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Summary: The modulation of cellular processes of apoptosis is essential for the normal physiology of cells and of fundamental importance for carcinogenesis. The disturbance of cellular control mechanisms of apoptosis is considered as the critical event in the initiation and the progression of tumours. Reduced apoptosis is further recognised as an important mechanism for the resistance of tumour cells to radio- and chemotherapy. The ductal adenocarcinoma of the pancreas belongs to those tumours which are nearly always resistant to a conventional chemotherapy. Based on actual results of the pathogenesis of pancreatic cancer and the previous findings referring the RANKL/OPG system, it was the aim of this thesis to examine the expression and modulation of OPG mRNA production and OPG protein secretion in human pancreatic cancer cell lines. The following adenocarcinoma cell lines PaTu 8988t, PaTu 8988s, PC-2 und Capan-2 were analysed and the effect of steroid hormones, cytokines and different drugs on the OPG expression was evaluated. The gene expression was determined using northern blot analysis and PCR, the protein secretion by ELISA. Osteoprotegerin (OPG) und Receptor activator of nuclear factor-κB ligand (RANKL) represent specific receptors and ligands of the TNF superfamily, the production of which could be proven up to now in different human tissues. Following the proof of OPG mRNA in pancreatic tissue with the help of a commercial northern blot, OPG could be detected in two of the four examined pancreatic carcinoma cell lines (PaTu 8988t und PC-2). The cell lines PaTu 8988s and Capan-2 on the other hand showed no production of OPG. In addition PaTu 8988t cells expressed RANKL mRNA. In the case of testosterone there was an inhibition dependent on dose and duration of OPG mRNA und protein expression in the pancreatic cancer cell line PaTu 8988t by 70-77%, whereas 5α-DHT and 17β-estradiol showed no effects. The adrenal androgen DHEA impeded the OPG production by 83%. In the case of PC-2 cells on the other hand the OPG expression was lowered by the gonadal androgen 5α-DHT. After the exposition of the cell line PaTu 8988t to the glucocorticoid dexamethasone there was a dose dependent inhibition of OPG mRNA levels and OPG protein production by 23%. Because of the important role of cytokines in the field of tumour immunology and the modulation of apoptosis in pancreatic cancer different cytokines (IL-1β, TNF-α and IL-8) were examined referring their regulation of OPG production. In contrast to many other cell types, these cytokines however showed no influence on OPG expression – except IL-8 –. The treatment with IL-8 resulted in a slight increase in the production of OPG mRNA production in PaTu 8988t cells. The HMG-CoA reductase inhibitors lovastatin and cerivastatin showed an inhibiting effect on the OPG mRNA and OPG protein expression in human pancreatic adenocarcinoma cell lines. Lovastatin as a statin of the first generation reduced the OPG mRNA and protein secretion in PaTu 8988t cells by 90%. In addition OPG mRNA and protein levels were reduced by Cerivastatin in PaTu 8988t cells by 27% and in PC-2 cells by 39%. The reduction through statins war dependent on dose and duration. The inhibition of the OPG production in pancreatic carcinoma cell lines was a further proof of the pleiotropic effects of statins, which should be clinically examined concerning their apoptosis modulation effects on pancreatic carcinoma cells. In conclusion these results in connective view with the results of other groups indicate that the pancreas represents both a site of production and a potential target of OPG, an important osteotropic and apoptosis-modulating cytokine of the TNF receptor superfamily. Future studies should not only investigate the precise interaction of OPG and TRAIL, but also their importance for the creation, progression and metastasis of the pancreatic carcinoma. These findings could be the basis of future clinical studies in which the pinpointed modulation of apoptosis could be implemented as therapeutical principle with patients with pancreatic cancer., Zusammenfassung: Die Modulation zellulärer