Search

Gonadotropin-inhibitory hormone (GnIH)is a hypothalamic neuropeptide belonging to the RFamide peptide family that was first discovered in quail by Tsutsui and co-workers in the year 2000. Since then, different GnIH orthologues have been identified in all vertebrate groups, from agnathans to mammals. These GnIH genes synthesize peptide precursors that encompass two to four C-terminal LPXRFamide peptides. Functional and behavioral studies carried out in birds and mammals have demonstrated a clear inhibitory role of GnIH on GnRH and gonadotropin synthesis and secretion as well as on aggressive and sexual behavior. However, the effects of Gnih orthologues in reproduction remain controversial in fish with both stimulatory and inhibitory actions being reported. In this paper, we will review the main findings obtained in our laboratory on the Gnih system of the European sea bass, Dicentrarchus labrax. The sea bass gnih gene encodes two putative Gnih peptides (sbGnih1 and sbGnih2), and is expressed in the olfactory bulbs/telencephalon, diencephalon, midbrain tegmentum, rostral rhombencephalon, retina and testis. The immunohistochemical study performed using specific antibodies developed in our laboratory revealed Gnih-immunoreactive (ir)perikarya in the same central areas and Gnih-ir fibers that profusely innervated the brain and pituitary of sea bass. Moreover, in vivo studies revealed the inhibitory role of centrally- and peripherally-administered Gnih in the reproductive axis of male sea bass, by acting at the brain (on gnrh and kisspeptin expression), pituitary (on gnrh receptors and gonadotropin synthesis and release)and gonadal (on androgen secretion and gametogenesis)levels. Our results have revealed the existence of a functional Gnih system in sea bass, and have provided evidence of the differential actions of the two Gnih peptides on the reproductive axis of this species, the main inhibitory role in the brain and pituitary being exerted by the sbGnih2 peptide. Recent studies developed in our laboratory also suggest that Gnih might be involved in the transduction of photoperiod and temperature information to the reproductive axis, as well as in the modulation of daily and seasonal rhythmic processes in sea bass., The works on sea bass Gnih system reviewed in this manuscript were supported by grants from the Junta de Andalucía (grant no. P10-AGR-05916) and University of Cádiz (Plan Propio, grants no. AT2014-020, AT2016-014, AT2017-009) to JAM-C. JAP-S was a postdoctoral fellow from the Junta de Andalucía. MA-G was recipient of a predoctoral fellowship from MINECO (BES-2011-047276). MC was recipient of a Marie Curie postdoctoral contract from the European Union’s Seventh Framework Programme (FP7/2007-2013, grant no. 331964).

This study evaluated the impact of continuous light (LL) within the photolabile period on advanced puberty in juvenile male European sea bass. The exposure to an LL regime for 1 month, from August 15 to September 15 (LLa/s), was compared to a constant simulated natural photoperiod (NP) and constant continuous light conditions year-round (LLy). Somatic growth, hormone plasma levels, rates of testicular maturation and spermiation, as well as the mRNA levels of some reproductive genes were analyzed. Our results demonstrated that both LLa/s and LLy treatments, which include LL exposure during the photolabile period, were highly effective in inhibiting the gametogenesis process that affects testicular development, and clearly reduced the early sexual maturation of males. Exposure to an LL photoperiod affected body weight and length of juvenile fish during early gametogenesis and throughout the first year of life. Interestingly, LL induced bi-weekly changes in some reproductive factors affecting Gnrh1 and Gnrh2 content in the brain, and also reduced pituitary fshβ expression and plasmatic levels of 11-KT, E2, Fsh throughout early gametogenesis. We suggest that low levels of E2 in early September in the LL groups, which would be concomitant with the reduced number of spermatogonial mitoses in these groups, might indicate a putative role for estrogens in spermatogonial proliferation during the early gonadal development of this species. Furthermore, a significant decrease in amh expression was observed, coinciding with low plasma levels of 11-KT under LL regimes, which is consistent with the idea that this growth factor may be crucial for the progress of spermatogenesis in male sea bass., This research was supported by grants Q5RS-2002-01801 (PUBERTIMING) from the UE, Generalitat Valenciana Grupos 04/80 and PROMETEO 2010/003-2014/051, Ministerio de Ciencia y Tecnología, with special action No. AGL2002-12470-E and AGL2009-11086 and AGL2016-75400-R from the Ministerio de Ciencia e Innovación (MICINN) of Spain. R.R. was supported by an FPI fellowship from MICINN.

