The seasonal termination of ovarian cycles in mares initiated near the time of the autumnal equinox is a significant managerial issue for horse breeders world-wide. Studies presented herein had two over-arching aims. In Aim I, objectives were to develop the principals needed to apply gonadotropin-releasing hormone (GnRH) therapeutics for routinely establishing pregnancies in the winter anovulatory mare. We first tested the hypothesis that continuous administration of native GnRH, beginning in either early February or March, would induce ovulation without reversion to an anovulatory state following treatment withdrawal. Continuous 28-d treatments elevated circulating luteinizing hormone (LH) and stimulated spontaneous ovulation much earlier than controls. However, mares treated only in February ceased ovarian cycles at termination of treatment. In contrast, mares administered GnRH in March continued to exhibit estrous cycles. Thus, we concluded that GnRH treatment must continue through March to ensure continued escape from winter anovulation. We then tested the hypothesis that the Julian day of conception could be accelerated in winter anovulatory mares treated continuously with native GnRH for 56 d beginning on February 1. Indeed, GnRH treatment caused a marked increase in the frequency of pregnancy compared to controls. Data illustrated that continuous administration of native GnRH is a practical and highly efficient option for managing seasonal anovulation. In Aim II, we examined hypothalamic distribution, adenohyphyseal receptor gene expression, and ligand functionality of RFRP3 in the mare during the breeding and non-breeding seasons. Hypothalamic RFRP3 mRNA was detected in the mare; however, neither hypothalamic expression of RFRP3 nor its anterior pituitary receptor differed between reproductive states. We then used equine adenohypophyseal cell culture to test the hypothesis that RFRP3 reduces the responsiveness of the equine gonadotrope to GnRH. Addition of RFRP3 to cell culture failed to counter the effects of GnRH. Finally, the effects of a RFRP3 receptor-signaling antagonist (RF9) were examined in winter anovulatory mares. A robust increase in circulating concentrations of LH relative to controls was observed in response to RF9 treatments, but treatments had no effect on adenohypophyseal responsiveness to GnRH. Data provide indirect evidence that antagonism of the RFRP3 system by RF9 may be at the GnRH neuronal level.

De nombreux agents chimiothérapeutiques sont gonadotoxiques et peuvent donc induire une insuffisance ovarienne précoce chez les jeunes patientes traitées. La protection pharmacologique de l'ovaire pendant la chimiothérapie à l'aide d'analogues de la Gonadotropin-Releasing Hormone (GnRHa) est une option intéressante de préservation de la fertilité de par son caractère non-invasif et la possibilité d’une récupération spontanée de la fonction ovarienne. Ces molécules sont des inhibiteurs bien connus de l'axe hypothalamo-hypophyso-gonadique, mais leur efficacité dans cette indication est, cependant, controversée et leurs mécanismes d'action sont mal compris. Par conséquent, nous avons investigué les mécanismes potentiels de protection ovarienne des GnRHa pendant la chimiothérapie sur modèle murin. Nous avons montré que le cyclophosphamide (Cy) induit une déplétion folliculaire aiguë et proportionnelle à la dose affectant à la fois les follicules quiescents et en croissance. Lorsqu'ils sont administrés seuls à différentes doses et sites, l'agoniste et l'antagoniste de la GnRH altèrent les cycles oestraux, mais ne bloquent ni la folliculogenèse ni la sécrétion de la Follicle-Stimulating Hormone (FSH) chez la souris. De plus, le Cy atteint les follicules primordiaux, que les souris aient été traitées avec les GnRHa ou non. Ces résultats suggèrent que les GnRHa n'inhibent pas l'axe hypophyso-gonadique aussi efficacement chez la souris que chez la femme. Par conséquent, nous avons développé de nouveaux modèles pour étudier les mécanismes potentiels de protection ovarienne des GnRHa. Afin de différencier les effets directs des GnRHa via leurs récepteurs ovariens ou indirects par inhibition de la sécrétion de gonadotrophines, l'effet de