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Although anti-Müllerian hormone (AMH) has classically been correlated with the regression of Müllerian ducts in male mammals, involvement of this growth factor in other reproductive processes only recently come to light. Teleost is the only gnathostomes that lack Müllerian ducts despite having amh orthologous genes. In adult teleost gonads, Amh exerts a role in the early stages of germ cell development in both males and females. Mechanisms involving the interaction of Amh with gonadotropin- and growth factor-induced functions have been proposed, but our overall knowledge regarding Amh function in fish gonads remains modest. In this study, we report on Amh actions in the European sea bass ovary. Amh and type 2 Amh receptor (Amhr2) are present in granulosa and theca cells of both early and late-vitellogenic follicles and cannot be detected in previtellogenic ovaries. Using the Pichia pastoris system a recombinant sea bass Amh has been produced that is endogenously processed to generate a 12–15 kDa bioactive mature protein. Contrary to previous evidence in lower vertebrates, in explants of previtellogenic sea bass ovaries, mature Amh has a synergistic effect on steroidogenesis induced by the follicle-stimulating hormone (Fsh), increasing E2 and cyp19a1a levels., This research was funded by the Spanish MICINN, grant numbers AGL2015-67477-C2- 1-R and RTI2018-094667-B-C22 and by EU, grant LIFECYCLE FP7-22719-1. The group is partially funded by the REPROBASS (PROMETEOII/2014/051) project from Generalitat Valenciana. C.Z. was supported by a postdoctoral Juan de la Cierva-Formación contract from the Spanish MINECO and A.M. by a PhD contract from GV (GRISOLIAP/2020/129).

Galanin (Gal) is a neuropeptide with multiple functions that is widely expressed in the central and peripheral nervous systems of vertebrates. Anatomical and functional evidence suggests a possible role in regulating reproduction in fishes. To test this possibility, we have isolated and characterized two gal alternative transcripts in European sea bass (Dicentrarchus labrax) that encode two prepropeptides, respectively of 29 (gal_MT853221) and 53 (gal_MT853222) amino acids. The two gal transcripts are highly expressed in brain, pituitary and gonads, and appear to be differentially regulated in males and females. In males, gal_MT853222 in the hypothalamus and gal_MT853221 in the pituitary were downregulated with the progression of spermatogenesis (stages I-III). Both transcripts are downregulated in testicles of 1-year (precocious) and 2-year spermiating males compared to immature fish of the same age. Gal peptides and receptors are expressed throughout ovarian development in the hypothalamic-pituitary-gonadal (HPG) axis of females. In the testis, immunoreactive Gal-29 and Gal-53 peptides were detected in blood vessels and Leydig cells during the spermatogenesis stages I-III but Gal immunostaining was barely undetected in more advanced stages. In the ovary, both peptides localized in interstitial cells and blood vessels and in theca cells surrounding the maturing oocytes. The immunolocalization of galanin in Leydig and theca cells suggests a possible role in steroid production regulation. The different pattern of gal expression and Gal localization in the testis and ovary may suggest the possibility that androgens and estrogens may also regulate Gal gene transcription and translation. Altogether, this study showed evidence for the possible involvement of locally produced Gal in gametogenesis and that its production is differentially regulated in male and female gonads., This research was funded by the Portuguese national funds from FCT (Foundation of Science and Technology, I.P.) through projects UIDB/04326/2020 and PTDC/MAR-BIO/3890/2012 and by the European Union Seventh Framework Programme AQUAEXCEL (Grant Agreement 262336, TNA project 318). AG and GM were supported by Spanish MINECO (AGL2015-67477-C2-1-R) and CSIC (201640E073). R.S.T.M and P.I.S.P were supported by FCT under contracts of Norma transitória (DL 57/2016/CP1361/CT0021 and DL57/2016/CP6321/CT0015, respectively) with Universidade do Algarve. We acknowledge Zélia Velez for the help during sampling, Deborah Power for IHC advice and Victoria Alvarado and Alicia Felip for providing hypothalamus and pituitary RNA panels.

