Nadine Le Belle

Differential Regulation of the Two Forms of Gonadotropin-Releasing Hormone (mGnRH and cGnRH-II) by Sex Steroids in the European Female Silver Eel (Anguilla anguilla)

The effect of steroids on the two gonadotropin-releasing hormone (GnRH) forms present in the eel (mammalian GnRH, mGnRH and chicken GnRH-II, cGnRH-II), as well as on gonadotropin (GTH), was studied using specific radioimmunoassays. Female silver eels received chronic treatments with various steroids (estradiol, testosterone, androstenedione, 5 alpha-androstane-3 beta, 17 beta-diol). Estradiol or the combination of estradiol and androgens induced increases in brain and pituitary mGnRH levels and pituitary GTH level, whereas androgens given alone had no significant effect. In contrast, androgens or their combination with estradiol reduced brain cGnRH-II levels (this form remaining undetectable in the pituitary), estradiol given alone having no significant effect. This work demonstrates that the two forms of GnRH undergo a differential regulation by steroids, with a positive estrogen-dependent feedback on mGnRH (as well as on GTH) and a negative androgen-dependent feedback on cGnRH-II. These data are in agreement with previous results obtained in experimentally matured female eels (induced by a gonadotropic treatment which stimulates the production of both estrogens and androgens) showing increases in mGnRH and GTH levels, as well as a decrease in cGnRH-II [1]. The positive feedback of steroids on the mGnRH-GTH axis adds credence to the hypothesis according to which mGnRH would be the main form involved in the control of the gonadotropic function. This positive feedback would play an important role, amplifying pubertal stimulation of the gonadotropic axis, in this fish species.

Dopamine Inhibits Luteinizing Hormone Synthesis and Release in the Juvenile European Eel: A Neuroendocrine Lock for the Onset of Puberty

Abstract In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.

Pituitary Growth Hormone Secretion in the Turbot, a Phylogenetically Recent Teleost, Is Regulated by a Species-Specific Pattern of Neuropeptides

In mammals, growth hormone (GH) is under a dual hypothalamic control exerted by growth hormone-releasing hormone (GHRH) and somatostatin (SRIH). We investigated GH release in a pleuronectiform teleost, the turbot (Psetta maxima), using a serum-free primary culture of dispersed pituitary cells. Cells released GH for up to 12 days in culture, indicating that turbot somatotropes do not require releasing hormone for their regulation. SRIH dose-dependently inhibited GH release up to a maximal inhibitory effect of 95%. None of the potential stimulators tested induced any change in basal GH release. Also, neither forskolin, an activator of adenylate cyclase, nor phorbol ester (TPA), an activator of protein kinase C, were able to modify GH release, suggesting that spontaneous basal release already represents the maximal secretory capacity of turbot somatotropes. In contrast, forskolin and TPA were able to increase GH release in the presence of SRIH. In this condition (coincubation with SRIH), pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated GH release, whereas none of the other neuropeptides tested (GHRHs; sea bream or salmon or chicken II GnRHs; TRH; CRH) had any significant effect. These data indicate that inhibitory control by SRIH may be the basic control of GH production in teleosts and lower vertebrates, while PACAP may represent the ancestral growth hormone-releasing factor in teleosts, a role taken over in higher vertebrates by GHRH.

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 FSHβ subunit was cloned, sequenced and together with earlier isolated eel LHβ and glycoprotein hormone α (GPα) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHβ and LHβ are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHβ and FSHβ subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHβ and GPα increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHβ 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 LHβ and GPα mRNA levels, without any significant effect on FSHβ. In contrast, T treatment induced a decrease in FSHβ 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.

Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback

We have previously developed a novel in vitro assay that utilises cultures of primed female stickleback kidney cells for the screening of potential androgenic and anti-androgenic environmental contaminants. Stickleback kidney cells are natural targets for steroid hormones and are able to produce a protein, spiggin, in response to androgenic stimulation. We undertook a combined in vivo/in vitro study where we used the magnitude of spiggin production as an endpoint to test the anti-androgenic properties of the pharmaceutical androgen antagonist flutamide and three environmental contaminants: the organophosphate insecticide fenitrothion, the urea-based herbicide linuron and the fungicide vinclozolin. In vitro, kidney cells were exposed to a range of concentrations [from 10(-14) M (2.5 pg/L) up to 10(-6) M (280 microg/L)] of the test compounds alone for determining agonist activities, or together with 10(-8) M (3 microg/L) dihydrotestosterone (DHT) for determining antagonist activities. An in vivo flow-through aquarium-based study was carried out in parallel. Female sticklebacks were exposed to a range of concentrations of the same chemicals alone or in combination with DHT (5 microg/L) for 21 days. All of the compounds significantly inhibited DHT-induced spiggin production in a concentration-dependent manner in both the in vitro (FN > or = FL > or = LN > VZ) and in vivo (FN > FL > or = VZ > LN) assays. Fenitrothion and flutamide inhibited spiggin production in vitro at a concentration as low as 10(-12) M (P < 0.05), while linuron and vinclozolin inhibited DHT-induced spiggin production at concentrations of 10(-10) M (P < 0.05) and 10(-6) M (P < 0.001) respectively. Similarly, fenitrothion and flutamide were the most potent chemicals in vivo and significantly reduced DHT-induced spiggin production at a concentration of 10 microg/L and 25 microg/L respectively (P < 0.01). Both linuron and vinclozolin induced a significant decrease in DHT-induced spiggin production at a concentration of 100 microg/L when tested in vivo. In addition, kidney cell primary culture was used to test the (anti-)androgenic effects of the major environmental contaminants: oestradiol (E2), nonylphenol (NP) and bisphenol A (BPA) for the first time in teleosts. We observed that these compounds were able to significantly inhibit spiggin production at high doses (E2: 270 microg/L; NP: 2.2 microg/L; BPA: 2.3 microg/L). When tested in the absence of DHT, none of the compounds showed a significant agonistic activity in either in vivo or in vitro assays. Overall, our data further demonstrate that kidney cell primary culture is a reliable and a sensitive screening tool for the detection of (anti-)androgenic compounds. In addition, our study represents the first attempt to develop a combined in vivo/in vitro screening strategy for assessing the effects of (anti-)androgenic endocrine disrupters.

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.

Long-Term Inhibitory Effects of Somatostatin and Insulin-Like Growth Factor 1 on Growth Hormone Release by Serum-Free Primary Culture of Pituitary Cells from European Eel (Anguilla anguilla)

To investigate the ability of hypothalamic and peripheral factors to directly regulate growth hormone (GH) release in a primitive teleost, the European eel (Anguilla anguilla L.), we used primary cultures of dispersed pituitary cells. When cultured for 12 days in a serum-free medium, pituitary cells continuously released large amounts of GH, which exceeded the initial cellular content. Somatotropin-release inhibiting hormone (SRIH-14) dose-dependently inhibited GH release (EC50 0.75 nM) up to a maximal inhibitory effect of 95%. No desensitization of somatotropes to SRIH was observed over the 12 days of culture. Use of receptor subtype-selective SRIH agonists suggests the existence on eel somatotropes of SRIH receptor(s) related to the mammalian sst2/sst3/sst5 class rather than to the sst1/sst4 class. Insulin-like growth factor 1 (IGF1) dose-dependently inhibited GH release (EC50 0.03 nM) up to a maximal inhibitory effect of 85%, without desensitization. IGF1 and IGF2 were equipotent in inhibiting GH release, whereas insulin was 1,000 times less active, suggesting the implication of a receptor related to the mammalian IGF type 1 receptor. These results indicate that eel somatotropes are active in vitro without any specific additional factors, and suggest the existence of a dominant inhibitory control of GH release in vivo. Two potential candidates for this chronic negative regulation are a neurohormone, SRIH and a circulating factor, IGF1. These data underline the early evolutionary origin of the molecular and functional SRIH-GH-IGF1 neuroendocrine axis in vertebrates.

Cortisol Selectively Stimulates Pituitary Gonadotropinβ -Subunit in a Primitive Teleost, Anguilla anguilla1

It has been suggested that in mammals, glucocorticoids, beside their stress-related inhibitory effects on reproductive function, may also play a stimulatory role at the onset of puberty. Using the juvenile female eel as a model, we investigated the potential stimulatory role of cortisol (F) on pituitary gonadotropin (GtH-II). GtH-II levels were measured by RIA, and messenger RNA (mRNA) levels for α- and GtH-IIβ -subunits were determined by dot blot using homologous probes. F treatment increased eel pituitary GtH-II content in vivo and in vitro. Using a long term, serum-free primary culture of pituitary cells, we studied the direct effect of F on GtH-II production. F increased the GtH-II cellular content in vitro in a dose- and time-dependent manner. The relative potencies of various corticosteroids on GtH-II were: triamcinolone acetonide > dexamethasone > F ≫ cortisone and aldosterone, indicating a glucocorticoid-specific receptor (GR). F stimulated GtH-II production through a selective increase in mRNA l...