Apoptoseprozesse ist nicht nur essenziell für die normale Zellphysiologie, sondern auch von fundamentaler Bedeutung in der Karzinogenese. Störungen zellulärer Kontrollmechanismen der Apoptose gelten als kritisches Ereignis in der Initiierung und Progression von Tumoren. Eine reduzierte Apoptoserate wird ferner als wichtiger Mechanismus für die Resistenz von Tumorzellen gegenüber einer Radio- und Chemotherapie angesehen. Das duktale Adenokarzinom des Pankreas gehört zu den Tumoren, die weitgehend resistent gegenüber einer konventionellen Chemotherapie sind. Basierend auf den bisherigen Befunden zur Pathogenese des Pankreas-karzinoms und den Vorarbeiten zum Thema RANKL-OPG-System war das Ziel der vorliegenden Arbeit, die Expression und Modulation der OPG-mRNA-Produktion und OPG-Proteinsekretion in humanen Pankreaskarzinomzelllinien zu untersuchen. Als Zellmodelle wurden die Adenokarzinomzelllinien PaTu 8988t, PaTu 8988s, PC-2 und Capan-2 analysiert und der Effekt von Steroidhormonen, Zytokinen und Medikamenten auf die OPG-Expression evaluiert. Die Genexpression wurde mittels Northern Blot und PCR, die Proteinsekretion mittels ELISA bestimmt. Osteoprotegerin (OPG) und Receptor activator of nuclear factor-κB ligand (RANKL) stellen spezifische Rezeptoren und Liganden der TNF-Superfamilie dar, deren Produktion bislang in verschiedenen humanen Geweben nachgewiesen werden konnte. Im Anschluss an den Nachweis von OPG-mRNA im Pankreasgewebe mittels eines kommerziellen Northern Blots konnte OPG auf mRNA- und Proteinebene in zwei der vier untersuchten Pankreaskarzinomzelllinien (PaTu 8988t und PC-2) nachgewiesen werden. Die PaTu 8988s- und Capan-2-Zellen zeigten dagegen keine Produktion von OPG. Zusätzlich exprimierten PaTu 8988t-Zellen auf mRNA-Ebene RANKL. Für Testosteron ergab sich eine dosis- und zeitabhängige Hemmung der OPG-mRNA- und -Proteinexpression in der Pankreaskarzinomzelllinie PaTu 8988t um 70 bis 77%, während 5α-DHT und 17β-Östradiol keine Effekte hatten. Das adrenale Androgen DHEA hemmte die OPG-Produktion um 83%. In den PC-2- Zellen hingegen senkte das gonadale Androgen 5α-DHT die OPG-Expression. Nach der Exposition der Zelllinie PaTu 8988t durch das Glukokortikoid Dexamethason kam es ebenfalls zu einer dosisabhängigen Hemmung der OPG-mRNA-Spiegel und der OPG-Proteinproduktion um 23%. Aufgrund der wichtigen Rolle von Zytokinen im Rahmen der Tumorimmunologie und der Modulation der Apoptose beim Pankreaskarzinom wurden unterschiedliche Zytokine (IL-1β, TNF-α und IL-8) hinsichtlich ihrer Regulation der OPG-Produktion untersucht. Im Gegensatz zu vielen anderen Zelltypen jedoch hatten diese Zytokine – IL-8 ausgenommen – keinen Einfluss auf die OPG-Expression. Die Behandlung mit IL-8 führte zu einer leichten Steigerung der OPG-mRNA-Produktion in PaTu 8988t-Zellen. Die HMG-CoA-Reduktase-Inhibitoren Lovastatin und Cerivastatin zeigten in Pankreaskarzinomzelllinien hemmende Effekte auf die OPG-mRNA- und OPG-Proteinexpression. Lovastatin als Statin der ersten Generation senkte die OPG-mRNA- und -Proteinsekretion in PaTu 8988t-Zellen um 90%. Des Weiteren wurden die OPG-mRNA- und -Proteinspiegel durch Cerivastatin in PaTu 8988t-Zellen um 27% und in PC-2-Zellen um 39% gesenkt. Diese Hemmung durch Statine war dosis- und zeitabhängig. Die Hemmung der OPG-Produktion in Pankreaskarzinomzelllinien ist ein weiterer Beleg für die pleiotropen Wirkungen der Statine, die klinisch hinsichtlich ihrer Apoptose modulierenden Effekte auf Pankreaskarzinomzellen genauer untersucht werden sollten. Zusammenfassend weisen die eigenen Ergebnisse in Zusammenschau mit den Arbeiten anderer Gruppen darauf hin, dass das Pankreas sowohl eine Produktionsstätte als auch ein potenzielles Ziel von OPG darstellt, einem wichtigen osteotropen und Apoptose modulierenden Zytokin der TNF-Rezeptorsuperfamilie. Zukünftige Studien sollten die genaue Interaktion von OPG und TRAIL sowie ihre Bedeutung für die Entstehung, Progression und Metastasierung des Pankreaskarzinoms analysieren. Diese Erkenntnisse könnten die Basis künftiger klinischer Studien sein, in denen die gezielte Modulation der Apoptose als therapeutisches Prinzip bei Patienten mit Pankreaskarzinomen eingesetzt werden kann.

