fish and S-small fish) reared in NP, CC or AP, and sampled at different times after photoperiod change. Comparison of the brain transcriptome under NP showed the larger fish with higher levels of circadian clock, glycolysis-related and fatty acid sensing genes, as well as differential expression of growth regulating neuropeptides - higher PACAP/PAC1 and lower somatostatin (SST). In the adipose/pancreatic tissue, downregulation of pancreatic hormones ¿ insulin (INS), STT, and glucagon (GCG) - and upregulation of lipolysis-related genes in L compared to S. When L were exposed to AP, the opposite expression was seen, as well as the activation of SST in the brain and SST and INS/GCG genes in the pancreatic tissue, indicating inhibition of lipolysis and growth stimulating signals. Exposure to CC downregulated growth promoting signals in L but had no effect on SST in the brain nor on SST or INS/GCG levels in pancreatic cells. This may explain the stimulation of lipolysis related genes and previously reported reduced growth/weight seen in fish under CC. Overall these results suggest that precocious puberty is associated to growth/metabolic networks which respond differently according to fish size and photoperiod ¿ activation of puberty seems to be associated with lipid storage and activation of SST and INS/GCG axis., Funded by the European Community's Seventh Framework Programme (FP7/ 2007- 2013) under grant agreement n° 222719 – LIFECYCLE.

The acquisition of reproductive competence requires the activation of the brain-pituitarygonad (BPG) axis, which in most vertebrates, including fishes, is initiated by changes in photoperiod. In the European sea bass long-term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males. In contrast, an early shift from long to short photoperiod (AP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. Here, we investigated if the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the BPG axis in the European sea bass. We found that the clock genes clock, npas2, bmal1 and the BPG genes gnrh, kiss and kissr share conserved transcription factor frameworks in their promoters, suggesting co-regulation. Other gene promoters of the BGP axis were also predicted to be co-regulated by the same frameworks. Co-regulation was confirmed through gene expression analysis of brains from males exposed to LL or AP photoperiod compared to natural conditions: LL fish had suppressed gnrh1, kiss2, galr1b and esr1, while AP fish had stimulated npas2, gnrh1, gnrh2, kiss2, kiss1rb and galr1b compared to NP. It is concluded that fish exposed to different photoperiods present significant expression differences in some clock and reproductive axis related genes well before the first detectable endocrine and morphological responses of the BPG axis., The research leading to these results was funded by European Community's Seventh Framework Programme (FP7/ 2007–2013) under grant agreement n° 222719—LIFECYCLE and by the Foundation for Science and Technology of Portugal (FCT), through project PEst-C/MAR/LA0015/2011 and Prometeo II/2014/051 from the Valencian Regional Goverment and CSD 2007–0002 (AQUAGENOMICS) Spanish Ministry of Science and Innovation (MICINN). RSTM was funded by the Foundation for Science and Technology of Portugal (FCT) under fellowship (SFRH/BPD/66742/2009).

Comunicación presentada en el XV Congreso Nacional de Acuicultura / I Congreso Ibérico de Acuicultura, celebrado en Huelva, España, del 13 al 16 de octubre de 2015, Galanin is a neuropeptide known for its appetite stimulating properties and for the regulation of energy metabolism. There are some evidence that in mammals it may have an important role in the regulation of puberty and reproductive functions but little is known about its possible function in the regulation of fish reproductive cycle. In this study, we have characterized the galaninergic system in sea bass, an important species for aquaculture, through cloning several forms of galanin (GAL) and galanin receptors (GALRs). Their expression was detected in brain and gonads of adult male and female sea bass, with sex dimorphism for some of the transcripts that had higher expression in testis than ovary. Through the normal first reproductive season of male sea bass, galanin forms presented a differential expression pattern in hypothalamus, while no differences throughout the cycle were detected for GALRs. In pituitary galanin expression was very low and high expression and regulation throughout the cycle was detected for type 1 receptors. These results support roles for the galaninergic system in the reproductive cycle of male sea bass, which will be interesting to explore in the future.

Trabajo presentado en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Castellón (España), del 23 al 25 de septiembre de 2015, 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., Funded by FCT – Portuguese Science and Technology Foundation through projects UID/Multi/04326/2013 and PTDC/MARBIO/3890/2012 and grant FCT SFRH/BPD/25247/2005 to PISP.

