Functional Activity of Recombinant Forms of Amh and Synergistic Action with Fsh in European Sea Bass Ovary.

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.

Advanced puberty triggered by bi-weekly changes in reproductive factors during the photolabile period in a male teleost fish, Dicentrarchus labrax L.

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 fshß 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.

Is the kisspeptin system involved in responses to food restriction in order to preserve reproduction in pubertal male sea bass (Dicentrarchus labrax)?

Previous works on European sea bass have determined that long-term exposure to restrictive feeding diets alters the rhythms of some reproductive/metabolic hormones, delaying maturation and increasing apoptosis during gametogenesis. However, exactly how these diets affect key genes and hormones on the brain-pituitary-gonad (BPG) axis to trigger puberty is still largely unknown. We may hypothesize that all these signals could be integrated, at least in part, by the kisspeptin system. In order to capture a glimpse of these regulatory mechanisms, kiss1 and kiss2 mRNA expression levels and those of their kiss receptors (kiss1r, kiss2r) were analyzed in different areas of the brain and in the pituitary of pubertal male sea bass during gametogenesis. Furthermore, other reproductive hormones and factors as well as the percentage of males showing full spermiation were also analyzed. Treated fish fed maintenance diets provided evidence of overexpression of the kisspeptin system in the main hypophysiotropic regions of the brain throughout the entire sexual cycle. Conversely, Gnrh1 and gonadotropin pituitary content and plasma sexual steroid levels were downregulated, except for Fsh levels, which were shown to increase during spermiation. Treated fish exhibited lower rates of spermiation as compared to control group and a delay in its accomplishment. These results demonstrate how the kisspeptin system and plasma Fsh levels are differentially affected by maintenance diets, causing a retardation, but not a full blockage of the reproductive process in the teleost fish European sea bass. This suggests that a hormonal adaptive strategy may be operating in order to preserve reproductive function in this species.

Gonadotropin Inhibitory Hormone Down-Regulates the Brain-Pituitary Reproductive Axis of Male European Sea Bass (Dicentrarchus labrax).

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.

LPXRFa peptide system in the European sea bass: A molecular and immunohistochemical approach.

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.

Kiss2 as a Regulator of Lh and Fsh Secretion via Paracrine/Autocrine Signaling in the Teleost Fish European Sea Bass (Dicentrarchus labrax).

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.

Differential activation of kiss receptors by Kiss1 and Kiss2 peptides in the sea bass.

Two forms of kiss gene (kiss1 and 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 and protein kinase A pathway. Synthetic sea bass Kiss1-15 and Kiss2-12 peptides activated Kiss receptors with different potencies, indicating a 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.

The forebrain-midbrain acts as functional endocrine signaling pathway of Kiss2/Gnrh1 system controlling the gonadotroph activity in the teleost fish European sea bass (Dicentrarchus labrax).

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.

Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

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 ß subunit (Fshß) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshß-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 Fshß subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids.

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17ß-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function.

Characterization of sea bass FSHß 5' flanking region: transcriptional control by 17ß-estradiol.

The sea bass follicle-stimulating hormone 5' flanking region (sbFSHß 5' FR) was cloned and characterized in order to study the molecular mechanisms underlying transcriptional regulation of the sbFSHß gene. Analysis of the ~3.5 kb of this region revealed the presence of several putative cis-acting elements, including steroid hormone response elements, cAMP response elements, pituitary-specific transcription factor response elements, activator protein-1 response elements and TATA sequence. Deleted constructs containing ~3.5 kb of the sbFSHß 5' FR fused to a luciferase reporter gene were transiently transfected into human embryonic kidney (HEK 293) and mouse mature gonadotrope (LßT2) cell lines. The sbFSHß 5' FR was efficiently expressed under basal conditions in LßT2 but not in HEK 293, pointing to both positive and negative regulatory elements. In order to elucidate the estrogen-mediated sbFSHß transcriptional activity, in vitro treatments with 17ß-estradiol were carried out on primary cultures of pituitary cells and LßT2 cells transiently expressing luciferase under the control of sbFSHß 5' FR. Overall, these results demonstrate that 17ß-estradiol inhibits sbFSHß gene expression directly at the level of the pituitary. However, it was also shown that estrogen did not induce changes of the sbFSH promoter-directed luciferase activity, suggesting that sbFSHß 5'FR (~3.5 kb) activity is cell type dependent and its estrogen regulation could require cis-acting elements located upstream of the promoter region, which is characterized in this article.