© 2022 The Authors., This study investigated the influence of long-term T and 11-KT administration (by means of an implant) on the synthesis and release of gonadotropins (Fsh, Lh) and spermatogenesis progression in juvenile male sea bass exposed to either a natural photoperiod (NP) or continuous light (LL). The results in the control group indicated a clear role for Fsh and 11-KT in the recruitment of type-A spermatogonia (SPGA) towards more advanced stages of spermatogenesis under the NP regime. Fsh promoted androgen synthesis and SPGA differentiation into spermatocytes, while no significant changes in pituitary Gnrh1 and plasma Lh levels were detected. On the other hand, the LL regime inhibited Fsh synthesis, and although no effects were observed on Gnrh1 and Lh, the progression of spermatogenesis was arrested. T and 11-KT administration increased the Gnrh1 and Lh content and reduced the pituitary Fsh content, regardless of the photoperiod regime, although the inhibitory effect of T on pituitary Fsh was stronger than that of 11-KT. Our results show that exogenous 11-KT administration in fish under the LL regime was able to partially restore the progression of spermatogenesis, thus stimulating gonadal development and eliciting spermiation in a few fish, while males that received T implants remained sexually immature under LL. In addition, our results provided new knowledge about the importance of Fsh bioactivity modulation in the regulation of this gonadotropin function, and in turn, on spermatogenesis control in fish., This research was financially supported by UE project No. Q5RS-2002-01801 (PUBERTIMING). Additional funding was provided by the Spanish Ministry of Science and Innovation (AGL2016-75400, PUBERTRAIT).

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.

15 pages, 6 figures, supplementary material https://doi.org/10.1007/s10126-019-09886-x, The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17β10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species, This work was supported by Spanish Government Consolider-Ingenio 2010 grant “Aquagenomics” (ref. CDS2007-0002) to SMK, FM, SZ and FP. BC was supported by an Aquagenomics contract; LR was supported by Aquagenomics postdoctoral contract; ND was supported by Aquagenomics predoctoral contract. NSB was supported by AGL2016-78710-R

In populations of 1-year-old male European sea bass (Dicentrarchus labrax), only large males are able to acquire for the first time a functional competence of their reproductive axis; in other words, to attain puberty. To examine the causes and mechanisms involved in the onset of puberty in this species, a size sorting sampling was carried out to obtain two experimental groups of small and large male fish exhibiting different growth rates. As expected, only large fish reached full spermiogenesis (stage V of testicular development) by the end of the experiment. Our study suggests that fish size is a permissive condition to ensure full effectiveness of the hormonal (Gnrh1, gonadotropins and sexual steroids) actions. Thus, though small fish had endocrine profiles similar to those of large fish, their amplitude was much lower, and was most likely the reason why functional competence of the reproductive axis was not achieved. Moreover, this work provides evidence of the involvement of kisspeptin and Gnrh1 systems in the onset of puberty in a marine teleost fish. It also indicates that very likely kisspeptin and Gnrh1 may regulate gonadotropins and sex steroids at specific stages of testicular development., Supported by an EU project LIFECYCLE (FP7-222719-1) and grants by the Valencian Regional Government (REPROBASS; PROMETEO/2010/003 and PROMETEOI/2014/051) awarded to S.Z. F.E. was supported by a JAE Predoctoral fellowship (CSIC).

Male European sea bass were used to investigate the effects of long-term exposure (14 months) to a maintenance ration on body weight gain, muscle lipid content and maturity. To this end, a group of animals were fed to visual satiety (CT), whereas a second group (EX) was provided an amount of feed each month equal to 0.35% of the CT group's biomass. To determine how energy balance affects metabolic factors, the circulating levels of Igf-1 and leptin and changes in mRNA levels of igf-1, igf-2 and igf-3 in the gonad were analyzed. Feed restriction impacted fish growth, and the specific growth rate was higher in the CT group than in the EX group. Moreover, the condition factor decreased over time in the EX group. Muscle lipid content, HSI, MFI and GSI also decreased in the EX group, which displayed increased apoptosis and low spermatogonial proliferation. Consequently, fish exposed to the maintenance ration showed lower rates of spermiation and delayed testicular development. Overall, fish in the EX group exhibited lower plasma levels of Igf-1 and leptin than those of the CT group. Of note, both plasma and expression levels of igf-1 in September and mRNA levels of igf-2 and igf-3 in February decreased significantly in the EX group, coinciding with the beginning of gametogenesis and full spermiation in this species, respectively. These results indicate that fish under a maintenance ration apparently use stored energy to support at least a basal metabolism and preserve their reproduction, although growth is disrupted. In this line, we suggest that leptin might act as a permissive factor for reproductive function, while Igf-1 might control spermatogenesis in sea bass., This research was financially supported by EU project FP7-222719-1 (LIFECYCLE), project PROMETEO/2010/003 (REPROBASS) from the Valencian Regional Government and CSD2007-00002 (AQUAGENOMICS) and AGL2016-75400-R from the Ministry of Science and Innovation (MICINN). S.E. was sponsored by a JAE fellowship from CSIC (Spain) and a CONICYT Becas Chile fellowship from Chile.

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.

