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Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1&ndash, 2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary L&beta, T2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, &beta, catenin activation and mouse luteinizing-hormone &beta, encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and L&beta, T2 cells. Cetrorelix inhibited the 3 &times, EC50 GnRH-activated calcium signaling at concentrations of 1 nM&ndash, 1 &micro, M, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM&ndash, M range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in L&beta, T2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 &micro, M GnRH-induced &beta, catenin activation, Lhb gene expression increase occurring upon L&beta, T2 cell treatment by 1 &micro, M GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

International audience; Visfatin and resistin appear to interfere with reproduction in the gonads, but their potential action at the hypothalamic-pituitary level is not yet known. The aim of the present study was to investigate the mRNA and protein expression of these adipokines in murine gonadotroph cells and to analyse the effects of different concentrations of recombinant mouse visfatin and resistin (0.01, 0.1, 1 and 10 ng mL(-1)) on LH secretion and signalling pathways in L beta T2 cells and/or in primary female mouse pituitary cells. Both visfatin and resistin mRNA and protein were found in vivo in gonadotroph cells. In contrast with resistin, the primary tissue source of visfatin in the mouse was the skeletal muscle, and not adipose tissue. Visfatin and resistin both decreased LH secretion from L beta T2 cells after 24 h exposure of cells (P < 0.03). These results were confirmed for resistin in primary cell culture (P < 0.05). Both visfatin (1 ng mL(-1)) and resistin (1 ng mL(-1)) increased AMP-activated protein kinase a phosphorylation in L beta T2 cells after 5 or 10 min treatment, up to 60 min (P < 0.04). Extracellular signal-regulated kinase 1/2 phosphorylation was transiently increased only after 5 min resistin (1 ng mL(-1)) treatment (P < 0.01). In conclusion, visfatin and resistin are expressed in gonadotroph cells and they may affect mouse female fertility by regulating LH secretion at the level of the pituitary.

Transgenic female mice overexpressing the hCGβ subunit (hCGβ+) and producing elevated levels of luteinizing hormone (LH)/hCG bioactivity present as young adults with enhanced ovarian steroidogenesis, precocious puberty, and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels, and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor-deficient (PRLR−/−) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGβ+ and PRLR−/− mice. The double-mutant hCGβ+/PRLR−/− females remained sterile with an ovarian phenotype similar to PRLR−/− mice, indicating that LH action, Lhcgr expression, and consequent luteinization are not possible without simultaneous PRL signaling. The high frequency of pituitary prolactinomas in PRLR−/− mice was not affected by transgenic hCGβ expression. In contrast, none of the hCGβ+/PRLR−/− females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGβ+ mice, was absent in double-mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signaling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.

International audience; Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258 micro g/L of clotrimazole for 7 days. Expression of pituitary gonadotropins ß subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesisrelated genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly upregulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and in inducing the expression of Fsh-regulated igf3 in Sertoli cells.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Rabconnectin-3α and the control of puberty Human genetics shows that low levels of rabconnectin-3α cause a loss of the neurons that produce gonadotropin-releasing hormone, revealing a new mechanism for incomplete puberty and infertility., Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes., Author Summary Investigation of neurodevelopmental disorders with abnormal puberty can reveal neuronal processes that control the initiation of puberty and subsequent reproductive function. We describe here a new syndrome observed in three brothers with incomplete puberty, central hypothyroidism, peripheral polyneuropathy, mental retardation, and abnormal glucose regulation. Molecular genetic investigation in this consanguineous family revealed an in-frame deletion of 15 nucleotides in the DMXL2 gene, which resulted in lower levels of its expression. We found that rabconnectin-3α, the synaptic protein encoded by DMXL2, is widely expressed in the brain and in the ends of the axons of neurons that produce gonadotropin-releasing hormone (GnRH). We then observed that neuron-specific deletion of one allele of Dmxl2 in mice reproduces the incomplete puberty seen in the human patients and results in decreased numbers of GnRH neurons in the hypothalamus. We also showed that rabconnectin-3α controls glucose-induced insulin secretion. These findings reveal a new mechanism of gonadotropin deficiency and identify rabconnectin-3α as a key controller of neuronal and endocrine homeostasis.