Trabajo presentado en el 11th International Symposium on Reproductive Physiology of Fish, celebrado en Manaus (Brasil), del 3 al 8 de junio de 2018

In this study, sex steroids (E2, T, and 11-KT) concentrations in blood of captive leopard grouper (Mycteroperca rosacea) and their relation togonad development were determined during a reproductive cycle. Each month, gonad samples from 24 fish were obtained to analyze gonadal maturation and blood was extracted to determine steroid levels. Throughout the year, ovaries showed a higher percentageof primary oocytes. In January, oocytes at early and late vitellogenesis stages increased progressively; from March to June, ovulation rates were high. In females, estradiol levels increased from March and decreased after the spawning season. Lower levels of testosterone were found during the spawning season and 11-KT remained without significant changes throughout the year. Males exhibit a significant increase in E2 from March until July; testosterone remained without significant variation throughout theyear, and 11-KT plasma levels increased from March until July and decreased after the spawning season. In this study, we verified the sex change of an organism and the concentrations of sexual steroids were determined and discussed., N.M. Kiewek Martínez is a recipient of a CONACYT fellowship and V. Gracia-López is a recipient of a grant from the International Foundation for Science.

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1–29 Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1–29 Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates., This work was supported by the Portuguese Foundation for Science and Technology [grant numbers SFRH/BPD/84033/2012, PTDC/MAR-BIO/3890/2012, UID/Multi/04326/2013]; the European Union Seventh Framework Programme [AQUAEXCEL grant number 262336]; the Spanish Ministry of Science and Innovation (MICINN) and the Spanish Ministry of the Economy and Competitiveness (MINECO) [KISSCONTROL project, AGL2009-11086]; the Spanish Ministry of the Economy and Competitiveness and the Regional Government of Valencia (REPROBASS project, PROMETEOII/2014/051).

This study assesses the effect of combining different photoperiod regimes on the sexual maturation and growth of male sea bass during the first two years of life. In two different trials, regimes of continuous light (LL) lasting 24 consecutive months (Trial 1) or shorter periods within the summer-early autumn months during the first and second annual cycles (Trial 2) were compared to control groups under a natural photoperiod (NP). In a third trial (Trial 3), fish kept under either NP or LL conditions during the first year were subsequently assigned a treatment involving exposure to NP or a constant long photoperiod (LP) throughout the second year. The rates of spermiating males, gonadal size (GSI) and growth under all these photoperiod regimes were analyzed. Exposure to LL reduced the rates of precocity in all trials (, The authors wish to thank C. Marín and X. Simó for their technical assistance in rearing and sampling the fish. The work of the authors in this article was supported by UE projects Q5RS-2002-01801 (PUBERTIMING) and Grants AGL2006-04672 (PUBERCONTROL) and CSD2007-00002 (AQUAGENOMICS) from the Spanish Ministry of Science and Innovation (MICINN) and PROMETEO/2010/003 (Generalitat Valenciana). RR was supported by a FPI fellowship from the MICINN (Spain).

Trabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017, In higher vertebrates, anti-Müllerian hormone (AMH) is required for involution of the Müllerian ducts during male sexual differentiation and for negatively regulating gonadal development in both sexes. AMH signals through a transmembrane AMH type II receptor (AMHR2) with serine-threonine kinase activity. Despite the absence of Müllerian ducts in teleosts, orthologues of mammalian AMH and AMHR2 have been described in some fish species, and a role of this hormone in sex determination and gonad differentiation has been demonstrated. In addition, it seems that Amh inhibits germ cell proliferation and differentiation, as well as steroidogenesis, in adult gonads of both sexes. However, the mechanism of Amh signaling and its implication in gonad development are poorly investigated. In European sea bass, a recent study showed higher expression levels of amh and amhr2 during early and final stages of spermatogenesis, and immunolocalization of Amh in Sertoli cells surrounding early germ-cell generations, in line with its role in germ cell proliferation and differentiation. Available information of amh and amhr2 expression during sea bass ovarian development shows an opposite profile for these two genes. Although this information may suggest a role of Amh in early stages of oogenesis, the specific mechanisms of Amh signaling during ovarian development still have to be investigated. As tool for this kind research recombinant sea bass Amh would be needed. We know from previous studies that this hormone is processed in a similar way to mammalian AMH, becoming a biologically active protein able to bind and activate the sea bass Amhr2. Here, we report the production of an His-tagged recombinant sea bass Amh in a Pichia pastoris expression system, where Amh is endogenously cleaved and secreted as a mature peptide into the culture media. Bioactivity of this recombinant sea bass Amh has been demonstrated using COS7 cells co-transfected with the sea bass amhr2 cDNA and the BRE-luc reporter plasmid. Localization of Amh in granulosa cells of vitellogenic ovarian follicles was shown using a specific antibody. To investigate how Amh regulates sea bass ovarian development, we performed an in-vitro tissue culture of vitellogenic ovaries treated with recombinant Amh. First, we analyze steroid release in the medium by using a specific EIA. Finally, several candidate genes regulated by Amh were analyzed by real time qPCR., Funded by MINECO (AGL2015-67477-C2-1-R, AGL2011-28890), EU (LIFECYCLE FP7- 22719-1) and GV (PROMETEOII-2014/051)

Trabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017, The pituitary gland synthesizes and secrets differently glycosylated forms of gonadotropins that differ in their oligosaccharide structure. In the case of FSH, several enzymes are involved in its post-translational glycosylation processing; particularly the sialyltransferases (STs) that transfer sialic acid residues into the FSH carbohydrate chain. Previous studies with recombinant sea bass Fsh showed that, as occurs in mammals, less sialylated isoforms exhibit higher in-vitro biological activity but shorter plasma half-life than their more sialylated counterparts. These facts suggest that gonadotropin heterogeneity represents an alternative mechanism of the pituitary gland to regulate the intensity and duration of gonadotropin stimuli in the gonads. In addition, the composition of the FSH carbohydrate moiety is likely regulated by the endocrine milieu, and the GnRH and gonadal sex steroids have been proposed as the main regulators of FSH microheterogeneity. Our aim in this study was to know the expression profile of several sea bass ST genes in male pituitary during a whole reproductive cycle and to elucidate whether treatments with gonadal steroids (Estradiol, Testosterone, Dihydroxyprogesterone) and LHRHa can affect the expression of these genes in a pituitary primary cell culture, and therefore to the potential sialylation of sea bass Fsh., Founded by MINECO (AGL 2015-67477-C2-1-R), CSIC (201640E073) and GV (PROMETEO II-2014/051)

Trabajo presentado en el 6th International Workshop on the Biology of Fish Gametes, celebrado en Vodňany (República Checa), del 4 al 7 de septiembre de 2017, Recent studies in European sea bass using a hemigonadectomy approach and transcriptomic analyses have revealed several genes potentially involved in the onset of puberty in female gonads. One of these genes is ncoa7 that encodes a nuclear receptor co-activator that, in mammals, increases the transcriptional activity of the estrogen receptor (Esr). This study is aimed at the molecular and functional characterization of the ncoa7 gene and its role during gametogenesis. We have cloned the complete cDNA of sea bass ncoa7 and studied its expression in different male and female tissues showing high expression in brain and gonads. Functional analyses using HEK293 cells co-transfected with Ncoa7 together with the estrogen, androgen and progesterone receptors, and exposed to E2, Testosterone or 17,20ß-P, showed that Ncoa7 is able to increase the transcriptional activity of all the different receptors, suggesting that can control different processes in early gametogenesis. Quantification of the expression of ncoa7, esr1, esr2a and esr2b in the ovary and testis of adult sea bass during a whole reproductive cycle showed different expression patterns among stages. In females, an increase of ncoa7 expression is observed during vitellogenesis coinciding with an increase of esr2b expression and E2 levels in the ovary, reinforcing the hypothesis that Ncoa7 is involved in the onset of puberty in female sea bass. In males, the highest expression of ncoa7 was observed in immature testes overlapping with the highest expression of esr1 and high levels of E2 in plasma. In addition, Ncoa7 and Esr1 colocalized in type A spermatogonia of immature testis, and further co-immunoprecipitation studies demonstrated that Ncoa7 was bound to Esr1 in these gonads. Finally, treatment of immature testis with E2 increased proliferation of type A spermatogonia as found by phospho-Histone H3 (Ser10) immunocytochemistry, what together with the previous results suggest that both factors may be involved in spermatogonia proliferation., Funded by AGL2015-67477-C2-1-R, Aquagenomics (CSD2007-00002), PROMETEOII-214/051.