Special issue Expanding the knowledge base of reproductive success: from genes to the environment.-- 11 pages, 7 figures, In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes, This work was supported by the Spanish Fundación Ramón Areces (to J.C.), and partially funded by the Spanish Ministry of Science and Innovation (MICINN) Aquagenomics project (CSD2007-00002 to S.Z.). M.B. was supported by a predoctoral fellowship (FPI) from MICINN

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.

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).

17 pages, 7 figures, 2 tables, additional files.-- The complete design was deposited at the Gene Expression Omnibus NCBI database GSE47400 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=exgnmkgcfbwnhkp&acc=GSE47400 and the platform that validates the microarray can be found at GPL13443.-- Erratum to: Identification of conserved genes triggering puberty in European sea bass males (Dicentrarchus labrax) by microarray expression profiling, BMC Genomics 18(1): 513 (2017), https://doi.org/10.1186/s12864-017-3909-x, Spermatogenesis is a complex process characterized by the activation and/or repression of a number of genes in a spatio-temporal manner. Pubertal development in males starts with the onset of the first spermatogenesis and implies the division of primary spermatogonia and their subsequent entry into meiosis. This study is aimed at the characterization of genes involved in the onset of puberty in European sea bass, and constitutes the first transcriptomic approach focused on meiosis in this species., [Results] European sea bass testes collected at the onset of puberty (first successful reproduction) were grouped in stage I (resting stage), and stage II (proliferative stage). Transition from stage I to stage II was marked by an increase of 11ketotestosterone (11KT), the main fish androgen, whereas the transcriptomic study resulted in 315 genes differentially expressed between the two stages. The onset of puberty induced 1) an up-regulation of genes involved in cell proliferation, cell cycle and meiosis progression, 2) changes in genes related with reproduction and growth, and 3) a down-regulation of genes included in the retinoic acid (RA) signalling pathway. The analysis of GO-terms and biological pathways showed that cell cycle, cell division, cellular metabolic processes, and reproduction were affected, consistent with the early events that occur during the onset of puberty. Furthermore, changes in the expression of three RA nuclear receptors point at the importance of the RA-signalling pathway during this period, in agreement with its role in meiosis., [Conclusion] The results contribute to boost our knowledge of the early molecular and endocrine events that trigger pubertal development and the onset of spermatogenesis in fish. These include an increase in 11KT plasma levels and changes in the expression of several genes involved in cell proliferation, cell cycle progression, meiosis or RA-signalling pathway. Moreover, the results can be applied to study meiosis in this economically important fish species for Mediterranean countries, and may help to develop tools for its sustainable aquaculture., This work was supported by Aquagenomics project (CSD2007-0002) from the Spanish Ministry of Science and Innovation to SZ and a MICINN project (AGL2011-28890) to AG. PM was supported by a grant from the University of Antofagasta MECE2 (ANT0806)

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).

The introduction of naked plasmid DNA in the somatic tissue of an adult organism was established as a system for transient gene transfer years ago. The genes contained in the plasmid are efficiently expressed and expression can be maintained over long periods of time. In the present work, we established this technique for an aquacultured marine fish, European sea bass (Dicentrarchus labrax). The target tissue was muscle and the delivery method used was direct injection of the plasmid DNA. The maximum expression of the introduced gene was detected 5 days after the injection with an optimal dose of 25 ¿g. The efficiency of this method had a clear dependence on water temperature and muscle condition. Different systems previously used in mammals for the improvement of DNA uptake in muscle cells have not worked properly in fish. However, we found that electroporation improved DNA uptake in fish reared at low temperatures. Use of this technique allowed us to evaluate the tetracycline-regulated gene expression system for its use in a marine fish for the first time. All together, these data provide the foundations for undertaking future applications for DNA vaccination or gene therapy in European sea bass.

Kisspeptins are now considered key players in the neuroendocrine control of puberty and reproduction, at least in mammals. Most teleosts have two kiss genes, kiss1 and kiss2, but their sites of expression are still poorly documented. As a first step in investigating the role of kisspeptins in the European sea bass, a perciform fish, we studied the distribution of kiss1 and kiss2-expressing cells in the brain of males and females undergoing their first sexual maturation. Animals were examined at early and late in the reproductive season. We also examined the putative expression of estrogen receptors in kiss-expressing cells and, finally, we investigated whether kisspeptins are expressed in the pituitary gland. We show that kiss1-expressing cells were consistently detected in the habenula and, in mature males and females, in the rostral mediobasal hypothalamus. In both sexes, kiss2-expressing cells were consistently detected at the level of the preoptic area, but the main kiss2 mRNA-positive population was observed in the dorsal hypothalamus, above and under the lateral recess. No obvious sexual differences in kiss1 and kiss2 mRNA expression were detected. Additional studies based on confocal imaging clearly showed that most kiss1 mRNA-containing cells of the mediobasal hypothalamus strongly express ERα and slightly express ERβ2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHβ-positive cells and never in LHβ-positive cells. © 2012 Wiley Periodicals, Inc., Grant sponsor: EU Project LIFECYCLE; Grant number: FP7-222719-1 (to O.K., S.Z.); Grant sponsor: JAE-Predoc (CSIC, Spain; to S.E.).