l'agent alkylant sur le développement folliculaire et la réserve ovarienne a été testé sur des follicules cultivés in vitro avec ou sans GnRHa et in vivo chez des souris déficientes en FSHb (Fshb-/-). Pour imiter la profonde inhibition de FSH observée chez la femme après traitement aux GnRHa, nous avons étudié la toxicité de la chimiothérapie chez les souris Fshb-/-. L’administration de gonadotrophines exogènes (pregnant mare serum gonadotropin, PMSG) induit une croissance folliculaire jusqu’au stade antral mais n’influence pas le nombre total de follicules au sein de l’ovaire. Le Cy induit une perte folliculaire significative dans le groupe contrôle et dans le groupe traité au PMSG. Aucune différence concernant la prolifération ni l'apoptose n'a été observée entre les groupes traités à la chimiothérapie. A ce jour, ce modèle murin représente le meilleur modèle pour étudier l'inhibition gonadotrope induite par les GnRHa observée chez la femme. Ces résultats suggèrent que la FSH n'est pas impliquée dans la protection ovarienne potentielle des GnRHa pendant la chimiothérapie. Afin d’évaluer les effets directs des GnRHa sur les follicules en croissance et quiescents, des follicules préantraux ou des ovaires de nouveau-nés (PND4) ont été cultivés avec ou sans GnRHa avant l'exposition au métabolite actif du Cy, le 4-hydroperoxycyclophosphamide (4HC). Nous avons d'abord montré que l'exposition in vitro aux GnRHa n'affectait ni la survie et le développement folliculaire, ni la maturation ovocytaire. Dans les follicules en croissance, le 4HC diminue significativement les taux de survie et de maturation; et retarde le développement folliculaire, indépendamment du traitement aux GnRHa. La chimiothérapie diminue le nombre de cellules de la granulosa par follicule tandis que la production d’adénosine monophosphate cyclique (AMPc) par million de cellules de la granulosa n'est pas modifiée, ni par le 4HC, ni par les GnRHa. La sécrétion d'oestradiol tend à être retardée dans le groupe traité à l’agoniste mais pas dans le groupe antagoniste. De même, dans les ovaires PND4, le 4HC induit une perte folliculaire importante et atteint directement les cellules de la granulosa des follicules ovariens. Aucune différence dans la distribution folliculaire, la prolifération ou l'apoptose n'a été observée entre les groupes traités avec le 4HC, peu importe la présence des GnRHa ou non. Pour conclure, en se basant sur des modèles murins robustes et originaux, notre travail remet en question l'efficacité des GnRHa pour préserver l'ovaire contre les dommages causés par la chimiothérapie que ce soit par une action directe sur l'ovaire, ou indirectement par l'absence de FSH. D'autres investigations seront nécessaires pour comprendre les mécanismes d'action potentiels des GnRHa sur l'ovaire et les voies impliquées. Des preuves expérimentales sont encore indispensables pour clore le débat sur cette option attrayante de préservation de la fertilité. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Chez les mammifères, la reproduction est regulée par des neurones spécifiques qui sécrètent le neuropeptide GnRH (Gonadotropin Releasing Hormone). Ces cellules naissent au stade prénatal dans la placode nasale et migrent dans l'hypothalamus, le long des nerfs olfactifs voméro-nasaux, pour devenir des membres à part entière de l'axe hypothalamo-hypophyso-gonadique. Un certain nombre de pathologies de la reproduction humaine sont associées à la perturbation soit de la migration neuronale des cellules à GnRH ou soit de la sécrétion de la GnRH.L'objectif général de ma thèse était d'identifier de nouveaux facteurs moléculaires régulant la migration des cellules à GnRH, leur ciblage axonale à l'éminence médiane, mais aussi leur neurosécrétion au cours de la vie reproductive.Les événements complexes du développement correcte du système à GnRH sont strictement régulés par l'expression spatio-temporelles des molecules de guidage et des molécules de la matrice extracellulaire, dont les fonctions, sont en partie médiées par leur liaison avec la β1-intégrine (Itgb1). La première partie de mon travail a été d’étudier le rôle biologique de ces protéines de surface dans la reproduction. La technologie Cre/loxP