Identification and stable expression of vitellogenin receptor through vitellogenesis in the European eel.

In teleosts, vitellogenin (Vtg) is a phospholipoglycoprotein synthesized by the liver, released into the blood circulation and incorporated into the oocytes via endocytosis mediated by the Vtg receptor (VTGR) to form the yolk granules. The VTGR is crucial for oocyte growth in egg-laying animals but is also present in non-oviparous vertebrates, such as human. The VTGR belongs to the low-density lipoprotein receptor superfamily (LDLR) and is also named very-low-density lipoprotein receptor (VLDLR). In this study, we identified and phylogenetically positioned the VTGR of a basal teleost, the European eel, Anguilla anguilla. We developed quantitative real-time PCR (qRT-PCR) and investigated the tissue distribution of vtgr transcripts. We compared by qRT-PCR the ovarian expression levels of vtgr in juvenile yellow eels and pre-pubertal silver eels. We also analyzed the regulation of ovarian vtgr expression throughout vitellogenesis in experimentally matured eels. The Vtg plasma level was measured by homologous ELISA experimental maturation. Our in silico search and phylogenetical analysis revealed a single vtgr in the European eel, orthologous to other vertebrate vtgr. The qRT-PCR studies revealed that vtgr is mainly expressed in the ovary and also detected in various other tissues such as brain, pituitary, gill, fat, heart, and testis, suggesting some extra-ovarian functions of VTGR. We showed that vtgr is expressed in ovaries of juvenile yellow eels with no higher expression in pre-pubertal silver eels nor in experimentally matured eels. This suggests that vtgr transcription already occurs during early pre-vitellogenesis of immature eels and is not further activated in vitellogenic oocytes. European eel Vtg plasma level increased throughout experimental maturation in agreement with previous studies. Taken together, these results suggest that vtgr transcript levels may not be a limiting step for the uptake of Vtg by the oocyte in the European eel.

A comparison of techniques for studying oogenesis in the European eel Anguilla anguilla.

A multi-technique approach was used to study the changes occurring in European eel Anguilla anguilla ovaries during hormonally-induced vitellogenesis. Aside from classic techniques used to monitor the vitellogenic process, such as ovary histology, fat content analysis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and vitellogenin enzyme linked immunosorbent assay (ELISA), a new technique, Fourier-transform infrared (FT-IR) microspectroscopy, was used to analyse A. anguilla ovaries. The results from the different techniques provided different ways of approaching the same process. Although it is considered a time consuming approach, of all the employed techniques, histology provided the most direct evidences about vitellogenesis. SDS-PAGE and ELISA were also useful for studying vitellogenesis, whereas fat analysis cannot be used for this purpose. The FT-IR analysis provided a representative IR spectrum for each ovarian stage (previtellogenic stage, early vitellogenic stage, mid-vitellogenic stage and late vitellogenic stage), demonstrating that it is a valid method able to illustrate the distribution of the oocytes within the ovary slices. The chemical maps obtained confirmed changes in lipid concentrations and revealed their distribution within the oocytes at different maturational stages. When the results and the accuracy of the FT-IR analysis were compared with those of the traditional techniques commonly used to establish the vitellogenic stage, it became evident that FT-IR is a useful and reliable tool, with many advantages, including the fact that it requires little biological material, the costs involved are low, analysis times are short and last but not least, the fact that it offers the possibility of simultaneously analysing various biocomponents of the same oocyte.

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

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

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

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

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

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

Potentiating effect of growth hormone on vitellogenin synthesis induced by 17 beta-estradiol in primary culture of female silver eel (Anguilla anguilla L.) hepatocytes.

Previous in vivo experiments have indicated a potentiating role of growth hormone (GH) during experimentally induced vitellogenesis by 17-beta-estradiol (E2) in the female silver eel (Anguilla anguilla L.). To investigate whether GH has direct hepatic actions, the effects of hypophysial-purified and recombinant QH on vitellogenin (Vg) synthesis in response to E2 were tested on primary cultures of hepatocytes. Hepatocytes were prepared from control or E2-primed eels. Addition of E2 alone into the culture medium induced both Vg synthesis and secretion in a dose- and time-related fashion. Bovine growth hormone (bGH) alone had no effect on the induction of Vg synthesis or secretion. Bovine GH enhanced the in vitro effects of F2 on both Vg synthesis and secretion, an effect attenuated by an in vivo E2 priming which was dose-dependent with an ED(50) of 5 ng/ml. To investigate the specificity of GH action, purified eel and salmon GH and salmon, trout, and tilapia prolactins (PRL), as well as recombinant trout and tilapia GH, were tested, and the responses were compared to bGH. Purified salmon and homologous eel GH potentiated the vitellogenic response to F2. Recombinant GH were highly efficacious, excluding the presence of active contaminants in the potentiating effect of GH preparations. The potentiating effect of recombinant trout GH on the vitellogenic response was reduced at high doses (above 20 ng/ml), suggesting a down-regulation of GH binding sites by GH itself. Salmon PRL has minimal activity, but not trout and tilapia PRL, indicating that PRL is not an important potentiating factor on Vg synthesis in our model. It is concluded that GH acts directly on the liver to potentiate E2 induction of eel hepatic Vg synthesis. The potentiating effect of GH appears to be time- and dose-dependent and modulated as a function of hormonal status (E2 priming) of the eel.

