17α-Ethinylestradiol exposure disrupts anxiety-like behaviours but not social preference in sea bass larvae (Dicentrarchus labrax).

Endocrine-disrupting chemicals (EDCs) are widespread pollutants known to interfere with hormonal pathways and to disrupt behaviours. Standardised behavioural procedures have been developed in common fish model species to assess the impact of various pollutants on behaviours such as locomotor activity and anxiety-like as well as social behaviours. These procedures need now to be adapted to improve our knowledge on the behavioural effects of EDCs on less studied marine species. In this context, the European sea bass (Dicentrarchus labrax) is emerging as a valuable species representative of the European marine environment. Here, we designed and validated a two-step procedure allowing to sequentially assess anxiety-like behaviours (novel tank test) and social preference (visual social preference test) in sea bass. Thereafter, using this procedure, we evaluated whether social behavioural disruption occurs in 2-month-old larvae after an 8-day exposure to a xenoestrogen, the 17α-ethinylestradiol (EE2 at 0.5 and 50 nM). Our results confirmed previous studies showing that exposure to 50 nM of EE2 induces a significant increase in anxiety-like behaviours in sea bass larvae. On the contrary, social preference seemed unaffected whatever the EE2 concentration, suggesting that social behaviour has more complex mechanical regulations than anxiety.

New Insights Into the Evolution of Corticotropin-Releasing Hormone Family With a Special Focus on Teleosts.

Corticotropin-releasing hormone (CRH) was discovered for its role as a brain neurohormone controlling the corticotropic axis in vertebrates. An additional crh gene, crh2, paralog of crh (crh1), and likely resulting from the second round (2R) of vertebrate whole genome duplication (WGD), was identified in a holocephalan chondrichthyan, in basal mammals, various sauropsids and a non-teleost actinopterygian holostean. It was suggested that crh2 has been recurrently lost in some vertebrate groups including teleosts. We further investigated the fate of crh1 and crh2 in vertebrates with a special focus on teleosts. Phylogenetic and synteny analyses showed the presence of duplicated crh1 paralogs, crh1a and crh1b, in most teleosts, resulting from the teleost-specific WGD (3R). Crh1b is conserved in all teleosts studied, while crh1a has been lost independently in some species. Additional crh1 paralogs are present in carps and salmonids, resulting from specific WGD in these lineages. We identified crh2 gene in additional vertebrate groups such as chondrichthyan elasmobranchs, sarcopterygians including dipnoans and amphibians, and basal actinoperygians, Polypteridae and Chondrostei. We also revealed the presence of crh2 in teleosts, including elopomorphs, osteoglossomorphs, clupeiforms, and ostariophysians, while it would have been lost in Euteleostei along with some other groups. To get some insights on the functional evolution of the crh paralogs, we compared their primary and 3D structure, and by qPCR their tissue distribution, in two representative species, the European eel, which possesses three crh paralogs (crh1a, crh1b, crh2), and the Atlantic salmon, which possesses four crh paralogs of the crh1-type. All peptides conserved the structural characteristics of human CRH. Eel crh1b and both salmon crh1b genes were mainly expressed in the brain, supporting the major role of crh1b paralogs in controlling the corticotropic axis in teleosts. In contrast, crh1a paralogs were mainly expressed in peripheral tissues such as muscle and heart, in eel and salmon, reflecting a striking subfunctionalization between crh1a and b paralogs. Eel crh2 was weakly expressed in the brain and peripheral tissues. These results revisit the repertoire of crh in teleosts and highlight functional divergences that may have contributed to the differential conservation of various crh paralogs in teleosts.

Effects of 17α-Ethinylestradiol (EE2) exposure during early life development on the gonadotropic axis ontogenesis of the European sea bass, Dicentrarchus labrax.

Exposure of young organisms to oestrogenic endocrine disrupting chemicals (EDCs) can elicit adverse effects, particularly on the reproductive function. In fish, as in other vertebrates, reproduction is controlled by the neuroendocrine gonadotropic axis, whose components are mainly regulated by sex steroids and may then be targets for EDCs. In the present study, we investigated the effects of a xenoestrogen exposure on the ontogenesis of the gonadotropic axis in European sea bass. After exposure of hatching larvae for 8 days to 17α-ethinylestradiol (EE2) (0.5 nM and 50 nM), gene expression for kisspeptins (kiss1, kiss2), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), gonadotropin beta subunits (lhß and fshß) and brain type aromatase (cyp19a1b) were measured using quantitative real-time PCR. Our results demonstrate that EE2 strongly stimulated the expression of brain type aromatase (cyp19a1b) in sea bass larvae. In addition, EE2 exposure also affected the mRNA levels of kiss1, gnrh1 and gnrh3 by inducing a downregulation of these genes during the early developmental stages, while no effect was seen in gnrh2, lhß and fshß. These results reinforce the idea that the larval development is a sensitive critical period in regard to endocrine disruption and that the gonadotropic axis in the developing sea bass is sensitive to xenoestrogen exposure.

