Radioimmunoassay of ovarian steroids in plasmas of ovulating female sea bass (Dicentrarchus labrax)

Radioimmunoassays of the free and conjugated fractions of plasmas from ovulating sea bass (Dicentrarchus labrax) have revealed the presence of several unusual polar steroids. Among them is 3 alpha,17 alpha,20 beta,21-tetrahydroxy-5 beta-pregnane.     

Daily rhythms of insulin and glucose levels in the plasma of sea bass Dicentrarchus labrax after experimental feeding

Significant and inverse circadian rhythms are demonstrated in glucose and plasma insulin in fish fed a natural diet. The highest glucose levels are found during the light period, around feeding time, and the insulin level peaks during the dark period. As a possible cause for the insulin rhythmicity, the daily variations in several plasma amino acids are considered. The feeding times could be a training factor for metabolic rhythms, which are maintained even during a fast of 7 days. The differences in the compositions of the diets could be responsible for the lack of circadian rhythms in fish fed on a commercial diet.     

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.     

Genetic, endocrine, and environmental components of sex determination and differentiation in the European sea bass (Dicentrarchus labrax L.)

The European sea bass (Dicentrarchus labrax L.) is a differentiated gonochoristic marine teleost of the family Moronidae (closely related to the hermaphrodites of the family Serranidae), where many juvenile males exhibit intratesticular oocytes, suggesting a certain sexual lability. Like most fish, the sea bass does not have recognizable heterochromosomes or sex-linked markers but there are clear parental effects on the sex ratios. The data available so far indicate that the proportion of females resulting from individual crossings may range from as little as 1 to about 70%. Sex differentiation proceeds in a caudo-cranial fashion and starts when fish reach 8-9 cm standard length (usually about 200 days post-hatching, dph, under typical rearing conditions), with females differentiating first. Both forms of aromatase have been cloned in this species and their temporal expression has been studied. Brain aromatase is detectable already in the larval stages but its involvement in sex differentiation is not yet clear. The ovarian form increases after 100 dph before ovarian differentiation, with high levels in females and basal levels in males. Thus, ovarian aromatase seems to be involved in female differentiation. On the other hand, androgen receptor (AR) gene expression levels show the opposite pattern, with higher levels in males than in females. It is not yet known whether androgens are necessary for testicular differentiation or rather they are the result of it. Of the several environmental factors tested, temperature is the only one that has been shown to be able to clearly influence sex ratios. Larval and juvenile sea bass reared in captivity at high temperature usually develop as males. Recent research suggests that the high incidence of males under aquaculture conditions is due to the high water temperature used, and that the effects of temperature would be mediated by an inhibition of aromatase mRNA expression and activity in genotypic females. However, other effects of temperature mediated through alterations in developmental rates cannot be discarded. This paper reviews the current knowledge on sex determination and differentiation in the sea bass and suggests some directions for future research.     

Oral administration of melatonin reduces food intake and modifies macronutrient selection in European sea bass (Dicentrarchus labrax, L.)

Seasonal and circadian changes have been observed in dietary selection and feeding behavior of fish. It is known that the light-dark cycle is the principal mediator of the production of pineal-derived melatonin in fish, and also that the digestive tract synthesizes and secretes melatonin and indeed is the principal extrapineal source of this secretor product, suggesting that melatonin could be involved in the processes of feeding and/or digestion. Fish are capable of regulating their energy intake from separate sources of macronutrients, but the mechanisms of this selection process are unknown. In the present paper, we explored the effect of melatonin administered orally to European sea bass on their selection of encapsulated macronutrients. Melatonin doses of 0.1, 0.5, and 2.5 mg/kg body weight were administered in gelatin capsules. The voluntarily ingested melatonin was absorbed into the fish's plasma, 45 min after the administration reaching a level depending on the dose that was up to 26 times greater than the controls with the highest dose of melatonin. The indole produced a dose-dependent inhibition of total food intake of 9, 26, and 34%, respectively, and also modified the pattern of macronutrient selection. Carbohydrate intake was significantly reduced (by 17, 33, and 42% for the three doses, respectively), but the observed reductions in fat and protein intake were not statistically significant. On the contrary, fat percentage significantly increased for the highest dose, but no changes were observed in the protein or carbohydrate percentages for any of the melatonin doses. In conclusion, orally administered melatonin affected both the amount of food consumed and the pattern of macronutrients selected. This is the first evidence for the existence of neurohumoral mediators involved in the selection of macronutrients in fish.     