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.

In higher vertebrates, anti-Mullerian hormone (AMH) is required for Mullerian duct regression in fetal males. AMH is also produced during postnatal life in both sexes regulating steroidogenesis and early stages of folliculogenesis. Teleosts lack Mullerian ducts, but Amh has been identified in several species including European sea bass. However, information on Amh type-2 receptor (Amhr2), the specific receptor for Amh binding, is restricted to a couple of fish species. Here, we report on cloning sea bass amhr2, the production of a recombinant sea bass Amh, and the functional analysis of this ligand-receptor couple. Phylogenetic analysis revealed that sea bass amhr2 segregates with Amhr2 from other vertebrates. This piscine receptor is capable of activating Smad proteins. Antibodies raised against sea bass Amh were used to study native and recombinant Amh, revealing proteins in the range of 66–70 kDa corresponding to the full length Amh. Once proteolytically treated, recombinant sea bass Amh gener...

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).

The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes (cyp19a1, fshr, lhr and star). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle.

Since the late 1980s, gonadotropins have been isolated and characterized in several fish species, but specific immunoassays for the follicle-stimulating hormone (FSH) have only been developed for a few. The present study reports the development and use of a specific and homologous competitive ELISA for measuring FSH in European sea bass (Dicentrarchus labrax) using a recombinant FSH and its specific antiserum. Recombinant European sea bass FSHβ and FSH heterodimer were produced in the methylotrophic yeast Pichia pastoris and a baculovirus expression system, respectively. Specific polyclonal antibodies, generated by rabbit immunization against recombinant FSHβ, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166οg/ml). The sensitivity of the assay was 0.5ng/ml [B 0-2SD], and the intra- and inter-assay coefficients of variation were 2.12% (n=10) and 5.44% (n=16) (B i/B 0 ∿45%), respectively. A high degree of parallelism was observed between the standard curve and serially diluted plasma and pituitary samples of European sea bass. The ELISA developed was used to study the plasma FSH profiles of mature males and females during the reproductive cycle, and those of immature juvenile males under different light regimes. The analysis showed that FSH increased significantly during the intermediate stages of spermatogenesis and during vitellogenesis. Analyses in immature juvenile males showed that the continuous light photoperiod significantly reduced plasma FSH levels, and consequently, testicular growth and precocious puberty. In conclusion, the immunoassay developed has proven to be sensitive, specific and accurate for measuring European sea bass FSH, and it represents a valuable tool for future studies on the reproductive endocrinology of this species. © 2011 Elsevier Inc., This study was supported by projects AGL2005-00796 (MEC), AQUAGENOMICS:CSD2007- 00002 (MICINN) and PROMETEO/2010/003 (GV).

Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species. © 2011 Elsevier Inc., The work of the authors summarized in this article was supported by grants BFU 2008‐00984 and AGL2009‐11086 (Ministerio de Ciencia e Innovación, Spain), projects P08‐CVI‐03788 (Junta de Andalucía, Spain) and PROMETEO/2010/003 (Generalitat Valenciana), and EU research contracts DEER FP7‐ENV‐2007‐1 and LIFECYCLE/FP7222719. CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).

In higher vertebrates, anti-Müllerian hormone (AMH) is required for Müllerian duct regression in fetal males. AMH is also produced during postnatal life in both sexes regulating steroidogenesis and early stages of folliculogenesis. Teleosts lack Müllerian ducts, but Amh has been identified in several species including European sea bass. However, information on Amh type-2 receptor (Amhr2), the specific receptor for Amh binding, is restricted to a couple of fish species. Here, we report on cloning sea bass amhr2, the production of a recombinant sea bass Amh, and the functional analysis of this ligand-receptor couple. Phylogenetic analysis revealed that sea bass amhr2 segregates with Amhr2 from other vertebrates. This piscine receptor is capable of activating Smad proteins. Antibodies raised against sea bass Amh were used to study native and recombinant Amh, revealing proteins in the range of 66-70 kDa corresponding to the full length Amh. Once proteolytically treated, recombinant sea bass Amh generates a 12 kDa C-terminal mature protein, suggesting that contrary to what has been described for other fish Amh proteins, this protein is processed in a similar way as mammalian AMH. The mature sea bass Amh is a biologically active protein able to bind sea bass Amhr2 and, surprisingly, also human AMHR2. In prepubertal sea bass testes, Amh was detected by immunohistochemistry mostly in Sertoli cells surrounding early germ-cell generations. During spermatogenesis, a weaker staining signal could be observed in Sertoli cells surrounding spermatocytes.

Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Several studies have demonstrated that the KISS/GPR54 system is expressed by gonadotrophs, but in vitro studies assessing the direct stimulatory effects of kisspeptin on gonadotropin secretion in the pituitary have provided conflicting results. In this study, we investigated whether kisspeptin directly influences the reproductive function of sea bass pituitary. First, the highly active peptides Kiss1-15 and Kiss2-12 were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results show that, first, Kiss2-12 induced luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion at full spermiation stage. Second, the distribution and nature of Kiss2 and its potential interactions with the gonadotropin-releasing hormone 1 (Gnrh1) system 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 hypophysiotropic neuropeptide or as an autocrine/paracrine factor.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone that plays a key role in the regulation of gonadal functions in vertebrates. The present study reports the monitoring of pituitary and plasma Fsh levels during sex differentiation and oogenesis in European sea bass (Dicentrarchus labrax) using a homologous immunoassay and an in vitro bioassay. Both assays were used complementarily for the first time in a fish species. High levels of Fsh bioactivity in plasma were found during the initial phases of sexual differentiation. Plasma and pituitary Fsh (quantity and bioactivity) levels and biological to immunological (B:I) ratios were higher in females than in males, suggesting sexual dimorphism in the synthesis and potency of Fsh. In females, the B:I ratios in adult were lower than during sex differentiation indicating that Fsh would be less biopotent in the adult stage. Plasma Fsh bioactivity levels increased during vitellogenesis, suggesting that Fsh would be involved in the regulation of the midphases of oogenesis, whereas luteinizing hormone would be responsible for the final events.

91 p., il., y bibliografía. Geir Lasse Taranger [et al.], Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesized to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

26 p., il. y bibliografía, Two large groups of fingerling (3.2 g) male European sea bass (around 75,000 individuals each) reared in net sea cages for a period of two years were exposed to two different photoperiods starting in June: either ambient photoperiod (37º 24′ N) (NP) or a constant long photoperiod (LP; 18 h light: 6 h dark, 18 L: 6D). Long days had limited effect on somatic growth at the time of incipient gonad development (IGD, 1st annual cycle; November–March), while they were effective at the time of full gonad development (FGD, 2nd annual cycle). Moreover, the annual patterns of morphological indices, i.e., Fulton's condition factor (CF), visceral fat index (VFI) and particularly gonadosomatic index (GSI), were delayed by one or two months as the result of the long days compared to the control group (NP). LP did not prevent the onset of puberty but delayed spermiation by one month, and modified accordingly the phase and/or amplitude of the annual rhythms of pituitary gonadotropin releasing hormone (sbGnRH) content and plasma levels of luteinising hormone (LH) and sex steroids, particularly that of 11-ketotestosterone (11-KT). In general, high levels of sbGnRH (≥4 ng/pit) were associated with elevated plasma LH (1–3 ng/ml at IGD and ≥8 ng/ml at FGD), and the sex steroid peaks usually preceded LH peak in both treatments (1–2 months earlier). Finally, full spermiation and the maximum GSI values coincided with the LH peak, highlighting the close relationship that exists between LH plasma levels and gonadal development., This research was supported by MCYT grant No. 1FD1997-1699-CO2-02/MAR to SZ, and by Generalitat Valenciana Grupos 04/80 to MC; I. Begtashi was funded by an AECI grant.

A specific chronology for puberty and changes at the brain-pituitary-gonad axis for sea bass are reviewed. Recent findings demonstrate that the Kisspeptin system, gonadotropin releasing hormones, follicle stimulating hormone, 11-ketotestosterone, and leptin are potential candidates for the onset of puberty of this fish species, stressing the importance of the daily and annual rhythms of some of these hormones. Environmental control of puberty is also reviewed, specifically the manipulations of constant photoperiods for altering or even suppressing the onset of puberty in sea bass. Recently, a possible narrow sensitive period for suppressing gonadogenesis in sea bass has been identified. © 2009 New York Academy of Sciences., Sea bass original data were obtained through European Union (EU) #Q5RS-2002-01801 (PUBERTIMING), Ministery of Education and Science (MEC) (AGL2000-12470-E, AGL200500796 and AGL200604672), and Generalitat Valenciana (GV) (ACOMP07262 and GV06/268) grants.