International audience; The involvement of γ-aminobutyric acid (GABA) in the control of prolactin (PRL) release was investigated in rainbow trout using both perifused pituitary fragments and pituitary cells in primary culture. In our perifusion system, infusion of GABA (10−6 to 10−4 M) caused an inhibition of PRL release (between 20 and 40%). Administration on perifused pituitary fragments of 3APS, a GABAa agonist, mimicked this inhibitory effect. Moreover, bicuculline, a specific antagonist of GABAa receptors, totally abolished GABA effect. When tested on cultured pituitary cells during 40h exposure, GABA (10−5 M) caused a significant decrease in PRL release (24.5%). Baclofen, a specific agonist for GABAb receptor tested at 10−6 and 10−5 M, also inhibited PRL released from cultured pituitary cells. These results demonstrate that GABA inhibits PRL release by acting directly on pituitary cells and that probably both types of GABA receptor (a and b) are involved in this regulation.

International audience; For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.

Insulin-like growth factor (IGF)-I, which is crucially involved in fish growth, differentiation, and reproduction, occurs in tilapia pituitary. IGF-I peptide, which is probably produced in hypothalamic perikarya, is present in axons of the neurohypophysis, and IGF-I mRNA and peptide are present in the adenohypophysis in adrenocorticotrophic hormone cells, melanocyte-stimulating hormone cells, with interindividual differences in growth hormone cells, and, starting with puberty, in gonadotrophic hormone (GTH) cells. Subordinate males showed a high IGF-I but a lower beta-luteinizing hormone expression, while in dominant males the opposite was found. IGF-I from the GTH cells may act as auto/paracrine regulators of GTH cell proliferation and enhance GTH synthesis and release during puberty and reproduction, depending on the social status.

Background: Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to “real-life,” environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal.\ud Objectives: We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein–coupled receptor 54) system.\ud Methods: KiSS-1, GPR54, and ERα (estrogen receptor α) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHβ (luteinizing hormone β) and ERα in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry.\ud Results: Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHβ and ERα in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary.\ud Conclusions: This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs. Epidermal growth factor (EGF) has been shown to inhibit division of pituitary somato-lactotrope cells occurring in parallel with EGF-mediated differentiation of these precursors into lactotrope cells. We show here that in somato-lactotrope pituitary cell line GH4C1, EGF triggers a PCD characterized by an apoptosis-like DNA fragmentation, insensitivity to broad-range caspase inhibitors, and absence of either cytochrome c or apoptosis-inducing factor release from mitochondria. Dying cells display loose chromatin clustering and numerous cytoplasmic vacuoles, a fraction of which are autophagic, thus conferring a heterogeneous phenotype to this PCD. Moreover, overexpression of cell death inhibitor Bcl-2 prevented not only the EGF-induced PCD but also its prodifferentiation effects, thus pointing to a mechanistic relationship existing between these two phenomena. Overall, the characterized differentiation-linked cell death represents an original form of caspase-independent PCD. The mechanisms underlying this PCD involve combinatorial engagement of discrete death effectors leading to a heterogeneous death phenotype that might be evolutionary related to PCD seen during the differentiation of some unicellular organisms.

Fatty acid composition of anterior pituitary cell membranes of rats deprived of essential fatty acids (EFA) and of rats receiving a standard diet was determined during postnatal development and in adults. Pregnant rats were fed an EFA-deficient diet and the offspring were fed the same diet after weaning. In parallel, effects of the diet on growth and on growth hormone (GH) responsiveness to GHRH stimulation were determined in control animals. Membrane content of arachidonic acid (20:4n-6) and of its elongation product adrenic acid (22:4n-6) increased regularly from day 2 to day 12 after birth. EFA-deficiency resulted on day 2 in increased oleic acid and in substitution of arachidonic and adrenic acids by corresponding elongation-desaturation products of oleic acid: eicosatrienoic (20:3n-9) and docosatrienoic (22:3n-9) acids. At the age of 24 days, n-9 series fatty acid reached the same level as in adult animals. Two-day-old EFA-deficient rats paradoxically exhibited a higher level of 20:4n-6 as compared to control rats. EFA-deficiency also decreased growth rate and GH pituitary responses to GHRH during the prepubertal period. These results suggest that changes in the lipid structure and in pituitary secretion properties elicited by EFA-deficiency depend upon the stage of development.

International audience; Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of p-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from S-35-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.