a été utilisée pour générer des souris conditionnelles GnRH spécifiques KO pour la β1-intégrine (GnRH-Itgb1-/-). La perte d’activité de la β1-intégrine altére la migration des neurones à GnRH, leur extension axonale à l’eminence mediane et la fertilité de ces souris. Ces résultats mettent en évidence que la β1-intégrine joue un rôle important dans le développement normal du système GnRH et dans l'acquisition de compétences reproductives normales chez les rongeurs.Dans la deuxième partie de ma thèse de doctorat, j'ai identifié de nouveaux facteurs moléculaires qui pourraient être responsables de l'apparition du syndrôme des ovaires polykystiques (SOPK). Cette maladie est présente chez près de 10 % des femmes. Il s’agit d’une hyperandrogénie associée à une oligo-anovulation chronique, une morphologie ovarienne polykystique et d'autres situations cliniques de transition d'un état endocrinien à un autre. Chez les patientes atteintes du SOPK, le niveau d'hormone antimüllérienne (AMH) est élevé et indique clairement que l'AMH pourrait est un marqueur possible dans le diagnostic et le traitement du SOPK. Une autre manifestation du syndrome est une élévation des sécrétions du GnRH provoquant une augmentation des taux de LH et un rapport LH/FSH élevés, qui stimulent la production d'androgènes ovariens. Toutefois, jusqu'à présent cette maladie a été considérée principalement comme une pathologie gonadique et des régulations possibles plus élevées au niveau du système nerveux central ou des interactions avec ce dernier n'ont pas été étudiées. En particulier, des informations concernant les effets extra-ovariens possibles de l'AMH sur l'axe hypothalamo-hypophyso-gonadique manquent actuellement. Mon projet de recherche a été d’étudier le rôle encore méconnu de l'AMH dans la régulation de la physiologie du système à GnRH. Mes études ont permis d’identifier un nouveau rôle extra-ovarien pour l'AMH, et notamment comme un puissant activateur de la neurosécrétion de la GnRH. / During aging, oxidative stress occurs characterized by an imbalance between the production reactive oxygen species and antioxidant capacity. It’s well recognized that acute exercise induces oxidative stress which response may be affected by aging. Only few studies have focused on the response of oxidative stress parameters to an acute exercise in relation with aging and they were not made in humans. The first aim of this work was to investigate the response of oxidative stress parameters to acute exercise in young and older subjects. Our results showed that aging has no effects on oxidative stress parameters at rest. However, in response to an acute physical exercise, our results showed that aging is characterized by an antioxidant defenses deficiency and an increase in free radical damage markers. On the other hand, regular physical activity is considered as an effective way to reduce free radical attacks and enhance the antioxidant defense. These adaptations to regular physical activity are often related to the level of physical activity and this has been shown in young subjects. The second aim of this work was to study oxidative stress parameters in elderly subjects with different physical activity levels. Our results showed a positive correlation between physical activity level and antioxidant potential. However, physical activity at high level increases free radical damage in older adults. In view of changes in oxidative stress parameters with aging, adaptations of those to regular physical activity could also be affected by aging phenomena. The aim of the third study of this work was to investigate the effects of aging and physical activity level on oxidative stress parameters responses to an acute exercise. To do so, we compared these parameters in two young subjects groups (active and sedentary) and two older adults groups (active and sedentary) before and after an acute exercise. Our results showed that, benefits of regular physical activity on the oxidative parameters stress were more pronounced in younger age groups compared to older groups. On the other hand, the effects of aging on oxidative stress parameters in the active groups were lower than those noted in the sedentary groups.