In einer klinischen Phase II-Studie wurde an 14 gesunden Männern über einen Behandlungszeitraum von 24 Wochen die Anwendung von 1000 mg Testosteron-Undecanoat i.m. in den Wochen 0, 6, 12 und 18 mit der Addition von 10 mg Norethisteronazetat täglich per os zum Zwecke der Kontrazeption untersucht. Zielparameter waren die Spermiensuppression und die Auswirkungen der Therapie auf die Sexualhormone, besonders die Gonadotropine. Zudem wurden die Nebeneffekte der Anwendung beobachtet. Es kam zu einer signifikanten Suppression der Gonadotropine und der Spermatogenese. Von 14 Probanden erreichten 12 eine Azoospermie und 2 eine schwere Oligospermie = 0,1x106/ml. An Nebeneffekten konnte eine signifikante Gewichtszunahme beobachtet werden, ein Abfall des HDL-Cholesterins und der alkalischen Phosphatase, eine Zunahme der Thrombozyten zu einigen Zeitpunkten, eine klinisch unbedeutende signifikante Erhöhung des Leberenzyms LDH und eine nichtsignifikante Erhöhung der GPT im Behandlungszeitraum.

As a major actor of the brain-pituitary-gonad axis, GnRH has received considerable attention, mainly in vertebrates. Biochemical, molecular, neuroanatomical, pharmacological and physiological studies have mainly focused on the role of GnRH as a gonadotrophin-releasing factor and have led to a detailed knowledge of the hypophysiotrophic GnRH system, primarily in mammals, but also in fish. It is now admitted that the corresponding neurons develop from the olfactory epithelium and migrate into the forebrain during embryogenesis to establish connections with the median eminence in tetrapods or the pituitary in teleost fish. However, all vertebrates possess a second GnRH system, expressing a variant known as chicken GnRH-II in neurons of the synencephalon, whose functions are still under debate. In addition, many fish species express a third form, salmon GnRH, whose expression is restricted to neurons of the olfactory systems and the ventral telencephalon, with extensive projections in the brain and a minor contribution to the pituitary. In vertebrates, GnRHs are also expressed in the gonads where they act on cell proliferation and steroidogenesis in males, and apoptosis of granulosa cells and reinititaion of meiosis in females. These functions could possibly represent the primitive roles of GnRH-like peptides, as an increasing number of studies in invertebrate classes point to a more or less direct connection between GnRH-producing sensory neurons and the gonads. According to recent studies, GnRHs appear as very ancient peptides that emerged at least in the cnidarians, the first animals with a nervous system. GnRH-like peptides have been partially characterized in several classes of invertebrates notably in molluscs, echinoderms and prochordates in which effects on the reproductive functions, notably gamete release and steroidogeneis, have been evidenced. It is possible that, with the increasing complexity of metozoa, GnRH neurons have lost their direct connection with the gonad to specialize in the control of additional regulatory centers such as the hypophysis in vertebrates or the optic gland in cephalopods. However, reminiscent effects of GnRH functions at the gonadal level would have persisted due to local production of GnRHs in the gonad itself. Altogether, these data indicate that GnRHs were involved in the control of reproduction long before the appearance of pituitary gonadotrophs.

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International audience; From day 23 of pregnancy, 24 gilts received either a medium (M, $n = 16$) or a high (H, $n = 8$) level of feeding calculated to meet 115% or 190% of energy for maintenance, respectively. During lactation, all H sows were fed ad libitum (H-AL) whereas M sows were fed either ad libitum (M-AL, $n = 8$) or were restricted (M-RE, $n = 8$) to the amount of feed ingested by H-AL sows. Increased feed intake during pregnancy increased live weight, backfat thickness, and estimated body lipid and protein on days 4 and 25 of lactation ($P < 0.05$). It also resulted in lower feed intake and higher lipid mobilisation during lactation ($P < 0.05$) without a detrimental influence on milk production. Activities of malic enzyme and glucose-6-phosphate dehydrogenase from neck fat samples were higher in H than M sows on day 4 ($P < 0.05$). They decreased during lactation in H sows ($P < 0.05$). Mean diameter of adipocytes decreased during lactation in the 3 groups ($P < 0.05$) but did not differ between groups on days 4 and 25. Plasma leptin on days 4, 11, 18 and 25 was higher in H than in M sows ($P < 0.05$) but was not influenced by lactational feed intake. Neither measured characteristics of gonadotrophin secretion on day 22, nor of ovarian activity on day 26, were significantly influenced by the level of feeding during pregnancy or lactation.; Influence de la consommation d'aliment pendant la gestation et la lactation sur la mobilisation des réserves adipeuses, le taux de leptine plasmatique et la fonction de reproduction de truies primipares allaitantes. à partir du 23e jour de gestation, 24 cochettes reçoivent un niveau alimentaire moyen (M, $n = 16$) ou élevé (H, $n = 8$) destiné à couvrir respectivement 115 et 190 % des besoins énergétiques d'entretien. Pendant la lactation, toutes les truies H sont nourries à volonté (H-AL) alors que les truies M sont nourries à volonté (M-AL, $n = 8$) ou rationnées (M-RE, $n = 8$) de façon à recevoir la même quantité d'aliment que celle consommée par les truies H. L'augmentation de la consommation d'aliment en gestation permet un accroissement du poids vif, de l'épaisseur de lard, du contenu en lipides et en protéines de la carcasse à 4 et 25 jours de lactation ($P < 0,05$). Cela induit une réduction de la consommation d'aliment en lactation et une augmentation de la mobilisation des réserves lipidiques ($P < 0,05$) sans diminution de la production laitière. Les activités de l'enzyme malique et de la glucose-6-phosphate deshydrogénase mesurées dans du tissu gras prélevé au niveau du cou sont plus élevées chez les truies H que chez les M, au jour 4 ($P < 0,05$). Elles diminuent pendant la lactation chez les truies H ($P < 0,05$). Le diamètre moyen des adipocytes diminue pendant la lactation dans les trois groupes ($P < 0,05$) mais ne diffère pas entre groupes aux jours 4 et 25 de lactation. La concentration de leptine plasmatique mesurée aux jours 4, 11, 18 et 25 est plus élevée chez les truies H que M et n'est pas influencée par le niveau alimentaire de lactation. Aucune des caractéristiques de la sécrétion des hormones gonadotropes au jour 22 et de l'activité ovarienne 4 jours plus tard n'est influencée significativement par le niveau alimentaire de gestation et de lactation.