30 p., 5 figures and bibliography, The European sea bass is a short-day breeder, a characteristic that is highly valued in aquaculture. A high percentage of males of this species mature precociously before reaching commercial size, resulting in economic losses for fish farmers. We investigated the effects of continuous light (LL) on the circadian variations of several reproductive hormones in males of this species in order to understand how the presumed absence of a melatonin rhythm caused by LL affects their daily profile. The study was conducted during four critical stages of the sea bass reproductive cycle (pre spermatogenesis (PSpg), spermatogenesis (Spg), spermiation (Spm), and post- spermiation (PSpm)). Every 3 h during a complete 24 h cycle, six fish kept under a natural photoperiod (NP) and six under LL were anaesthetized, measured, weighed, and bled. The pituitary was removed and frozen at -80ºC. The pituitary content of sea bream gonadotrophin-releasing (sbGnRH) and luteinizing hormone (LH), as well as plasma content of LH, testosterone, and 11-ketotestosterone (11-KT) were analyzed by ELISA. The percentage of spermiating males (precocity) per group was determined by periodic abdominal massages of the animals. Our results confirm LL treatment, maintained from the early stages of development onward, effectively reduces the percentage of precocious male sea bass. As has already been described for caged sea bass, plasma LH showed a clearly marked nocturnal rise near midnight during Spg and Spm during NP, but which was absent under LL. Pituitary sbGnRH and LH content and plasma LH concentration, under both NP and LL conditions, increased during the second half of the reproductive cycle, while sexual steroids were higher at the beginning of the cycle. LL inhibited steroid secretion, especiallytestosterone secretion, during Spg. In summary, without photoperiod cue, as accomplished by continuous exposure to LL, circadian variations of reproductive hormones appeared altered, causing irregularities in the reproductive process of male sea bass. These findings may have a practical application in aquaculture, namely by applying LL treatment in an effort to reduce the presence of precocious males in a stock., This research was funded by the Spanish MEC project No. AGL 2006-04672 and by a grant awarded by the Generalitat Valenciana (AE05/066 and AE07/098) to M.C.E. M.J.B.C. is the recipient of a Juan de la Cierva contract from the Spanish Ministerio de Educación y Ciencia.

There is little information regarding pollutant levels in farmed fish, and the risks associated to consumption. This study was designed to assess levels of exposure to metals, organochlorinated compounds, polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APEs) in farmed sea bass Dicentrarchus labrax from five aquacultures located in Southern Europe. Additionally, several biochemical responses (metallothionein, 7-ethoxyresorufin O-deethylase, vitellogenin) were determined as complementary tools. The obtained data indicate that pollutants exposure in farmed fish is similar to the levels reported in wild specimens from the area. Nonetheless, some biochemical responses were observed in the studied organisms, viz. metallothionein induction in Cu exposed organisms, and 7-ethoxyresorufin O-deethylase (EROD) and vitellogenin induction in PAHs and APEs exposed ones. The study further supports the usefulness of the biomarker approach as a first screening method to discriminate between basal and high levels of exposure in cultured fish. © 2007 Elsevier Ltd. All rights reserved., Denise Fernandes -PhD student at Algarve University- acknowledges a PhD fellowship (SFRH/BD/6123/2001) from the Portuguese Fundacao para a Ciencia e Tecnologia (FCT) of the Ministry of Science and Technology of Portugal.

The objective of this study was to investigate the effect of exposure to continuous light (LL) during the pregametogenesis (4-month duration) and gametogenesis (6-month duration) periods on early male sexual maturation in the sea bass. A simulated natural photoperiod group (SNP) and a long-term exposure group to LL during 12 months were kept as controls. Somatic growth, gonadal development, plasma testosterone (T) and 11-ketotestosterone (11-KT) and the expression of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary were analysed as indicators of maturation. Growth in all treatments was independent of photoperiod. Nevertheless, all LL treatments were effective in reducing the number of early maturing males (< 3% with < 0.2% GSI versus 22% with 0.8% GSI in SNP). This inhibition was not complete but the effect was comparable among all LL treatments thus suggesting that a potential photo-labile period might exist in the sea bass. In the SNP group, the T and 11-KT plasma levels as well as the gene expression values of the three gonadotropin subunits (i.e., LHβ, FSHβ and glycoprotein (GP) α) peaked in February concomitantly with the increase of the GSI (≥ 0.51%) and the accumulation of LH protein in the pituitary (≥ 14.6 μg) and declined at post-spermiation stage (GSI ≤ 0.12% and LH protein ≤ 10.2 μg/pit). Exposure to different LL treatments induced a differential response particularly on the 11-KT levels and the gene expression of the FSHβ subunit. We suggest that the unbalanced production of this androgen regulated by FSHβ might be limiting the stimulation of germ cell proliferation at the testicular level and thus arresting early puberty male sea bass. © 2008 Elsevier B.V. All rights reserved., This research was supported by a UE project No. Q5RS-2002-01801 (PUBERTIMING), Generalitat Valenciana Grupos 04/80 and a MCYT Special Action No. AGL2002-12470-E to M.C. A.F. was supported by a research contract from PUBERTIMING