Pituitary levels of three forms of GnRH in the male European sea bass (Dicentrarchus labrax, L.) during sex differentiation and first spawning season

In the present study, levels of three GnRH forms [seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH)] were analyzed in the pituitary of male sea bass during sex differentiation and the first spawning season. Plasma levels of gonadotropin (GTH-2), testosterone (T), and 11-ketotestosterone (11-KT) were determined during the same periods. All GnRH forms were present in the pituitary. sbGnRH levels were 9-fold higher than cGnRH-II and 17-fold higher than sGnRH levels. The highest GnRHs levels were detected in November 1995, when fish were 9 months old and when the gonads started to differentiate. Levels of the three forms decreased and remained low during the first spawning season, with the exception of sbGnRH, which showed a significant increase in November 1996. Plasma GTH-2 levels were lowest in November 1995, later increasing 2.5 times during the next months. During the first spawning season, plasma GTH-2 levels peaked in December 1996, 1 month after the peak of sbGnRH. During sex differentiation, plasma T levels were high in November 1995 but decreased over the next months, while levels of 11-KT remained low and unchanged. During the first spawning season, both steroids peaked in January 1997. These results suggest a possible role for all three GnRH forms in achieving gonadal differentiation, while sbGnRH may be the most relevant form in the regulation of the first spawning season in male sea bass. Moreover, GTH-2 and 11-KT may play important roles in gonadal maturation, since plasma GTH-2 and 11-KT levels were high throughout the period of spermiation.     

Immunoaffinity purification and partial characterization of sea bass (Dicentrarchus labrax) growth hormone

Growth hormone (GH) was isolated from sea bass (Dicentrarchus labrax) pituitary extract by a simple one-step procedure involving immunoaffinity chromatography. A monoclonal antibody raised against chicken GH and found to immunostain very specifically the GH cells in the pituitary of the sea bass was coupled to CNBr-activated Sepharose 4B. Sea bass pituitary extracts were run on the affinity column, and the eluted material was analyzed on reversed-phase HPLC and found to consist of one single peak. The yield of purified hormone was 2.4 mg/g pituitary. Two monomeric forms (MW = 20,000 and 22,000 Da) of sea bass GH were identified by gel electrophoresis. Gel electrofocusing revealed apparent isoelectric points of 6.15, 6.50, and 6.95. Amino acid composition is consistent with other vertebrate GHs. The immunological relatedness was tested by immunoblotting using antisera raised against GH of different species. Polyclonal antisera raised against the isolated hormone exhibited a specific labeling of the GH cells in sea bass pituitary sections as well as of the immunoblotted purified GH.     

Long-term exposure to continuous light inhibits precocity in European male sea bass (Dicentrarchus labrax, L.): hormonal aspects

The effect of long-term exposure to continuous light on the hormonal aspects of the reproductive axis was investigated in juvenile male sea bass (Dicentrarchus labrax, L.) during the first annual cycle. Four-month-old fish were exposed to a simulated natural photoperiod (NP) and a continuous light (24 h) regime (LL) under natural conditions of temperature (13.3-25.8 degrees C). A dot-blot technique was used to analyse gonadotropin (the common glycoprotein alpha, GPalpha; the follicle stimulating hormone beta, FSHbeta; and the luteinizing hormone beta, LHbeta, subunits) mRNA levels in the pituitary during the experiment. A homologous ELISA was used to determine pituitary sea bream gonadotropin-releasing hormone (sbGnRH) and LH and plasma LH levels; gonadal and plasma sex steroids concentrations were determined by specific immunoassays. LL significantly inhibited the expression of all three gonadotropins subunits in the pituitary. However, no significant differences on plasma LH levels were observed between NP and LL groups throughout the period of the experience. Long-term exposure to LL regime was extremely effective in inhibiting gonadal growth and hence precocious maturation as well as the accumulation of Testosterone (T) and 11-ketotestosterone (11-KT) in the gonads compared to the control group. 11-KT plasma levels remained low and unchanged in the LL group during the study. This work describe important alterations of the endocrine system, particularly at the pituitary-gonad axis provoked by exposure to continuous illumination and discusses the mechanism by which precocity in male sea bass is generated.     

The retina is a target for GnRH-3 system in the European sea bass, Dicentrarchus labrax

The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass.     