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, The gonadotropins, follicle stimulating hormone (Fsh) and the luteinizing hormone (Lh), are central endocrine regulators of reproduction in vertebrates. They are heterodimeric glycoproteins secreted by the pituitary gland that act by binding specific receptors located in the gonads, controlling different steps of gametogenesis and steroidogenesis. The structural duality of these hormones is well defined, but so far their individual roles are not clear in many fish species due to the lack of tools for their study. In European sea bass (Dicentrarchus labrax) the isolation of the genes coding for the gonadotropin subunits and receptors allowed firstly to study their expression profiles. Later, to overcome the limitation of native hormones, recombinant dimeric gonadotropins have been generated and used for functional studies in vitro and in vivo. In addition, the production of recombinant gonadotropin beta-subunits provided adequate antigens to raise antibodies, which were the basis for the development of detection methods for native gonadotropins. In this way, sea bass is one of the few teleost species where the amount of circulating and hypophysiary gonadotropins can be quantified. All the sea bass recombinant gonadotropins produced are functionally active, showing no cross-reaction between receptors. However, the production rates and in vivo stability of these recombinant forms showed remarkable differences depending on the cell expression system and DNA construct used, leading in some cases to the rapid clearance from circulation of the exogenously administered hormone. For this reason, gene-based therapies using somatic transfer of gonadotropin genes have also been tested as an alternative for hormone delivery in vivo, resulting in different studies on gonadotropin actions in spermatogenesis., Funded by MINECO (AGL 2015-67477-C2-1-R), CSIC (201640E073) and GV (PROMETEO II-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, Gonadotropin-inhibitory hormone (GnIH) suppresses reproduction in several vertebrate species by acting at both the brain and pituitary levels. New evidences obtained in birds and mammals suggest that, in addition to the brain, GnIH may also be produced in testis and ovary and can regulate gonadal steroidogenesis and gametogenesis. Sex steroids are essential for gonadal function and cyclicity and play a key role in referring the sexual status to the brain and pituitary. However, the function of GnIH in gonadal physiology has received scanty attention in fish. One of the aims of this study was to evaluate the effects of peripheral sbGnIH-1 and sbGnIH-2 implants on gonadal development and steroidogenesis along the reproductive cycle of male sea bass. Implants of sbGnIH-1 and sbGnIH-2 peptides did not affect plasma levels of the maturation-inducing steroid 17,20¿-dihydroxy-4-pregnen-3-one. Although similar profiles of plasma testosterone (T) and 11-kestostestosterone (11-KT) were observed in controls and GnIH-implanted animals, with a peak in January, both GnIHs decreased T and 11-KT plasma levels in November and December. In February, control fish only exhibited late meiotic and full spermiogenic testicles containing mostly sperm. In contrast, both sbGnIH-1 and sbGnIH-2 treated fish also exhibited a significant percentage of testicles containing abundant type A spermatogonia and partial spermatogenesis. In addition, we determined the effects of peripheral GnIH implants on plasma FSH and LH levels, as well as on brain and pituitary expression of reproductive hormones genes (gnrh1, gnrh2, gnrh3, kiss1, kiss2, gnih, lhbeta, fshbeta) and their receptors (gnrhr II-1a, gnrhr II-2b, kiss1r, kiss2r and gnihr) at the spawning period (February). Treatment with sbGnIH-2 increased brain gnrh2, gnih, kiss1r and gnihr transcripts. Moreover, both sbGnIH-1 and sbGnIH-2 treatments decreased lhbeta expression and plasma LH levels, but only sbGnIH-1 reduced plasmatic FSH. Finally, we have shown that both GnIHs prevented the characteristic increase in nocturnalism observed in sea bass during the reproductive season. Taken together, our results indicate that GnIH may regulate the reproductive axis of sea bass acting not only at the brain and pituitary level but also on gonadal physiology and reproductive behaviour., Funded by grants from Junta de Andalucía (P10-AGR-5916) and the European Union (FP7/2007-2013, grant no. 331964) to JAM-C

Gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin synthesis and release from the pituitary of birds and mammals. However, the physiological role of orthologous GnIH peptides on the reproductive axis of fish is still uncertain, and their actions on the main neuroendocrine systems controlling reproduction (i.e., GnRHs, kisspeptins) have received little attention. In a recent study performed in the European sea bass, we cloned a cDNA encoding a precursor polypeptide that contained C-terminal MPMRFamide (sbGnIH-1) and MPQRFamide (sbGnIH-2) peptide sequences, developed a specific antiserum against sbGnIH-2, and characterized its central and pituitary GnIH projections in this species. In this study, we analyzed the effects of intracerebroventricular injection of sbGnIH-1 and sbGnIH-2 on brain and pituitary expression of reproductive hormone genes (gnrh1, gnrh2, gnrh3, kiss1, kiss2, gnih, lhbeta, fshbeta), and their receptors (gnrhr II-1a, gnrhr II- 2b, kiss1r, kiss2r, and gnihr) as well as on plasma Fsh and Lh levels. In addition, we determined the effects of GnIH on pituitary somatotropin (Gh) expression. The results obtained revealed the inhibitory role of sbGnIH-2 on brain gnrh2, kiss1, kiss2, kiss1r, gnih, and gnihr transcripts and on pituitary fshbeta, lhbeta, gh, and gnrhr-II-1a expression, whereas sbGnIH-1 only down-regulated brain gnrh1 expression. However, at different doses, central administration of both sbGnIH-1 and sbGnIH-2 decreased Lh plasma levels. Our work represents the first study reporting the effects of centrally administered GnIH in fish and provides evidence of the differential actions of sbGnIH-1 and sbGnIH-2 on the reproductive axis of sea bass, the main inhibitory role being exerted by the sbGnIH-2 peptide., The research leading to these results has received funding from the Junta de Andalucía (P10-AGR-05916), the European Union’s Seventh Framework Programme (FP7/2007-2013, grant agreement no. 331964) to J.A.M.C. and Generalitat Valenciana (PROMETEOII/2014/051) to S.Z. J.A.P.-S. is a fellow FPI from the Junta de Andalucı´a. M.C. was a recipient of a Marie Curie contract from UE

The onset of puberty is characterized by activation of the brain-pituitary-gonad axis. However, the molecular and endocrine mechanism involved in the process 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 photoperiod manipulation in combination with size-sorting. Two treatment groups, a puberty accelerating photoperiod (AP) group and a continuous light (LL) group, were studied from August to February. Our results indicate that AP and LL are able to entrain the rhythms of both kiss1 and gnrh2 mRNA levels in the brain, while kiss2 and gnrh1 mRNA expression does not seem to be directly affected by the photoperiod, at least during testicular growth. It is likely that AP and LL photoperiod regimes affected both plasma Fsh and 11-KT profiles, which might explain, respectively, the phase shift and reduction of testes maturation seen under these conditions. We therefore hypothesize that the unbalanced production of this androgen regulated by circulating Fsh might be limiting the stimulation of germ cell proliferation in European sea bass males. In summary, our study establishes that photoperiod modulates the expression of kiss1 and gnrh2 in the forebrain-midbrain, which may be involved in the translation of the light stimulus to activate the reproductive axis., This work was supported by EU (LIFECYCLE, FP7-222719-1) and Generalitat Valenciana (PROMETEOII/2014/051) projects awarded to S.Z. F.E. was supported by a JAE Predoctoral fellowship (CSIC).