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Chez le mammifère la fonction de reproduction est sous le contrôle des neurones hypothalamiques à GnRH-I. Au cours du développement embryonnaire ces neurones migrent de la fosse nasale vers le cerveau. De nombreuses études s’intéressent aux facteurs impliqués dans leur migration, mais l’influence de leur environnement cellulaire est très peu étudiée. Nous avons émis l’hypothèse que les neurones à GnRH-I d’origine extra-cérébrale possèdent un environnement gliale nécessaire à leur migration, connaissant le rôle de ces cellules dans l’ontogenèse neuronale du cerveau. Nos résultats montrent que 1) les neurones à GnRH-I sont associés à des cellules gliales au cours de leurs migrations nasale et télencéphalique 2) ces cellules gliales sont des progéniteurs des cellules gliales olfactives engainantes qui se différencient dans les régions rostrales au cours de la migration neuronale. 3) ces cellules expriment des gènes codant pour des facteurs impliqués dans la migration de ces neurones. 4) le transcriptome de ces cellules gliales est perturbé en présence d’un perturbateur endocrinien œstrogèno-mimétique, et touche des familles de gènes impliquées dans les molécules d’adhésions cellulaires nécessaire à la migration et à la régulation de l’activité des neurones à GnRH-I. / GnRH-I cells control reproduction functions in mammals. These cells are extra cerebral since they come from the nasal pit and migrate to the forebrain during embryonic development. Numerous studies have described the influence of different molecules on the migration of GnRH-1 neurons, however, the role of microenvironment cells remains poorly understood. Considering the role of glial cells in the forebrain’s neuronal migration, we had hypothesized that extra-cerebral GnRH-I neurons possess a glial environment necessary for their migration from the nose to the brain. Our results demonstrated that 1) GnRH-I neurons are associated with glial cells during their migration in the nasal septum and forebrain 2) These glial cells are progenitors of olfactory ensheathing cells, and differentiated within the rostral regions during neuronal migration. 3) These cells express genes encoding factors involved in GnRH-I neurons migration 4) Glial cells transcriptome are disrupted with estrogen-mimicking endocrine disruptor, and affects gene families involved in cell adhesion molecules necessary for migration and activity regulation of GnRH-I neurons

of The aim of this study was to develop and evaluate two protocols for estrus synchronization and/or induction in 12-14 months old beef heifers, which would allow the use of timed artificial insemination (TAI) after a short period of estrus detection (48h). The first experiment consisted of evaluating whether eCG was a necessary component in the BioRep system to be used in heifers. To address this question 64 heifers (Bos taurus x Bos indicus) were randomly divided into 2 groups: eCG (treatment n=32) and no eCG (controle n=32). A second experiment was performed with the objective of increasing the number of animals in the eCG group to evaluate the consistency of the results obtained in the first experiment (n=92). On day 0 animals from group treatment received a vaginal pessary containing 250mg of medroxyprogesterone acetate (MPA) for 7 days, along with an intramuscular (IM) injection containing 2.5mg of estradiol benzoate (EB). On day 6 animals were treated with 250IU of eCG (IM) and i~g of cloprostenol sodium in the vulvar submucosa (VSM). After MPA withdrawal heifers were checked for estrus manifestation for 48h and were inseminated 12 hours after estrus detection. Heifers which did not manifest estrus during the 48h observation received i~g of GnRH (IM) and were inseminated 16 to 24h later by TAI. Heifers in the control group received a similar treatment except for the eCG injection which was not used. Because results from treatment group on first and second experiments were not different, pooled conception (55.2%) and pregnancy (46.2%) rates were compared to control rates (23.07% and 25.0% respectively) showing a significant improvement of those rates. We conclude that the use of eCG associated with MPA improves pregnancy rates of 12-14 months old beef heifers. Our second objective was to evaluate the use of progestogens in a system of estrus induction/synchronization using eCG-GnRH-MPA-PGGnRH (eMGPG) in 12-14 months old beef heifers. Bos taurus heifers, mostly Red Angus, were