To study the control of prolactin secretion in fish, an in-vitro technique using a monolayer cell culture system of rainbow trout pituitary glands was developed. Such secretion was characterized by measurement of both prolactin release and prolactin mRNA content using a trout prolactin cDNA as a probe. This cell culture technique, already used to study the regulation of gonadotrophin secretion in rainbow trout, was further validated by measuring total DNA and protein content. Both parameters appeared to be stable after 2 days of culture. Studying the effect of somatostatin (SRIF) on prolactin cells indicated that a maximal inhibitory effect (62%) was observed after 24 h of treatment. Significant inhibition of prolactin release was obtained for SRIF doses ranging from 50 nm to 1 μm. However, in the same experiment, SRIF was much more potent as an inhibitor of growth hormone release. Short-term (

Using a specific antiserum raised against synthetic neuropeptide-Y (NPY), the distribution of NPY-like immunoreactivity in the brain and pituitary of the trout Oncorhynchus mykiss has been examined with the indirect immunofluorescence and peroxidase-antiperoxidase methods. The highest density of NPY-immunoreactive elements was found in the basal telencephalon and hypothalamus. In particular, NPY-immunoreactive neurons were located in the nucleus entopeduncularis and the preoptic nucleus. NPY-immunoreactive fibers were observed throughout the trout brain. The preoptic nucleus, the suprachiasmatic nucleus, and the nucleus entopeduncularis were densely innervated. In addition, NPY-positive fibers were detected in the nucleus lateralis tuberis and in the distal and intermediate lobes of the pituitary. The NPY-like peptide of the trout brain was characterized by combining HPLC analysis and radioimmunological detection. Serial dilutions of trout hypothalamus and pituitary extracts produced displacement curves that were parallel to the standard curve. HPLC analysis resolved a major peak which was slightly less hydrophobic than porcine NPY. The possible effect of NPY, either alone or in combination with a GnRH antagonist, on gonadotropin (GtH) release from trout pituitaries was investigated using a perifusion system technique. Graded concentrations of synthetic NPY induced a dose-dependent stimulation of GtH release. The stimulatory activities of NPY and various short chain analogs on GtH release were compared: the order of potency was NPY greater than NPY-(2-36) greater than NPY-(16-36) greater than NPY-(25-36). This result suggests that the biological determinant of NPY is located in the C-terminal part of the molecule. Administration of a short pulse of NPY or GnRH (10(-7) M each) induced a marked stimulation of GtH release. Prolonged infusion of the GnRH antagonist D-Phe2-6,Pro3-GnRH induced a significant reduction of GnRH-evoked GtH secretion. In addition, the GnRH antagonist blocked NPY-induced GtH release. The widespread distribution of NPY in the trout brain suggests the involvement of this neuropeptide in a variety of physiological functions. The present data support the view that NPY, released by nerve terminals in the distal lobe of the pituitary, may act presynaptically on GnRH fibers to modulate GtH release.

International audience; In rainbow trout, a variable in vivo pituitary sensitivity to GnRH has been previously observed, depending on the stage of oogenesis. The purpose of the present work was to study, in vitro, the role of oestradiol (E2) and 17α-hydroxy,20β-dihydroprogesterone (17α20βP), respectively, involved in vitellogenesis and in oocyte maturation, upon this variability. The study was performed using primary cultures of whole pituitary cells from animals at different stages of oogenesis and subjected to increasing doses of salmon GnRH (sGnRH) after a 3-day pretreatment with control medium or medium supplemented with the steroid at levels corresponding to those circulating at the time of particular events of the sexual cycle (maturation and vitellogenesis). In control cultures, pituitary GtH responsiveness to sGnRH was maximal at ovulation, since at this time the gonadotrophs were able to respond to 10−9 M sGnRH, whereas during vitellogenesis and preovulatory stages, the minimal effective dose of sGnRH ranged between 10−6 and −8 M. We have demonstrated that 17α20βP has a positive or negative effect by acting directly on pituitary cell responsiveness to sGnRH, depending on the stages at which it is applied; its effect is positive during early vitellogenic and preovulatory stages whereas it is negative at the time of ovulation. E2 also increased pituitary responsiveness to sGnRH when applied during early vitellogenesis at low doses, corresponding to circulating levels at the time of ovulation; higher levels of E2, corresponding to circulating levels found during the last stages of vitellogenesis, did not modify pituitary responsiveness but increased cell GtH content. Results obtained are discussed in relation to hormonal changes (gonadotropin and steroid hormones) observed by different authors during the sexual cycle.

