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Dans l’hypophyse, Pax7 spécifie l’état du lobe intermédiaire grâce à son action pionnière. Cette action implique dans un premier temps une liaison de cette protéine à certains sites d’hétérochromatine et, par la suite, le remodelage de cette hétérochromatine en euchromatine. Le remodelage de l’épigénome par Pax7 permet au facteur de transcription Tpit d’entrainer l’activation du transcriptome mélanotrope par opposition au destin hypophysaire corticotrope entraîné par Tpit en l’absence de Pax7. Pour étudier l’action pionnière de Pax7, différents membres du laboratoire Drouin ont utilisé un modèle de cellules corticotropes. L’expression ectopique du facteur pionnier Pax7 dans ces cellules permet leur transition d’un phénotype corticotrope à mélanotrope. En plus de l’isoforme prédominante de Pax7 exprimée dans hypophyse avec lequel les travaux de Budry et Mayran ont étaient faits, il existe trois autres isoformes de Pax7 qui diffèrent par quelques acides aminés (Q et GL) dans le domaine PAIRED liant l’ADN. Nous voulions savoir quels étaient les domaines protéiques de Pax7 impliqués pour son activité pionnière. Nous avons d’abord étudié les propriétés des différents isoformes naturelles de Pax7 présentant des variations dans le domaine PAIRED que ce soient pour leurs capacités de facteur transcription ou de facteur pionnier dans un modèle de cellules de corticotrope. Nous avons constaté que les quatre isoformes de Pax7, ainsi que Pax3, le facteur le plus homologue à Pax7 dans la famille Pax, présentent une capacité similaire à lier la chromatine fermée. Cependant, Pax3 et les isoformes de Pax7 (GL+) sont incapables d’ouvrir la chromatine sur un sous-groupe d’enhancers pionniers nécessaire à l’activation du transcriptome mélanotrope. Par la suite, par analyse structure-fonction, nous avons identifié une région de Pax7 importante pour sa liaison à soixante pour cent des sites présents dans l’hétérochromatine qui sont ciblés par l’action pionnière et une région importante pour l’ouverture de chromatine aux sites des pionniers sensibles à la variation GL. Collectivement, nos données ont montré que les acides aminés GL dans le domaine PAIRED en N-terminal et la partie C-terminale de la protéine Pax7 sont critiques pour l’ouverture de la chromatine à des enhancers spécifiques présents dans l’hétérochromatine dont l’activation est nécessaire au destin mélanotrope. D’autre part, les analyses de structure-fonctions nous ont permis d’identifier une région critique de Pax7 nécessaire à son interaction avec l’hétérochromatine sur ces même enhancers., In the pituitary, Pax7 specifies the intermediate lobe state through a pioneer factor action that involves the binding of this protein to certain heterochromatin sites and, subsequently, the remodeling of this heterochromatin into euchromatin. Epigenome remodeling by Pax7 thus allows the transcription factor Tpit to determine the melanotrope fate as opposed to the corticotrope pituitary fate that is driven by Tpit in absence of Pax7. To study the pioneering action of Pax7, Budry et al, and Mayran et al, used a model of corticotrope cells which, following the expression of Pax7, transit from a corticotrope phenotype to melanotic phenotype thanks to its pioneering action. In addition to the predominant Pax7 isoform expressed in pituitary with which the work of Budry and Mayran were done, there are three other Pax7 isoforms that differ by a few amino acids (Q and GL) in the DNA-binding paired domain. We wanted to know what protein domains of Pax7 was involved in its pioneering activity. We have investigated the properties of the different Pax7 isoforms with respect to their transcriptional and pioneer capabilities in a model of corticotrope cells. We found that all four Pax7 isoforms together with the closely related factor Pax3, exhibit a similar ability to bind closed chromatin. However, Pax3 and the isoforms of Pax7 (GL+) failed to open the chromatin at a subset of enhancer necessary to activate the melanotrope transcriptome. In addition, we have identified by structure-function analysis a region of Pax7 that appears important for Pax7 binding at 60 percent of heterochromatin sites that are targeted for pioneering and one region important for chromatin opening to the pioneer sites GL-sensible. Collectively, our data suggest that the amino acids GL in the paired domain and the C-terminal part are critical for the chromatin opening to heterochromatin sites necessary to melanotrope transcriptome. On the other hand, structure-functions analyses allowed us to identify a critical region of Pax7 required for the interaction with heterochromatin on these same enhancers.

International audience; We report a case of malignant melanoma revealed by a metastasis to the pituitary gland. The tumor was misdiagnosed as a pituitary adenoma and aggressive transsphenoidal surgery was complicated by a cerebrospinal fluid fistula. Nine weeks later, the patient presented multiple leptomeningeal and brain metastases spreading from the sellar region. Regarding these observations, we conducted a systematic review of the literature in order to investigate clinicoradiological features that should lead clinicians to suspect pituitary metastasis and how it should impact the surgical management.

International audience; Hypercortisolism usually occurs in women of childbearing age. Even if hypercortisolism theoreticallyleads to infertility, due to altered mechanisms of GnRH secretion, pregnancy can happen.Endocrinologists should be aware of the management of this situation in this context, as it may leadto serious comorbidities for both the mother (gestational diabetes, hypertension...) and the fetus(intra-uterine growth retardation, early birth...). Cushing’s syndrome can also happen duringpregnancy: the comorbidities are the same, but the diagnosis is usually very difficult to perform dueto hormonal changes especially due to hyperestrogenism. This review will detail the mechanismsleading to infertility in women with Cushing’s syndrome, the risks of hypercortisolism duringpregnancy, the methods to diagnose Cushing’s syndrome during pregnancy, and the methods ofcontraception.; Le syndrome de Cushing se définit par un hypercortisolisme endogène ou exogène. L’augmentationde sécrétion du cortisol va avoir un impact négatif sur la sécrétion pulsatile de GnRH.L’hyperandrogénie pouvant être associée va également avoir un impact sur la GnRH, ces 2mécanismes aboutissant à un sur-risque d’infertilité. En cas de grossesse survenant chez unepatiente présentant un syndrome de Cushing, de nombreuses conséquences vont pourvoir êtreobservées chez la mère (avec un sur-risque de diabète gestationnel, hypertension artérielle,éclampsie... en sus des complications propres à l’hypercortisolisme) et chez le fœtus (retard decroissance intra-utérin, prématurité...). Il s’agit cependant d’un évènement rare, de diagnosticdifficile, du fait de nombreuses modifications hormonales induites par la grossesse sur le cortisol.L’objectif de cette revue est de détailler les mécanismes de l’infertilité, les méthodes diagnostiquesde syndrome de Cushing pendant la grossesse, les conséquences materno-fœtales, ainsi que lesmoyens de contraception pouvant être proposés.

Le craniopharyngiome est une tumeur épithéliale bénigne prenant naissance dans le cerveau, plus précisément au niveau de l’hypophyse ou de la tige pituitaire. Cette maladie bien que rare, affecte une personne sur cinquante mille. Elle peut être découverte à n’importe quel âge de la vie puisque cette pathologie silencieuse à ses débuts, passe souvent inaperçue en raison de symptômes peu spécifiques. Il s’agit toutefois d’une maladie grave puisqu’elle peut entrainer à terme des séquelles endocriniennes, visuelles, ou neuro intellectuelles. Le craniopharyngiome nécessite une prise en charge à la fois médicale et thérapeutique. Dans un premier temps, le traitement consiste en l’exérèse la plus complète possible de la tumeur mais la proximité immédiate de l’hypophyse avec des structures essentielles en fait une chirurgie très délicate et non dénuée de risque. Outre les risques inhérents à l’opération, le retrait de la tumeur conduit à un dysfonctionnement hypothalamo-hypophysaire plus ou moins complet.

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1&ndash, 2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary L&beta, T2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, &beta, catenin activation and mouse luteinizing-hormone &beta, encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and L&beta, T2 cells. Cetrorelix inhibited the 3 &times, EC50 GnRH-activated calcium signaling at concentrations of 1 nM&ndash, 1 &micro, M, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM&ndash, M range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in L&beta, T2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 &micro, M GnRH-induced &beta, catenin activation, Lhb gene expression increase occurring upon L&beta, T2 cell treatment by 1 &micro, M GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

The anterior pituitary is a master endocrine gland producing 6 hormones (GH, TSH, ACTH, PRL, FSH and LH) that regulate essential life processes such as growth (GH), metabolism (TSH), stress response (ACTH), puberty and reproduction (FSH/LH and PRL), thyroid function (TSH) and lactation (PRL). In the human, mutations in SHH, BMP4 and FGF8 genes encoding morphogenes and GLI2, LHX3, LHX4, HESXJ, SOX2 and SOX3 genes encoding transcription factors implicated in pituitary ontogenesis are responsible for isolated (IGHD) or combined (CPHD) pituitary hormone deficit(s) that can be syndromic. The incidence of those deficits is close to 1/8,000 births. So far, mutations reported in about thirty genes explain only 25% of the cases.The first goal of this thesis was to identify new genes responsible for pituitary hormone deficits through a candidate gene approach, and by whole-exome sequencing coupled with homozygosity mappini, using a large cohort of more than 3000 DNA samples collected by the laboratory since 1997. The candidate gene approach, led to the identification of variations in NOG, VAXJ and GSXJ, all involved in mouse pituitary ontogenesis, and we identified potentially key genes for pituitary development.The other aim of this work was to characterize the function of LHX4, GHRHR, GL/2 and GSXJ through the functional evaluation of the variants identified in the laboratory. The implication of new disease-causing genetic variants in LHX4, GHRHR and GL/2 allowed us to delineate the phenotypic spectrum associated with mutations for each gene, and also to precise the role of LHX4 in the complex molecular network required for proper pituitary development.A better understanding of the molecular mecanisms involved in pituitary formation should lead to new ways for management of this heterogenous condition, including genetic counseling and therapeutic decisions in affected families, L'antehypohyse est une glande endocrine maitresse produisant 6 hormones (GH, TSH, ACTH, PRL, FSH et LH) qui regulent des processus vitaux comme la croissance et le metabolisme (GH et TSH), la reponse au stress (ACTH), la puberte et la reproduction (FSH, LH et PRL), la fonction thyroidienne (TSH) et la lactation (PRL). Chez l'Homme, des mutations des genes SHH, BMP4 et FGF8, codant pour des morphogenes, et de GLI2, LHX3, LHX4, HESXJ, SOX2 et SOX3, codant pour des facteurs de transcription impliques dans l'ontogenese pituitaire sont responsables de deficit(s) isole (IGHD) ou multiples (CPHD) en hormones antehypophysaires, pouvant etre syndromique(s). L'incidence de ces deficits est d'environ 1/8000 naissances. A ce jour, des mutations rapportees dans une trentaine de genes permettent d'expliquer cette affection chez seulement 25% des patients.Le premier objectif de ce travail de these etait d'identifier de nouveaux genes responsables de deficit antehypophysaire par une approche « genes candidats », et par des analyses d'exome couplees a une cartographie par homozygotie a partir d'une cohorte de plus de 3000 ADNs colliges par le laboratoire depuis 1997. L'approche « genes candidats » nous a permis de mettre en evidence des variations dans les genes NOG, VAX1 et GSX1, impliques dans l'ontogenese pituitaire murine, et identifier d'autres genes potentiellement cles dans le developpement de l'hypophyse. L'autre objectif etait de caracteriser la fonction de LHX4, GHRHR, GLI2 et GSX1 a travers l'etude fonctionnelle des variants identifies au laboratoire. L'implication de nouveaux variants genetiques de LHX4, GHRHR et GLI2 a permis de preciser le spectre phenotypique associe aux mutations de chacun de ces genes, et aussi de preciser le role de LHX4 dans le reseau moleculaire complexe necessaire a la formation de l'hypophyse. La comprehension des mecanismes moleculaires a l'origine du developpement pituitaire devrait permettre a terme une meilleure prise en charge de cette pathologie tres heterogene, incluant le conseil genetique des familles concernees et le traitement des patients

