Sylvie Baloche

Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol

In freshwater or seawater female silver eel, the release of gonadotropin (GTH) accumulated in the pituitary under estradiol (E2) influence could be stimulated by a conjugated treatment with a mammalian gonadoliberin agonist (GnRH-A = des-Gly10, (D-Ala6)-LH-RH ethylamide) and a blocker of dopamine receptor (pimozide). Furthermore, despite the GTH release, no reduction or even a significant increase in pituitary GTH levels were noted, indicating a stimulation of GTH synthesis. In consequence of the endogenous GTH release, a stimulation of ovarian development was induced, as demonstrated by the gonadosomatic index and histological study. Similar results were obtained with a combined treatment with GnRH-A and an inhibitor of catecholamine synthesis (L-alpha-methyl-3,4-dihydroxyphenylalanine). In contrast, no effect was produced by GnRH-A, pimozide, or L-alpha-methyl-DOPA, given alone. The results suggest that a double neuroendocrine mechanism (a lack of GnRH production and a dopaminergic inhibition of GnRH action) is involved in the prepubertal blockage of eel gonadotropic function before the reproductive migration.

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.

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Changes in the levels of plasma vitellogenin (Vg), estradiol (E2) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 µg kg−1 body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E2 and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml−1) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E2 (0.62 ± 0.13 ng ml−1 and T (0.33 ± 0.30 ng ml−1), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 µg ml−1 during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml−1 for E2 and 0.1 ng ml−1 for T); cGTH treatment did not significantly increase plasma E2 level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml−1 and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml−1 after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E2 to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10−6 to 10−5 M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E2 receptor.In vivo, androgens given alone at 50 µg kg−1 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E2-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E2.

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%)].

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.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshβ and lhβ expression, plasma 17β-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshβ, lhβ, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

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 FSHβ and LHβ 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 LHβ, 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.

Involvement of thyroid hormones in the control of larval metamorphosis in Sicyopterus lagocephalus (Teleostei: Gobioidei) at the time of river recruitment

After oceanic migration, post-larvae of the amphidromous Sicyopterus lagocephalus recruit to rivers in Reunion Island. As they enter the river mouth, post-larvae undergo many morphological, physiological and behavioural changes. These drastic changes, which allow them to change feeding regime and to colonise the juvenile and adult freshwater habitat, are defined as metamorphosis. The endocrine control of these changes has never been investigated in Gobioid fish. Here, we investigated whether thyroid hormones (TH) influence metamorphosis in recruiting S.lagocephalus. An analytical study was first performed on a cohort of 2400 fish caught at post-larval stage 1 and maintained for 37 days after capture in a flume tank (fluvarium), which replicates as closely as possible the natural conditions. Biometrical parameters (total and standard lengths, corner of mouth angle, body mass and condition factor) and whole-body thyroxine (T(4)) and triiodothyronine (T(3)) contents were measured on fish, sampled at regular intervals during these 37 days (192 fish). TH levels, measured by radioimmunoassays, were highest when morphological changes, such as the change in the position of the mouth, were most important. An experimental approach was then used to test the effect of the hormonal treatment (T(4) or thiourea, TU, a TH inhibitor) on biometrical parameters of 576 post-larvae. The change in the position of the mouth was significantly accelerated in the T(4)-treated post-larvae, while it was significantly delayed in the TU-treated post-larvae, compared to controls. Our study suggests that S.lagocephalus post-larva undergoes a true metamorphic event under the control of thyroid hormones at the time of its recruitment into the river.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17β-estradiol and growth hormone

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.