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L'étude des régulations hormonales, reponsables du contrôle de grandes fonctions telles que le développement, la croissance et la reproduction, présente chez l'anguille un intérêt tout particulier du fait du cycle biologique original de ce poisson. Les objectifs d'une telle étude sont à la fois fondamentaux (connaissance de la biologie de l'espèce, notion d'espèce modèle, écophysiologie, évolution) et appliqués (caractérisation des populations, maîtrise du cycle biologique). Cependant, la complexité et la méconnaissance actuelle de ce cycle apportent des difficultés supplémentaires (absence de référence naturelle pour les caractéristiques physiologiques et environnementales des dernières étapes du cycle) qui s'ajoutent à celles concernant classiquement la mise en oeuvre d'une telle étude chez une espèce particulière (nécessité de la mise au point des outils spécifiques d'investigation), espèce de surcroît sauvage (hétérogénéité physiologique et génétique des animaux étudiés) et e n danger (limitation du nombre d'animaux expérimentaux). C'est la mise en oeuvre de tout un ensemble de méthodes d'investigation, allant de la mise au point des outils moléculaires (purifications d'hormones, obtention d'anticorps, dosages immunologiques, clonage, obtention de sondes...) à la réalisation d'expériences in vitro (cultures de cellules, incubation ou périfusion d'organes) et in vivo (ablations de glandes, traitements hormonaux, prélèvements sanguins...) juqu'aux études sur le terrain, qui permet une approche complète et intégrée de l'endocrinologie de ce poisson.

Abstract Background The three vertebrate pituitary glycoprotein hormones (GPH) are heterodimers of a common α and a specific β subunit. In human, they are located on different chromosomes but in a similar genomic environment. We took advantage of the availability of genomic and EST data from two cartilaginous fish species as well as from two lamprey species to identify their repertoire of neurotrophin, lin7 and KCNA gene family members which are in the close environment of gphβ. Gphα and gphβ are absent outside vertebrates but are related to two genes present in both protostomes and deuterostomes that were named gpa2 and gpb5. Genomic organization and functional characteristics of their protein products suggested that gphα and gphβ might have been generated concomitantly by a duplication of gpa2 and gpb5 just prior to the radiation of vertebrates. To have a better insight into this process we used new genomic resources and tools to characterize the ancestral environment before the duplication occurred. Results An almost similar repertoire of genes was characterized in cartilaginous fishes as in tetrapods. Data in lampreys are either incomplete or the result of specific duplications and/or deletions but a scenario for the evolution of this genomic environment in vertebrates could be proposed. A number of genes were identified in the amphioxus genome that helped in reconstructing the ancestral environment of gpa2 and gpb5 and in describing the evolution of this environment in vertebrates. Conclusion Our model suggests that vertebrate gphα and gphβ were generated by a specific local duplication of the ancestral forms of gpa2 and gpb5, followed by a translocation of gphβ to a new environment whereas gphα was retained in the gpa2-gpb5 locus. The two rounds of whole genome duplication that occurred early in the evolution of vertebrates generated four paralogues of each gene but secondary gene losses or lineage specific duplications together with genomic rearrangements have resulted in the present organization of these genes, which differs between vertebrate lineages.

In humans, as in the other mammals, the neuroendocrine control of reproduction is ensured by the brain-pituitary gonadotropic axis. Multiple internal and environmental cues are integrated via brain neuronal networks, ultimately leading to the modulation of the activity of gonadotropin-releasing hormone (GnRH) neurons. The decapeptide GnRH is released into the hypothalamic-hypophysial portal blood system and stimulates the production of pituitary glycoprotein hormones, the two gonadotropins luteinizing hormone and follicle-stimulating hormone. A novel actor, the neuropeptide kisspeptin, acting upstream of GnRH, has attracted increasing attention in recent years. Other neuropeptides, such as gonadotropin-inhibiting hormone/RF-amide related peptide, and other members of the RF-amide peptide superfamily, as well as various nonpeptidic neuromediators such as dopamine and serotonin also provide a large panel of stimulatory or inhibitory regulators. This paper addresses the origin and evolution of the vertebrate gonadotropic axis. Brain-pituitary neuroendocrine axes are typical of vertebrates, the pituitary gland, mediator and amplifier of brain control on peripheral organs, being a vertebrate innovation. The paper reviews, from molecular and functional perspectives, the evolution across vertebrate radiation of some key actors of the vertebrate neuroendocrine control of reproduction and traces back their origin along the vertebrate lineage and in other metazoa before the emergence of vertebrates. A focus is given on how gene duplications, resulting from either local events or from whole genome duplication events, and followed by paralogous gene loss or conservation, might have shaped the evolutionary scenarios of current families of key actors of the gonadotropic axis.