International audience; Juvenile rainbow trout received gonadotropic hormone (GtH) treatment to investigate the response of the immature gonad and its effect on the onset of rising pituitary GtH levels, an early sign of initiation of sexual maturation in the teleost fishes. Brief exposure to GtH evoked increases in plasma androgens and significantly increased pituitary GtH in males; the accumulation of pituitary GtH was not observed in female trout which showed significant but smaller increases in plasma androgen resulting from GtH administration. There was no evidence of gonadal negative feedback in intact immature fish since pituitary GtH and plasma androgens remained at basal levels in castrate juvenile trout. Since the castrate male trout failed to respond to GtH injections, the data suggest that the immature gonad produces a steroid capable of triggering pituitary GtH production by hypothalmo-pituitary feedback in males.

International audience; Changes in the maturational gonadotropin (c‐GtH) levels during 24 h in carp males, as a result of the presence or absence of other carp specimens, were investigated. Carp males were cannulated to ventral aortae. Gonadotropin levels were radioimmunologically determined. It has been found that in males two peaks of c‐GtH occur strictly in 2 periods during the day in which spawning takes place under natural conditions (early morning and late evening). Being in contact or not with another male or hypophysed female did not affect circadian changes in c‐GtH levels in investigated males.

International audience; A testicular feed back on the hypothalamo-pituitary system was studied in male rainbow trout (Salmo gairdneri) after bilateral castration and steroid or testicular supplementation. Experiments were carried out at various periods of the spermatogenetic cycle ; in March-April (resting stage), June (initiation of spermatogenesis), October (end of spermatogenesis and before spermiation), and December (running stage). In March and April, plasma t-GTH is low in intact males (< 2 ng/mi) and castration induced a threefold rise of GTH. Testosterone (T) given by a silastic capsule inserted in the body cavity of the castrate did not change plasma t-GTH in comparison with the intact control. When T is given in the same manner in intact males GTH is significantly increased. In June, a significant t-GTH rise was observed after castration (P < 0.05), this rise was prevented by injection of T and estradiol ()2.E When 2E and T were given to intact males plasma t-GTH was significantly decreased. In October plasma t-GTH was high (13 ng/ml) and was not modified by castration ; injected T or E2 had no effects on plasma t-GTH either in intacts or castrates. In December, a sevenfold increase of t-GTH is observed after castration. This was partly suppressed by intraperitoneal injection of T and E2 and declined after stereotaxic implantation of 11-ketotestosterone into the pituitary. It was not significantly suppressed by intraperitoneal injection of testicular extracts. It is suggested that at this period steroids are the main gonadal factors involved in the feed back at the hypothalamo-pituitary system level.

International audience; Une technique d'hypophysectomie par voie operculaire particulièrement simple et rapide, est décrite chez le Guppy Poecalia reticulata. La pénicilline administrée pendant la cicatrisation diminue la mortalité postoperatoire.A quick easy surgical technique of hypophysectomy using opercular approach is described in the Guppy Poecilia reticulata. The addition of penicillin to the water during the healing phase reduces poset-operative lethality.

The lactogenic properties of extracts of the pituitary glands of salmon and trout were evaluated by using the organ culture technique with rabbit mammary explants. Crude extracts and fractions obtained after chromatography on Ultrogel and selected for their capacity to compete with ovine prolactin in a rabbit mammary gland radioreceptor assay were added to the culture medium. The criteria of lactogenesis were lactose synthetase activity, casein synthesis, measurements of the concentration of casein messenger RNA and the histology of mammary glands. All these tests led to the conclusion that salmon and trout pituitary glands contain a prolactin-like principle capable of initiating milk synthesis in the rabbit mammary cell.

