Bruno Quérat

Phylogenetic Analysis of the Vertebrate Glycoprotein Hormone Family Including New Sequences of Sturgeon (Acipenser baeri) β Subunits of the Two Gonadotropins and the Thyroid-Stimulating Hormone

Abstract The β 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 β 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.

Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbetas (35-37%) than to LHbetas (26-30%), whereas sea bass LHbeta is more similar to LHbetas (40-53%) than to FSHbetas (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.

Distinct Expression Patterns of Glycoprotein Hormone-α2 and -β5 in a Basal Chordate Suggest Independent Developmental Functions

The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common alpha- and specific beta-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-alpha2 (GPA2) and-beta5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH-alpha and -beta. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.

Androgens stimulate gonadotropin-II b-subunit in eel pituitary cells in vitro

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 α and GtH-II β-subunits (dot-blot). Testosterone stimulated GtH-II production in a dose- and time-dependent manner by selectively increasing mRNAs for GtH-II β-subunit but not α-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 β appears closer to the regulation of mammalian follice stimulating hormone-β (FSHβ) than that of salmonid GtH-II β or mammalian luteinizing hormone-β (LHβ)-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.

Cloning and sequence analysis of the cDNA for the pituitary glycoprotein hormone α-subunit of the European eel

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.

Emergence and evolution of the glycoprotein hormone and neurotrophin gene families in vertebrates

BackgroundThe 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.ResultsAn 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.ConclusionOur 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.

Rapid Communication: A MicroRNA-132/212 Pathway Mediates GnRH Activation of FSH Expression

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.

Sequence and regulation of European eel prolactin mRNA

cDNA clones encoding the European eel (Anguilla anguilla L.) prolactin were isolated from a pituitary cDNA library constructed in gamma gt10, using a rainbow trout Prl cDNA fragment as a probe. Four different inserts were subcloned into the pGEM 3Z plasmid after PCR amplification. The 1082 bp-long nucleotide sequence revealed an open reading frame of 627 bp encoding a 24 amino acid-long signal peptide followed by a 185 amino acid-long mature protein. Comparison studies showed 60-70% homology with other known teleost fish prolactins and 30-45% with non-teleost fish, amphibian, reptilian, avian and mammalian prolactins. In situ hybridization studies using labelled prolactin RNA probe showed a strong signal in the rostral pars distalis of the pituitary gland. We next examined the physiological regulation of this prolactin synthesis in vivo using Northern blot analysis and prolactin cDNA probe labelled by random priming. The pituitary prolactin mRNA level was markedly decreased 3 weeks after transfer of eels from freshwater to sea water. Implants of thyroid hormones left for up to three weeks were ineffective on prolactin mRNA. Estradiol administered as implant, alone or in combination with 500 micrograms testosterone, was also unable to significantly alter the pituitary mRNA level for prolactin in the freshwater silver eels whatever the dose used (20-500 micrograms) and whatever the duration of treatment (from 4 days to 10 weeks).

Looking for the bird Kiss: evolutionary scenario in sauropsids

BackgroundThe 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.ResultsSauropsid 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.ConclusionThese 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-Induced Silencing and LIM-Homeodomain Protein-Induced Activation Are Mediated by a Bi-Functional Response Element in the Rat GnRH Receptor Gene

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.