Gonadotrope cell identity genes emerge in a stepwise process during mouse pituitary development. Cga, encoding for the α-subunit of TSH, LH, and FSH, is initially detected at E11.5 followed by Gnrhr and steroidogenic factor Sf1 at E13.5, specifying cells engaged in a gonadotrope cell fate. Lhb and Fshb appear at E16.5 and 17.5, respectively, typifying differentiated gonadotrope cells. Using the αT1-1, αT3-1 and LβT2 cell lines recapitulating these stages of gonadotrope differentiation, DNA methylation at Gnrhr and Sf1 was investigated. Regulatory regions were found hypermethylated in progenitor αT1-1 cells and hypomethylated in differentiated LβT2 cells. Abundance of RNA polymerase II together with active histone modifications including H3K4me1, H3K4me3, and H3K27ac were strictly correlated with DNA hypomethylation. Analyses of epigenomic modifications and chromatin accessibility were further extended to Isl1, Lhx3, Gata2, and Pitx2, highlighting alternative usages of specific regulatory gene domains in progenitor αT1-1, immature αT3-1, and mature LβT2 gonadotrope cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The GnRH neurohormone is the main activator of the pituitary gonadotropins, LH and FSH. Here we investigated the contribution of microRNAs in mediating GnRH activation. We first established that miR-125b targets several actors of Gαq/11 signalling pathway, without altering Gαs pathway. We then showed that a Gαs-mediated, PKA-dependent phosphorylation of NSun2 methyltransferase leads to miR-125b methylation and thereby induces its down-regulation. We demonstrated that NSun2 mRNA is a target of miR-132 and that NSun2 may be inactivated by the PP1α phosphatase. Time-course analysis of GnRH treatment revealed an initial NSun2-dependent down-regulation of miR-125b with consecutive up-regulation of LH and FSH expression. Increase of miR-132 and of the catalytic subunit of PP1α then contributed to NSun2 inactivation and to the return of miR-125b to its steady-state level. The Gαq/11-dependent pathway was thus again silenced, provoking the down-regulation of LH, FSH and miR-132. Overall, this study reveals that a regulatory loop that tends to maintain or restore high and low levels of miR-125b and miR-132, respectively, is responsible for gonadotrope cells desensitization to sustained GnRH. A dysregulation of this loop might be responsible for the inverted dynamics of these two miRNAs reported in several neuronal and non-neuronal pathologies.

GnRH plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. However, the potential involvement of microRNAs (miRNAs) on this activation has still to be explored. In this study, we investigated the role of miRNA-132 and miRNA-212, two tandemly expressed miRNAs that target the same transcripts, on GnRH-induced FSH expression. We first showed that the GnRH stimulation of FSH secretion was reduced and Fshb mRNA abolished by blocking miR-132/212 action in rat pituitary cells. In mouse LβT2 gonadotrope cells, the GnRH stimulation of Fshb mRNA was also demonstrated to be dependent on miR-132/212 and reproduced by overexpressing one or both miRNAs. We then showed that the miR-132/212-mediated action of GnRH involved a posttranscriptional decrease of sirtuin 1 (SIRT1) deacetylase. The lower level of SIRT1 deacetylase correlated with an increase in the acetylated form of Forkhead Box O1 (FOXO1), a transcriptional repressor of Fshb. Interestingly, we show that the acetylated mimicking mutant of FOXO1 was localized outside the nucleus, thus alleviating its repressive effect on Fshb transcription. Overall, we demonstrate that the GnRH stimulation of Fshb expression is dependent on miR-132/212 and involves a SIRT1-FOXO1 pathway. This is the first demonstration of an obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene.

Thyroid-stimulating hormone (TSH) is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

