Vg mRNA induction in an endangered fish species (Anguilla anguilla) from the Loire estuary (France).

Estuarine zones are extremely fragile due to increasing stress from anthropogenic activities. Among those, the Loire estuary (France) is potentially exposed to various contaminants including Endocrine Disruptors Compounds (EDCs) able to impact the reproduction physiology of fish. The European eel (Anguilla anguilla), endangered fish species, is apparently not relevant, in its yellow stage, to monitor the effects of endocrine disruption. Despite this weakly responsiveness, this study aimed to investigate whether European eel from the Loire estuary may still be the subject of estrogenic disruption quantifying the hepatic Vg gene expression according to gender and sexual stage. Vitellogenin (Vg) appears as a valuable biomarker of EDCs, as well as for exposure and effects. Quantitative real-time Reverse Transcription Polymerase Chain Reaction (q RT PCR) was used in this study to amplify responses of hepatic Vg transcripts. European eels were sampled in May 2009 (N=57) and November 2010 (during the downstream migration, N=10) in two sites of the Loire estuary with different ecological conditions and contamination pressures (upstream: Varades; downstream: Nantes). Reproductive (gender, sexual maturity stage) and biometric parameters of collected eels were determined. A laboratory exposure of silver male to steroid hormones (Testosterone (T), 11-KetoTestosterone (11-KT), Estradiol (E2)) was conducted in parallel to validate the q RT PCR approach on hepatic Vg mRNA. Results demonstrated the responsiveness of exposed silver male eels, since hepatic mRNA Vg induction was observed in E2 treated males compared to control specimens. In the field, results of female silver eels reflected large inter-individual differences in the activation of hepatic Vg at silvering. However, while only female silver eels should express hepatic Vg mRNA, quantifiable levels were also detected in a proportion of 38% of the other individuals sampled, normally not inclined to express it, those being undifferentiated eels, yellow females, yellow and silver males. According to each sexual stage, no difference of expression was observed between eels from the two sampling sites. Histological results as well as low Vg mRNA levels detected do not permit a conclusion as to a potential effect of endocrine disruption.

Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHß and LHß transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHß, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.

Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis.

European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)].

Cortisol mobilizes mineral stores from vertebral skeleton in the European eel: an ancestral origin for glucocorticoid-induced osteoporosis?

Endogenous excess cortisol and glucocorticoid (GC) therapy are a major cause of secondary osteoporosis in humans. Intense bone resorption can also be observed in other vertebrates such as migratory teleost fish at the time of reproductive migration and during fasting when large amounts of calcium and phosphate are required. Using a primitive teleost, the European eel, as a model, we investigated whether cortisol could play an ancestral role in the induction of vertebral skeleton demineralization. Different histological and histomorphometric methods were performed on vertebral samples of control and cortisol-treated eels. We demonstrated that cortisol induced a significant bone demineralization of eel vertebrae, as shown by significant decreases of the mineral ratio measured by incineration, and the degree of mineralization measured by quantitative microradiography of vertebral sections. Histology and image analysis of ultrathin microradiographs showed the induction by cortisol of different mechanisms of bone resorption, including periosteocytic osteolysis and osteoclastic resorption. Specificity of cortisol action was investigated by comparison with the effects of sex steroids. Whereas, testosterone had no effect, estradiol induced vertebral skeleton demineralization, an effect related to the stimulated synthesis of vitellogenin (Vg), an oviparous specific phospho-calcio-lipoprotein. By contrast, the cortisol demineralization effect was not related to any stimulation of Vg. This study demonstrates GC-induced bone demineralization in an adult non-mammalian vertebrate, which undergoes natural bone resorption during its life cycle. Our data suggest that the stimulatory action of cortisol on bone loss may represent an ancestral and conserved endocrine regulation in vertebrates.

Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids.

Two full-length cDNA encoding putative dopamine D2-like receptors were cloned from the brain of female European eel. The deduced protein sequences, termed D2A- and D2B-R, exhibit closer phylogenetic relationships to vertebrate D2 receptors compared with D3 and D4 or D1 receptors. The two protein sequences share 100% identity within the transmembrane domains containing the highly conserved amino acids involved in dopamine binding. Accordingly, an apparent single population of sites on eel brain membranes bound [(3)H]spiperone, a D2-R-specific antagonist, with a K(d) of 0.2 +/- 0.04 nM. However, D2A- and D2B-R significantly differ within the amino terminus and the third intracellular loop. As analyzed by quantitative PCR and in situ hybridization, both receptor transcripts were found, with different relative abundance, in the majority of brain areas and in the pituitary, whereas in the retina, olfactory epithelium, spinal cord, and adipose tissue, only D2A-R gene was expressed. Because sex steroid hormones recently have been shown to regulate eel brain dopamine systems, we analyzed the effect of steroids on the amount of D2-R transcripts by quantitative PCR and in situ hybridization. In eels treated with testosterone, the gene expression of the D2B-R, but not D2A-R, was increased in a region-dependent manner. The effect of testosterone on D2B-R transcript levels was mimicked by dihydrotestosterone, a nonaromatizable androgen, whereas estradiol had no stimulatory action, evidencing an androgen receptor-dependent mechanism. Although functionality of the two receptors awaits determination of D2-R proteins, we hypothesize that differences in the tissue expression pattern and hormonal regulation of eel D2A- and D2B-R gene expression could represent selective forces that have contributed to the conservation of the duplicated D2-R.

How cadmium could compromise the completion of the European eel's reproductive migration.

The European eel (Anguilla anguilla L.) is severely threatened with extinction. Surprisingly, even though their unusual life cycle makes them particularly vulnerable to pollution, the possible contribution of contamination remains especially poorly known. Here we have investigated the possible effect of cadmium (Cd), a widespread nonessential metal, on eel reproductive capacities. Both control and Cd precontaminated female silver eels were experimentally matured and forced to swim in metal-free conditions to mimic their reproductive migration. Cd pre-exposure was found to strongly stimulate the pituitary-gonad-liver axis of maturing female silver eels leading to early and enhanced vitellogenesis. This was followed by a strong phenomenon of oocyte atresia and eel mortality. These phenomena occurred before oocytes could reach full maturation and were associated with a large entry of both vitellogenin and Cd into the ovaries. Indeed, a redistribution of previously stored cadmium, even from the low Cd levels of control eels, was observed during sexual maturation. Atresia and mortality phenomena were also associated with an overexpression of the pituitary gene encoding the growth hormone, a marker of physiological stress and energy reserves exhaustion. Significantly, these devastating effects of Cd were observed in organisms that presented liver and kidney Cd concentrations still below those observed in eels from Cd contaminated hydrosystems. Our research shows how common levels of cadmium contamination could disrupt endocrine pathways implicated in gonad maturation and subsequently impair reproductive capacity of eel future genitors.

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.

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.