Development of an exposure protocol for toxicity test (FEET) for a marine species: the European sea bass (Dicentrarchus labrax).

Regulatory assessment of the effects of chemicals requires the availability of validated tests representing different environments and organisms. In this context, developing new tests is particularly needed for marine species from temperate environments. It is also important to evaluate effects that are generally poorly characterized and seldom included in regulatory tests. In this study, we designed an exposure protocol using European sea bass (Dicentrarchus labrax) larvae. We examined classical toxicological values (LCx) as well as behavioral responses. By comparing different hatching and breeding strategies, we defined the optimal conditions of exposure as non-agitated conditions in 24- or 48-well microplates. Our exposure protocol was then tested with 3,4-dichloroaniline (3,4-DCA), a recommended reference molecule. Based on our results, the 96 h LC50 for 3,4-DCA corresponded to 2.04 mg/L while the 168 h LC50 to 0.79 mg/L. Behavioral analyses showed no effect of 3,4-DCA at low concentration (0.25 mg/L). In conclusion, the present work established the basis for a new test which includes behavioral analysis and shows that the use of sea bass is suitable to early-life stage toxicity tests.

Fish life-history traits are affected after chronic dietary exposure to an environmentally realistic marine mixture of PCBs and PBDEs.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants that have been shown to affect fish life-history traits such as reproductive success, growth and survival. At the individual level, their toxicity and underlying mechanisms of action have been studied through experimental exposure. However, the number of experimental studies approaching marine environmental situations is scarce, i.e., in most cases, individuals are exposed to either single congeners, or single types of molecules, or high concentrations, so that results can hardly be transposed to natural populations. In the present study, we evaluated the effect of chronic dietary exposure to an environmentally realistic marine mixture of PCB and PBDE congeners on zebrafish life-history traits from larval to adult stage. Exposure was conducted through diet from the first meal and throughout the life cycle of the fish. The mixture was composed so as to approach environmentally relevant marine conditions in terms of both congener composition and concentrations. Life-history traits of exposed fish were compared to those of control individuals using several replicate populations in each treatment. We found evidence of slower body growth, but to a larger asymptotic length, and delayed spawning probability in exposed fish. In addition, offspring issued from early spawning events of exposed fish exhibited a lower larval survival under starvation condition. Given their strong dependency on life-history traits, marine fish population dynamics and associated fisheries productivity for commercial species could be affected by such individual-level effects of PCBs and PBDEs on somatic growth, spawning probability and larval survival.

Pituitary gonadotropins FSH and LH are oppositely regulated by the activin/follistatin system in a basal teleost, the eel.

European eels are blocked at a prepubertal silver stage due to a deficient production of pituitary gonadotropins. We investigated the potential role of activin/follistatin system in the control of eel gonadotropins. Through the development of qPCR assays for European eel activin ß(B) and follistatin, we first analyzed the tissue distribution of the expression of these two genes. Both activin ß(B) and follistatin are expressed in the brain, pituitary and gonads. In addition, a striking expression of both transcripts was also found in the retina and in adipose tissue. The effects of recombinant human activins and follistatin on eel gonadotropin gene expression were studied using primary cultures of eel pituitary cells. Activins A and B strongly stimulated FSHß subunit expression in a time- and dose-dependent manner. In contrast, activin reduced LHß expression, an inhibitory effect which was highlighted in the presence of testosterone, a known activator of eel LHß expression. No effect of activin was observed on other pituitary hormones. Follistatin antagonized both the stimulatory and inhibitory effects of activin on FSHß and LHß expression, respectively. Activin is the first major stimulator of FSH expression evidenced in the eel. These results in a basal teleost further support the ancient origin and strong conservation of the activin/follistatin system in the control of FSH in vertebrates. In contrast, the opposite regulation of FSH and LH may have emerged in the teleost lineage.

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda).

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the 'receptive areas' via the CNS to the chromatophore effectors.

Development of real-time RT-PCR assays for eel gonadotropins and their application to the comparison of in vivo and in vitro effects of sex steroids.