Background: The neuropeptide Kiss and its receptor KissR are key-actors in the brain control of reproduction in mammals, where they are responsible for the stimulation of the activity of GnRH neurones. Investigation in other vertebrates revealed up to 3 Kiss and 4 KissR paralogs, originating from the two rounds of whole genome duplication in early vertebrates. In contrast, the absence of Kiss and KissR has been suggested in birds, as no homologs of these genes could be found in current genomic databases. This study aims at addressing the question of the existence, from an evolutionary perspective, of the Kisspeptin system in birds. It provides the first large-scale investigation of the Kisspeptin system in the sauropsid lineage, including ophidian, chelonian, crocodilian, and avian lineages., Results: Sauropsid Kiss and KissR genes were predicted from multiple genome and transcriptome databases by TBLASTN. Phylogenetic and syntenic analyses were performed to classify predicted sauropsid Kiss and KissR genes and to re-construct the evolutionary scenarios of both gene families across the sauropsid radiation.Genome search, phylogenetic and synteny analyses, demonstrated the presence of two Kiss genes (Kiss1 and Kiss2 types) and of two KissR genes (KissR1 and KissR4 types) in the sauropsid lineage. These four genes, also present in the mammalian lineage, would have been inherited from their common amniote ancestor. In contrast, synteny analyses supported that the other Kiss and KissR paralogs are missing in sauropsids as in mammals, indicating their absence in the amniote lineage. Among sauropsids, in the avian lineage, we demonstrated the existence of a Kiss2-like gene in three bird genomes. The divergence of these avian Kiss2-like sequences from those of other vertebrates, as well as their absence in the genomes of some other birds, revealed the processes of Kiss2 gene degeneration and loss in the avian lineage., Conclusion: These findings contribute to trace back the evolutionary history of the Kisspeptin system in amniotes and sauropsids, and provide the first molecular evidence of the existence and fate of a Kiss gene in birds.

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH β-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

Background and Aims: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary., Methods: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created., Results: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context., Conclusion: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology., (Copyright © 2012 S. Karger AG, Basel.)

The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.

Background: The three vertebrate pituitary glycoprotein hormones (GPH) are heterodimers of a common α and a specific β subunit. In human, they are located on different chromosomes but in a similar genomic environment. We took advantage of the availability of genomic and EST data from two cartilaginous fish species as well as from two lamprey species to identify their repertoire of neurotrophin, lin7 and KCNA gene family members which are in the close environment of gphβ. Gphα and gphβ are absent outside vertebrates but are related to two genes present in both protostomes and deuterostomes that were named gpa2 and gpb5. Genomic organization and functional characteristics of their protein products suggested that gphα and gphβ might have been generated concomitantly by a duplication of gpa2 and gpb5 just prior to the radiation of vertebrates. To have a better insight into this process we used new genomic resources and tools to characterize the ancestral environment before the duplication occurred., Results: An almost similar repertoire of genes was characterized in cartilaginous fishes as in tetrapods. Data in lampreys are either incomplete or the result of specific duplications and/or deletions but a scenario for the evolution of this genomic environment in vertebrates could be proposed. A number of genes were identified in the amphioxus genome that helped in reconstructing the ancestral environment of gpa2 and gpb5 and in describing the evolution of this environment in vertebrates., Conclusion: Our model suggests that vertebrate gphα and gphβ were generated by a specific local duplication of the ancestral forms of gpa2 and gpb5, followed by a translocation of gphβ to a new environment whereas gphα was retained in the gpa2-gpb5 locus. The two rounds of whole genome duplication that occurred early in the evolution of vertebrates generated four paralogues of each gene but secondary gene losses or lineage specific duplications together with genomic rearrangements have resulted in the present organization of these genes, which differs between vertebrate lineages.

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.

The glycoprotein hormone alpha subunit and two beta subunits were cloned from the ventral lobe of the pituitary gland of an elasmobranch fish, Scyliorhinus canicula. The mature alpha subunit was 96 amino acids long and showed 64-76 amino acid residues in common with alpha subunit sequences of representatives of sarcopterygians (tetrapods and dipnoi) and actinopterigyans (chondrostei and teleostei). The Scyliorhinus beta 1 subunit was 115 amino acid long and had characteristics specific to FSH beta subunits and, in particular, the two potential N-linked glycosylation sites in conserved positions. The beta 2 sequence was 112 amino acids long. The Scyliorhinus beta 2 subunit had only one potential N-linked glycosylation site at the same position as that in LH. None of the two beta subunits from Scyliorhinus displayed the two amino acid insertions shared by TSH beta subunit sequences between the fifth and the sixth cysteines as compared to actinopterygian and sarcopterygian gonadotropins. These data indicate that Scyliorhinus beta 1 and beta 2 subunits are orthologous to FSH and LH beta subunits, respectively. It is concluded that the two FSH and LH lineages were already individualized at the emergence of chondrichthyans.

The beta subunits of the two gonadotropins (GTH1 and GTH2) and of the thyroid-stimulating hormone (TSH) of a chondrostean fish, Acipenser baeri, were cloned. These new sequences and selected representative members of beta subunits of vertebrate glycoprotein hormones, including tetrapod follicle-stimulating hormones (FSH) and luteinizing hormones (LH), allowed us to infer the phylogenetic relationships within this family. Both distance matrix and maximum parsimony methods were used on both nucleotide and amino acid sequences, with bootstrapping evaluation over 1000 replicates. The four trees obtained had highly similar topologies. In each case, three monophylogenetic lineages, TSH, GTH1-FSH, and GTH2-LH were clearly identified. The three monophylogenetic lineages were supported by 21-23 specific characters at the amino acid level, out of a total of 121 characters. The resolved topologies within each monophyletic hormone cluster were congruent with the known phylogenetic relationships between the related species. The inferred parental relationships within gonadotropins are in agreement with data concerning their biological functions. The present study demonstrates that GTH1 and GTH2 are the actinopterygian homologues of tetrapod FSH and LH, respectively.