International audience; To study the regulation of gonadotropin secretion in rainbow trout in vitro, a method for preparing primary cultures of dispersed pituitary cells is described. Cells were dispersed by collagenase 0.1% in Hank's saline solution for 20 hr at 12 degrees and a high yield of viable cells was obtained. Attempts to improve cell functioning were made by varying culture conditions (density of cells initially plated, age of the culture). Cell functioning was assessed by their ability to respond to increasing doses of mammalian and salmon GnRH. Pituitaries were collected from spermiating males whose pituitaries are known to be sensitive to mammalian GnRH in vivo. Using 96-well plates, optimal conditions for good biological activity, are initial plating with 6.2 X 10(4) cells, incubation with GnRH for 24 hr on the third day after plating. In these conditions mammalian analog and salmon GnRH induced an increase in GtH release for doses ranging from 10(-9) to 10(-6) M. The GtH released during the GnRH incubation period does not decrease the sensitivity of the system since addition of 20 ng of GtH at the beginning of incubation does not modify the response profile.

International audience; The efficiency of different hormonal treatments to induce ovulation of carp at low temperatures (13–15°C) was tested. “Priming” with a low dose (0.6 mg/kg) of carp pituitary extract was found necessary for a subsequent successful treatment with 17α-hydroxy-20β-dihydroprogesterone (17α-20β P) (2 mg/kg) 1 day later. The eggs produced by this method showed satisfactory fertilization (75–96%) and hatching success (70%).On the other hand, normal hypophysation (5.4 mg/kg) following priming resulted only in partial ovulation and oocyte resorption at this temperature. Priming was shown to increase the gonadotropin level in plasma from about 2 to 35 ng/ml and to induce germinal vesicle migration toward the periphery of oocytes. Neither 17α-20β P nor desoxycorticosterone alone or in association gave any positive result in the absence of a preliminary priming.

Le cycle reproducteur des poissons est sous la dependance de l'hypophyse qui elabore et secrete les hormones gonadotropes. Ces hormones interviennent lors du deroulement de la gametogenese et sont produites activement pendant la maturation ovocytaire puis l'ovulation. La technique d'induction de ponte dite technique d'hypophysation consiste en un apport exogene d'hormone gonadotrope par injection d'hypophyses pour provoquer le deroulement de phases du cycle sexuel qui ne se produisent pas spontanement en condition de captivite comme l'ovulation.

International audience; The effect of implanting an extra pituitary containing large amounts of gonadotropic hormone (GtH), combined or not with a luteinizing hormone releasing-hormone analogue (LHRHa) treatment, on GtH levels and gonadal development was investigated in juvenile host fish. The extrapituitaries were collected from mature spermiating fish or from immature fish treated with testosterone. In recipient males and females circulating plasma GtH levels increased following transplantation of both types of pituitaries. Elevated GtH levels presumably triggered steroid synthesis by the immature male gonad since pituitary GtH content was observed to accumulate in recipient males and not in females. However, the potency of the two kinds of pituitaries seemed different since spermatogenesis was stimulated only in some recipient males bearing a mature adult pituitary. This divergence could be due to a different sensitivity to endogenous gonadotropin-releasing hormone (GnRH). Only mature extrapituitaries might be highly sensitive to GnRH, as suggested by results obtained in juvenile host fish after LHRHa treatment. At the end of the 6-week experimental period, this LHRHa treatment stimulated spermatogenesis and induced a significant increase in pituitary GtH content only in juvenile hosts transplanted with a mature pituitary. Such a result was not observed in juvenile hosts submitted to a LHRHa treatment combined or not with the transplantation of juvenile testosterone-treated pituitary. However, previous works have shown that pituitaries collected from immature testosterone-treated fish are sensitive to GnRH. In the present experiment, the amount of GnRH-induced GtH release might have been too low to initiate spermatogenesis during the 6-week experimental period.