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) I. Immunocytochemical characterization of glucagon- and PP-related peptides

PP-, PYY-, and glucagon-immunoreactive cells were immunocytochemically identified in the pancreatic islets of Dicentrarchus labrax (sea bass). PYY cells also reacted with anti-PP serum. The specificity control showed that preabsorption of PP antiserum by PYY peptide abolished the immunostaining, while the reaction did not change when the PYY antiserum was preabsorbed by PP. These results suggested the existence of a PP/PYY molecule in the sea bass islets. The islet distribution of PP/PYY-immunoreactive cells differed markedly. Thus, in the principal islet and some intermediate islets few PP/PYY-immunoreactive cells are present (type I islets), whereas in the smaller and some intermediate ones they are numerous (type II islets). Adjacent sections stained by peroxidase-antiperoxidase (PAP) technique and individual sections stained by immunofluorescence double staining showed the coexistence of glucagon and PP/PYY-like immunoreactivities. Both islet types contained cells with PP/PYY coexisting with glucagon peptide, while cells showing solely glucagon immunoreactivity were found in type I islets only.     

Effects of a natural parasitical infection (Lernanthropus kroyeri) on the immune system of European sea bass, Dicentrarchus labrax L

The immune response of European sea bass, Dicentrarchus labrax , to a natural infection by the copepod parasite Lernanthropus kroyeri was evaluated for the first time in vivo . The results clearly demonstrated the triggering of the fish immune system by the parasite. Lysozyme activity and alternative complement pathway were involved in the early action against the parasitical infection, whilst classical complement and respiratory burst (RB) activity took over in the later stages of infection. It was hypothesized that the levels of alternative and classical complement and RB stimulation indexes may determine the resistance capacity of the fish to the parasite. It is not clear how parasites can survive despite the strong immunological arsenal deployed by the fish. The continual increase of prevalence and severity of parasite infection suggested that the parasite's mechanism of evasion of the immune system was extremely successful. The contrasting decrease in the negative effects of parasites on the fish health (such as gills anaemia) suggested that an equilibrium between the parasites and their hosts was reached in chronic infection. These dynamic interactions between parasites and fish hosts were probably the main determinant of host specificity.     

Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age,sex, and the annual reproductive cycle

Cytochrome P450 aromatase activity (AA) was measured in different tissues of the sea bass (Dicentrarchus labrax L.) using a tritiated water release assay that was previously optimized and validated for this species. In adult fish entering the reproductive season, AA was highest on a per mg protein basis, in the brain (2.04+/-0.4 pmol/mg prot/h; mean+/-SEM), followed by the ovary (0.59+/-0.1) and was detectable in visceral fat (0.21+/-0.05), liver (0.08+/-0.009), and head kidney (0.03+/-0.004). However, AA was negligible in the rest of the tissues tested: heart, testis, muscle, and spleen. Consistent with results obtained in other species, dissection of the brain into its major constitutive parts revealed that AA was concentrated in areas implicated in the control of reproduction, including the olfactory bulb, telencephalon, and hypothalamus (range: 2.6-16.2 pmol/mg prot/h), as well as the pituitary gland (6.2-9.3 pmol/mg prot/h). Lower AA was noted in the optic bulb, cerebellum, and medulla. However, in contrast to some previously published reports concerning the content and distribution of neural aromatase in fish, males consistently exhibited higher AA than females. In one-year-old juvenile fish completing the process of gonadal sex differentiation, brain AA (0.63 pmol/mg prot/h) was similar in both sexes and ten times lower than that measured in the brain of first time spawners (6.52 pmol/mg prot/h), in this case with males showing an overall higher (24%) activity than females. When surveyed throughout the year, brain AA dramatically changed during the reproductive cycle. Maximum average values of approximately 7 pmol/mg prot/h were obtained that coincided with the spawning season. The peak in brain AA was preceded by two and one months by the peak of plasma testosterone and the peak of the gonadosomatic index, respectively. This is the first measurement of the distribution of the activity of a steroidogenic enzyme in the sea bass, an established model in comparative endocrinology. Together, these results demonstrate sex- and seasonally-related variations in AA and establish the basis for further comparative studies of certain androgen-mediated actions through locally formed estrogen in both central and peripheral targets.     

Identification of hormone-producing cells of the endocrine pancreas of the sea bass, Dicentrarchus labrax, by ultrastructural immunocytochemistry

The hormone-producing cells of the endocrine pancreas of the sea bass Dicentrarchus labrax have been identified by ultrastructural immunocytochemistry. The glucagon cells have "clear" cytoplasm and contain characteristic electron-dense polygonal granules surrounded by a "halo" of electron-lucent material. The insulin cells have numerous, tightly packed, electron-dense granules that are almost twice as large as the peripherally located granules of the somatostatin cells. The pancreatic polypeptide cells have granules with variable electron density. When specific antisera are applied in the peroxidase-anti-per-oxidase immunocytochemical method at the electron microscope level, each of the four types of granule is identified by the resultant overlying immunoreaction deposit. Especially in older fish, a fifth, nonclassified type of cell has been identified within the endocrine pancreatic tissue. These cells have many ramifying processes and contain a mixture of the granules of the four endocrine cell types as well as granules from the exocrine tissue. It is suggested that these cells may be undertaking macrophage activities. A distinct patterned arrangement of the endocrine cell types in both small pancreatic islets and Brockmann bodies is observed. There is a central core of insulin and somatostatin cells surrounded by an outer peripheral layer of glucagon and pancreatic polypeptide cells. A definite functional interrelationship is suggested by this arrangement.     