The aim of this study was to determine the causes of the high heterogeneity, in the number and the length, of the thyrotropin (TSH) beta mRNA in the European eel. Northern blot analysis showed that removal of the poly(A) tail did not affect this heterogeneity. PCR amplification on reverse-transcribed pituitary RNAs (RT) showed the main source of heterogeneity to be a highly variable region in the 3' untranslated region (UTR). PCR amplification of the 3' UTR from RTs and genomic DNAs demonstrated that the high variability reflected polymorphism within the eel TSH beta gene. Isolation and sequencing of 3' UTR amplification fragments showed that the variable region comprised more or less exact repetitions of a 26-42-bp fragment. The number of repetitions varied from one allele to another. This variable region could be characterized as a minisatellite. In conclusion, instability of a minisatellite in the 3' UTR of the TSH beta gene generated the multiple and widely differing TSH beta mRNAs.

The effects of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) on alpha and thyroid-stimulating hormone (TSH) beta subunit mRNA pituitary levels were examined in a teleost, the European silver eel. Northern blot analysis showed that the number and length of mRNAs encoding TSH beta varied among individuals, a variability apparently not related to thyroidal status. When several bands were present, their intensities were summed for quantitative analysis. Increasing circulating thyroid hormones (THs) by implantation of T3 or T4 significantly decreased TSH beta mRNA levels. Depression of circulating THs by thiourea treatment increased alpha and TSH beta mRNA levels. In vitro studies showed that T3 and T4 decrease TSH beta mRNA levels in primary cultures of eel pituitary cells. In conclusion, in vivo and in vitro experiments indicate that T3 and T4 exert a negative feedback action on pituitary TSH beta mRNA level in the European eel and that this effect might be exerted, at least partly, through a direct action on the pituitary., (Copyright 1997 Academic Press. Copyright 1997 Academic Press)

Primary cultures of juvenile eel (Anguilla anguilla) pituitary cells were used to study the direct effects of sex steroids on gonadotropin (GtH-II) cell content and release (radioimmunoassay) as well as on mRNAs levels for alpha and GtH-II beta-subunits (dot-blot). Testosterone stimulated GtH-II production in a dose- and time-dependent manner by selectively increasing mRNAs for GtH-II beta-subunit but not alpha-subunit. This positive effect was also induced by non-aromatizable androgens (androstanediol and dihydrotestosterone) but not by estradiol, indicating an androgen-specific effect in the eel. The androgen-specific stimulation of eel GtH-II beta appears closer to the regulation of mammalian follicle stimulating hormone-beta (FSHbeta) than that of salmonid GtH-II beta or mammalian luteinizing hormone-beta (LHbeta)-subunits. Comparison with previous in vivo experiments suggests multiple sites of action of sex steroids on the brain-pituitary gonadotropic axis for the positive feedback on GtH-II synthesis in this juvenile fish.

We obtained the sequence of eel thyrotropin beta-subunit cDNA. About 1,100 bp long, it encodes a 147-amino acid protein including a 20-residue signal peptide. We analyse homologies between angTSH beta and the other known beta-subunits taking in account the ability of mammal gonadotropins (GTH) to stimulate teleost thyroid. A peptide corresponding to mammalian CAGYC (implicated in subunit association) is original and different from its eel beta GTH2 counterpart.

The gonadotrophic function of the European eel (Anguilla anguilla L.) at the silver stage is very weak: gonadotrophin-releasing hormone (GnRH) secretion is deficient and, moreover, dopamine inhibition overrides GnRH action. At the silver stage, oestradiol stimulates the biosynthesis of the type-II gonadotrophin (GTH-II). To study the molecular mechanism of this activation further, we examined the effect of testosterone and oestradiol administration on pituitary levels of mRNA encoding GTH-II alpha and beta subunits. Corresponding eel cDNA probes and Northern blot analysis were used. After 2 weeks, testosterone and oestradiol implantation resulted in a strong increase in mRNA encoding the GTH-II beta subunit (7-fold and 25-fold, respectively) and in a slight, but non-significant, rise in the a subunit mRNA level (1.8-fold and 1.5-fold, respectively). Co-implantation of these two steroids suggested a potentiation of their effects on the beta subunit (104-fold) while an additive effect was indicated on the alpha mRNA level (2.9-fold). Effects were detectable within 4 days and were maximal 4 weeks after implantation. These results indicate that in the European eel at the silver stage, gonadal steroids stimulate differentially the expression of GTH-II subunit genes at a pretranslational level.