International audience; Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% α helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.

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Cet article fait partie du dossier : L’augmentation de la prolificité des truies : acquis, progrès envisageables et conséquences; National audience

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L'efficacite de differents analogues du GnRH et/ou du pimozide pour induire l'ovulation chez l'omble chevalier est etudie a deux temperatures: 5°C, une temperature propice pour l'ovulation spontanee de l'omble chevalier et 10°C, une temperature inhibant l'ovulation chez l'omble chevalier. A 5°C tous les differents analogues du GnRH etudies sont capables d'induire et de synchroniser les ovulations. A 10°C, une preparation a diffusion prolongee du D-Trp 6 LH-RH a 20 μg.kg -1 et une forme a diffusion instantanee du D-Arg 6 sGnRH a 100 μg.kg -1 sont capables d'induire l'ovulation chez 80 % des femelles en une quinzaine de jours (contre 2 % chez les temoins). Le pimozide seul ou associe a une faible dose de D-Arg 6 sGnRH est capable d'induire quelques ovulations a 10°C, suggerant qu'une inhibition de type dopaminergique de la secretion gonadotrope puisse sc produire a 10°C. La survie des oeufs provenant des poissons traites au GnRHa a 5°C est comparable a celle des temoins (73 % ) excepte dans le groupe des poissons traites avec du GnRHa associe a du pimozidc (45 %). A 10°C, la survie des oeufs est significativement inferieure a celle des temoins a 5°C, sauf dans le groupe de poissons traites uniquement au pimozide. Aux deux temperature, la survie des oeufs de chaque ponte est correlee negativement avec la teneur en gonadotropine plasmatique de la femelle genitrice (valcur maximale observee dans les quatre jours suivant le traitement), sauf dans les groupes recevant la preparation a diffusion prolongee du D-Trp 6 LH-RH.

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International audience; The rainbow trout (Oncorhynchus mykiss) ovarian gonadotropin (GTH II) receptor was solubilized by extraction with the nonionic detergent 1% Triton X-100 in the presence of 20% glycerol. The hormone-binding characteristics of the soluble receptors were similar to those of membrane-bound receptors: the Scatchard plot of the equilibrium binding data produced a straight line, suggesting that the solubilized GTH II receptors, like membrane-bound receptors, contained a single class of high affinity 125I-sGTH II binding sites with an association constant of 2-5 x 10(10) M-1 (Ka = 1.4-2 x 10(10) M-1 for membrane-bound receptor). The maximal binding capacity was very low and varied from 7 to 17 fmol/mg proteins (about 5 fmol/mg ovarian membrane protein). The soluble receptor was purified by a simple and rapid immunoaffinity chromatography. The sGTH II-solubilized receptor complex was adsorbed to anti-sGTH II beta-subunit gammaglobulins covalently linked to Sepharose 4B and then eluted with an acidic buffer. About 50% of the binding activity present in the Triton X-100 extract was recovered in the pH 4 eluate. The other binding sites were eluted as a hormone-receptor complex and/or a damaged form. The free purified receptor presented a Ka of 1.3 x 10(10) M-1 in agreement with those found in membrane preparation and solubilized extract.

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International audience; Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.

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; Manipulation of the opportunity to spawn was used to investigate the relationship between endocrine events, egg viability and spawning behaviour in female rainbow trout. Females were prevented from spawning by isolating them from males and gravel for up to 21 days after ovula- tion. Blood samples were taken before pairing with a male, at the onset of nesting activity, and at the completion of spawning. Plasma hormone levels of gonadotropin (GtH) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20P) were measured by specific radioimmunoassays. There were no qualitative or quantitative differences in the spawning behaviour of females paired on the day of ovulation or 7. 14, or 21 days after ovulation. There was a general decrease in the viability of eggs with increasing retention times. In females paired on the day of ovulation, or after 7 or 14 days, GtH levels increased with the onset of nesting behaviour and declined as fish reached the post-spawning condition. By day 21, GtH levels before pairing were significantly higher than prepairing levels in the other three treatment groups, and did not increase at the onset of nesting, or decrease in post-spawning fish. Plasma 17,20P remained high in prepairing and nesting samples of all four groups and declined to low levels in fish in post-spawning condition. In females paired on the day of ovulation there was a significant increase in 17,20P from the prepairing to the nesting stage. These results suggest that 1,20P plays a key role in the synchronization of behavioural and maturational events at the time of spawning.