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.

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, Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Contrary to placental mammals, where only one KISS1 and one KISS1R gene are present, a second ancient paralog of Kiss1, referred to as Kiss2, has been described in different vertebrate lineages and species, including European sea bass (Dicentrarchus labrax). Both genes are expressed in the brain and pituitary of this teleost species, but their differential roles in the central control of fish reproduction are only beginning to be elucidated.In this study, we investigated whether the brain represents the only site of kisspeptin action on reproductive function, and also the potential actions of kisspeptin at the pituitary level could be considered. First, we examined the effects of intracerebroventricular injections of the highly active sea bass peptides Kiss1-15 and Kiss2-12 on spermiating male sea bass. Physiological saline, Kiss1-15, or Kiss2-12 were injected into the third ventricle. Blood samples were collected at different times after injection to analyze the effects of kisspeptins on the release of gonadotropins (Lh and Fsh) and androgens (testosterone and 11-ketotestosterone). Sperm samples were also collected to study the effects on the sperm quality. Our results showed that Kiss2-12 provoked a marked effect on plasma luteinizing hormone (Lh) levels, which in turn had a strong stimulatory effect on the release of androgens and on sperm quality parameters.On the other hand, both synthetic peptides, Kiss1-15 and Kiss2- 12, were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results showed that Kiss2-12 induced Lh and folliclestimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion. Furthermore, the distribution and origin of Kiss2 and its potential interactions with the gonadotropins in the pituitary were analyzed using dual fluorescence immunohistochemistry. Kiss2 cells were found in the proximal pars distalis and colocalized with gonadotropin immunoreactive cells. In summary, our results provide, for the first time in a teleost species, functional and neuroanatomical evidence that Kiss2 may act through different routes to directly modulate the activity of gonadotrophs, either as a brain neuropeptide or as an autocrine/paracrine factor in the pituitary., Funded by grants: LIFECYCLE FP7-22719-1; AGL2011-28890; PROMETEOII-214/051.

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 comprises the developmental period that leads to the first successful reproduction. In European sea bass (Dicentrarchus labrax) intensive rearing results in a high percentage of precocious maturation one year before the physiological onset. Recent studies in European sea bass using an hemigonadectomy approach and transcriptomic analyses have revealed several genes potentially involved in the onset of puberty in female gonads. In these studies histological observation of the developmental stage achieved by the non-extracted gonad and plasma profiles of different reproductive hormones were used to classify the fish into precocious or non-precocious. RNA samples from precocious and non-precocious extracted ovaries were subsequently hybridized on a sea bass specific microarray for the identification of differentially expressed genes. One of these genes is ncoa7 that encodes a nuclear receptor co-activator that, in mammals, increases the transcriptional activity of the estrogen receptor. This study is aimed at the molecular and functional characterization of the ncoa7 gene. We have cloned the complete cDNA of sea bass ncoa7 and studied its expression in different tissues showing high expression in brain and gonads. Functional analyses using HEK293 cells co-transfected with Ncoa7 together with the estrogen, androgen and progesterone receptors, and exposed to E2, Testosterone or 17,20ß-P, show that Ncoa7 is able to increase the transcriptional activity of all the different receptors, suggesting that can control different processes in early gametogenesis. Finally, quantification of the expression of ncoa7 in the ovary and testis of adult sea bass during a whole reproductive cycle showed different expression patterns among stages, reinforcing the hypothesis that Ncoa7 could be involved in the onset of puberty in sea bass., Funded by AGL2015-67477-C2-1-R, Aquagenomics (CSD2007-00002), PROMETEOII-214/051