A cDNA library constructed using mRNAs isolated from pituitary glands of estradiol-treated eels was screened with a cDNA fragment for the rat glycoprotein hormone alpha-subunit. Three out of 10,000 cDNA clones were revealed and subcloned in pUC13 for characterization and sequencing. All three had the same nucleotide sequence except for a single, silent change in the coding sequence for one of them, and for the location of the poly(A) tail. Analysis of the deduced amino acid sequence strongly suggests that these cDNA clones encode the precursor for the eel common glycoprotein hormone alpha-subunit. This precursor would therefore consist of a 93 amino acid apoprotein preceded by a 24 amino acid long signal peptide. Alignment with glycoprotein hormone alpha-subunits from fish and mammals reveals high homology, ranging from 60 to 90%. Particularly, the ten cysteines and the two putative N-linked glycosylation sites were at the same position. Comparison between fish and mammals shows also that two regions are highly conserved, comprising about half of the protein length. This high conservation rate through evolution argues for the importance of these regions in the conservation of biological properties of the alpha-subunits. In contrast, other regions are highly variable and could be responsible for the immunological specificity. Northern blot analysis of pituitary RNA from control and estradiol-treated eels showed that estradiol treatment strongly increases the pituitary content of mRNA encoding the glycoprotein hormone alpha-subunit.

Oestradiol treatment enhances type-II gonadotrophin (GTH-II) synthesis in the European eel (Anguilla anguilla) at the silver stage. As a first step in studying the molecular mechanisms involved in this stimulation, we cloned and characterized the cDNA encoding the beta subunit of eel GTH-II. A cDNA library was constituted in lambda gt10 from oestradiol-treated eels. It was screened using an oligodeoxyribonucleotide mixed probe designed to be complementary to a highly conserved region of cDNAs from several LH-related beta subunits. Several clones were obtained and four were subcloned in pUC13 and sequenced. The longest clones comprised a 420 bp coding sequence, plus 5' and 3' untranslated regions of 36 and 172 bp respectively. Comparison with GTH-II from other teleost fish permitted the localization of the putative cleavage site of a 24 amino acid signal peptide. The resulting 116 amino acid apopeptide had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 70-80% with GTH-II from other fish, 45% with LH from mammals and birds, 38% with mammalian FSH and only 35% with fish GTH-I. Preliminary results indicated a strong positive effect of oestradiol treatment on the level of the putative GTH-II beta-subunit mRNA. This supports our proposal that the European eel provides a suitable model for studying the positive regulation of gonadotrophin synthesis by gonadal steroids.

Testosterone (T) and 17 beta-estradiol (E2) plasma levels and metabolic clearance rates (MCR) were measured to investigate their ovarian production in immature silver eel. The dynamics of T and E2 metabolism were studied in catheterized eels using single injections of 0.2 to 0.5 microCi 3H-labeled steroid. The distribution volumes, biological half-life and MCR of nonconjugated tracers were calculated on the basis of a two-compartment model. At the end of the experiments, radioactivity was measured in different organs and tissues to localize the site of T and E2 catabolism. The volume of the inner compartment was 3.4% for T and E2. The outer compartment was larger for T (6.4%) than that for E2 (4.3%). The biological half-life was three to four times shorter for T (14.5 hr) than that for E2 (48.5 hr). The MCR for T (1.71 ml/kg body wt/hr) was higher than for E2 (0.51 ml/kg body wt/hr). Plasma levels were determined, using radioimmunoassay, on samples taken before injections of radiolabeled steroid. Free or protein-bound hormone levels were 0.12 and 0.31 ng/ml for T and E2, respectively. Conjugated T and E2 levels were, respectively, 0.13 and 0.23 ng/ml. Production rates were determined as the product of the MCR and the plasma concentration of the nonconjugated hormone. No significant differences were observed between the production rates of T and E2 (0.24 ng/kg body wt/hr). The liver was the principal site of metabolism for both hormones, which were excreted via the enterohepatic route. E2 injection gave rise to no metabolite in the plasma whereas after T injection a metabolite was produced, the concentration of which increased as a function of time. Its chromatographic properties were different from that of E2 or androstenedione, suggesting that no significant peripheral aromatization or 17-oxidoreduction occurs in the immature silver eel.

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.