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; Adult goldfish put under controlled-raising photoperiod were subjected to a progressive rise in rearing temperature between February and May, or to a sudden rise in February when gametogenesis is initiated or in May when it is in progress. Plasma gonadotropin (c-GtH) was measured by radioimmunoassay. When temperature rose from 10 °C to 20 or 30 °C, a significant increase of immunoreactive c-GtH was observed. In a control group maintained at 10 °C from February to May, the cGtH level increased significantly, but remained significantly lower than the level of the group subjected to a progressive rise in rearing temperature and kept under the same photoperiod. This suggests an effect of both temperature and photoperiod on c-GtH release.; Des poissons rouges adultes placés en photopériode artificielle et croissante sont soumis à une élévation progressive de leur température d’élevage entre février et mai. D’autres subissent une élévation de température rapide, soit en février lors de l’initiation de la gamétogenèse ou en mai lorsque les animaux sont en pleine gamétogenèse. La gonadotropine plasmatique (c-GtH) est dosée par une méthode radioimmunologique. Lorsque la température s’élève de 10 à 20 ou 30 °C, il se produit un accroissement significatif de la c-GtH immunoréactive. Dans les groupes d’animaux maintenus sous photopériode croissante et soumis à des températures d’élevage constantes (10°C) ou croissantes (passage de 10 à 20 °C entre février et mai) les niveaux circulant de c-GtH augmentent significativement, mais l’augmentation est plus marquée pour le lot soumis à l’élévation de température (P < 0,005). Ceci suggère la possibilité d’une action combinée de la température et de la photopériode sur la sécrétion de c-GtH.

The influence of testosterone on plasma and pituitary levels of gonadotrophin (GTH) as well as on gonadal development was studied in immature rainbow trout. Among the animals receiving a testosterone-cocoa butter implant (200 micrograms) at the age of 5 months, gonadal puberty occurred 8 months later in half of the males (opposite to the controls which remained immature) and the beginning of oocyte maturation was observed in only one female. These animals were characterized by a higher pituitary GTH level. Owing to the multivariate statistical analyses made, it was possible to provide evidence for the presence of two populations with different reactions to the same steroid treatment. They also confirmed the existence of a positive testosterone feedback, in the male, leading to a precocious gonadal development. The pituitary GTH load obtained with 200 micrograms of testosterone seemed to be related to the age of first maturation. The secretion of an appropriate level of GTH resulting in the stimulation of gametogenesis required the availability of a relatively large pituitary GTH level and seemed to be possible because the animals were already in the pubertal period. The fact that the highest pituitary GTH level of the treated lot was found in the only female showing a beginning of sexual maturation suggests that testosterone may also act in females.

International audience; This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10−7 M) induced a 20–30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10−7 M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3–7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.

During an experimental period of 12 days, the onset of spermiation, quantity of collectable milt, spermatocrit, plasma and pituitary gonadotropin hormone (GtH) were compared in control and LHRH analogue (LHRHa)-treated prespawning landlocked salmon. Des-Gly 10 -[D-Ala 6 ]-LHRH ethylamide (D-Ala 6 -LHRH EA) was administered either by intraperitoneal multiple injections in saline or 40% propylene glycol or by a single intraperitoneal implant in silicone rubber. [6-(D-2-naphthylalanine)] LHRH (D-Nal (2) 6 -LHRH) was administered in a single cholesterol pellet. All the methods of administering LHRHa effectively accelerated the onset of spermiation and increased the volume of collectable milt. Study of the spermatocrit indicated that this increase is associated with an advancement in the production of spermatozoa and is not just due to sperm dilution. Furthermore, this increase in milt volume is related to an increase in plasma GtH. On the other hand, LHRHa treatments did not provoke any change in pituitary GtH content.

As in the female (Breton et al., 1988), plasma gonadotropin levels (GtH) in male roach were always the lowest the day of aggregation on the spawning areas, eventhough they were elevated 3 days earlier. The levels increased during spawning and remained at a high level in most of the animals captured out of the visible spwaning areas, 3 weeks after the beginning of the spawning activity. During the same period the GnRH contents did not significantly vary in the hypothalamic lobes, the di-met-and myelencephalon. On the contrary there were marked variations in the pituitary gland and the telencephalon in which the GnRH levels were the highest the day of aggregation when GtH plasma levels were the lowest. In these two structures they decreased during spawning in correlation with the increase of the plasma GtH levels. These results are discussed in comparison with those obtained in the female and in relation with the possible endocrine and paracrine roles of the GnRH in these structures and mainly in the telencephalon., Comme chez la femelle (Breton et al., 1988, les niveaux de gonadotropine (GtH) plasmatique sont toujours les plus faibles le jour du rassemblement sur les frayères alors qu'ils étaient encore élevés 3 jours avant. Ils augmentent et sont élevés pendant le frai et le restent chez la plupart des animaux capturés hors des zones visibles de frai, 3 semaines après le début de celle-ci. Au cours de la période étudiée, les contenus en GnRH ne varient pas significativement dans l'hypothalamus, le di, le met et le myélencéphale. Par contre, on constate d'importantes modifications dans l'hypophyse et le téléencéphale dans lesquels le GnRH est le plus élevé le jour de l'aggrégation quand la GtH plasmatique est au contraire la plus faible. Dans ces deux structures, ils décroissent pendant le frai corrélativement à l'augmentation des niveaux de GtH plasmatique. Ces résultats sont discutés, en comparaison avec ceux obtenus chez la femelle, par rapport aux relations GtH-GnRH et aux rôles possibles endocrines et paracrines du GnRH de ces structures et plus spécialement du téléencéphale.