Remerciements :INRA, UMR PRC 0085, Plateforme CIRE, 37380 NouzillyStephane Fabre and Alan McNeilly for helpful discussionYves Combarnous for helpful suggestions, Maxime Capelle and Marie Champion for technical assistanceElodie Chaillou for critical reading of the manuscript; Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance (SPR) and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). SPR and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C (VWC) domain of TSP-1 is likely responsible for this BMP-2/4 binding activity, an assertion based on sequence similarity that TSP-1 shares with the VWC domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via the blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

The anterior pituitary is a master endocrine gland producing 6 hormones (GH, TSH, ACTH, PRL, FSH and LH) that regulate essential life processes such as growth (GH), metabolism (TSH), stress response (ACTH), puberty and reproduction (FSH/LH and PRL), thyroid function (TSH) and lactation (PRL). In the human, mutations in SHH, BMP4 and FGF8 genes encoding morphogenes and GLI2, LHX3, LHX4, HESXJ, SOX2 and SOX3 genes encoding transcription factors implicated in pituitary ontogenesis are responsible for isolated (IGHD) or combined (CPHD) pituitary hormone deficit(s) that can be syndromic. The incidence of those deficits is close to 1/8,000 births. So far, mutations reported in about thirty genes explain only 25% of the cases.The first goal of this thesis was to identify new genes responsible for pituitary hormone deficits through a candidate gene approach, and by whole-exome sequencing coupled with homozygosity mappini, using a large cohort of more than 3000 DNA samples collected by the laboratory since 1997. The candidate gene approach, led to the identification of variations in NOG, VAXJ and GSXJ, all involved in mouse pituitary ontogenesis, and we identified potentially key genes for pituitary development.The other aim of this work was to characterize the function of LHX4, GHRHR, GL/2 and GSXJ through the functional evaluation of the variants identified in the laboratory. The implication of new disease-causing genetic variants in LHX4, GHRHR and GL/2 allowed us to delineate the phenotypic spectrum associated with mutations for each gene, and also to precise the role of LHX4 in the complex molecular network required for proper pituitary development.A better understanding of the molecular mecanisms involved in pituitary formation should lead to new ways for management of this heterogenous condition, including genetic counseling and therapeutic decisions in affected families, L'antehypohyse est une glande endocrine maitresse produisant 6 hormones (GH, TSH, ACTH, PRL, FSH et LH) qui regulent des processus vitaux comme la croissance et le metabolisme (GH et TSH), la reponse au stress (ACTH), la puberte et la reproduction (FSH, LH et PRL), la fonction thyroidienne (TSH) et la lactation (PRL). Chez l'Homme, des mutations des genes SHH, BMP4 et FGF8, codant pour des morphogenes, et de GLI2, LHX3, LHX4, HESXJ, SOX2 et SOX3, codant pour des facteurs de transcription impliques dans l'ontogenese pituitaire sont responsables de deficit(s) isole (IGHD) ou multiples (CPHD) en hormones antehypophysaires, pouvant etre syndromique(s). L'incidence de ces deficits est d'environ 1/8000 naissances. A ce jour, des mutations rapportees dans une trentaine de genes permettent d'expliquer cette affection chez seulement 25% des patients.Le premier objectif de ce travail de these etait d'identifier de nouveaux genes responsables de deficit antehypophysaire par une approche « genes candidats », et par des analyses d'exome couplees a une cartographie par homozygotie a partir d'une cohorte de plus de 3000 ADNs colliges par le laboratoire depuis 1997. L'approche « genes candidats » nous a permis de mettre en evidence des variations dans les genes NOG, VAX1 et GSX1, impliques dans l'ontogenese pituitaire murine, et identifier d'autres genes potentiellement cles dans le developpement de l'hypophyse. L'autre objectif etait de caracteriser la fonction de LHX4, GHRHR, GLI2 et GSX1 a travers l'etude fonctionnelle des variants identifies au laboratoire. L'implication de nouveaux variants genetiques de LHX4, GHRHR et GLI2 a permis de preciser le spectre phenotypique associe aux mutations de chacun de ces genes, et aussi de preciser le role de LHX4 dans le reseau moleculaire complexe necessaire a la formation de l'hypophyse. La comprehension des mecanismes moleculaires a l'origine du developpement pituitaire devrait permettre a terme une meilleure prise en charge de cette pathologie tres heterogene, incluant le conseil genetique des familles concernees et le traitement des patients

International audience; Visfatin and resistin appear to interfere with reproduction in the gonads, but their potential action at the hypothalamic-pituitary level is not yet known. The aim of the present study was to investigate the mRNA and protein expression of these adipokines in murine gonadotroph cells and to analyse the effects of different concentrations of recombinant mouse visfatin and resistin (0.01, 0.1, 1 and 10 ng mL(-1)) on LH secretion and signalling pathways in L beta T2 cells and/or in primary female mouse pituitary cells. Both visfatin and resistin mRNA and protein were found in vivo in gonadotroph cells. In contrast with resistin, the primary tissue source of visfatin in the mouse was the skeletal muscle, and not adipose tissue. Visfatin and resistin both decreased LH secretion from L beta T2 cells after 24 h exposure of cells (P < 0.03). These results were confirmed for resistin in primary cell culture (P < 0.05). Both visfatin (1 ng mL(-1)) and resistin (1 ng mL(-1)) increased AMP-activated protein kinase a phosphorylation in L beta T2 cells after 5 or 10 min treatment, up to 60 min (P < 0.04). Extracellular signal-regulated kinase 1/2 phosphorylation was transiently increased only after 5 min resistin (1 ng mL(-1)) treatment (P < 0.01). In conclusion, visfatin and resistin are expressed in gonadotroph cells and they may affect mouse female fertility by regulating LH secretion at the level of the pituitary.

Transgenic female mice overexpressing the hCGβ subunit (hCGβ+) and producing elevated levels of luteinizing hormone (LH)/hCG bioactivity present as young adults with enhanced ovarian steroidogenesis, precocious puberty, and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels, and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor-deficient (PRLR−/−) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGβ+ and PRLR−/− mice. The double-mutant hCGβ+/PRLR−/− females remained sterile with an ovarian phenotype similar to PRLR−/− mice, indicating that LH action, Lhcgr expression, and consequent luteinization are not possible without simultaneous PRL signaling. The high frequency of pituitary prolactinomas in PRLR−/− mice was not affected by transgenic hCGβ expression. In contrast, none of the hCGβ+/PRLR−/− females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGβ+ mice, was absent in double-mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signaling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.

Pituitary dysfunction is a late-delayed side effect of cranial radiotherapy. The object of this study was to evaluate radiation induced pituitary toxicity of proton beam therapy in a cohort of adult chondrosarcoma patients. The files of 113 patients were reviewed retrospectively. Mean age at the beginning of radiotherapy was 43 years old (18 to 76). Mean dose delivered to the tumor was 67 Gy and mean dose delivered to the pituitary gland was 59 Gy. Mean post radiotherapy follow up time was 7 years. 78 patients (69 %) presented with a pituitary dysfunction including hyperprolactinemia (40 %), thyrotrophic insufficiency (36 %), corticotropic insufficiency (31 %) and gonadotropic insufficiency (25 %). Pituitary anomalies were developed in more or less 4 years and no significant differences were shown between the axes for this delay. No cardiovascular risk factor was associated with pituitary insufficiency. Pituitary toxicity of radiotherapy is frequent after 4 years in this study. The high prevalence of hyperprolactinemia on diagnosis pinpoints a possible hypothalamic origin of post radiotherapy pituitary insufficiency. A prospective follow up is necessary to complete these observations.; L’insuffisance antéhypophysaire est une complication tardive de la radiothérapie des tumeurs cérébrales et de la base du crâne. L’objectif de cette étude était d’évaluer la toxicité antéhypophysaire dans une cohorte de patients adultes ayant eu une radiothérapie pour des chondrosarcomes de la base du crâne. Les dossiers de 113 patients ont été revus de façon rétrospective. L’âge moyen lors de la radiothérapie était de 43 ans (18 à 76). L’irradiation a délivré une dose moyenne de 67 Gy à la tumeur, et 59 Gy à l’hypophyse. La durée moyenne du suivi endocrinologique était de 7 ans (4 à 17). Une anomalie du bilan hypophysaire est survenue chez 78 patients (69%) à 4 ans de la radiothérapie. La prévalence des anomalies était de 40 % pour l’hyperprolactinémie, de 36 % pour l’insuffisance thyréotrope, de 31 % pour l’insuffisance corticotrope et 25 % pour l’insuffisance gonadotrope. Le délai d’apparition des anomalies pour les différents axes n’était pas significativement différent. En analyse multivariée, les facteurs de risque pouvant augmenter l’atteinte artérielle secondaire à la radiothérapie (sexe, âge, tabagisme, hypertension artérielle, dyslipidémie ou diabète sucré) n’étaient pas associés à un sur-risque d’insuffisance hypophysaire. La toxicité hypothalamo-hypophysaire de la radiothérapie est donc fréquente après 4 ans dans cette étude. Du fait de l’hyperprolactinémie se pose la question d’une atteinte hypothalamique première. Un suivi prospectif est nécessaire pour confirmer ces résultats et proposer des recommandations.

International audience; Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258 micro g/L of clotrimazole for 7 days. Expression of pituitary gonadotropins ß subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesisrelated genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly upregulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and in inducing the expression of Fsh-regulated igf3 in Sertoli cells.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

In order to determine the time of puerperium when the pituitary- ovarian axis of the suckler zebu cow (Bos indicus) is functional, a single dose of 100 μg synthetic GnRH was injected intramuscularly in 60 suckler zebu cows divided into six groups. The animals were treated 7, 10, 15, 20, 25 and 30 days after parturition. Ten milliliters of milk were sampled from each animal as follows: before GnRH injection (A: 1st sample range), on days 5, 6, 7, 8 (B: 2nd sample range) and 20, 21, 22, 23, 24 (C: 3rd sample range) after GnRH injection. In each group, samples obtained before GnRH injection were used as control. Overall, 600 milk samples were used to determine progesterone concentrations by radioimmunoassay. Among the animals treated with GnRH 15 days after parturition, 60% responded to the treatment with a mean progesterone concentration of 2.67 ± 1.89 nmol/L. This response increased along with the length of the post-partum period. Thirty days after calving, all animals responded well to the treatment (mean progesterone concentration: 4.09 ± 1.09 nmol/L) and presented a significant reduction (p < 0.05) in the interval from calving to conception compared to animals treated on days 7 and 10 after calving. This study shows that, as early as 15 days post-partum, the pituitary-ovarian axis of suckler zebu cows is sensitive to exogenous GnRH and that such treatment may improve reproductive performances.