A likely endocrine control mechanism for sexual differentiation in size-graded populations of European sea bass (Dicentrarchus labrax) is proposed by evaluating the brain expression and pituitary content of two forms of gonadotropin-releasing hormone (GnRH), namely sea bream (sbGnRH) and salmon (sGnRH), the pituitary expression of one subtype of GnRH receptor (dlGnRH-R-2A) and the three gonadotropin (GtH) subunits, namely glycoprotein α (GPα), follicle-stimulating hormone β (FSHβ) and luteinizing hormone β (LHβ), as well as the pituitary and plasma LH levels between 50 and 300 days post-hatching (dph). Four gradings were conducted between 2 and 8 months after hatching, resulting in a population of large and small individuals, having 96.5% females (female-dominant population) and 69.2% males (male-dominant population), respectively, after the last grading. The onset of gonadal differentiation was different in the two sexes, and coincided with a peak of expression of sbGnRH or sGnRH. Furthermore, the expression of these GnRHs was correlated with the expression of dlGnRH-R-2A. Sex-related differences in the brain and pituitary content of sbGnRH were also found at the time of sexual differentiation. Moreover, the observed sexual dimorphism at the transcriptional or synthesis level of these GnRH forms suggests that a different neuro-hormonal regulation is operating according to sex. At the onset of sex differentiation, FSHβ transcriptional activity reached maximal values, which were maintained until the completion of the process. The present study suggests a role for sbGnRH, sGnRH and the dlGnRH-R-2A during gonadal differentiation, possibly through enhancement of FSHβ gene expression. In males, a different endocrine regulation seems to exist also during spermiogenesis and spermiation, when gene transcription, peptide synthesis and release of LH are of greater importance., This work was supported by a EU grant under the Quality of Life and Management of Living Resources programme (contract Q5RS-2000-31365) to S.Z. and C.C.M. and a MEC and GV grants (AGL2002-10024E and GV04B-591, respectively) to S.Z. G.M. was supported by a doctoral I3P fellowship from the CSIC, Spain. We thank L. Rodríguez, S. Ibañez, and J. Beltran for assistance in tissue preparation and result analysis. We are indebted to F. Piferrer and A. Felip for critically reviewing the manuscript.

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.

9 pages, 4 figures, 1 table, The intensive culture of the Senegalese sole (Solea senegalensis) is hampered by the low or null fertilization rates exhibited by the first generation (F1) of reared males. To investigate the regulation of the reproductive processes in this species by the pituitary gonadotropins follicle-stimulating and luteinizing hormones (Fsh and Lh, respectively), we developed a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for Lh measurements. Quantification of the Fsh and Lh plasma levels in cultured sole using the Lh ELISA developed here, and a previously developed ELISA for Fsh, indicated that in both males and females circulating Fsh steadily increased during autumn and winter and prior to the major spawning in spring, whereas an Lh surge occurred specifically during spawning. The increase in Fsh was associated with a rise of plasma levels of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β (E2), but that of Lh was concomitant with a strong decline of the levels of E2 in females and of 11-KT in males, possibly reflecting a rapid steroidogenic shift promoting the final maturation of gametes. Comparison of the plasma levels of gonadotropins and steroids between wild and F1 fish during autumn and spring revealed that F1 males showed significantly lower plasma Lh titres compared to wild males, whereas the levels of T and 11-KT were similar or more elevated in the F1 fish. These data suggest that an impaired Lh secretion during spawning, and perhaps altered Lh-mediated mechanisms in the testis, may be underlying causes for the low reproductive performance of Senegalese sole F1 males. © 2015 Elsevier Inc, This work was funded by the Spanish Fundación Ramón Areces (to J.C.), and partially supported by the Spanish Ministry of Science and Innovation (MICINN) Aquagenomics project (CSD2007-00002 to S.Z.) and the Spanish National Institute for Agronomic Research (INIA) project RTD2011-00050 (to N.D). Participation of E.F. was financed by a predoctoral fellowship from INIA