1. 1. The development of an in vitro brain-pituitary perfusion technique allowed both the measurement of gonadotropin release and the recording of electrical activity in various brain areas of the rainbow trout. 2. 2. GnRH sensitive neurons were found in the nucleus preopticus (NPO). 3. 3. Gonadotropin release was evoked after microinjections of LHRH-A into the pituitary or into the NPO. 4. 4. Norepinephrine has been demonstrated to have a stimulatory effect on gonadotropin release.

Goldfish serum gonadotropin (GTH) concentrations were determined by radioimmunoassay 20 min after injection of goldfish brain extracts or synthetic luteinizing hormone-releasing hormone (LH-RH) into the third ventricle in the ventrobasal hypothalamic region. Extracts of the cerebellum and medulla were ineffective. Synthetic LH-RH, and extract of the lateral hypothalamus each significantly increased plasma GTH levels. The results indicate that the hypothalamus of the sexually mature female goldfish contains gonadotropin releasing hormone activity, and that releasing hormone is effective when administered via the intrahypothalamic route.

International audience; The present study investigates the effects of seasonally changing photoperiod regimes on the levels of serum gonadotropin, oestradiol-17/3, and vitellogenin (as indicated by phosphoprotein phosphorus and calcium measurements) on the time of spawning of female rainbow trout (Salmo gairdneri). This included a control regime and also seasonal cycles in which the increasing and decreasing components of the photoperiod were maintained but compressed into 6- and 9-month periods. Spawning in the control fish occurred in mid- January at the same time as fish in outside ponds under ambient conditions, whereas under the 6- and 9-month cycles it was advanced by approximately 12 and 6 weeks, respectively. Under both the control and experimental regimes, the primary change was an increase in serum levels of gonadotropin during the early stages of ovarian development, after which levels were reduced. This was followed by increased oestradiol-17β levels which became particularly pronounced during the period of exogenous vitellogenesis. These changes in oestradioL17β appeared to stimulate vitellogenin production and release by the liver as evidenced by increases in serum calcium and phosphoprotein phosphorus levels. Subsequently, vitelogenin levels remained high until after spawning was complete. During the later stages of ovarian development oestradiol levels fell, thus signalhng, probably by a negative feedback mechanism, a further and larger increase in gonadotropin levels presumably required for the control of fmal oocyte maturation and ovulation. This sequence of endocrine changes was similar under the shortened photoperiod cycles, although modifications were apparent in both their timing and duration, resulting in earlier spawning times.

Plasma and pituitary gonadotropin (GtH) levels, and the gonadotropin releasing hormone (GnRH) in differents areas of the brain, were measured using radioimmunoassays during the prespawning and the spawning period in a wild population of roach (Rutilus rutilus). GtH pituitary levels did not vary. On the contrary GtH plasma levels showed variations during the prespawning period and were minimum on the day of arriva1 on the spawning areas, Do. During spawning they increased in ovulated females but also in unovulated animals and returned to low values, 1 month after. During this period GnRH varied inversely in all brain structures and was always negatively correlated with the blood plasma GtH levels. This suggests that all the brain GnRH would be involved in the control of the GtH secretion during spawning. This was not the case during the prespawning period, during which only hypothalamic and pituitary GnRH levels were correlated with GtH. There were also marked variations in the telencephalic GnRH extra hypothalamic GnRH. It is hypothetized that it could be a link between environmental factors and the hypothalamo-pituitary cornplex., Les niveaux de gonadotropine (GtH) hypophysaires et plasmatiques ainsi que les contenus en « gonadotropin releasing hormone » (GnRH) de l'hypothalamus et différentes aires cérébrales ont été mesurés par dosage radioimmunologique. L'expérience s'est déroulée pendant la période précédant le frai et durant la période de frai d'une population sauvage de gardons (Rutilus rutilus). Les contenus hypophysaires en GtH ne varient pas, contrairement aux niveaux plasmatiques de cette hormone, dont les concentrations sont toujours les plus faibles le jour du rassemblement sur les aires de frai. Ils augmentent chez les femelles ovulées, mais aussi chez les poissons non ovulés. Pendant le frai les contenus en GnRH varient en sens inverse de la GtH dans toutes les structures cérébrales et sont corrélés négativement aux niveaux plasmatiques de GtH. On retrouve des variations similaires de la GtH et du GnRH hypophysaire et hypothalamique avant le frai. Mais, durant cette période, bien que les contenus en GnRH télencephalique montrent des variations importantes, ils ne sont pas correlés aux niveaux de GtH plasmatique. Ces résultats sont discutés et plus spécialement les rôles éventuels du GnRH extrahypothalamique. Il est fait l'hypothèse d'un rôle de médiation des facteurs de l'environnement par le GnRH télencephalique.