Gonadotrope Zellen in der anterioren Hypophyse sind innerhalb der Hypothalamus- Hypophysen-Gonaden-Achse von besonderer Bedeutung, da diese die Kommunikation zwischen dem Gehirn und den Gonaden vermitteln und damit eine funktionelle Verbindung innerhalb der Reproduktionsachse darstellen. Neuere Ergebnisse deuten darauf hin, dass gonadotrope Zellen weiblicher Mäuse in Form eines Netzwerkes organisiert sind, das Plastizität zeigt und sich dadurch den veränderten endokrinen Bedingungen unterschiedlicher physiologischer Zustände anpasst. Allerdings ist bisher wenig darüber bekannt, wie sich diese Zellen auf molekularer Ebene den unterschiedlichen hormonellen Bedingungen funktionell anpassen. In dieser Dissertation habe ich eine binäre genetische Strategie benutzt, die das Markieren der gonadotropen Zellen von Mäusen mit einem Fluoreszenzprotein erlaubt. Dieses Mausmodell ermöglichte mir das Anreichern der gonadotropen Zellen unter Verwendung der fluoreszenzaktivierenden Zellsortierung und anschließender mRNA-Sequenzierung. Unter Verwendung dieser Methode habe ich die Expressionsprofile der gonadotropen Zellen unter Berücksichtigung des Geschlechtes und in verschiedenen Entwicklungs- und Hormonstadien analysiert und miteinander verglichen werden. Hierzu habe ich das Transkriptom gonadotroper Zellen aus juvenilen Männchen bzw. Weibchen, aus zyklierenden Weibchen im Diestrus bzw. im Proestrus, und aus laktierenden Weibchen bzw. aus adulten Männchen untersucht. meine Daten zeigen, dass gonadotrope Zellen in jedem analysierten Stadium ein einzigartiges Expressionsprofil mit 100-500 Genen, die nur in einem bestimmten Stadium exprimiert werden, aufweisen. Zudem konnte ich zeigen, dass in diesen Zellen umfangreiche Genexpressionsprofiländerungen mit bis zu 2200 differenziell exprimierten Genen während der Entwicklung und beim Vergleich interschiedlicher Hormonprofile stattfinden. Unter Verwendung der Kyoto Enzyklopädie (Gen und Genomanalyse) konnte ich zeigen, dass von den differentiell exprimierten Genen jene prominent exprimiert waren, die unter anderem im GnRH-Signalweg, im Ca2+-Signalweg und im MAPK-Signalweg eine wichtige Rolle einnehmen. Meine Daten zeigen einen hohen Grad an molekularer Plastizität innerhalb der gonadotropen Zellpopulation. Neuere Ergebnisse deuten auf einen Zusammenhang zwischen den Transienten Rezeptor Potential (TRP) Kationenkanäle und der Hypophysenphysiologie hin. Bisher ist jedoch nicht bekannt, ob und wie TRP-Kanäle zur Funktion der gonadotropen Zellen beitragen. Meine Untersuchungen zeigen, dass 14 von 28 TRP-Kanälen, die im gesamten Mausgenom kodiert sind, in den gonadotopen Zellen der Maus exprimiert werden, wobei die höchsten Expressionswerte für TRPC5 bei juvenilen Weibchen gefunden wurden. Die TRP-Kanal-Expression in diesen Zellen weisteine beträchtliche Plastizität auf und hängt sowohl vom Geschlecht als auch vom Entwicklungs- und Hormonstatus des Tieres ab. Wir kombinierten dann verschiedene genetische Strategien, um die TRPC5-Kanalfunktion in den gonadotropen Zellen von juvenilen Weibchen zu charakterisieren. Wir konnten zeigen, dass der TRPC5-Agonist Englerin A ein zytosolisches Ca2+-Signal und einen Zellstrom in diesen Zellen aktiviert, der bei TRPC5-defizienten Mäusen fehlt. Weiterhin konnten wir eine TRPC5-Aktivierung in den gonadotropen Zellen nachweisen, die durch die Stimulation des GnRHR induziert wird. Bisher konnte ich allerdings keine Abnormalitäten in reproduktionsphysiologischen Parametern wie dem Einsetzen der Pubertät wie auch dem Körpergewicht in TRPC5-defizienten weiblichen Mäusen im Vergleich zu den Kontrollen feststellen. Die Ten-eleven translocation (TET) Enzyme nehmen eine zentrale und komplexe Rolle in der Festlegung der Genexpressionsänderung während der Entwicklung ein. Wir zeigen hier, dass Tet1, Tet2 und Tet3 in murinen gonadotropen Zellen exprimiert werden und dass Tet1 die Expression der luteinisierenden Hormon β-Untereinheit (Lhb) unterdrückt, während Tet2 die Expression des Lhb-Gens verstärkt. Das Tet1-Level sinkt einhergehend mit Zelldifferenzierung und bei Exposition zum regulatorischen GnRH. Unsere Arbeit identifiziert neue regulatorische Elemente und einen neuartigen epigenetischen Signalweg bezüglich der zentralen Reproduktionregulation von Säugetieren. Zusammenfassend konnte ich zeigen, dass murine gonadotrope Zellen erhebliche Plastizität auf molekularer Ebene aufweisen, um sich den unterschiedlichen physiologischen Bedürfnissen anzupassen. Wir fanden, dass Tet1 und Tet2 unterschiedliche Rollen bei der Regulierung der Expression von Lhb einnehmen. Zudem konnten wir zeigen, dass GnRH den TRPC5-Kanal über den GnRHR aktiviert. Gonadotropes in the anterior pituitary gland are of particular importance within the hypothalamic-pituitary-gonadal axis because they provide a means of communication and thus a functional link between the brain and the gonads. Recent results indicate that female gonadotropes may be organized in the form of a network that shows plasticity and adapts to the altered endocrine conditions of different physiological states. However, little is known about functional changes on the molecular level within gonadotropes during these different conditions. In this study I capitalized on a binary genetic strategy in order to fluorescently label murine gonadotrope cells. Using this mouse model allows to produce an enriched gonadotrope population using fluorescence activated cell sorting to perform mRNA sequencing. By using this strategy, I analyzed and compared the expression profile of murine gonadotropes in different genders and developmental and hormonal stages. I found that gonadotropes taken from juvenile males and females, from cycling females at diestrus and at proestrus, from lactating females, and from adult males each have unique gene expression patterns with approximately 100 to approximately 500 genes expressed only in one particular stage. I also demonstrated extensive gene-expression profile changes with up to 2200 differentially expressed genes when comparing female and male development, juveniles and adults, and cycling females. Differentially expressed genes were significantly enriched in the GnRH signaling, Ca2+ signaling, and MAPK signaling pathways by Kyoto Encyclopedia of Genes and Genomes analysis. My data provide an unprecedented molecular view of the primary gonadotropes and reveal a high degree of molecular plasticity within the gonadotrope population. Recent results have implicated Transient Receptor Potential (TRP) cation channels in pituitary physiology, however, if and how TRP channels contribute to gonadotrope function is not known. I found that 14 out of 28 TRP channels encoded in the mouse genome are expressed in murine gonadotropes with highest expression levels found for TRPC5 in juvenile females. We show that TRP channel expression in these cells exhibits considerable plasticity and that it depends on gender as well as on the developmental and hormonal status of the animal. We then combined different genetic strategies to characterize TRPC5 channel function in gonadotropes from juvenile females. We show that the TRPC5 agonist Englerin A activates a cytosolic Ca2+ signal and a whole-cell current in these cells, which is absent in TRPC5-deficient mice. We further show a TRPC5 activation in gonadotropes via stimulation of the GnRHR. However, I also observed normal puberty onset and body weight in TRPC5-deficient female mice. The Ten-eleven translocation (TET) enzymes play central and complex roles in determining the changing patterns of gene expression during development. We report here that Tet1, Tet2 and Tet3 expressed in murine gonadotropes, and that Tet1 represses the expression of the luteinizing hormone β-subunit (Lhb) gene, whereas Tet2 enhances the expression of the Lhb gene. Tet1 levels drop with cell differentiation and exposure to the regulatory gonadotropin-releasing hormone. Our work thus exposes new regulatory elements and a novel epigenetic pathway in the central regulation of mammalian reproduction. In summary, I found that murine gonadotropes show great plasticity to adapt different physiological needs. We found Tet1 and Tet2 played distinct roles in regulating the expression of the gene Lhb. We also found a TRPC5 activation in gonadotropes via stimulation of the GnRHR.

Rabconnectin-3α and the control of puberty Human genetics shows that low levels of rabconnectin-3α cause a loss of the neurons that produce gonadotropin-releasing hormone, revealing a new mechanism for incomplete puberty and infertility., Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes., Author Summary Investigation of neurodevelopmental disorders with abnormal puberty can reveal neuronal processes that control the initiation of puberty and subsequent reproductive function. We describe here a new syndrome observed in three brothers with incomplete puberty, central hypothyroidism, peripheral polyneuropathy, mental retardation, and abnormal glucose regulation. Molecular genetic investigation in this consanguineous family revealed an in-frame deletion of 15 nucleotides in the DMXL2 gene, which resulted in lower levels of its expression. We found that rabconnectin-3α, the synaptic protein encoded by DMXL2, is widely expressed in the brain and in the ends of the axons of neurons that produce gonadotropin-releasing hormone (GnRH). We then observed that neuron-specific deletion of one allele of Dmxl2 in mice reproduces the incomplete puberty seen in the human patients and results in decreased numbers of GnRH neurons in the hypothalamus. We also showed that rabconnectin-3α controls glucose-induced insulin secretion. These findings reveal a new mechanism of gonadotropin deficiency and identify rabconnectin-3α as a key controller of neuronal and endocrine homeostasis.

Die Entwicklung der Hypophyse ist ein komplexer Prozess, an dem viele Transkriptionsfaktoren beteiligt sind. Bedeutende Funktionen haben die Bone Morphogenetic Proteins 2, 4 und 7. Ziel der Arbeit war es, an Patienten mit angeborener kombinierter Hypophyseninsuffizienz diese 3 Gene auf Mutationen zu untersuchen. Dabei wurden die Exons, Exon-Intron-Übergänge sowie die 3`- und 5`-untranslatierten Regionen sequenziert. Neu identifizierte Varianten wurden anschließend in einer gesunden Kontrollkohorte untersucht, um pathogenetisch nicht relevante Genvarianten auszuschließen. Um die funktionelle Relevanz der Varianten beurteilen zu können, wurden phylogenetische Analysen und Untersuchungen mittels Programmen zum Einfluss auf die Proteinstruktur und -funktion durchgeführt. Neben bekannten Varianten, konnten wir neue Varianten mit und ohne Aminosäureaustausch identifizieren. Eine neue Mutation im kodierenden Bereich von BMP4 erschien dabei besonders relevant. Wir konnten sie nur bei einem Patienten nachweisen, der zusätzlich zur kombinierten Hypophyseninsuffizienz Skelettveränderungen aufwies. Die hohe Konservierung und diese spezifische Klinik legen eine funktionelle Relevanz nahe.

