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(Oberflächen-) Gewässer weltweit sind mit geringen Mengen (ng/L bis wenige µg/L) humaner Pharmazeutika belastet. Diclofenac (DCF; nicht-steroidal, entzündungshemmend) und Metoprolol (MTP; ß-Blocker) gehören entsprechend ihres hohen Verbrauchs zu den am häufigsten gefundenen Substanzen. Deren biologische Aktivität ist nicht auf den Menschen beschränkt. Gut konservierte Enzyme innerhalb der Vertebraten legen Auswirkungen auf Nicht-Zielorganismen wie Fische nahe, die bisher in Langzeituntersuchungen mit umweltrelevanten Konzentrationen unzureichend untersucht wurden. In der vorliegenden Arbeit wurden die physiologischen Effekte von DCF und MTP auf die Nil-Tilapie (Oreochromis niloticus), einem der wichtigsten Aquakulturfische weltweit, untersucht. In vitro konnte anhand primärer Hepatozyten gezeigt werden, dass bereits umweltrelevante Konzentrationen von DCF zu einer erhöhten Genexpression verschiedener Schlüsselenzyme der Detoxifizierung führten. Nach MTP-Exposition waren die Veränderungen weniger eindeutig. Beide Substanzen induzierten die Vitellogenin Genexpression, nur DCF jedoch bereits in umweltrelevanter Konzentration. In vivo wurden in zwei Langzeit-Expositionsversuchen die physiologischen Effekte vom befruchteten Ei bis 80 Tage nach Schlupf in O. niloticus untersucht. Beide Substanzen hatte keinen Einfluss auf Schlupferfolg und Überleben, das Wachstum war nach 80 Tagen nach Schlupf leicht reduziert. Die deutlichsten Auswirkungen waren histopathologische Veränderungen der Kiemen, veränderte Genexpressionen der Gonadotropine und eine erhöhte Expression von Vitellogenin. Die Ergebnisse legen eine stärkere östrogene Aktivität von DCF im Vergleich zu MTP nahe. Zusammenfassend sind die Bedenken gegenüber den Einzelsubstanzen eher gering, negative Auswirkungen auf die Reproduktion und sich verstärkende Effekte bei zeitgleicher Exposition gegenüber DCF und MTP lassen sich jedoch nicht ausschließen und sollten im Weiteren untersucht werden. Surface waters worldwide are contaminated with low levels (ng/L up to few µg/L) of human pharmaceuticals. Diclofenac (DCF; non-steroidal, anti-inflammatory) and metoprolol (MTP; ß-blocker) are highly consumed and therefore commonly detected. Their biological activity is not restricted to humans. Well conserved enzymes within the vertebrates suggest effects on non-target organisms such as fish, poorly studied in long-term exposure experiments using environmentally relevant concentrations. In the presented work, physiological effects of DCF and MTP on the Nile tilapia (Oreochromis niloticus), an important aquaculture fish species, were studied. Using primary hepatocytes, it was shown in vitro that environmentally relevant concentrations of DCF increased the gene expression of different key enzymes of the detoxification, while MTP exposure had a less clear effect. Both substances induced vitellogenin gene expression, but only after DCF exposure this was significantly elevated already at the environmentally relevant concentration. In vivo, two long-term exposure studies on the physiological effects from the fertilized egg until 80 days post-hatch were evaluated. Both substances did not affect hatching success and survival, while growth was slightly reduced after 80 days post-hatch. Histopathological alterations of the gills, changed gene expression patterns of the gonadotropins and induced vitellogenin gene expression were the most dominant findings. The results indicate a stronger estrogenic mode of action of DCF compared to MTP. Overall, the risk due to a single substance exposure seems to be relatively low but adverse effects on reproduction and additive effects during simultaneous exposure to DCF and MTP cannot be excluded and should be investigated further.