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Submitted by Adelino Canario (acanario@ualg.pt) on 2014-09-19T10:37:13Z No. of bitstreams: 1 Identity of a tilapia pheromone released by dominant males that primes females for reproduction.pdf: 2355520 bytes, checksum: 312a44e09178d73f756c03364c6a8d7f (MD5) Approved for entry into archive by Carla Janeira (cjaneira@ualg.pt) on 2014-09-22T09:16:33Z (GMT) No. of bitstreams: 1 Identity of a tilapia pheromone released by dominant males that primes females for reproduction.pdf: 2355520 bytes, checksum: 312a44e09178d73f756c03364c6a8d7f (MD5) Made available in DSpace on 2014-09-22T09:16:34Z (GMT). No. of bitstreams: 1 Identity of a tilapia pheromone released by dominant males that primes females for reproduction.pdf: 2355520 bytes, checksum: 312a44e09178d73f756c03364c6a8d7f (MD5) Previous issue date: 2014-08-21

Póster presentado en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, [Introduction] The activation of the hypothalamus-pituitary-gonadal (HPG) axis at the onset of puberty is a key requirement for enabling the production of sex steroids and gametogenesis to proceed. In vertebrates, the central role of kisspeptins and its receptors as key regulators of the activation of gonadotropin-releasing hormone (GnRH) production and pulsatile release and/or gonadotropin (LH/FSH) release at puberty is well demonstrated. However, the signals that trigger this process are far from being identified. In the European sea bass (Dicentrarchus labrax), a species with photolabile pubertal activation, precocious puberty of larger males is thought to be associated to differences in metabolic status signaling to the brain and anticipating the HPG axis activation. One of the candidate neuroendocrine systems that may be involved in translating an inducing signal to the neuroendocrine axis is the galinergic system. We have recently characterized four galanin receptors (GALR1a, GALR1b, GALR2a and GALR2b) in sea bass and highlighted the photolabile regulation of brain GALR1b gene expression and androgen induced gene expression in the testes of pre-pubertal fish. The aim of this study was to isolate the sea bass galanin gene, to identify novel receptors and to characterize their evolution and its possible roles in sea bass reproductive function., [Methods] Vertebrate and invertebrate genome and protein databases were searched using sea bass galanin receptors and the Nile tilapia galanin gene. Multiple sequence alignments and phylogenetic analysis were done to identify and cluster the different receptors. Primers were designed to amplify the sea bass transcripts for a novel galanin receptor and for the galanin peptide. Their expression in different tissues was characterized by quantitative PCR., [Results and Discussion] We have isolated from sea bass brain two transcripts for galanin (a longer form encoding the full-length GAL protein and a shorter splice variant) and one transcript for a novel and uncharacterized galanin receptor (GALR1-like). Vertebrate GALRs phylogenetic analysis highlights the existence of two separate clades (GALR1/GALR-like and GALR2/3) that appear to have arisen from a common ancestral gene and the novel receptor is present from lamprey to higher vertebrates, except in mammals. Expression for the two GAL transcripts and the novel GALR was detected in several tissues of male and female sea bass, with apparent sex dimorphisms., [Conclusion] We have identified and characterized a novel GALR in vertebrates. Sexual dimorphic expression of galanin forms and its receptors suggests a role in the reproductive function of sea bass and possibly in other fish species.

Comunicación presentada en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014, [Introduction] Puberty onset is initiated by the activation of the hypothalamus-pituitary-gonadal (HPG) axis to produce gonadotropins and its timing is influenced by internal and external factors, including photoperiod. However, how these external signals are integrated and which gene networks are triggered to initiate this process is still unknown. Thus, the aim of this study was to identify the gene networks that respond to either puberty inhibiting (continuous light-CC) or accelerating (short days- SD) photoperiod regimes at the early events of puberty onset in sea bass, as a step to understand the high incidence of precocious puberty of male sea bass in culture. To achieve this aim, the global transcriptomic changes occurring in pre-pubertal sea bass brain and adipose tissue were characterized throughout the first month after changing photoperiod regimens known to influence puberty onset in sea bass., [Methods] Immature sea bass of two sizes ¿ large fish, the majority expected to go into puberty, and small fish, not expected to go into puberty - were either maintained in natural photoperiod or changed from natural photoperiod to photoperiod regimens known to delay (continuous light) or accelerate (long to short days) gametogenesis progression. SuperSAGE (Serial Analysis of Gene Expression) libraries (72) were constructed from brain or adipose tissue of fish exposed to the three photoperiod regimens for one to 38 days and sequenced by next generation SOLiD4 sequencing. Transcript tags were extracted, counted and their expression profiles statistically analyzed by pairwise comparisons between photoperiod regimens with false discovery rates < 0.05. The differentially expressed transcripts were mapped to the sea bass genome and available cDNAs using a multi-step Blast approach and mapped to cell pathways for enrichment analysis., [Results and Discussion] Gene annotation allowed matching 35-54% of the differentially expressed tags to known proteins and 20-24% to anonymous DNAs, with a global annotation rate up to 78%. Genes related to the brain circadian clock, to reproduction, to metabolism and neurotransmitter signaling were identified as differentially expressed between photoperiods in large or small fish in brain and/or adipose tissue., [Conclusion] In this study we successfully identified key genes and signaling pathways that are changed centrally and peripherally during the early events in response to photoperiod changes highlighting candidate genes and pathways that may be involved in triggering the onset of puberty in sea bass., Funded by the European Community's Seventh Framework Programme (FP7/ 2007- 2013) under grant agreement n° 222719 – LIFECYCLE.