International audience; The involvement of γ-aminobutyric acid (GABA) in the control of prolactin (PRL) release was investigated in rainbow trout using both perifused pituitary fragments and pituitary cells in primary culture. In our perifusion system, infusion of GABA (10−6 to 10−4 M) caused an inhibition of PRL release (between 20 and 40%). Administration on perifused pituitary fragments of 3APS, a GABAa agonist, mimicked this inhibitory effect. Moreover, bicuculline, a specific antagonist of GABAa receptors, totally abolished GABA effect. When tested on cultured pituitary cells during 40h exposure, GABA (10−5 M) caused a significant decrease in PRL release (24.5%). Baclofen, a specific agonist for GABAb receptor tested at 10−6 and 10−5 M, also inhibited PRL released from cultured pituitary cells. These results demonstrate that GABA inhibits PRL release by acting directly on pituitary cells and that probably both types of GABA receptor (a and b) are involved in this regulation.

Numerous models have been proposed to explain the mechanisms of tumor development and progression. Nevertheless some aspects such as the nature and hierarchy of primary and secondary alterations are still debated. To answer these questions, we decided to focus on the tumoral progression of human prolactin pituitary tumors. These monoclonal tumors are usually benign but some present an aggressive or malignant phenotype. To identify the molecular events involved in tumoral progression of human PRL towards aggressive phenotype we used an integrative genomics approach (microarrays, high-throughput sequencing) coupling analysis of transcriptome, genome (variation in the number of chromosomal copy polymorphisms) and miRNome from the same human tumor. Using this strategy we identified and prioritized specific alterations of aggressive and / or malignant tumors in a model explaining the tumor progression of human prolactin pituitary tumors. We have shown that under-expression of micro-RNA miR-183, miR-744 and miR-98 stimulates proliferation through overexpression of their targets KIAA0101, TGFB1 and E2F2 specifically in aggressive tumors. This leads to the acquisition of chromosomal damage (loss of chromosome 11 and gain of chromosome 1q) which allowed the activation of the metastatic processes. Finally, this work shows that the integrative genomic multi-dimensional approach can provide new clues for the characterization of tumor phenotypes, diagnosis of aggressive tumors and prediction of tumor behavior, De nombreux modèles ont été proposés pour expliquer les mécanismes de développement et de progression tumorale, néanmoins certains aspects comme la nature et la hiérarchie des altérations primaires et secondaires sont encore discutés. Pour répondre à ces questions, nous nous sommes intéressés à la progression tumorale des tumeurs hypophysaires à prolactine humaines. Ces tumeurs d'origine monoclonale sont souvent bénignes mais certaines présentent un phénotype agressif voire malin. Afin d'identifier les mécanismes impliqués dans la progression vers le phénotype agressif nous avons utilisé des techniques de génomique intégrative (puces à ADN, séquençage haut débit) couplant l'étude du transcriptome, du génome (variation du nombre de copie chromosomique, polymorphismes) et du miRNome à partir des mêmes échantillons tumoraux. Par cette stratégie nous avons identifié et hierarchisé des altérations spécifiques des tumeurs agressives et / ou malignes dans un modèle expliquant la progression tumorale des tumeurs hypophysaires à prolactine humaines. Nous avons montré que la sous-expression des microARNs miR-183, miR-744 et miR-98 stimule la prolifération via la surexpression de leurs cibles KIAA0101, TGFB1 et E2F2 spécifiquement dans les tumeurs agressives. Ceci entraine l'acquisition d'altérations chromosomiques (perte du chromosome 11 et le gain du chromosome 1q) permettant l'activation de la dissémination métastatique. Enfin, ce travail montre que l'approche de génomique intégrative multidimensionnelle permet d'apporter de nouveaux éléments pour la caractérisation des phénotypes tumoraux, le diagnostic des tumeurs agressives et la prédiction du comportement tumoral

Objective: Knowledge about hormone receptor expression turned out to be essential for the management of pituitary adenomas, especially for prolactinoma and GH-secreting adenomas. For this reason we analyzed the expression of hormone receptors of 36 resected pituitary adenomas by immunohistochemistry.[for full text, please go to the a.m. URL], 64. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Numerous models have been proposed to explain the mechanisms of tumor development and progression. Nevertheless some aspects such as the nature and hierarchy of primary and secondary alterations are still debated. To answer these questions, we decided to focus on the tumoral progression of human prolactin pituitary tumors. These monoclonal tumors are usually benign but some present an aggressive or malignant phenotype. To identify the molecular events involved in tumoral progression of human PRL towards aggressive phenotype we used an integrative genomics approach (microarrays, high-throughput sequencing) coupling analysis of transcriptome, genome (variation in the number of chromosomal copy polymorphisms) and miRNome from the same human tumor. Using this strategy we identified and prioritized specific alterations of aggressive and / or malignant tumors in a model explaining the tumor progression of human prolactin pituitary tumors. We have shown that under-expression of micro-RNA miR-183, miR-744 and miR-98 stimulates proliferation through overexpression of their targets KIAA0101, TGFB1 and E2F2 specifically in aggressive tumors. This leads to the acquisition of chromosomal damage (loss of chromosome 11 and gain of chromosome 1q) which allowed the activation of the metastatic processes. Finally, this work shows that the integrative genomic multi-dimensional approach can provide new clues for the characterization of tumor phenotypes, diagnosis of aggressive tumors and prediction of tumor behavior, De nombreux modèles ont été proposés pour expliquer les mécanismes de développement et de progression tumorale, néanmoins certains aspects comme la nature et la hiérarchie des altérations primaires et secondaires sont encore discutés. Pour répondre à ces questions, nous nous sommes intéressés à la progression tumorale des tumeurs hypophysaires à prolactine humaines. Ces tumeurs d'origine monoclonale sont souvent bénignes mais certaines présentent un phénotype agressif voire malin. Afin d'identifier les mécanismes impliqués dans la progression vers le phénotype agressif nous avons utilisé des techniques de génomique intégrative (puces à ADN, séquençage haut débit) couplant l'étude du transcriptome, du génome (variation du nombre de copie chromosomique, polymorphismes) et du miRNome à partir des mêmes échantillons tumoraux. Par cette stratégie nous avons identifié et hierarchisé des altérations spécifiques des tumeurs agressives et / ou malignes dans un modèle expliquant la progression tumorale des tumeurs hypophysaires à prolactine humaines. Nous avons montré que la sous-expression des microARNs miR-183, miR-744 et miR-98 stimule la prolifération via la surexpression de leurs cibles KIAA0101, TGFB1 et E2F2 spécifiquement dans les tumeurs agressives. Ceci entraine l'acquisition d'altérations chromosomiques (perte du chromosome 11 et le gain du chromosome 1q) permettant l'activation de la dissémination métastatique. Enfin, ce travail montre que l'approche de génomique intégrative multidimensionnelle permet d'apporter de nouveaux éléments pour la caractérisation des phénotypes tumoraux, le diagnostic des tumeurs agressives et la prédiction du comportement tumoral