Gonadotropin‐inhibitory hormone (GnIH) is a neuropeptide that suppresses reproduction in birds and mammals by inhibiting GnRH and gonadotropin secretion. GnIH orthologs with a C‐terminal LPXRFamide (LPXRFa) motif have been identified in teleost fish. Although recent work also suggests its role in fish reproduction, studies are scarce and controversial, and have mainly focused on cyprinids. In this work we cloned a full‐length cDNA encoding an LPXRFa precursor in the European sea bass, Dicentrarchus labrax. In contrast to other teleosts, the sea bass LPXRFa precursor contains only two putative RFamide peptides, termed sbLPXRFa1 and sbLPXRFa2. sblpxrfa transcripts were expressed predominantly in the olfactory bulbs/telencephalon, diencephalon, midbrain tegmentum, retina, and gonads. We also developed a specific antiserum against sbLPXRFa2, which revealed sbLPXRFa‐immunoreactive (ir) perikarya in the olfactory bulbs‐terminal nerve, ventral telencephalon, caudal preoptic area, dorsal mesencephalic tegmentum, and rostral rhombencephalon. These sbLPXRFa‐ir cells profusely innervated the preoptic area, hypothalamus, optic tectum, semicircular torus, and caudal midbrain tegmentum, but conspicuous projections also reached the olfactory bulbs, ventral/dorsal telencephalon, habenula, ventral thalamus, pretectum, rostral midbrain tegmentum, posterior tuberculum, reticular formation, and viscerosensory lobe. The retina, pineal, vascular sac, and pituitary were also targets of sbLPXRFa‐ir cells. In the pituitary, this innervation was observed close to follicle‐stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) cells. Tract‐tracing retrograde labeling suggests that telencephalic and preoptic sbLPXRFa cells might represent the source of pituitary innervation. The immunohistochemical distribution of sbLPXRFa cells and fibers suggest that LPXRFa peptides might be involved in some functions as well as reproduction, such as feeding, growth, and behavior, Grant sponsor: Junta de AndalucÍa; Grant number: P10-AGR-05916 (toJ.A.M.-C.); J.A.P.-S. is a fellow FPI from the Junta de Andalucía

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.

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, 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 forebrainmidbrain (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.

In this study, we report the cloning of three transcripts for leptin receptor in the European sea bass, a marine teleost of economic interest. The two shortest variants, generated by different splice sites, encode all functional extracellular and intracellular domains but missed the transmembrane domain. The resulting proteins are therefore potential soluble binding proteins for leptin. The longest transcript (3605 bp), termed sblepr, includes all the essential domains for binding and transduction of the signal. Thus, it is proposed as the ortholog for the human LEPR gene, the main responsible for leptin signaling. Phylogenetic analysis shows the sblepr clustered within the teleost leptin receptor group in 100% of the bootstrap replicates. The neuroanatomical localization of sblepr expressing cells has been assessed by in situ hybridization in brains of sea bass of both sexes during their first sexual maturation. At histological level, the distribution pattern of sblepr expressing cells in the brain shows no clear differences regarding sex or reproductive season. Transcripts of the sblepr have a widespread distribution throughout the forebrain and midbrain until the caudal portion of the hypothalamus. A high hybridization signal is detected in the telencephalon, preoptic area, medial basal and caudal hypothalamus and in the pituitary gland. In a more caudal region, sblepr expressing cells are identified in the longitudinal torus. The expression pattern observed for sblepr suggests that in sea bass, leptin is very likely to be involved in the control of food intake, energy reserves and reproduction., This research was supported by UE project FP7-222719-1 (LIFECYCLE), project PROMETEO/11/2014/051 (REPROBASS) from the Valencian Regional Government and CSD2007-00002 (AQUAGENOMICS) from the Ministry of Science and Innovation (MICINN), Spain. S.E was sponsored by a JAE fellowship from CSIC (Spain) and a CONICYT predoctoral fellowship from Chile.