International audience; The mechanisms and the mediators relaying Fsh action on testicular functions are poorly understood. Unlike in mammals, in fish both gonadotropins (Fsh and Lh) are able to efficiently stimulate steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, it is crucial to understand if Fsh effects are mediated through the production of steroids. To address this issue we performed transcriptome studies after in vitro incubations of rainbow trout testis explants in the presence of Fsh alone or in combination with trilostane, an inhibitor of Δ4- steroidogenesis. Trilostane significantly reduced or suppressed the response of many genes to Fsh (like wisp1, testis gapdhs, cldn11, inha, vt1 or dmrt1) showing that, in fish, important aspects of Fsh action follow indirect pathways and require the production of Δ4-steroids. What is more, most of the genes regulated by Fsh through steroid mediation were similarly regulated by Lh (and/or androgens). In contrast, the response to Fsh of other genes was not suppressed in the presence of trilostane. These latter included genes encoding for anti-mullerian hormone, midkine a (pleiotrophin related), angiopoietine-related protein, cyclins E1 and G1, hepatocyte growth factor activator, insulin-like growth factor 1b/3. A majority of those genes were preferentially regulated by Fsh, when compared to Lh, suggesting that specific regulatory effects of Fsh did not depend on steroid production. Finally, antagonistic effects between Fsh and steroids were found, in particular for genes encoding key factors of steroidogenesis (star, hsd3b1, cyp11b2-2) or for genes of the Igf system (igf1b/3). Our study provides the first clear evidence that, in fish, Fsh exerts Δ4-steroid-independent regulatory functions on many genes which are highly relevant for the onset of spermatogenesis.

Follicle-stimulating hormone (FSH) is a master endocrine regulator of mammalian reproductive functions. Hence, it is used to stimulate folliculogenesis in assisted reproductive technologies (ART), both in women and in breeding animals. However, the side-effects that hormone administration induces in some instances jeopardizes the success of ART. Similarly, the luteinizing hormone (LH) is also of paramount importance in the reproductive function because it regulates steroidogenesis and the LH surge is a pre-requisite to ovulation. Gaining knowledge as extensive as possible on gonadotropin-induced biological responses could certainly lead to precise selection of their effects in vivo by the use of selective agonists at the hormone receptors. Hence, over the years, numerous groups have contributed to decipher the cellular events induced by FSH and LH in their gonadal target cells. Although little is known on the effect of gonadotropins on microRNA expression so far, recent data have highlighted that a microRNA regulatory network is likely to superimpose on the signaling protein network. No doubt that this will dramatically alter our current understanding of the gonadotropin-induced signaling networks. This is the topic of this review to present this additional level of complexity within the gonadotropin signaling network, in the context of recent findings on the microRNA machinery in the gonad.

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International audience

International audience

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.

National audience

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.

International audience

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.

International audience

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.

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.

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.

GnRH receptors and GnRH-like activity in the rat testis

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.

Gonadotropic activity is relatively low in the female freshwater (FW) eel (A. anguilla ): pituitary GTH was about 15 ng equivalent cGTH/mg fresh tissue. Plasma GTH was always less than 1 ng equivalent cGTH/ml and often undetectable (< 0.2 ng equivalent cGTH/ml). Ovarian steroidogenesis in FW fish is similarly low; plasma concentration of both free testosterone (T) and free estradiol (E sub(2)) were less than 0.3 ng/ml as measured by RIA. Treatment with E sub(2) in FW fish caused a great increase in pituitary GTH, up to 1000 times control values, without changing plasma GTH. Stimulation of gonadotropic activity and ovarian development occur during the eel's migration to the Sargasso Sea. Adaptation to seawater (SW) caused a great increase of plasma E sub(2) concentration without modifying that of T. However, adaptation to SW did not cause any detectable change in gonadotropic activity or ovarian development., La fonction gonadotrope (estimée par dosage radioimmunologique (RIA) pour la sous-unité 3 de l'hormone gonadotrope de carpe (cGTH)) est peu active chez l'anguille femelle d'eau douce ; la teneur hypophysaire en GTH est de l'ordre de 15 ng d'équivalent cGTH/mg tissu frais et la teneur plasmatique est toujours inférieure à 1 ng d'équivalent cGTH/ml et souvent indétectable (

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.

Plasma gonadotrophin (GTH) and 11-oxotestosterone levels have been studied in relation to sperm production during the last weeks of the spermiation period. The level of 11-oxotestosterone decreased in parallel to sperm production (coefficient of correlation: p = 0.59; P less than 0.01) and reached its lowest values (mean +/- SE = 0.33 +/- 0.19 ng/ml; n = 11) when spermiation was over. No significant variations were found in GTH levels; however, there was a slight positive correlation between sperm production and GtH levels (p = 0.39; P less than 0.05).