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are key factors in the brain-pituitary-gonad axis and understanding their regulation remains essential for future management of eel reproduction. In this regard, we developed quantitative real-time RT-PCR (qrtRT-PCR) assays for the expression of European eel LHbeta, FSHbeta and GPalpha subunits, using the Light Cycler system. The qrtRT-PCR was adapted to permit detection of the three gonadotropin subunit mRNAs in individual pituitaries and in dispersed pituitary cells. The validated assays were applied to investigate the effects of sex steroids (estrogens and androgens) on gonadotropin subunit expression, in vivo in steroid-injected eels, and in vitro by steroid treatments of primary cultures of eel pituitary cells. In vivo, a stimulation of LHbeta mRNA was observed after estradiol (E2) treatments, while testosterone (T) or the non-aromatizable androgen dihydrotestosterone (DHT) had no effect. Concerning FSHbeta expression, slight but non-significant decreases were observed after both E2 and androgen treatments. Different results were obtained in vitro: E2 induced an increase in FSHbeta mRNA levels but had no effect on LHbeta expression. In contrast, androgens (T and DHT) stimulated LHbeta expression while no significant variation was observed on FSHbeta mRNA levels following androgen treatment. Concerning the GPalpha mRNA, no significant effect of sexual steroids was observed in vivo or in vitro. This demonstrated specific direct actions of steroids on gonadotropin subunit expression. The differences observed between in vivo and in vitro experiments may be explained by the involvement of cerebral control, including GnRH and dopamine neurons, and their specific regulation by sex steroids. The data indicate that sex steroid feedbacks on gonadotropins are exerted via multiple pathways, indirectly at the brain level and directly on pituitary gonadotrope cells.

Thyroid hormone-induced demineralisation of the vertebral skeleton of the eel, Anguilla anguilla.

The role of thyroid hormones (TH) in bone remodelling is controversial. Indeed, in humans, while they are necessary for normal growth and development, their overproduction can induce important mineral bone loss and osteoporosis. Intense bone resorption is a natural phenomenon also observed in some teleosts, during reproductive migration and fasting. Our work aimed at investigating the effects of chronic treatments with TH (thyroxin, T4 or triiodothyronine, T3) on bone resorption in a migratory fish, the European eel (Anguilla anguilla), a representative species of an ancient group of teleosts (Elopomorphs). The incineration method showed that TH induced a significant mineral loss in eel vertebral skeleton. Histology and histophysical (qualitative and quantitative microradiographs) methods were then applied to vertebral sections to determine which types of resorption were induced by TH. Quantitative image analysis of microradiographs showed that TH significantly increased the porosity of the vertebrae, demonstrating the induction of a severe bone loss. Histology revealed the appearance of large osteoclastic lacunae, indicating a stimulation of osteoclastic resorption. Quantitative image analysis of ultrathin microradiographs showed a significant increase of the size of osteocytic lacunae, indicating a stimulation of periosteocytic osteolysis. Finally, quantitative microradiographs indicated a significant fall of mineralisation degree. TH treatments did not stimulate the production of the calcium-bonded lipo-phospho-protein vitellogenin, indicating that TH-induced bone demineralisation was not mediated by any indirect effect on vitellogenesis. Our study demonstrates that TH may participate in the mobilisation of bone mineral stores in the eel, by inducing different types of vertebral bone resorption, such as osteoclastic resorption and periosteocytic osteolysis. These data suggest that the stimulatory action of TH on bone resorption may be an ancient regulatory mechanism in vertebrates.

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development.

Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event.

Silvering (transition from yellow to silver eel) has been traditionally considered as a metamorphosis in view of the numerous morphological, physiological and behavioral changes preparing the eel for the oceanic migration. However, some changes, such as increases in gonad weight and steroidogenesis, suggest that silvering could also be considered as a pubertal event. In order to assess which endocrine axis may be involved in the induction of silvering, we compared the profiles of pituitary and peripheral hormones during the transition from yellow to silver female eels. A strong activation of the gonadotropic axis was shown during silvering. Follicle-stimulating hormone (FSH) mRNA levels increased during the early stages of silvering, followed by a later increase in luteinizing hormone (protein and mRNA) levels. In addition, plasma levels of sexual steroids (estradiol, E2; testosterone, T, and 11-ketotestosterone) and of vitellogenin significantly increased. In contrast, thyrotropin mRNA levels did not change and no or weak variations in plasma thyroid hormones were observed, indicating no or moderate change of the thyrotropic axis during silvering. Similarly, the somatotropic axis was not activated, as shown by pituitary growth hormone expression (protein and mRNA) and plasma levels. In addition, we studied the effects of chronic treatments of female yellow eels with thyroid hormone (thyroxine, T4) and sex steroids (T and E2) on biometrical parameters characteristics of silvering. T induced an increase in eye size and a reduction of digestive tract, whereas T4 and E2 had no effect. These hormonal profiles and experimental data lead to the conclusion that eel silvering should be considered as an onset of puberty rather than a 'genuine' metamorphosis.