Comunicación presentada en The International Symposium on Genetics in Aquaculture XII, celebrado en Santiago de Compostela, España, del 21 al 27 de junio de 2015, Two forms of kiss gene (kiss1and kiss2) have been described in the teleost sea bass. This study assesses the cloning and characterization of two Kiss receptor genes, namely kissr2 and kissr3 (known as gpr54-1b and gpr54-2b, respectively), and their signal transduction pathways in response to Kiss1 and Kiss2 peptides. Phylogenetic and synteny analyses indicate that these paralogs originated by duplication of an ancestral gene before teleost specific duplication. The kissr2 and kissr3 mRNAs encode proteins of 368 and 378 amino acids, respectively, and share 53.1% similarity in amino acid sequences. In silico analysis of the putative promoter regions of the sea bass Kiss receptor genes revealed conserved flanking regulatory sequences among teleosts. Both kissr2 and kissr3 are predominantly expressed in brain and gonads of sea bass, medaka and zebrafish. In the testis, the expression levels of sea bass kisspeptins and Kiss receptors point to a significant variation during the reproductive cycle. In vitro functional analyses revealed that sea bass Kiss receptor signals are transduced both via the protein kinase C (PKC) and protein kinase A (PKA) pathway. Synthetic sea bass Kiss1-15 and Kiss2-12 peptides activated Kiss receptors with different potencies, indicating differential ligand selectivity. Our data suggest that Kissr2 and Kissr3 have a preference for Kiss1 and Kiss2 peptides, respectively, thus providing the basis for future studies aimed at establishing their physiologic roles in sea bass., Supported by GV (GV06/268) and (ACOMP2010), Programa PROMETEO (Prometeo/2010/003; PrometeoII/2014/051) and EU grant 655 FP7222719 (LIFECYCLE). A.F was supported by a Ramón y Cajal and F.E by a JAE-Predoc CSIC

Kisspeptins are key players in the neuroendocrine control of puberty and other reproductive processes in mammals. Several studies have demonstrated that the KISS/GPR54 system is expressed by gonadotrophs, but in vitro studies assessing the direct stimulatory effects of kisspeptin on gonadotropin secretion in the pituitary have provided conflicting results. In this study, we investigated whether kisspeptin directly influences the reproductive function of sea bass pituitary. First, the highly active peptides Kiss1-15 and Kiss2-12 were used to stimulate dispersed sea bass pituitary cells obtained from mature males. Our results show that, first, Kiss2-12 induced luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) release, whereas Kiss1-15 had no effect on gonadotropin secretion at full spermiation stage. Second, the distribution and nature of Kiss2 and its potential interactions with the gonadotropin-releasing hormone 1 (Gnrh1) system 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 hypophysiotropic neuropeptide or as an autocrine/paracrine factor.

Some teleost species, including European sea bass, harbor two different kisspeptin coding genes: kiss1 and kiss2. Both genes are expressed in the brain, but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, we have 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 was injected into the third ventricle. To establish the gene expression cascade involved in the action of kisspeptins, the expression of the two sea bass kisspeptin receptor genes (kiss1r and kiss2r) and the three sea bass Gnrh genes (gnrh1, gnrh2, and gnrh3) were analyzed in the forebrain-midbrain and the hypothalamus. In addition, the protein levels of hypothalamic and pituitary Gnrh1 were measured. 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). The present results provide the first evidence that the effects of Kiss2 on central regulation of reproductive function involve the neuroendocrine areas of the forebrain-midbrain in teleost fish. The marked effect of Kiss2 on kiss2r and gnrh1 expression in the forebrain-midbrain and on Gnrh1 release suggest that this neuronal system is involved in the neuroendocrine regulation of gonadotroph activity. This hypothesis was confirmed by a surge of plasma Lh in response to Kiss2, which presumably has a strong stimulatory effect on testosterone release, and thus on sperm quality parameters.

Some teleost species, including European sea bass, harbor two different kisspeptin coding genes: kiss1 and kiss2. Both genes are expressed in the brain, but their differential roles in the central control of fish reproduction are only beginning to be elucidated. In this study, we have 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 was injected into the third ventricle. To establish the gene expression cascade involved in the action of kisspeptins, the expression of the two sea bass kisspeptin receptor genes (kiss1r and kiss2r) and the three sea bass Gnrh genes (gnrh1, gnrh2, and gnrh3) were analyzed in the forebrain-midbrain and the hypothalamus. In addition, the protein levels of hypothalamic and pituitary Gnrh1 were measured. 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). The present results provide the first evidence that the effects of Kiss2 on central regulation of reproductive function involve the neuroendocrine areas of the forebrain-midbrain in teleost fish. The marked effect of Kiss2 on kiss2r and gnrh1 expression in the forebrain-midbrain and on Gnrh1 release suggest that this neuronal system is involved in the neuroendocrine regulation of gonadotroph activity. This hypothesis was confirmed by a surge of plasma Lh in response to Kiss2, which presumably has a strong stimulatory effect on testosterone release, and thus on sperm quality parameters.