International audience; A study was conducted to determine the specificity of the steroids able to cause a stimulation of pituitary gonadotropic hormone (GtH) levels in sexually immature rainbow trout. Low pituitary GtH levels characteristic of juvenile trout were rapidly elevated following treatment with estrogen and aromatizable androgenic steroid hormones, but nonaromatizable androgens and progestogenic steroids were ineffective. An aromatase inhibitor, 1,4,6-androstatrien-3,17-dione, significantly reduced the pituitary GtH increase in response to treatment with testosterone. A central effect of steroid hormones on pituitary GtH was confirmed by the fact that pituitary steroid implants did not increase liver weight as observed after perivisceral hormone treatment. The possible participation of sex steroids in a mechanism of initiation of gonad recrudescence or onset of pubertal gonadal development in salmonids is discussed.

International audience; The plasma levels of estradiol-17β (E2), 17α, 20β-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (~ 45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (~ 2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (~ 0.7 ng/ml) and increased dramatically at GVBD (- 148 ng/ml). The levels of 17,20-P remained high at ovulation (~ 142 ng/ml) and then dropped significantly within 24 h. to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.

A method has been developed suitable for the study of the binding of salmon (Oncorhynchus tsawytscha) gonadotropic hormone (GtH) to brown trout (Salmo trutta) ovarian particulate fractions. Several proteolytic enzyme inhibitors were tested to preserve the binding capacity of particulate fractions (1000, 20,000, and 110,000g) from yolky oocytes. The best preservation was obtained with trypsin inhibitor. The effects on binding of the pH, time, and temperature of the incubation were investigated. Binding increased linearly with amount of particulate fraction, and is saturable. In both the 20,000 and 110,000g fractions, there are two classes of receptors: one with a high-affinity constant (3.14 and 0.95 10(10) M-1 for the 20,000 and 110,000g fractions, respectively) and a low capacity (1.76 and 0.63 fmol/g of ovary), the other with a higher-affinity constant (3.44 and 1.78 10(9) M-1) but a higher capacity (7.37 and 7.42 fmol/g of ovary). GtH binding was not affected by ovine luteinizing hormone (oHL), ovine follice-stimulating hormone (oFSH), and human chorionic gonadotropin (hCG), but was partially inhibited by 1 micrograms of bovine thyroid-stimulating hormone (bTSH) and salmon prolactin (sPRL). The results are discussed in relation to the biological specificity of fish GtH and to the unsolved problem concerning the number of gonadotropins in fish.

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.

The effects of LH-RH and analog on the stimulation of gonadotropin (GtH) secretion have been studied in order to use them as priming, before inducing maturation with 17α-hydroxy-20β-dihydroprogesterone (17α-20βP) in the carp Cyprinus carpio . One injection of LH-RH (2 μg/kg body weight) or LH-RH analog (0.3 μg/kg) induces a transient rise in the plasma GtH concentration, but subsequent 17α-20βP treatment does not result in an increase in the number of oocytes with germinal vesicle breakdown or in ovulation. Two and three in ections of LH-RH or analog at the same dosages are followed by a stimulation of GtH secretion (to 30 ng/ml) which is maintained for 6–9 h, and lasts longer with LH-RH analog than with LH-RH. The same magnitude in the increase of GtH secretion is obtained with lower doses of LH-RH analog. Injection of 17α-20βP then increases the number of oocytes undergoing germinal vesicle breakdown and sometimes partial ovulation. Futher studies on the mechanism of action of GtH during priming hypophysation treatment are needed in order to develop a sequential treatment using: (1) priming by LH-RH or analog, (2) 17α-20βP induced maturation treatment.

Photoperiod and gonadotropin control of the reproductive cycle in the female rainbow trout

International audience; A hypophysial homogenate injection increased blood gonadotropin level in adult female carp and maintained it at that level for three days after injection. The blood gonadotropin level in adult female carp seems to be dependent on the physiological condition of the ovary expressed by the relation of old oocytes with a completed vitellogenesis process to the new ones, and the relation of the oocytes with the nucleus shifted to the periphery to those with a centrally placed nucleus.