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) II. Immunocytochemical study of insulin and somatostatin peptides.

Insulin (INS)- and somatostatin (SST)-immunoreactive cells were demonstrated by light immunocytochemistry in the endocrine pancreas of sea bass (Dicentrarchus labrax). INS-immunoreactive cells were identified using bovine/porcine, bonito, and salmon (s) INS antisera; the immunostaining was abolished when each antiserum was preabsorbed with its respective peptide but not with unrelated peptides. These cells also reacted with mammal (m) SST-28 (4-14) antiserum. The immunoreaction did not change when this antiserum was preabsorbed by bovine INS. INS-immunoreactive cells were located in the central part of the endocrine areas of the principal, intermediate, and small islets. Two SST-immunoreactive cell types (D1 and D2) were revealed. D1 cells, immunoreactive to SST 14 (562) and sSST-25 antisera, were located next to the glucagon-immunoreactive cells in the peripheral part of the endocrine areas. D2 cells, immunoreactive to SST-14 (562), SST-14 (566), and mSST-28 (4-14) antisera, were found in apposition to the INS-immunoreactive cells. The specificity controls showed that D1 cells expressed sSST-25-like peptides, while D2 cells might contain SST-14 and/or mSST-28-like peptides. The close topographic association between the different SST-immunoreactive cells and both glucagon- and insulin-immunoreactive cells might indicate the existence of a specific paracrine regulation of each endocrine cell type in the sea bass endocrine pancreas.     

Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax)

Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40–85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50–1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0–50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein α (GPα) and 13.6 kDa for FSHβ subunits. After separation by SDS–PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPα and FSHβ). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17β (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPα and FSHβ), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.     

Thyrotropin-releasing hormone-immunoreactive system in the brain and pituitary gland of the sea bass (Dicentrarchus labrax, Teleostei)

The immunohistochemical distribution of thyrotropin-releasing hormone-like immunoreactivity (TRH-ir) in the brain and pituitary of the sea bass (Dicentrarchus labrax) was examined on cryostat sections of tissues perfuse fixed in a formaldehyde-glutaraldehyde mixture. TRH-ir fibres were found in many areas of the brain: dorsal and ventral telencephalon, preoptic and tuberal hypothalamus, thalamus, midbrain tegmentum, optic tectum, and medulla oblongata. In the hypothalamus the densest area of innervation was the nucleus anterioris tuberis and medial nucleus recessus lateralis, where small TRH-ir cell bodies were also found. In the pituitary gland, TRH-ir fibres were numerous in the posterior neurohypophysis, and these appeared to form varicosities between groups of melanocorticotropic cells of the pars intermedia. No clear relationship was seen between TRH-ir fibres and the thyrotropic cells, or any other cell type of the pars distalis. In the brain stem a notable feature was the prominent innervation of groups of motoneurons by beaded TRH-ir fibres. These observations suggest that in teleost fishes the role of TRH may be related to pars intermedia function, rather than the thyrotropin- or prolactin-releasing function established in tetrapods. In addition the tripeptide may act as a central neurotransmitter involved in sensory and autonomic motor integration.     

Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water

The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na+/K+ -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1alpha (ef1alpha) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prlwas 4.5-foldhigher in FWthan in SW, whereas pituitary sl was 1.8-fold higher in SW- compared with FW-adapted fish. Gill Na+/K+ -ATPase specific activity was 2.3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6.5-fold lower in SW- than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FWeuryhaline teleosts.     

Seasonal changes in gonadal steroids of a monogamous versus a polyandrous shorebird

We examined the relationship between circulating levels of gonadal steroids (testosterone, progesterone, and estradiol) and breeding behavior in semipalmated sandpipers (Calidris pusilla) and red-necked phalaropes (Phalaropus lobatus) breeding sympatrically at La Pérouse Bay, 40 km east of Churchill, Manitoba. Semipalmated sandpipers are territorial and monogamous. Both parents incubate equally. Red-necked phalaropes are nonterritorial and polyandrous. Only male phalaropes care for eggs and young. Gonadal steroid hormone profiles were not reversed in the sex-role-reversed species. There was little difference in testosterone profiles between males of the territorial and nonterritorial species.