El proceso reproductivo en peces, al igual que en el resto de vertebrados, está regulado por una cascada hormonal en la que están implicados el cerebro, la hipófisis y las gónadas, conocido como eje CHG. Dos de las principales hormonas implicadas en el control de la reproducción son las gonadotrofinas FSH (hormona estimuladora del folículo) y LH (hormona luteinizante), ambas producidas en la hipófisis y liberadas tras el estímulo con las hormonas liberadoras de gonadotrofinas (GnRH) y con acción a nivel gonadal. Desde la perspectiva de la producción animal en general, y de la acuicultura en particular, es esencial conocer en la especie a domesticar los mecanismos básicos de control endocrino. Este conocimiento básico es de gran utilidad para poder manipular la reproducción a conveniencia y al mismo tiempo poder desarrollar estrategias terapéuticas para solventar problemas de fertilidad que en ocasiones presentan los stocks de reproductores. Sin embargo, como consecuencia de la limitación en la disponibilidad de herramientas para su estudio, para una gran mayoría de las especies cultivables de peces se desconocen los mecanismos particulares que controlan su reproducción. En esta Tesis se optimiza una alternativa nueva de administración de gonadotrofinas recombinantes, la tecnología conocida como transferencia génica somática. En los capítulos 3 y 4 se inyectaron adultos y juveniles de lubina con construcciones de DNA codificante de las gonadotrofinas de cadena única Lh y Fsh, respectivamente, y se evaluó el incremento de estas hormonas en el torrente sanguíneo. En ambos experimentos las inyecciones de DNA se compararon con la inyección directa de la gonadotrofina recombinante correspondiente. Estos capítulos muestran que las construcciones de DNA se incorporan a las células musculares en el lugar de inyección, y las hormonas codificadas son sintetizadas y liberadas al torrente sanguíneo. Los capítulos 3 y 4 confirman que la transferencia génica somática es una herramienta poderosa para realizar estudios funcionales in vivo con gonadotrofinas, siendo además útil como terapia hormonal en peces, presentándose como alternativa a los tratamientos tradicionales con hCG o GnRH.

Trabajo presentado en Aquaculture Europe 2014, celebrado en San Sebastián (España) del 4 al 17 de octubre de 2014, Puberty comprises the developmental period in which an immature juvenile becomes capable of sexual reproduction for the first time. This process involves functional activation of the brain-pituitary-gonad axis (Taranger et al., 2010). Under farming conditions, the sea bass populations show biased sex ratios with more than 70% of males. About 20 to 30% of these males mature precociously in the first year of life, instead of the second (Carrillo et al., 1995). These precocious males show lower growth rate than the non-precocious during their second year (Felip et al., 2006), before reaching marketable size, resulting in economic loses to the fish farmer. The present study aimed to define non-lethal markers for early prediction of precocity rates in a population, helping to the management of stocks, and as a knowledge base to understand which are the physical/physiological conditions that lead a fish to mature precociously.

Trabajo presentado en Aquaculture Europe 2014, celebrado en San Sebastián (España) del 4 al 17 de octubre de 2014, Supported by Spanish MINECO (AGL2008-02937, AGL2011-28890, CSD2007-00002) and the EU 7th FP project LIFECYCLE

Trabajo presentado en la 27th Conference of European Comparative Endocrinologists, celebrada en Rennes (Francia), del 25 al 29 de agosto de 2014, The study investigates the effects of long-term (14 months) feeding restriction, during the second year of life of prepubertal male sea bass, on the first reproduction cycle and the relationships between nutritional status and plasma IGF-I and leptin levels. The experiment was done in triplicate and consisted of two groups: control fish (CT) fed until visual satiety and experimental fish (EX) which received food near to maintenance ration (0.35% body weight). EX fish had lower growth and muscle lipid content than CT, as well as lower gonadosomatic indeces, low rates of spermiation and a delay in its accomplishment. Nevertheless, all experimental fish achieved full maturation. EX exhibited a moderate delay of testicular maturation compared with CT, likely related to the smaller number of proliferating spermatogonial cells and larger proportion of apoptotic bodies found in this group. Plasma leptin levels of both groups appear correlated to the amount of fat stored. In EX fish, plasma IGF-I levels decreased during the experimental period. Although no correlation was observed between plasma IGF-I and leptin levels, current data suggest that in male sea bass IGF-I has a crucial role through pregametogenesis and post-gametogenesis notably during cell proliferation, while leptin could act as a permissive signal so that puberty occurs in due time.

Trabajo presentado en la 27th Conference of European Comparative Endocrinologists, celebrada en Rennes (Francia), del 25 al 29 de agosto de 2014