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

<p>Abstract</p> <p>Background</p> <p>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 <it>gphβ</it>. <it>Gphα </it>and <it>gphβ </it>are absent outside vertebrates but are related to two genes present in both protostomes and deuterostomes that were named <it>gpa2 </it>and <it>gpb5</it>. Genomic organization and functional characteristics of their protein products suggested that <it>gphα </it>and <it>gphβ </it>might have been generated concomitantly by a duplication of <it>gpa2 </it>and <it>gpb5 </it>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.</p> <p>Results</p> <p>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 <it>gpa2 </it>and <it>gpb5 </it>and in describing the evolution of this environment in vertebrates.</p> <p>Conclusion</p> <p>Our model suggests that vertebrate <it>gphα </it>and <it>gphβ </it>were generated by a specific local duplication of the ancestral forms of <it>gpa2 </it>and <it>gpb5</it>, followed by a translocation of <it>gphβ </it>to a new environment whereas <it>gphα </it>was retained in the <it>gpa2</it>-<it>gpb5 </it>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.</p

Direct Evidence for the Co-Expression of URP and GnRH in a Sub-Population of Rat Hypothalamic Neurones: Anatomical and Functional Correlation

Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence

Androgen receptor signaling regulates follicular growth and steroidogenesis in interaction with gonadotropins in the ovary during mini-puberty in mice

In females, androgens contribute to ovarian diseases such as polycystic ovarian syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Therefore, we analyzed the potential actions of androgens on the ovary and their possible interaction with gonadotropins during this period in mice. We used molecular-based studies and pharmacological approaches in vivo and on cultured ovaries. We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Overall, this work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, at the interplay with LH and FSH signaling. Its early action may, thus, contribute to the implementation of early ovarian function with possible impacts on reproductive function

Nanoparticules et fonction de reproduction

L’objectif principal du projet "NANOVHYP" était d’évaluer la toxicité des nanoparticules de noir de carbone sur la fonction endocrine de l’hypophyse et des gonades femelles, les ovaires. Les modèles utilisés étaient des cellules ou des souris exposées par instillation intra-trachéale, simulant l’inhalation

Effet du male sur la secretion gonadotrope (LH) de la brebis (Ovis Aries L). Analyse de l'interaction olfactive.

Diplôme : Dr. d'Universit

Effet du male sur la secretion gonadotrope (LH) de la brebis (Ovis Aries L). Analyse de l'interaction olfactive.

Diplôme : Dr. d'Universit

Nanoparticules et fonction de reproduction

L’objectif principal du projet "NANOVHYP" était d’évaluer la toxicité des nanoparticules de noir de carbone sur la fonction endocrine de l’hypophyse et des gonades femelles, les ovaires. Les modèles utilisés étaient des cellules ou des souris exposées par instillation intra-trachéale, simulant l’inhalation

Étude des mécanismes contrôlant l'efficacité et la spécificité de la signalisation du récepteur de la GnRH (identification et rôle de la protéine partenaire SET)

La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l intermédiaire d un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d interaction du RGnRH, jusqu alors inconnus, avec l idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d identifier le premier partenaire d interaction du RGnRH : la protéine SET. Par des expériences de GST pull down , nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l AMPc la première démonstration d un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d amplifier l action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l expression de SET est fortement augmentée dans l hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien.Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Etude des mécanismes de la désensibilisation beta-Adrénergique dans le myomètre de ratte en fin de gestation (implication des GRK (G protein-coupled receptor kinase))

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Effect of short exposure to the ram on later reactivity of anoestrous ewes to the male effect

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