The hypothalamus represents the most important component of the central nervous system (CNS) used for integrating afferent signaling information and regulating the homeostasis of body temperature, water and electrolyte balance of the extracellular space, circadian rhythm and energy metabolism. The most important hypothalamic structure (nucleus) for regulating the above mentioned physiological functions is the Nucleus paraventricularis (PVN) of the preoptic region, which is located closely to the third brain ventricle. The PVN consists of many neurosecretory cells and shows numerous efferent and afferent neuronal connections to other structural components of the brain. In addition to the PVN, the Nucleus praeopticus medianus (MnPO), which is located close to the Commisura anterior, displays a critical integrative function for regulating water and electrolyte balance, as well as regulating body temperature. Besides other peptides and classical neurotransmitters, the gaseous transmitter nitric oxide (NO), produced by the neuronal nitric oxide synthase (nNOS) amongst other substances, plays an essential neuromodulatory role in regulating water and electrolyte balance and body temperature. The apelinergic system, consisting of several active fragments of the preproapelin (apelin36, apelin17 and apelin13) and the G-protein coupled receptor, APJ, has been reported to also centrally modulate body temperature and water and electrolyte balance. Expression of apelin peptides and their receptors has been shown in peripheral organs and the CNS, in particular in the hypothalamus. Despite many expression studies having been undertaken, the detailed mapping of the APJ protein distribution in hypothalamus and thalamus remains to be determined. (1) The present thesis therefore includes, for the first time, a detailed semi-quantitative immunhistochemical mapping of the APJ protein on coronal hypothalamic rat brain slices and confirms published APJ mRNA expression in structures like the PVN and Nucleus supraopticus (SON). However, for many structures and/or nuclei like the MnPO or the medial devision of the bed nucleus of the stria terminalis, the N. praecommisuralis and the N. dorsomedialis, APJ protein has been described for the first time in single neurons or pseudopodia. (2) The expression of nNOS has been reported in both the PVN and MnPO previously. Additionally it has been postulated that apelinergic effects, for example on the cardiovascular system, could be mediated by NO. Therefore, the present work analyzed the possible immunhistochemical codistribution and colocalization of APJ and nNOS in the hypothalamus. In this study the MnPO, amongst other brain structures, showed distinct codistribution of nitrergic neurons and APJ immunopositive fiber structures. Quantitative analysis of the PVN and SON perikarya revealed about 40 % and, 50% cellular colocalization of both molecules. Due to this prominent colocalization and codistribution in the PVN and MnPO, NO could be involved as a downstream signaling molecule in mediating central apelinergic functions. (3) Because of the distinctive expression of APJ in the MnPO and PVN, specificly the primary neuroglial cultures of both nuclei and additionally primary intermediate lobe pituitary (HZL) cultures, with high native expression of APJ, these cultures were used to analyze apelin induced intracellular signal transduction in vitro. Changes of intracellular calcium concentrations ([Ca2+]i), which are important second or third messengers, were monitored in single cells by calcium binding fluorophors fura-2 (=calcium imaging). Neurons and astrocytes of the MnPO and PVN specific cultures exhibited 2-9 % specific microglial cells in the MnPO and 12% specific microglial cells in the PVN. Of the MnPO and PVN cell cultures, 12 % directly responded with changes in ([Ca2+]i ) when stimulated with the pyroglutamate modified form of apelin13 (PyrAp13) (10 -6 mol/l). As described for other neuropeptides, PyrAp13 was able to positively and negatively modulate the influx of calcium caused by the classical neurotransmitter glutamate in mainly non-nitregic cells, but also nitregic cells. This modulatory function of PyrAp13 could provide evidence for the cellular basis of apelin as a novel CNS neuromodulator. Besides analysis of [Ca2+]I as a downstream signal of the apelinergic system, some reports showed involvement of extracellular regulated kinase (ERK)1/2 in apelinergic signaling in peripheral or transfected cell systems. In the present work no activation of ERK1/2 was detected in primary HZL cultures with highly native APJ expression, using Western Blot analysis. The lack of ERK1/2 expression may be due to already high levels of phosphorylation before stimulation. (4) As a next important component of the present work, in vivo parameters were monitored in rats by radiotelemetry after intracerebroventricular (i.c.v.) micro application of PyrAp13 (20 nmol). (A) Microinjection of PyrAp13 i.c.v. induced no significant changes in bacterial lipopolysaccharide (LPS)-induced sickness behavior, including anorexia, adipsia and lethargy. However, it significantly reduced LPS-induced fever 3-6 and 6-9 h after injection. Moreover, it also led to a significant reduction in plasma levels of tumor necrosis factor- α (TNF-α), a prominent proinflammatory cytokine, 2 h after injection. (B) Despite a high sequence of similarity between the APJ and the Angiotensin II (AngII) receptor and a proven interaction of both in vitro, i.c.v. microinjected PyrAp13 exhibited no influence on water intake in vivo or nuclear translocation of the transcription factor and neuronal activity marker c-Fos, induced by i.c.v. AngII. (C) Furthermore i.c.v. PyrAp13 showed no influence on telemetrically recorded parameters, such as body temperature, locomotor activity and food/water intake of animals., Der Hypothalamus repräsentiert die wichtigste Komponente des Zentralnervensystems (ZNS) zur Integration afferenter Signalinformationen und zur Regulation der Homöostase von Körperkerntemperatur, Wasser- und Elektrolythaushalt des Extrazellularraumes, circadianer Rhythmik sowie Energiehaushalt. Das wichtigste Kerngebiet (Nucleus) des Hypothalamus zur Steuerung der genannten physiologischen Funktionen stellt der in der supraoptischen Region, in unmittelbarer Nähe zum dritten Hirnventrikel gelegene Nucleus paraventricularis (PVN) dar. Der PVN enthält zahlreiche neurosekretorische Zellen und weist darüber hinaus zahlreiche efferente und afferente neuronale Verknüpfungen zu anderen Struktureinheiten des Gehirns auf. Zusätzlich zum PVN kommt auch dem im Bereich der Commissura anterior gelegenen Nucleus praeopticus medianus (MnPO) eine bedeutende, integrative Funktion bei der Regulation des Wasser- und Elektrolythaushaltes sowie der Körperkerntemperatur zu. Bei der Regulation dieser Prozesse spielt neben klassischen und peptidergen Neurotransmittern auch das gasförmige Signalmolekül Stickstoffmonoxid (NO), produziert unter anderem von der neuronalen Stickstoffmonoxid-Synthase (nNOS), eine wesentliche neuromodulatorische Rolle. Auch für das apelinerge System, bestehend aus den verschiedenen, aktiven Fragmenten des Preproapelins (Apelin36, Apelin17 und Apelin13) und dem G-Protein gekoppelten Rezeptor APJ, wurde bereits ein zentral vermittelter Einfluss unter anderem auf die Temperaturregulation und auf die Kontrolle des Wasser- und Elektrolythaushaltes beschrieben. Eine Expression sowohl der Apelinpeptide als auch des Rezeptors konnte bisher in peripheren Organen sowie im ZNS, und dabei hauptsächlich im Hypothalamus, gezeigt werden. Trotz zahlreicher Expressionsstudien fehlte jedoch bisher eine detaillierte Kartierung der Verteilung des Rezeptorproteins in Hypothalamus und Thalamus. (1) Anhand der in der vorliegenden Arbeit durchgeführten immunhistochemischen Markierungen an coronalen Gehirnschnitten der Ratte wurde deshalb erstmalig eine detaillierte, semiquantitative Kartierung der Expression des APJ Proteins im Hypothalamus erstellt. Diese Untersuchung bestätigt einerseits die bereits auf mRNA-Ebene publizierte APJ-Expression in Strukturen wie PVN und Nucleus supraopticus (SON), konnte jedoch darüber hinaus in vielen Nuclei und/oder deren Substrukturen wie u.a. dem MnPO, dem medialen Teil des „Bed Nucleus“ der Stria terminalis, dem N. praecommissuralis und dem N. dorsomedialis eine APJ-Expression zum ersten Mal auf Proteinebene für einzelne Neurone bzw. deren Pseudopodia nachweisen. (2) Sowohl für den PVN als auch den MnPO wurde bereits eine Expression von nNOS in früheren Studien beschrieben. Zusätzlich dazu konnte in der Fachliteratur eine Vermittlung apelinerger Effekte über NO etwa anhand der Modulation kardiovaskulärer Parameter in der Peripherie postuliert werden. Deshalb wurde in der vorliegenden Arbeit eine mögliche Co-Distribution und -Lokalisation von APJ und nNOS anhand immunhistochemischer Markierungen im Hypothalamus der Ratte untersucht. Dabei konnte u.a. im MnPO eine ausgeprägte Co-Distribution nitrerger Neurone und APJ-immunpositiver Faserstrukturen festgestellt werden. Bei der quantitativen Analyse der Expression von nNOS und APJ in Perikaryen des PVN und des SON hingegen war sogar mit mit ca. 40 % bzw. ca. 50 % markante zelluläre Co-Expression der beiden Moleküle nachweisbar. Aufgrund der ausgeprägten Co-Lokalisation und Co-Distribution in PVN und MnPO könnte somit NO als nachgeschaltetes Signalmolekül auch an der Vermittlung zentraler, apelinerger Wirkmechanismen beteiligt sein. (3) Zur in vitro Untersuchung Apelin-induzierter, intrazellulärer Signaltransduktions-mechanismen wurden aufgrund der ausgeprägten APJ-Rezeptorexpression sowohl MnPO- und PVN-spezifische neurogliale Primärkulturen als auch eine primäre Hypophysenzwischen-lappenkultur (HZL) herangezogen. Änderungen der intrazellulären Calciumkonzentration ([Ca2+]i) als wichtigen second oder third messenger konnten für einzelne Zellen durch den Einsatz calciumbindener Fluorophore wie Fura-2 kontinuierlich erfasst werden (=Calcium-Imaging). Sowohl für Neurone als auch Astrozyten der MnPO- und PVN-spezifischen Kulturen ergab sich bei 2-9 % der Zellen, in PVN-spezifischen Mikrogliazellen hingegen bei 12 % eine direkte Responsivität in Form einer transienten Erhöhung der [Ca2+]i nach Stimulation mit der pyroglutamylierten Form von Apelin13 (PyrAp13) (10-6 mol/l). Wie bereits für andere Neuropeptide beschrieben, vermochte PyrAp13 darüber hinaus den Calciumeinstrom des klassischen Neurotransmitters Glutamat in nitrergen und vor allem nicht-nitrergen Zellen sowohl positiv als auch negativ zu modulieren. Diese modulatorische Wirkung könnte somit möglicherweise die zelluläre Grundlage für physiologische Funktionen des apelinergen Systems als neuer Neuromodulator im ZNS darstellen. Neben der Analyse der [Ca2+]i als dem Apelin13 nachgeschalteten Signal wurde in einigen Publikationen auch über die Involvierung der MAP-Kinasen extracellular regulated kinase 1/2 (ERK1/2) im apelinergen Signalweg in peripheren oder transfizierten Zellsystemen berichtet. In der Primärkultur des HZLs mit hoher nativer APJ-Expression konnte in der vorliegenden Arbeit nach apelinerger Stimulation anhand von Western Blot Analysen jedoch keine Aktivierung der ERK1/2 beobachtet werden, möglicherweise bedingt durch eine bereits hohe Phosphorylierung vor der peptidergen Stimulation. (4) Als weiterer wichtiger Aspekt der wissenschaftlichen Arbeit wurden in vivo physiologische Parameter nach intracerebroventrikulärer (i.c.v.) Applikation von PyrAp13 (20 nmol) in der Ratte anhand telemetrischer Datenaufzeichnung untersucht. (A) I.c.v. mikroappliziertes PyrAp13 zeigte dabei keinen signifikanten Einfluss auf durch bakterielles Lipopolysaccharid (LPS, 100 μg/kg Körpergewicht) induzierte Komponenten des sickness behavior wie Anorexie, Adipsie und Lethargie. Jedoch führte es zu einer signifikanten Reduktion des LPS-induzierten Fiebers 3-6 und 6-9 Std. post injectionem und einem signifikanten erniedrigten Plasmaspiegel an Tumornekrosefaktor-α (TNF-α) als prominenten proinflammatorischen Cytokin 2 Std. nach der Injektion. (B) Trotz der hohen Sequenzähnlichkeit von APJ und dem Angiotensin II (AngII)-Rezeptor und einer in vitro bereits gezeigten Interaktion mit diesem, zeigte i.c.v. mikroappliziertes PyrAp13 jedoch in vivo keinen Einfluss auf durch die zentrale AngII-Applikation induzierte Trinkwasseraufnahme bzw. nukleäre Translokation des neuronalen Aktivitätsmarkers und Transkriptionsfaktors c-Fos. (C) Weiterhin zeigte PyrAp13 i.c.v. selbst beim Vergleich der Injektion vor der Aktivitätsphase mit der Inaktivitätsphase der Versuchstiere keinen Einfluss auf die circadiane Rhythmik telemetrisch erfasster Parameter wie Körpertemperatur, lokomotorische Aktivität und Futter- bzw. Wasseraufnahme.