Synthesis of vitellogenin by eel (Anguilla anguilla L.) hepatocytes in primary culture: requirement of 17 beta-estradiol-priming.

Control and 17 beta-estradiol-primed eels were used to investigate the hormonal requirement of vitellogenesis in an immature fish, the eel. A primary culture of isolated liver cells from female silver eels was developed. The hepatocytes were maintained as monolayers on poly-L-lysine coated dishes for up to 12 days in a defined medium alone or supplemented with 17 beta-estradiol (E2, from 10(-8) to 10(-5) M). The amounts of vitellogenin (Vg) in the cells and secreted into the medium were measured at 2-day intervals using a homologous vitellogenin ELISA. Different E2-priming conditions were determined before hepatocyte isolation (one injection of 250 micrograms of E2 21 days, 17 days, or 24 hr). The vitellogenic response of hepatocytes to E2 stimulation was studied in relation to the duration of the E2-priming. After 8 days of culture, when hepatocytes from control eels were used, Vg was undetectable both in cells and in culture media, even if the culture was performed in the presence of E2 10(-5) M. However, Vg was detectable both in cells and in culture media of hepatocytes from E2-primed eels. If the priming was performed 24 hr before the culture, the Vg synthesis significantly increased (P < 0.001) in the presence of E2 10(-5) M after 10 days of culture but remained low. When the culture was performed 17 or 21 days after the priming, the level of the vitellogenic response was higher than after a short priming. In particular, with hepatocytes from 21-day E2-primed eel, the concentration of secreted Vg was 1.5 times higher than in control dishes (P < 0.01), in the presence of E2 10(-8) M after 12 days of culture. Higher doses of E2 (10(-5) M) increased Vg 2.7-fold over control values (P < 0.01) after 4 days of culture. In control dishes, cultured without steroid, the amounts of secreted and intracellular Vg remained unchanged over 12 days of culture (respectively, 72.8 +/- 2.7 ng/10(6) cells/48 hr and 28.7 +/- 2.7 ng/10(6) cells). These results show that cultured hepatocytes retain their functional capacity by synthesizing a specific protein, Vg, in the presence of E2 and there are dose- and time-related effects of E2 on in vitro Vg synthesis. The induction of hepatic vitellogenesis in vitro requires a preliminary in vivo E2-priming.

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels.

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.

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

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

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

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

[Changes in corticosterone and aldosterone concentrations in various tissues of Xenopus laevis tadpoles during the metamorphosis]. / Variations des concentrations en corticostérone et en aldostérone dans divers tissus du têtard de Xenopus laevis pendant la métamorphose.

During the metamorphosis of Xenopus laevis tadpoles, tissue concentrations of corticosterone and aldosterone did not change significantly in forelegs and hindlegs; they increased in tail, liver, skin and intestine. The rise of corticosteroid concentrations appeared in tissues which were deeply transformed during the first part of the climax, when plasma levels of corticosteroids also increased. Highest tissue levels, attained at the mid-climax, were maintained at least until the end of metamorphosis although plasma concentrations of two steroids were then abruptly fallen. Tissues able to retain corticosteroids reacted as Vertebrate "target tissues" and transformations which took place in them could be dependent, at least partially, on corticosteroids.

[Radioimmunoassay, in Ascidiella aspersa, of a gonadoliberin (GnRH)- like factor with an apparent molecular weight higher than that of the mammalian decapeptide]. / Dosage radioimmunologique, chez Ascidiella aspersa, d'un facteur de type gonadolibérine (GnRH) de poids moléculaire apparent supérieur à celui du décapeptide mammalien.

A radiommunoassay (RIA) for mammalian gonadoliberin (mGnRH) showed the presence of a GnRH-like factor in the neural complex of a Protochordate, Ascidiella aspersa (about 0,6 pg eq mGnRH/complex). The slope of the displacement curves was slightly lower than with mGnRH indicating antigene differences. No cross reactive material was found in mantle and siphonal area. The KD on Sephadex G25 was 0.45 versus 0.90 with mGnRH. That suggests that the molecular weight of the Ascidian GnRH-like factor is higher than that of known Vertebrate GnRH's, possibly due to a different processing of the precursor.