International audience; In fish there are few data on the gonadotropin-releasing hormone (Gn-RH) neurosecretory activity, which could explain long- and short-term variations of the gonadotropin secretion. There is no biological species specificity between mammal and fish Gn-RH; although there is a structural difference, they are, on the contrary, characterized by a high immunological specificity which does not allow measurement of fish Gn-RH using radioimmunoassay for LH-RH. We have synthesized salmon Gn-RH according to the formula recently proposed by Sherwood (N. Sherwood, L. Eiden, M. Brownstein, J. Spies, J. Rivier, and W. Vale, 1983. Proc. Natl. Acad. Sci. USA 80, 2794-2798). Its activity has been tested by its ability to stimulate the gonadotropin hormone (GtH) secretion in vivo in testosterone-implanted juvenile rainbow trout, and for the recognition of synthesized Gn-RH (s-Gn-RH) perykaria by a specific antibody raised against the s-Gn-RH in regions of the brain described as containing LH-RH immunoreactive-like material. A radioimmunoassay has been developed for the salmon Gn-RH, and its specificity to measure trout Gn-RH has been tested. Using this assay, the brain and pituitary Gn-RH contents have been measured throughout the final phases of maturation and ovulation. Brain Gn-RH increases from the end of vitellogenesis (8.9 +/- 0.76 ng/brain) to ovulation (more than 15 ng/brain). Pituitary Gn-RH is lower (1.58 +/- 0.69 ng/pituitary) at the end of vitellogenesis and follows a similar profile as in the brain, except for a significant decrease just prior the beginning of oocyte maturation. The correlations between Gn-RH levels and GtH pituitary and plasma levels show that total brain Gn-RH is never correlated to the GtH, suggesting that the increase in the brain Gn-RH content is related to a Gn-RH system closely related to maturation and ovulation, which remains to be investigated. On the contrary, pituitary Gn-RH levels are well correlated with pituitary and plasma GtH levels, indicating that pituitary Gn-RH levels might represent a good index of the Gn-RH neurosecretory activity in the fish hypothalamohypophysial complex, given the absence of a portal system in teleost.

International audience; Le méthallibure administré en bains à la dose de 2 p.p.m. entraîne chez le poisson rouge (Carassius auratus) une inhibition de la spermatogenèse qui intéresse toutes les cellules germinales à l'exception des spermatogonies A. Lorsque les mâles soumis à ce traitement reçoivent en même temps des injections de poudre hypophysaire brute de Carpe, la spermatogenèse est maintenue et même fortement stimulée. Après la suppression du traitement au méthallibure, la spermatogenèse se rétablit complètement en 20 jours, mais la restauration est plus importante si les animaux reçoivent des injections de poudre hypophysaire brute de Carpe.

SUMMARY The level of gonadotrophin measured by radioimmunoassay in fully mature male goldfish was about 15 ng carp gonadotrophic hormone (c-HG)/ml plasma and 30 μg c-HG/g pituitary (wet weight). After treatment for 35 days with methallibure (added to the water at the rate of 2 p.p.m./day), the level of gonadotrophin in the blood was very low and remained at the pretreatment level in the pituitary. This suggested that releasing factor might control either synthesis or discharge of gonadotrophin from the pituitary. These results have also shown that type B spermatogonia and meiosis were gonadotrophin dependent but that the development of spermatocytes into spermatids did not require high concentrations of gonadotrophin. Spermiation was suppressed when the concentration of c-HG dropped below 10 ng/ml plasma.

International audience; Il existe une confusion importante dans la notion de spécificité d’action des hormones gonadotropes de Poissons. Cette notion a été précisée en utilisant deux méthodes différentes : d’une part la comparaison des activités biologiques de différents extraits hypophysaires de Poissonssur un même récepteur testiculaire, d’autre part la détermination de leur comportement immunologique vis-à-vis d’anticorps hautement spécifiques dirigés contre l’hormone gonadotrope de Carpe c-HG et o-LH. La spermatogenèse du Carassin, Carassius auratus et du Guppy, Poecilia reticutata hypophysectomisés ne peut être regénérée quantitativement qu’avec des hormones gonadotropes ou extraits hypophysaires autologues d’une espèce donneuse très proche de l’espèce receveuse. La technique de compétition isotopique vis-à-vis d’anticorps spécifiques d’une entité moléculaire définie, donne des résultats comparables. Seuls des extraits hypophysaires de Carpe et d’autres Cyprinidés peuvent déplacer de l’hormone de Carpe marquée à l’iode 131 I (c-HGI 131) du complexe c-HGI131, anti c-HG. Les hormones mammaliennes o-LH, o-FSH et HCG n’ont en aucun cas montré d’activité sur la spermatogenèse du Carassin et du Guppy ni d’affinité vis-à-vis d’anticorps dirigés spécifiquement contre une hormone gonadotrope de Poisson. Ces résultats confirment l’existence d’une spécificité zoologique d’action des hormones gonadotropes hypophysaires entre les Mammifères et les poissons teléostéens et à l'intérieur même des différentes familles de téléostéens

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.