L'axe hypothalamo-hypophyso-surrénalien (HPA) permet de maintenir l'homéostasie de l'organisme face à divers stress. Qu'ils soient de nature psychologique, physique ou inflammatoire/infectieux, les stress provoquent la synthèse et la libération de CRH par l'hypothalamus. Les cellules corticotropes hypophysaires perçoivent ce signal et en réaction, produisent et sécrètent l'ACTH. Ceci induit la synthèse des glucocorticoïdes (Gc) par le cortex surrénalien; ces stéroïdes mettent le système métabolique en état d’alerte pour la réponse au stress et à l’agression. Les Gc ont le rôle essentiel de contrôler les défenses de l'organisme, en plus d'exercer une rétro-inhibition sur l'axe HPA. L'ACTH est une petite hormone peptidique produite par le clivage d'un précurseur: la pro-opiomélanocortine (POMC). À cause de sa position critique dans la normalisation de l'homéostasie, le contrôle transcriptionnel du gène Pomc a fait l'objet d'études approfondies au cours des dernières décennies. Nous savons maintenant que la région promotrice du gène Pomc permet une expression ciblée dans les cellules POMC hypophysaires. L'étude du locus Pomc par des technologies génomiques m'a permis de découvrir un nouvel élément de régulation qui est conservé à travers l'évolution des mammifères. La caractérisation de cet enhancer a démontré qu'il dirige une expression restreinte à l'hypophyse, et plus particulièrement dans les cellules corticotropes. De façon intéressante, l'activité de cet élément dépend d'un nouveau site de liaison recrutant un homodimère du facteur de transcription Tpit, dont l'expression est également limitée aux cellules POMC de l'hypophyse. La découverte de cet enhancer ajoute une toute nouvelle dimension à la régulation de l'expression de POMC. Les cytokines pro-inflammatoires IL6/LIF et les Gc sont connus pour leur antagonisme sur la réaction inflammatoire et sur le promoteur Pomc via l'action des facteurs de transcription Stat3 et GR respectivement. L'analyse génomique des sites liés ii par ces deux facteurs nous a révélé une interrelation complexe et a permis de définir un code transcriptionnel entre ces voies de signalisation. En plus de leur action par interaction directe avec l’ADN au niveau des séquences régulatrices, ces facteurs interagissent directement entre eux avec des résultats transcriptionnels différents. Ainsi, le recrutement de GR par contact protéine:protéine (tethering) sur Stat3 étant lié à l'ADN provoque un antagonisme transcriptionnel. Inversement, le tethering de Stat3 sur GR supporte une action synergique, tout comme leur co-recrutement à l'ADN sur des sites contigus ou composites. Lors d'une activation soutenue, ce synergisme entre les voies IL6/LIF et Gc induit une réponse innée de défense cellulaire. Ainsi lors d'un stress majeur, ce mécanisme de défense est mis en branle dans toutes les cellules et tissus. En somme, les travaux présentés dans cette thèse définissent les mécanismes transcriptionnels engagés dans le combat de l'organisme contre les stress. Plus particulièrement, ces mécanismes ont été décrits au niveau de la réponse globale des corticotropes et du gène Pomc. Il est essentiel pour l'organisme d'induire adéquatement ces mécanismes afin de faire face aux stress et d'éviter des dérèglements comme les maladies inflammatoires et métaboliques., The hypothalamo-pituitary-adrenal (HPA) axis regulates homeostasis in various conditions of stress contributing to both the stress response and its termination. Psychological, physical or inflammatory/infectious stresses all prompt the synthesis and secretion of hypothalamic CRH. The pituitary corticotrope cells receive this signal and in turn, secrete ACTH which triggers the synthesis of glucocorticoids (Gc) by the adrenal cortex; these steroids induce a general state of alertness in order to fight or flight aggressions and stresses. Glucocorticoids have the critical role to restrict the stress response by exerting a negative feedback on the HPA axis. ACTH is a small peptidic hormone produced after cleavage of a precursor protein: pro-opiomelanocortin (POMC). Due to its critical role in homeostasis, transcriptional control of the Pomc gene has been intensely studied during the last decades. Previous investigations identified a promoter region that is sufficient for expression of Pomc in the appropriate pituitary cells. Genome-wide studies of the Pomc locus led me to discover a novel regulatory element that is conserved throughout mammalian evolution. The activity of this enhancer is restricted to the pituitary, and more precisely to the corticotrope lineage. Interestingly, its activity depends on a novel transcription factor binding motif that binds homodimers of Tpit, a transcription factor that is only found in pituitary POMC cells. The discovery of this enhancer adds a new dimension in the control of pituitary Pomc expression. The IL6/LIF pro-inflammatory cytokines and the glucocorticoids are well known for their antagonism in control of the inflammatory response; at the Pomc promoter, their action is mediated by the transcription factors Stat3 and GR, respectively. The analysis of genomic sites bound by these two factors revealed a complex relationship and led us to define a transcription regulatory code linking these signalling pathways. In addition to their direct DNA interaction with cognate regulatory sequences, these factors iv interact with each other with different outcomes. Thus, the recruitment of GR on DNAbound Stat3 through protein:protein contacts (tethering) results in transcriptional antagonism. Conversely, Stat3 tethering to GR produces synergism; this is also the case when the two factors are co-recruited to DNA on contiguous or composite binding sites. Prolonged activation of the IL6/LIF and Gc pathways elicits a synergistic innate cell defense response in all cells and tissues. In summary, this doctoral work has defined transcriptional mechanisms that mediate and control the stress response. In particular, pituitary components of the stress response were defined at the level of the Pomc gene and as a global response of corticotrope cells. This response is critical for appropriate organism defense during stresses such as those produced in inflammatory and metabolic diseases.

We recently have seen the development of continuously glucose monitoring systems (CGM-systems). These systems measure the subcutaneous glucose concentration and are after calibrating able to determine the blood glucose concentration. Beside a pure documentation function CGM-systems can be combined with an insulin pump to form a closed-loop system, which can independently manage the glucose concentration of diabetes patients. So these patients can sustain a normoglycemic blood glucose concentration without increasing the risk of hypoglycemic episodes. Due to the subcutaneous measuring site we hypothesized that rapid changes in blood glucose will result in a temporal latency between the blood glucose and the subcutaneous glucose concentration. To date this phenomenon has not been investigated in a systematical and clinical study. Therefore the goal of this dissertation is to analyze CGM-systems in the setting of a rapid on-set insulin-induced hypoglycemia. We designed an experimental, prospective, clinical study which includes patients who are suspected to suffer from hypopituitarism. As a part of the normal diagnostic procedure a hypoglycemia will be induced in these patients (Insulin Tolerance Test). We recorded the hypoglycemia by venous, capillary and CGM measurements of blood glucose concentration. In addition we included endocrine (ACTH, cortisol, GH, epinephrine) and psychometric parameters (“Edinburgh hypoglycaemia Scale”). We analyzed the data of 40 normoglycemic patients. In comparison to the venous approach the CGM-Systems has a lag-time of 19,66 min to detect a blood glucose concentration of 60 mg/dl. The lag-time between venous measurements and capillary point-of-care-testing is 2,85 min respectively. In a multiple regression analysis we could identify age as a predictor of the lag-time (R2=0,22). In summary this study shows that subcutaneous CGM-Systems can detect a rapid on-set hypoglycemia only with a certain delay. The results of this study are relevant for the further development of algorithms for closed-loop systems.

Objective: Intracranial granular cell tumours (GCT) of the sellar or suprasellar region are very rare, arising from the pituitary stalk or the neurohypophysis. Corresponding to WHO grade I tumour, GCT is generally considered a benign condition. However, data on large patient series and long-term follow-up[for full text, please go to the a.m. URL], 63. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie (JNS)

Die vorliegenden Arbeit zeigt, welche Faktoren einen deutlichen Einfluss auf das Auftreten postoperativer Infektionen bei Prozessen im Sellabereich haben. Außerdem werden etablierte Infektionsparameter, wie Temperatur, Blutkörperchensenkungsgeschwindigkeit (BSG), Leukozytenzahl, und neuere Parameter wie das C-Reaktive-Protein (CRP), das Interleukin 6 (IL-6) und das Lipopolysaccharid-bindendes-Protein (LBP) bezogen auf den postoperativen Verlauf, Sensitivität und Spezifität zur Detektion von Infektionen untersucht. Methoden und Patienten: Es handelte sich um eine prospektive Längschnittuntersuchung bei Patienten, die %sich einer Operation an einem raumfordernden Prozess in der Sellaregion an der Neurochirurgischen Universitätsklinik Erlangen operiert wurden. Folgende Parameter wurden bei den Patienten nach den in der Klinik etablierten Standards systematisch untersucht: die Körpertemperatur mittels Infrarottmypanometrie, die BSG nach Westergren, die Leukozytenanzahl mittels Impedanz-Durchflusszytometrie, das CRP im Serum mittels Immunturbidimetrie, das IL-6 und das LBP im Serum mittels ELISA. Präoperativ erhobene Befunde, klinische Verläufe und gemessenen Parameter wurden dokumentiert und mit den aufgetreten Infektionen verglichen. Ergebnisse: Im Zeitraum von einem Jahr wurden 173 Patienten untersucht. Postoperativ kam es bei 13,5 % zu einer Sinusitis, bei 4,7 % zu einer Meningitis und bei 11,1 % zu einer Parainfektion (nicht-operationsassoziierten Infektion). Die Mortalität betrug 0 %. Eine Sinusitis begünstigende Faktoren waren hormonaktive Tumoren wie Akromegalie oder M. Cushing, eine positive Infektionsanamnese, ein Mikroadenom und eine präoperative thyreotrope oder somatotrope Hypophyseninsuffizienz. Prädisponierende Faktoren für eine Meningitis waren ein Makroadenom, ein hormoninaktives Hypophysenadenom, eine Voroperation im gleichen Gebiet, eine lumbale Drainage, sowie die präoperative adrenocortico- oder gonadotrope Hypophyseninsuffizienz.Parainfektionen traten häufig bei Patienten mit einer präoperativen gonado- oder adrenocorticotropen Hypophyseninsuffizienz auf. Bei dem Verlauf der Infektionsparameter Temperatur, BSG und Leukozytenzahl zeigt sich, dass die Temperatur und die Leukozytenzahl am ersten postoperativen Tag bei allen Patienten anstieg. Bei Patienten ohne Infektion fiel sie schnell wieder ab, während dies bei den Patienten mit einer Meningitis oder Parainfektion nicht der Fall war. Bei CRP, LBP und IL-6 kam es ähnlich wie bei der Temperatur und der Leukozytenzahl zu einem signifikanten Anstieg am ersten postoperativen Tag. Bei Patienten ohne Infektion normalisierte sich das LBP und IL-6 bereits am 3. postoperativen Tag und das CRP am 7. postoperativen Tag. Patienten mit einer Meningitis dagegen erreichten die höchsten Serumkonzentrationen von CRP, IL-6 und LBP am 3. postoperativen Tag. Insgesamt wies das IL-6 die höchste Sensitivität und das LBP die höchste Spezifität bei gleichzeitig schnellem Ansprechen auf das Auftreten oder Sistieren einer Noxe auf. Praktische Schlußfolgerungen: Eine peri- und postoperative Antibiotikaprophylaxe wird bei Risikopatienten aufgrund der deutlich höheren Infektionsraten empfohlen. Weiterhin muss der Einsatz lumbaler Drainagen kritisch hinterfragt werden und die Handhabung streng aseptisch erfolgen. Vor allem schwere Infektionen bei sellären Operationen werden durch die Verlaufsbeurteilung aus sehr sensitiven Parametern wie dem CRP und dem IL-6 und einem sehr spezifischen Parameter wie dem LBP frühzeitig erfasst. Durch die Verknüpfung aus den beobachteten Einflussfaktoren und der Verlaufsbestimmung der Infektionsparameter werden Infektionen früher erkannt und deren Folgen verhindert. Background and Goals: In this investigation, factors which have a strong impact on the occurrence of postoperative infections in sellar surgery are shown. Moreover, well-known infection parameters such as body temperature, erythrocyte sedimentation rate (ESR), white blood count (WBC) as well as the new parameters such as C-reactive protein (CRP), interleukin 6 (IL-6) and lipopolysaccharid-binding-protein (LBP) were analyzed in respect to their postoperative course, sensitivity and specificity in order to detect infections. Methods (Patients, Clincal Material and Methods) This is a prospective longitudinal clincal examination on patients, who were operated for a sellar lesion at the Neurochirurgische Universitätsklinik Erlangen. According to our standard clinical procedure, the following parameters were sytematically investigated: body temperature using infrared tympanomometry, ESR by Westergren, WBC using impedance-flow-cytometry, CRP in the serum using immunturbidimetry, IL-6 and LBP in the serum using ELISA. Preoperative findings, the clinical course and the test results of the infection parameters were documented and compared with the infections. Results and Observations In the space of one year 173 patients were examined. Postoperative complications included sinusitis in 13.5 %, meningitis in 4.7 % and parainfections (non-surgery-associated infection) in 11.1 % of the cases. The mortality was 0 %. Sinusitis was encouraged through hormone active lesions such as acromegaly or M. Cushing, a positive history of infection, a microadenoma and a preoperative pituitary insufficiency of the thyroid or the growth hormone axis. % Meningits was associated with a macroadenoma, a hormone active pituitary adenoma, an operation in the same region, a lumbar drainage and pituitary insufficiency of the adrenal or gonadal axis. Parainfections occurred more often with preoperative gonado- or adrenocorticotropic pituitary insufficiency. The course of the infection parameters, body temperature, ESR and WBC showed that the temperature and the WBC rose on the 1st postoperative day in all patients. In patients without infection, they declined rapidly, whereas this did not appear in patients with meningitis or parainfection. Concerning CRP, LBP and IL-6 there was a similar significant increase as in the temperature and WBC on the 1st postoperative day. In patients without infection, IL-6 and LBP were normal on the 3rd day and CRP on the 7th day. Patients with meningitis, however, reached the maximum levels of CRP, IL-6 and LBP on the 3rd postoperative day. All in all IL-6 had the highest sensitivity and LBP the highest specificity. Both parameters respond quickly to the occurence and disappearance of an infection. Conclusions A peri- and postoperative antibiotic prophylaxis is indicated in patients with predisposing factors for infection. Moreover, the use of a lumbar drain must be questioned and it should only be handled under aseptic conditions. In particular, severe infections in sellar surgery are detected by observing the clinical course of the combination of a very sensitive parameter such as IL-6 and CRP and a very specific parameter such as LBP. In summary these parameters as well as the clinical findings make it possible to detect infections early and to prevent their sequelae.