Administration of follicle-stimulating hormone in vivo triggers testicular recrudescence of juvenile European sea bass (Dicentrarchus labrax).

Follicle-stimulating hormone (Fsh) is thought to act early in the process of spermatogenesis; however, its action in fish has not yet been clearly established. In the present work, we analyzed the effects of recombinant Fsh in sea bass (Dicentrarchus labrax) spermatogenesis according to two different approaches: direct injection of recombinant single-chain Fsh hormone (scFSH) and injection of scFSH coding sequence. Both approaches were efficient in increasing plasma Fsh at 7 and 15 days, respectively, after injection. The Fsh increment caused a significant increase in plasma 11-ketotestosterone levels and induced dramatic changes at the testicular level. Fsh-treated groups showed an increase in germ cell proliferation at Day 7, and cysts of spermatocytes and spermatids were observed at the end of the experiment. After treatment with Fsh, a suppression in amh transcripts and an increase of lhr transcripts were detected at Day 7 and Day 15, respectively, and an increment in fshr expression became evident at Day 23. These results show that Fsh initiates germ cell proliferation, triggering spermatogenesis in sea bass via androgen production and regulation of spermatogenesis-related genes.

Expression of kisspeptins and kiss receptors suggests a large range of functions for kisspeptin systems in the brain of the European sea bass.

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.

Expression of kisspeptins in the brain and pituitary of the European sea bass (Dicentrarchus labrax).

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 ERß2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHß-positive cells and never in LHß-positive cells.

Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost.

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

Development of a homologous enzyme-linked immunosorbent assay for European sea bass FSH. Reproductive cycle plasma levels in both sexes and in yearling precocious and non-precocious males.

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 FSHß 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 FSHß, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166 µg/ml). The sensitivity of the assay was 0.5 ng/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.

Comparative insights of the kisspeptin/kisspeptin receptor system: lessons from non-mammalian vertebrates.

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.

Determination of Fsh quantity and bioactivity during sex differentiation and oogenesis in European sea bass.

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.

Seasonal changes in gonadal expression of gonadotropin receptors, steroidogenic acute regulatory protein and steroidogenic enzymes in the European sea bass.

The endocrine regulation of gametogenesis, and particularly the roles of gonadotropins, is still poorly understood in teleost fish. This study aimed to investigate transcript levels of both gonadotropin receptors (FSHR and LHR) during an entire reproductive cycle in male and female sea bass (Dicentrarchus labrax). To have a more comprehensive understanding of how different key factors interact to control sea bass gonadal function, changes in the transcript abundance of two important steroidogenic enzymes, P450 11beta-hydroxylase (CYP11B1) and P450 aromatase (CYP19A1), and the steroidogenic acute regulatory protein (StAR), were also studied. These expression profiles were analysed in relation to changes in the plasma levels of important reproductive hormones and histological data. Expression of the FSHR was connected with early stages of gonadal development, but also with the spermiation/maturation-ovulation periods. The expression profile of the LHR seen in both sexes supports the involvement of LH in the regulation of the final stages of gamete maturation and spermiation/ovulation. In both sexes StAR expression was strongly correlated with LHR expression. In females high magnitude increments of StAR expression levels were observed during the maturation-ovulation stage. In males, gonadotropin receptors and CYP11B1 mRNA levels were found to be correlated. In females, the expression profiles of FSHR and CYP19A1 and the changes in plasma estradiol (E2) indicate that the follicular production of E2 could be under control of FSH through the regulation of aromatase expression. This study supports the idea that FSH and LH may have different roles in the control of sea bass gonadal function.

Effects of continuous light on the reproductive system of European sea bass gauged by alterations of circadian variations during their first reproductive cycle.

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 the 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 another six fish kept under LL were anaesthetized, measured, weighed, and bled. The pituitary was removed and frozen at -80 degrees C. 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 that 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, increased during the second half of the reproductive cycle, while sexual steroids were higher at the beginning of the cycle. LL inhibited steroid secretion, especially testosterone 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.