Female goldfish were acclimated to an 8 h light photoperiod and 14–15°C, and, in separate experiments, injected daily for 18 and 14 days with low doses of a partially purified salmon gonadotropin (sGtH) or a highly purified carp gonadotropin (cGtH), respectively at either the onset of light, or 4 or 8 h after the onset of light. The response to GtH injection was measured by the gonadosomatic index (GSI) and by histoiogical analyses of oocy te development. There was a response to sGtH injected at 8 h and to cGtH at 4 h and 8 h after the onset of light. Female goldfish acclimated to a 16 h light photoperiodand injected with low doses of cGtH at either the onset of light or at 4, 8, 12 or 16 h after the onset of light for 14 days had an increased GSI in response to the cGtH injected at 12 h after the onset of light. These results demonstrate that there is a daily variation in responsiveness of the ovary of goldfish to GtH.

International audience; To define the optimal combination time and LRH-A (luteinizing releasing hormone analogue) administration route for inducing spermiation in carp, two experiments were carried out. In the first experiment, carp kept in raceways in running recycled water in August (at a water temperature of 15–20°C) were injected intraperitoneally with pimozide (PIM) at 1 or 10 mg/kg body weight as well as with Des-Gly10 [D-Tryp6] LH-RH-ethylamide (LRH-A) at 0.1, 1 or 10 μg/kg; these products were given 6 h apart. The response was evaluated by the volume of milt and the number of spermatozoa emitted and by the gonadotropin (GTH) concentration in the plasma. At doses of 0.1, 1 and 10 μg/kg LRH-A, the total milt production was increased 2, 4 and 6-fold respectively when these treatments were combined with 1 mg PIM. In the second experiment, adult male carp, held in cages in a pond in a natural environment (at a water temperature of 11–14°C) at the beginning of May, received an intraperitoneal injection of PIM (1 mg/kg body weight) and a silicone rubber implant of LRH-A (Des-Gly10 [D-Ala6] - LH-RH-ethylamide) at dose of 10 μg/kg body weight simultaneously. Other groups of fish received PIM plus a blank implant. All of the treatments were given at either 6 a.m. or 8 p.m. When given at 6 a.m., the treatment induced a similar rise in plasma GTH (12–15 ng/ml) 24 h later in the groups treated with PIM and LRH-A implant alone or in combination. When the same treatment was given at 8 p.m., no response was observed 24 or 48 h later. Spermiation was not stimulated in any of the groups.

Serum immunoreactive gonadotropin (GTH) levels were measured in sexually mature (gravid) female foldfish at 24 hr before and 24 and 72 hr after brain or pituitary stereotaxic implantation of either a blank pellet (cocoa butter only) or a pellet containing one of the anti-estrogens, clomiphene citrate or ICI 46474. Both pituitary implantation and implantation in the nucleus lateral tuberis (NLT) region of a pellet containing ICI 46474 caused a significant increase in serum GTH levels, with the former site having a more pronounced effect. Implantation of ICI 46474 in the nucleus preopticus (NPO) region, the posterodorsal hypothalamus, or the optic tectum (OT) had no significant effects on serum GTH. Implantation of clomiphene citrate in the pituitary caused a significant increase in serum GHT levels, but implantation in the NPO region or OT had no effects. The results demonstrate that the pituitary gland and, to a lesser extent, the NLT region are sites for negative feedback effects of estrogens in sexually mature (gravid) female goldfish.

To test the antifertility effect of various steroids, fish were fed a diet containing steroids at two stages of the reproductive cycle: during spermatogenesis (June to November, experiment A) and during spermiation (November to February, experiment B). The fish in experiment A were given testosterone (T), methyltestosterone (MT), estradiol-17beta (E2), hexestrol (H), progesterone (Pg), and FGA (Fluorogestone acetate) at doses of 0.05 and (or) 0.5 mg/kg of food. In experiment B, T, MT, and E2 were given at a dose of 0.5 mg/kg of food. The fish received a daily ration amounting to 1% of their body weight. In experiment A, inhibition of testicular growth was total with 0.5 mg MT and 0.5 mg E2 and partial with 0.5 mg T/kg of food. In these groups, there was no noticeable change in plasma gonadotropin (GTH) compared with the controls, except that the GTH rise observed in the other groups in September was prevented. This suggests that steroids act directly at the level of the gonad. In experiment B, no noticeable regression of the gonad occurred after steroid treatment. A big GTH rise was seen in the E2-treated group, but this was not followed by stimulation of spermiation. The total amount of sperm (50%) and spermatozoa collected during the experiment tended to be lower in the steroid-treated fish than in the controls. Spermatozoal fertilizing ability was significantly decreased (P < 0.005) by the testosterone treatment.