The embryology of the human adrenal and the pathogenesis of the hypoplasia adrenal congenital are complex and not well known. We studied the histological and functional characteristics of the fetal human adrenal in normal fetuses aged 12 to 36 weeks development (WD) and presented three cases of congenital adrenal hypoplasia with pituitary abnormalities. In the human fetal adrenal cortex, only the cells of the definitive zone proliferate from the 12th WD. We observe the expression of an adherence protein (NCAM) and of two steroidogenesis enzymes (3β-HSD and P450 c21) in the definitive zone cells, attesting to the capacity of these cells to synthesize mineralocorticoids and/or cortisol. In the fetal zone, only P450 c21 immunoreactivity is detected. The adrenal medulla is formed by immature neuroblasts (CgA -, NCAM+) which migrate and proliferate from the periphery toward the center of the gland, where they differentiate in mature neuroblasts (CgA+). In the two cases of congenital adrenal hypoplasia of anencephalic type without DAX 1 and SF-1 mutation, the cause is probably from pituitary origin and related to absence of gonadotrope cells. In the third case of congenital adrenal hypoplasia, never described, combining a retard intrauterine development, a congenital adrenal hypoplasia, sexual ambiguity, pituitary abnormalities. The absence of mutation of known genes (DAX -1, SF -1, SRY, FGF9, SOX2, SOX3, SOX5 and SOX9) suggests the existence of new genes regulating early differentiation of the adrenal, the gonad and the pituitary., L’embryologie de la surrénale humaine et la pathogénie de l’hypoplasie congénitale des surrénales sont mal connues et complexes. L’une pouvant expliquer l’autre, nous présentons, à partir de 119 fœtus humains normaux de12 à 36 semaines de développement (SD), une étude cinétique histologique et moléculaire de la surrénale fœtale et nous décrivons 3 cas d’hypoplasie surrénalienne. Après un rappel des mécanismes moléculaires connus, régulant ensemble l’embryologie surrénalienne, gonadique et hypophysaire et ceux de l’hypoplasie surrénale congénitale, nous présentons nos résultats. Dans la corticosurrénale humaine fœtale normale, les cellules du cortex permanent prolifèrent et, dès la 12ème SD, expriment la NCAM, la 3β-HSD et la P450 c21. Elles ont la capacité de synthétiser des minéralocorticoïdes et/ou du cortisol. Les cellules du cortex fœtal ne prolifèrent pas et expriment ni la 3β-HSD, ni la NCAM. La médullosurrénale est formée par des neuroblastes immatures (CgA-, NCAM+) qui migrent et prolifèrent de la périphérie vers le centre de la glande, où ils se différencient en neuroblastes matures (CgA+). Dans les deux cas d’hypoplasie surrénale de type anencéphalique, avec absence de mutation de DAX-1 et de SF-1, la dysembryoplasie surrénalienne est probablement d’origine hypophysaire, par absence de cellules gonadotropes. Dans le 3ème cas, jamais décrit, associant un RCIU, une hypoplasie surrénalienne congénitale, une ambiguïté sexuelle, une absence de différenciation des cellules antéhypophysaires, l’absence de mutation de gènes connus (DAX-1, SF-1, SRY, FGF9, SOX2, SOX3, SOX5 et SOX9) suggère l’existence de nouveaux gènes régulant la différenciation précoce de ces trois glandes endocrines.

Diplôme : Dr. d'Université

Das Kraniopharyngeom ist eine dysontogenetische Mittellinienfehlbildung mit raumforderndem Charakter. Die Ätiopathogenese ist noch ungeklärt. Bildgebende Verfahren (Sonographie, MRT) ermöglichen bereits pränatal eine Diagnostik. Das neonatale Kraniopharyngeom ist sehr selten. Bisher finden sich in der Literatur nur wenige Fallberichte von denen operative Verläufe sowie klinische Langzeitverläufe bekannt sind. Es bleibt eine Kontroverse hinsichtlich Radikalität und Zeitpunkt der Operation, nicht aber dahingehend, dass die operationsmikroskopische Versorgung die Therapie der Wahl des neonatalen Kraniopharyngeoms darstellt. Die Nähe zur hypophysären-hypothalamischen Achse bedingt eine hohe Komorbidität der Kinder prä- und postoperativ., Craniopharyngioma are embryogenic malformations of the sellar area. With an overall incidence of 0.5–2 new cases per million population per year, 30–50% of all cases occur in childhood.As the survival rate after craniopharyngioma diagnosed during childhood and adolescence is high, prognosis and quality of life in survivors mainly depend on adverse late effects such as hypopituitarism and obesity. Appropriate laboratory diagnostics of endocrine deficiencies and sufficient hormonal substitution have significant impact on prognosis. A review of the literature reveal only some few cases of neonatal craniopharyngioma. Modern picture-giving proceeds such as MRI enable a early diagnosis. On the one hand there is a controversy concerning the radicalness and the time of operation on the other hand the microsurgical resection is meant to be the goldstandard in the therapy of neonatal craniopharyngiomas.

The embryology of the human adrenal and the pathogenesis of the hypoplasia adrenal congenital are complex and not well known. We studied the histological and functional characteristics of the fetal human adrenal in normal fetuses aged 12 to 36 weeks development (WD) and presented three cases of congenital adrenal hypoplasia with pituitary abnormalities. In the human fetal adrenal cortex, only the cells of the definitive zone proliferate from the 12th WD. We observe the expression of an adherence protein (NCAM) and of two steroidogenesis enzymes (3β-HSD and P450 c21) in the definitive zone cells, attesting to the capacity of these cells to synthesize mineralocorticoids and/or cortisol. In the fetal zone, only P450 c21 immunoreactivity is detected. The adrenal medulla is formed by immature neuroblasts (CgA -, NCAM+) which migrate and proliferate from the periphery toward the center of the gland, where they differentiate in mature neuroblasts (CgA+). In the two cases of congenital adrenal hypoplasia of anencephalic type without DAX 1 and SF-1 mutation, the cause is probably from pituitary origin and related to absence of gonadotrope cells. In the third case of congenital adrenal hypoplasia, never described, combining a retard intrauterine development, a congenital adrenal hypoplasia, sexual ambiguity, pituitary abnormalities. The absence of mutation of known genes (DAX -1, SF -1, SRY, FGF9, SOX2, SOX3, SOX5 and SOX9) suggests the existence of new genes regulating early differentiation of the adrenal, the gonad and the pituitary., L’embryologie de la surrénale humaine et la pathogénie de l’hypoplasie congénitale des surrénales sont mal connues et complexes. L’une pouvant expliquer l’autre, nous présentons, à partir de 119 fœtus humains normaux de12 à 36 semaines de développement (SD), une étude cinétique histologique et moléculaire de la surrénale fœtale et nous décrivons 3 cas d’hypoplasie surrénalienne. Après un rappel des mécanismes moléculaires connus, régulant ensemble l’embryologie surrénalienne, gonadique et hypophysaire et ceux de l’hypoplasie surrénale congénitale, nous présentons nos résultats. Dans la corticosurrénale humaine fœtale normale, les cellules du cortex permanent prolifèrent et, dès la 12ème SD, expriment la NCAM, la 3β-HSD et la P450 c21. Elles ont la capacité de synthétiser des minéralocorticoïdes et/ou du cortisol. Les cellules du cortex fœtal ne prolifèrent pas et expriment ni la 3β-HSD, ni la NCAM. La médullosurrénale est formée par des neuroblastes immatures (CgA-, NCAM+) qui migrent et prolifèrent de la périphérie vers le centre de la glande, où ils se différencient en neuroblastes matures (CgA+). Dans les deux cas d’hypoplasie surrénale de type anencéphalique, avec absence de mutation de DAX-1 et de SF-1, la dysembryoplasie surrénalienne est probablement d’origine hypophysaire, par absence de cellules gonadotropes. Dans le 3ème cas, jamais décrit, associant un RCIU, une hypoplasie surrénalienne congénitale, une ambiguïté sexuelle, une absence de différenciation des cellules antéhypophysaires, l’absence de mutation de gènes connus (DAX-1, SF-1, SRY, FGF9, SOX2, SOX3, SOX5 et SOX9) suggère l’existence de nouveaux gènes régulant la différenciation précoce de ces trois glandes endocrines.

