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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.

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).

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).

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

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.