International audience; To investigate a possible effect of osmotic pressure on prolactin (PRL) release in rainbow trout, we developed a technique for in vitro perifusion of trout pituitaries. Changes in osmotic pressure similar to those observed in fish plasma during transfer experiments did not induce significant modifications of PRL release. In contrast, high-amplitude variation of osmotic pressure resulted in clear modifications of PRL secretion: hyperosmotic medium caused a reduction in PRL release, while infusion of hyposmotic medium induced a transitory increase in PRL release. By using different concentrations of mannitol, we found that the modifications of prolactin secretion could not be ascribed to alterations of the ionic composition of the medium but actually resulted from variations in the osmotic pressure of the incubation medium. In further experiments osmotic pressure was decreased from 300 to 220 mOsm/kg or from 400 to 300 mOsm/kg; a similar transitory increase in PRL release was observed. Measurement of gonadotropin (GtH) in the perifusion effluent medium showed that PRL and GtH secretion followed similar patterns. Thus, our results suggest a possible mechanical effect of wide changes in osmotic pressure on pituitary cell membranes. These data indicate that the rainbow trout differs notably from nonsalmonid teleost species thus far studied in the lack of sensitivity of its PRL cells to osmotic pressure.

A rise in rearing temperature stimulated gonadotropic hormone (c-GTH) secretion in Carassius auratus . This plasma c-GTH increase was not followed by gonadal development. No matter what the season, a rise of water temperature temporarily blocked gonadal development; however, high temperature inhibition of gametogenesis was reversible because after 9 months at 30 C, the fish showed completely and apparently normal gametogenesis. Photoperiod did not influence pituitary secretion and the mean plasma c-GTH level. However, the existence of circadian plasma c-GTH cycles indicated that photoperiod might affect gonadotropic secretion. Long winter photoperiods also strongly stimulated oogenesis. Reduced food intake for 3 months did not cause gonadotropic secretion to decrease with any of the photoperiod or temperatures used. On the other hand, at 20 C with long and short-day regimes there was a significant increase (P

Urea (8 M ) treatment of salmon gonadotropin results in the dissociation of the hormone into two subunits. These subunits were separated using DEAE-cellulose chromatography and gel filtration on Ultrogel ACA 54, and studied for their molecular weights, electrophoretic, biological, and immunological properties. Molecular weights of the α and β subunits were, respectively, 12,500 and 17,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. In anionic polyacrylamide gel electrophoresis, 7.5%, the α subunit, migrated more rapidly ( R f = 0.67) than the native hormone s-GtH ( R f = 0.38), and the β subunit did not migrate. The biological activities of the α and β subunits were less than 10% of the GtH. Reassociation allowed a 30% recovery of the initial biological activity. The immunological properties studied on two radioimmunological systems specifically directed against s- and cGtH, show that immunological determinants were connected with the β subunit, which also possesses characteristics of zoological specificity.

Les teneurs en gonadotropine (c-GtH) plasmatique ont été mesurées sur des poissons rouges élevés à des températures constantes ou croissantes comprises entre 10 et 30 °C de février à juillet. Aux températures d'élevage égales ou supérieures à 17 °C, les niveaux de c-GtH plasmatiques restent pendant 2 mois (avril et mai) significativement (P