International audience; For continued studies of GnRH receptor regulation in the winter flounder, we have developed an in vitro system consisting of cultured pituitary cells dissociated by collagenase. Using immunocytochemical staining methods for gonadotropin, growth hormone, and prolactin, these cell types were represented at the levels of 25, 20, and 19.5% of total pituitary cell population, respectively. Receptors for GnRH were characterized in intact monolayered attached pituitary cells, maintained in RPMI culture medium. The cell GnRH receptor characteristics were compared with those previously described using pituitary homogenates. The cells were capable of binding GnRH in a similar manner on Day 2 or Day 3 of culture, indicating the integrity of GnRH receptors. The specificity of binding was demonstrated since only high doses of cold GnRHa competed with 125I-GnRHa uptake, different peptides being without effect. The specific binding is saturable and the data suggest the presence of a single class of high-affinity (apparent Ka = 1.50 x 10(9) M-1), high-capacity sites (binding capacity = 25.03 fmol/2.5 x 10(5) cells or 242.23 x 10(3) sites/gonadotroph) which is in accordance with the characteristics of GnRH receptors present in homogenates of pooled male and female pituitary glands. All these observations suggest that such an in vitro pituitary cell system would be appropriate for studying GnRH receptor characteristics under different physiological conditions.

Hintergrund und Ziele: Eine erhöhte Amplitude der pulsatilen LH-Sekretion, ein erhöhter LH/FSH-Quotient und eine Störung des Kohlenhydratstoffwechsels im Sinne einer Insulinresistenz sind charakteristische Veränderungen bei Patientinnen mit Hyperandrogenämie. Es ist nicht bekannt, ob diese charakteristischen Veränderungen des Sekretionsmusters der Gonadotropine Ursache oder Folge der erhöhten Androgenspiegel sind. Untersucht wurde der Effekt einer Androgenrezeptorblockade mit dem nicht-steroidalen Antiandrogen Flutamid auf die Gonadotropinsekretion und Glukosetoleranz bei Patientinnen mit Hyperandrogenämie und gesunden Probandinnen. Methoden: Sieben Patientinnen mit Hyperandrogenämie und fünf Probandinnen wurde Flutamid über 7 Tage in einer Dosis von 750 mg/d verabreicht. Blutproben wurden über acht Stunden im Abstand von zehn Minuten unmittelbar vor Beginn der Flutamidgabe (Tag 1), sowie an Tag 2 und 6 der Behandlung vorgenommen. Pulsationsprofile wurden für LH, FSH und Prolaktin erstellt. An den weiteren Tagen wurde täglich eine Hormonbasisdiagnostik vorgenommen. An Tag 0 und 7 wurde ein oraler Glukosetoleranztest über drei Stunden mit fünfzehnminütiger Blutentnahme durchgeführt, bestimmt wurden Glucose, Insulin, Testosteron und Estradiol. Ergebnisse und Beobachtungen: In der Mehrzahl der untersuchten Patientinnen und Probandinnen kam es unter Flutamid zu einem Anstieg der LH-Pulsfrequenz und zu einem Absinken der LH-Pulsamplitude und einem Absinken der LH-Konzentrationen. FSH verhielt sich gleichsinnig zu LH. Die Muster der Prolaktinsekretion veränderten sich nicht signifikant. Die Plasmaspiegel von Testosteron, freien Testosteron und DHEA-S fielen ab, Estradiol blieb konstant. Die Insulinempfindlichkeit zeigt keine charakteristischen Veränderungen. Praktische Schlussfolgerungen: Erhöhte Androgene führen zu einer Verlangsamung der LH-Pulsfrequenz, gefolgt von einem Anstieg der LH-Pulsamplitude und einem Anstieg der LH-Konzentration, welche die Androgen-Sekretion des Ovars stimuliert und damit zu einer Situation führt, die sich selbst unterhält. Die Androgen-Blockade mit Flutamid führt zu einem Abfall der LH-Pulsamplitude, zu einem Abfall der LH-Konzentration und damit zu einer verminderten Stimulation des Ovars zur Androgensekretion. Eine Veränderung des Kohlenhydratstoffwechsels im Sinne einer Insulinresistenz ist bei Frauen mit Hyperandrogenämie häufig zu finden. Die erhöhten Androgenwerte sind nicht die Ursache der Insulinresistenz, weshalb eine Therapie mit Antiandrogenen die Insulinresistenz auch nicht bessert. Introduction: Elevated amplitude of pulsatile LH secretion increased LH/FSH ratio and insulin resistance are characteristic endocrine abnormalities in hyperandrogenemic women. The cause-effect relationship between these abnormal endocrine parameters and hyperandrogenemia , however, is still incompletely understood. Examined is therefore the effect of androgen receptor blockade on pulsatile gonadotropin secretion and glucose tolerance in hyperandrogenemic women and normal probands using flutamide, an antiandrogen devoid of intrinsic endocrine activity. Material and methods: Seven women with hyperandrogenemia and five normal ovulating females were treated for seven days with a daily dose of 750 mg of flutamide. For the study of pulsatile gonadotropin sectretion, blood samples were collected over 10 minute intervals for eight hours on day 1 (before treatment), day 2 and day 6 of treatment using a sampling device that permitted the continous withdrawal of blood via a heparinized catheter. LH, FSH and Prolactin were determined in all samples, the basal endocrine parameters daily. An oral glucose tolerance test was performed on day 0 and repeated on day 7. Results: Treatment with flutamide resulted in a decrease in LH pulse amplitude and an increase in LH-pulse frequency. This was associated with a decline of the plasma concentrations of LH, FSH, testosterone free testosterone, androstendione and DHEA-S. Serum levels of prolactin, estradiol and SHBG, in contrast did not change. During oGTT, administration of flutamide did not result in a change of the glucose /insulin ratio. Discussion: Increasing androgens reduce LH pulse frequency. This is followed by an increase of LH pulse amplitude and mean LH levels in plasma, which in turn stimulate androgen secretion from the ovary leading to a selfperpetuating effect. The androgen blockade caused by flutamide leads to a decrease of LH pulse amplitude, a decrease of LH plasma levels and subsequently to a decreased ovarian stimulation to androgen secretion. Insulin resistance is a frequent finding in hyperandrogenemic women. Increased androgen plasma levels are not the cause of insulin resistance, which is the reason why insulin resistance in hyperandrogenemia cannot be ameliorated by antiandrogens. It is suggested that endogenous opiates play a critical role in the process leading to hyperinsulinemia in hyperandrogenemia.

International audience; Environmental exposure to endocrine disrupting compounds (EDCs) during development could adversely affect reproductive function via effects on the hypothalamo-pituitary-gonadal axis (HPG). The HPG axis is regulated by many steroid sensitive inputs, including the KiSS-1 gene product kisspeptin. We have previously shown that KiSS-1 mRNA and protein are expressed within gonadotrophs in the foetal ovine pituitary and that KiSS-1 mRNA expression is decreased following gestational exposure to EDCs. The current study aimed to determine whether KiSS-1 mRNA expression is also changed in pituitary glands of adult animals exposed to EDCs in-utero. Tissue was obtained from 12 month old male and female sheep born to mothers pastured on fields fertilised with inorganic fertiliser (C) or sewage sludge (T) throughout pregnancy (n=12 per group). mRNA was extracted, reverse transcribed and KiSS-1 mRNA expression quantified by QrtPCR (relative to β-actin). Data were analysed using two-way ANOVA. Results - KiSS-1 mRNA expression was significantly (P

Insulin-like growth factor (IGF)-I, which is crucially involved in fish growth, differentiation, and reproduction, occurs in tilapia pituitary. IGF-I peptide, which is probably produced in hypothalamic perikarya, is present in axons of the neurohypophysis, and IGF-I mRNA and peptide are present in the adenohypophysis in adrenocorticotrophic hormone cells, melanocyte-stimulating hormone cells, with interindividual differences in growth hormone cells, and, starting with puberty, in gonadotrophic hormone (GTH) cells. Subordinate males showed a high IGF-I but a lower beta-luteinizing hormone expression, while in dominant males the opposite was found. IGF-I from the GTH cells may act as auto/paracrine regulators of GTH cell proliferation and enhance GTH synthesis and release during puberty and reproduction, depending on the social status.

Background: Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to “real-life,” environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal.\ud Objectives: We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein–coupled receptor 54) system.\ud Methods: KiSS-1, GPR54, and ERα (estrogen receptor α) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHβ (luteinizing hormone β) and ERα in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry.\ud Results: Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHβ and ERα in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary.\ud Conclusions: This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

Diplôme : Dr. d'Université

Using primary cultures of whole dispersed pituitary cells collected from rainbow trout at different stages of spermatogenesis, basal and GnRH-induced GtH release and cell GtH content were studied in control and steroid-pretreated cultures. Steroid pretreatments were performed for 3 days with 11-ketotestosterone (11KT) and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha 20 beta p) at levels corresponding to those circulating at the time of spermiation (50 and 20 ng/ml, respectively). In control cultures, basal GtH release and cell GtH content increased with the stage of spermatogenesis in a characteristic pattern as predicted from in vivo results concerning plasma and pituitary GtH contents. The pituitary response to salmon GnRH (sGnRH) also varied as indicated by the decrease in the minimal effective dose of GnRH able to induce a significant GtH release with the advancement of spermatogenesis: 10(-7) M at the spermatocyte stage, 10(-9) M at prespermiation and spermiation. Steroid pretreatments were shown to have a direct effect on pituitary gonadotrophs and particularly on pituitary response to sGnRH, depending on the stage at which they are applied. At the beginning of spermatogenesis both of them induced an increase in GtH release and at prespermiation they have a slight negative effect, significant only with 17 alpha 20 beta P. At spermiation they have no effect except for 17 alpha 20 beta P which increased the response to 10(-8) M of sGnRH. Results are discussed in relation to hormonel changes (gonadotropin and steroid) observed by different authors during the sexual cycle.

International audience

Leptin is an adipose hormone implicated in the control of energetic balance and growth hormone (GH) secretion. Its receptors ObRa and ObRb are expressed in the rat somatotroph cells and leptin regulates ghrelin receptor expression in the pituitary. In our obese DIO rats model, the alteration of plasma GH levels, identified in this pathology, is not related to pituitary resistance to leptin. In the Lou/C rats, GH is involved in the resistance to obesity, with leptin and ghrelin hormones playing a major role in the pituitary control of its secretion. In vitro, ObRb internalisation is constitutive but leptin inhibits the traffic of this receptor from the Golgi apparatus to the plasma membrane. This internalisation is required for STAT3 activation and thus this cellular process could be involved in leptin resistance development., La leptine est une hormone adipocytaire impliquée notamment dans le contrôle de la balance énergétique et de la sécrétion d'hormone de croissance (GH). Ses récepteurs ObRa et ObRb sont exprimés dans les cellules somatotropes de rat, et elle régule l'expression hypophysaire du récepteur de la ghréline. Dans notre modèle de rats obèses DIO, l'altération des taux plasmatiques de GH caractéristique de cette pathologie n'est pas liée à la résistance hypophysaire à la leptine. Chez les rats Lou/C, la GH participe à la résistance à l'obésité, les hormones ghréline et leptine jouant un rôle prépondérant dans le contrôle hypophysaire de sa sécrétion. In vitro, l'internalisation de ObRb est constitutive mais la leptine inhibe l'adressage de ce récepteur de l'appareil de Golgi vers la membrane plasmique. Cette internalisation est indispensable pour l'activation de STAT3 et ce processus pourrait donc participer à l'établissement de la résistance à la leptine.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.