Trabajo presentado en el 17th International Congress of Comparative Endocrinology (ICCE 2013), celebrado en Barcelona (España) del 15 al 19 de julio de 2013, Fish puberty is a developmental period that is rhythmically programmed and covers the transition from a juvenile to a mature adult. In the European sea bass long term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males, while a shift from long to short photoperiod (CP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. One possibility is that the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the brain- pituitary-gonad (BPG) axis. In order to shed light on these regulatory mechanisms, the promoter sequences of circadian clock genes (CLOCK, NPAS2, ARTLN) were compared with those of GnRH, KISS and KISSR and found that they share between themselves and other 173 gene promoters conserved transcription factor (TF) frameworks (same sets of equally spaced TF binding sites). Cluster analysis according to functional role showed an enrichment of genes linked to regulation of hormones and reproduction in general. Gene expression analysis in the brain of male European sea bass exposed to LL, CP and natural photoperiod (NP) provided evidence for co-regulation of the predicted genes by the different light regimes. LL fish, compared to other photoperiods, had low expression of sbGnRH, KISS1, GALR1b and ER¿, suggesting a suppressed BPG axis. Conversely, up-regulation of NPAS2, sbGnRH, cGnRH, KISS1, KISSr1b and GALR1b was detected within the first 48 hours in CP fish. These results show that clock and reproductive axis related genes are strongly modulated by light, prior to the first detectable signs of endocrine brain-pituitary-gonadal axis activation.

This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro-olfactogram. Receptor mechanisms were assessed by cross-adaptation with amino acids (L-cysteine, L-phenylalanine and 1-methyl-L-tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1-methyl-L-tryptophan and Lphenylalanine (food-related odorants) in the lower epithelium, and for taurocholic acid (conspecificderived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC-mediated transduction (IC50; 15–55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) (using SQ-22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non-PLC and non-AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non-PLC and non-AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.

Trabajo presentado en el 17th International Congress of Comparative Endocrinology (ICCE 2013), celebrado en Barcelona (España) del 15 al 19 de julio de 2013., Galanin (GAL) is an appetite-inducing neuropeptide also recognized to regulate a number of other physiological functions in mammals, including growth and reproduction. GAL stimulatesfoodintakeinteleostfishandaroleinthecontrolofreproduction issupportedby sexual dimorphic GAL-immunoreactivity in the brain of some species. GAL functions are mediated by three G-protein coupled receptor subtypes (GALR) of which little is known in fish. In this study, four GALR genes were identified in the genome of the European sea bass (Dicentrarchus labrax. Phylogenetic analysis indicate they may have arisen from fish- specific duplications from the ancestral of GalR1 and GalR2 mammalian genes and in accordance they were designated GALR1a, 1b, 2a and 2b, respectively. An orthologous for GALR3 appears to be absent from sea bass and other fish genomes that were searched. Transcripts for all GAL receptors were detected in different regions of the sea bass brain, testis and ovary. Significant changes in the GALR genes expression were detected in the brain and / or gonads of sexually immature sea bass in response to treatment with the sex steroids estradiol-17ß or 11-ketotestosterone for 12 to 24h, giving support to the hypothesis of possible roles in the control of reproduction that will be of interest to further explore., Work supported by a European Community's 7th Framework Programme (FP7/ 2007-2013), grant agreement 222719 - LIFECYCLE, and project PEst- C/MAR/LA0015/2011 from the Portuguese Science Foundation (FCT). PP and RM were in receipt from FCT (SFRH/BPD/25247/2005 and of Post-doctoral grants SFRH/BPD/66742/2009, respectively)

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (

Estrogen actions are mainly mediated by specific nuclear estrogen receptors (ERs), for which different genes and a diversity of transcript variants have been identified, mainly in mammals. In this study, we investigated the presence of ER splice variants in the teleost fish gilthead sea bream (Sparus auratus), by comparison with the genomic organization of the related species Takifugu rubripes. Two exon2-deleted ERα transcript variants were isolated from liver cDNA of estradiol-treated fish. The ΔE2 variant lacks ERα exon 2, generating a premature termination codon and a putative C-terminal truncated receptor, while the ΔE2,3* variant contains an in-frame deletion of exon 2 and part of exon 3 and codes for a putative ERα protein variant lacking most of the DNA-binding domain. Both variants were expressed at very low levels in several female and male sea bream tissues, and their expression was highly inducible in liver by estradiol-17β treatment with a strong positive correlation with the typical wild-type (wt) ERα response in this tissue. These findings identify novel estrogen responsive splice variants of fish ERα, and provide the basis for future studies to investigate possible modulation of wt-ER actions by splice variants.