GnRH receptors and GnRH-like activity in the rat testis

Cette étude a pour but de mesurer les teneurs en FSH et LH plasmatique des produits mâles et femelles et la croissance testiculaire des mâles issus de 16 pères Lacaune sélectionnés d’après leur index de prolificité en fermes (8 prolifiques H et 8 non prolifiques B) et de mères Romanov et F1 Lacaune X Romanov. Elle porte sur 161 agneaux dont 100 mâles et 61 femelles. Les taux de FSH et de LH sont mesurés sur un seul prélèvement de sang à 3, 5, 7, 9 et 11 semaines ; le diamètre testiculaire est mesuré à 7, 10 et 14 semaines. Chez les mâles, le taux de FSH est maximum à 5 semaines (3,6 ng/ml) ; il est indépendant du type de naissance, du type génétique des agneaux et de l’index de prolificité de leur père. Intra-type génétique, le diamètre testiculaire est indépendant de l’index du père. Chez les femelles, le maximum de FSH est plus élevé (8,2 ng) et atteint dès 3 semaines. L’effet de l’index du père est très significatif à 3 et 5 semaines et l’effet du type génétique très significatif à 3 semaines, alors que les effets du type de naissance et du poids vif ne sont jamais significatifs. Le taux de FSH à 5 semaines des 58 agnelles croisées est corrélé de façon significative avec leur taux d’ovulation mesuré à 9-10 mois. Ces résultats indiquent que le taux de FSH des agnelles pourrait constituer un critère précoce pour indexer les béliers. En ce qui concerne les concentrations en LH plasmatique des agneaux mâles et femelles, tous les effets (type de naissance, type génétique et index père) sont non significatifs., The purpose of this study was to examine the plasma FSH and LH levels of male and female lambs and the testicular growth of males issued from 16 Lacaune rams selected on their prolificacy index in farms (8 prolific and 8 non prolific rams) and from Romanov and F1 Lacaune X Romanov ewes. A total of 161 lambs (100 males and 61 females) was observed. Plasma FSH and LH were measured on single blood samples collected at 3, 5, 7, 9 and 11 weeks of age. The testicular diameter was measured at 7, 10 and 14 weeks. In males, plasma FSH reached a maximum at 5 weeks (3,6 ng/ml) ; it was independent of birth type, lamb genotype and sire prolificacy index. Within a genotype, testicular diameter was independent of the sire’s index. In females, the maximum concentration of plasma FSH was higher than that of males (8.2 ng/ml) and was reached as early as 3 weeks of age. The effect of sire prolificacy index was very significant (P < 0.01) at 3 and 5 weeks, when effects of birth-type and liveweight are never significant. At 5 weeks of age, FSH plasma concentration of the 58 crossbred ewe lambs was related to their ovulation rate observed at 10 months of age. These results show that plasma FSH levels in ewelambs could constitute an early criterion of ram selection. Plasma LH in males and females was independent of birth type, lamb genotype and sire prolificacy index.

To determine whether increases in gonadotropin levels are capable of altering thyroid function in goldfish, plasma thyroid hormone levels were measured following induced changes in endogenous gonadotropin secretion and injection of carp gonadotropin. Radio-frequency lesions placed in the nucleus preopticus periventricularis or monosodium- l -glutamate-induced lesions of the posterior nucleus lateralis tuberis (NLT) of the hypothalamus were capable of stimulating significant increases in plasma gonadotropin levels, but were without effect on plasma triiodothyronine (T 3 ) or thyroxine (T 4 ) at time intervals ranging from 5 hr to 10 days. Likewise, injections of a superactive analog of gonadotropin-releasing hormone resulted in profound increases in gonadotropin levels without associated changes in thyroid hormones. No changes in the circulating levels of T 4 or T 3 were observed in response to injection of purified carp gonadotropins whereas injection of bovine thyrotropin or carp pituitary extracts stimulated significant increases in T 4 . Radiofrequency lesions of the pituitary stalk or of the anterior NLT also resulted in significant increases in circulating levels of T 4 , but not of T 3 , at 10 and 30 hr postlesion. These results demonstrate that direct acute stimulation of circulating thyroid hormone levels is not an intrinsic action of endogenous goldfish gonadotropin and that activation of the reproductive system, leading to ovulation in some cases, is without effect on blood total thyroid hormone levels. Additionally, these results confirm that hypothalamic inhibition of the pituitary-thyroid axis exists in this teleost fish and demonstrate that interruption of this inhibition results in a time-dependent, high-magnitude increase in circulating thyroxine levels.

Dosage radioimmunologique des gonadotropines de poissons. Bases biologiques de l'aquaculture

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.

Daily changes in carp gonadotropin levels in adult female carp and daily changes in carp oocyte sensitivity to carp hypophysial homogenate, in vitro and in vivo, were investigated. A total of three series of experiments were carried out. Gonadotropin levels were radioimmunologically determined. The results of series 1 and 2 experiments were subjected to statistical analysis with the use of cosinors circle and ellipse of errors. It has been found that in the mature female carp in the pre-spawning period with the light periods being long (L:D = 16:8) the apogee for gonadotropin occurs 10 hr after the onset of the light period. The sensitivity of the oocytes, in terms of the percentage of mature oocytes (after GVBD) following a 24-hr incubation of ovarian fragments with the hypophysial homogenate, reached the highest value at 1300, i.e. 9 hr after the onset of the light period. It was also found that the injections of carp hypophysial homogenate made at 0900 were much more efficient in inducing ovulation than those at 2100.

The effects of injection of prostaglandin (PG) E1, PGE2, and PGF2? into the third ventricle on serum gonadotropin (GTH) concentrations in the goldfish were tested. Blood samples were taken at 30 min postinjection for radioimmunoassay of serum GTH. PG dosages of 0.5 and 1.0 ?g were ineffective. However, PGE2 and PGF2? at the 2.0-?g dosage significantly suppressed serum GTH. PGE1 at a 2.0-?g dosage had no effect. There were no effects on serum GTH when 2.0 ?g of PGE1, PGE2, or PGF2? were injected intraperitoneally. The results indicate that PGE2 and PGF2? suppress gonadotropin secretion by some action, presumably on the hypothalamus. However, action of PGE2 and PGF2? by diffusion from the site of injection to some other brain site or the pituitary cannot be eliminated in the present study.