At various stages throughout the annual reproductive cycle, female rainbow trout (Salmo gairdneri) were fitted with a catheter in the dorsal aorta. They were bled via the catheter at frequencies of once every 30, 60, or 240 min over periods of 5 to 24 hr. Gonadotropin (GtH), estradiol-17 beta (E(2)17 beta). and estrone levels were measured in the plasma samples. At early ovarian recrudescence (March), short-term (1-2 hr), high-amplitude (delta GtH = up to 100 ng/ml), episodic pulses of GtH were recorded in the plasma of 12 of the 26 studied fish. In the others, GtH levels remained low and constant. No synchronization was found among the individual GtH profiles. E(2)17 beta levels in the same fish were low and constant while estrone was not detectable. At early stages of exogenous vitellogenesis (June), plasma GtH (1-3 ng/ml) and E(2)17 beta (0.5-1.5 ng/ml) levels were low and constant. At advanced stages of exogenous vitellogenesis (September-October), fluctuating GtH levels were found again in most of the females; basal GtH concentrations were only slightly higher than those recorded in June. The fluctuations consisted of short-term (1-3 hr) random GtH pulses of moderate amplitude (delta GtH = up to 5 ng/ml), occurring at a relatively high frequency (up to 5 per 12 hr). Although no regular synchronous daily pattern of GtH was noted, most of the GtH pulses were observed during the photophase and early scotophase. The appearance of GtH pulsatility during exogenous vitellogenesis was accompanied by a large increase in plasma E(2)17 beta up to levels ranging from 6 to 30 ng/ml. In contrast to the GtH profiles, the E(2)17 beta profiles showed continuous and progressive variations, superimposed the abrupt GtH pulses, and a high degree of synchronization. E(2)17 beta levels increased during the photophase and reached maxima toward and during early scotophase.

A method of purifying trout gonadotropic hormone is discussed. The technique consists of the following stages : alcoholic extraction, two successive gel filtrations on Sephadex G5o- and G-ioo, chromatography on DEAE-cellulose and ultrogel A¢SC.A The biological activity of fractions obtained is measured using as assay the maturation in vitro of trout oocytes. The preparation obtained has a molecular weight of about 2,7500 determined by behavior on Sephadex-G 1 00 and analytical ultracentrifugation for KS20Sw of 2.45 Polyacrylamide gel electrophoresis shows two strips of electrophoretic mobility, of 0.50 and 0.58. Analysis of the amino acid composition of the preparation indicates a number of 160 AA residues per mole. These different values are compared to the corresponding ones for c-GtH, s-GtH and mammalian gonadotropins.

Pituitary extracts of 14 species of fish from the North Atlantic were tested in rainbow trout and carp gonadotropin radioimmunoassays (RIA). The poor cross-reaction and low gonadotropin content measured in the pituitary extracts indicate that RIA used for trout and carp gonadotropins are unsuitable for these species. The degree of specificity in the two systems does not reflect the phylogenetic relationships of the tested species. It is suggested that fish gonadotropins are species specific so that a sensitive RIA must be developed for each family investigated. A species specificity between fish and mammalian gonadotropins was suggested by work on goldfish spermatogenesis ( Billard et al. , 1970 ; Billard and Escaffre, 1973 ). Specificity among some groups of freshwater fish has also been indicated by other work on gonadotropins ( Burzawa-Gerard and Fontaine, 1972 ; Fontaine et al. , 1972 ; Breton et al. , 1973 ). However during the final stages of gametogenesis (ovulation and spermiation) the species specificity appears to be less pronounced since human chorionic gonadotropin or heterologous pituitary preparations are potent treatments ( cf. reviews by Pickford and Atz, 1957 ; Barnabe and Rene, 1972 ; Kuo et al. , 1973 ; Shehadeh et al. , 1973 ; De Vlaming, 1974 ). In the present study the immunological cross-reaction between pituitary preparations of a variety of fish species was tested in order to extend the investigation of species specificity and to examine the possibility of gonadotropin measurement in other species using carp and trout radioimmunoassay (RIA) systems already available ( Breton et al. , 1971 ; Breton and Billard, 1977 ).