randomly divided into 2 groups: eMGPG (n=85) and eGPG (n=85). Heifers on group eMGPG received 400IU of eCG (IM; day -3) with the objective of inducing an increase in follicular growth, leading to higher responsiveness of follicles to GnRH. Seventy-two hours after eCG injection animals were given GnRH (100g; IM; day 0) and a vaginal pessary containing 250mg of MPA for 7 days. At MPA withdrawal (day 7) 5mg of Dinoprost was injected into the VSM. Fourty-eight hours after pessary withdrawal estrus detection followed by AI was performed. Heifers which did not manifest estrus received 100g of GnRH (IM) and were inseminated 16 to 24h later by TAI. Females from group eGPG received a protocol similar to group eMGPG, except for MPA which was not used. Percentage of estrus manifestation on groups eMGPG and eGPG after prostaglandin injection were 23.5% (20/85) and 22.3% (19/85) respectivelly (p>0.05). Similarly, conception and pregnancy rates on groups eMGPG (65.0%; 13/20 and 37.6%; 32/85%) and eGPG (68.4%; 13/19 and 28.2%; 24/85) were not statistically different. To evaluate the response of follicles with different sizes after eCG administration, 12-14 months old heifers were randomly divided into groups of animals containing follicles of 5.0 (small), 8.5 (medium) and >10.0mm (large) to receive 400IU of eCG (IM) during diestrus. Follicular diameters on day 3 of the control of follicular growth by ultrasound were 13mm, 10mm and 8mm on groups of large, medium and small follicles, respectively. We conclude that the use of eCG prior to the regular hormonal protocol, with the objective of increasing the follicular responsiveness to GnRH does not replace a source of progestagen during the synchronization/induction of estrus in 12-14 months old beef heifers.Furthermore, some females despite the prior administration of eCG depending upon their stage of follicular development do not reach a diameter compatible with GnRH responsiveness / O objetivo do presente estudo, foi o de desenvolver e avaliar dois protocolos de sincronização e ou indução de estro em novilhas de corte com 12 a 14 meses de idade, que possibilite o emprego da inseminação em tempo fixo (IATF) após um curto controle de estro (48h). O primeiro trabalho experimental consistiu na avaliação da necessidade de eCG no sistema BioRep quando utilizado em novilhas. Para tanto, foram empregadas 64 novilhas (Bos taurus x Bos indicus) divididas ao acaso em dois grupos: eCG (tratamento- n =32) e s/eCG (controle -n=32). Em uma segunda etapa o grupo tratamento foi implantado em um número maior de fêmeas (n=92), avaliando a consistência dos resultados obtidos no pré-experimento. No dia 0, as novilhas do grupo tratamento receberam um pessário vaginal com 250mg de MAP por sete dias, e uma injeção intra-muscular (IM) de 2,5mg de BE. No sexto dia, foram aplicados 250UI IM de eCG e 104μg Cloprostenol sódico na sub-mucosa vulvar (smv). Após a retirada do MAP, as novilhas tiveram seu estro controlado por um período de 48h, sendo inseminadas 12h após a manifestação do mesmo. As novilhas que não manifestaram o estro no período de controle, receberam 100μg IM de GnRH e, após 16 a 24h, foram inseminadas por IATF. As novilhas do grupo controle receberam o mesmo tratamento anterior, sem a aplicação do eCG. Os índices de concepção e prenhez nos grupos tratamento (55,2% e 46,2%), mostrarem-se similares e ao serem agrupados foram significativamente superiores aos do grupo controle (23,07% e 25,0%) respectivamente (P<0,05). Conclui-se que o emprego de eCG, associado ao MAP, permite melhorar os índices de prenhez em novilhas de 12 a 14 meses de idade. Por sua vez, o segundo experimento teve por objetivo avaliar o emprego de progestágeno em um sistema de indução/sincronização de estro, utilizando eCG-GnRH-MAP-PG-GnRH (eMGPG) para novilhas de corte de 12 a 14 meses. Novilhas Bos taurus, predominante Red Angus, foram divididas aleatoriamente em dois grupos: eMGPG (n=85) e eGPG (n=85). As novilhas do grupo eMGPG receberam 400UI de eCG (IM; dia -3) com a finalidade de provocar um aumento de crescimento folicular, tornando-os mais responsivos ao GnRH. Após 72h, os animais receberam uma injeção de GnRH (100μg; IM; dia 0) e um pessário vaginal contendo 250mg de acetato de medroxi-progesterona (MAP) por sete dias. Na retirada do MAP (dia 7), foi aplicada uma dose de 5mg de Dinoprost, via submucosa vulvar. Nas 48h seguintes à retirada dos pessários, foi realizado o controle da manifestação de estro e inseminação artificial (IA). As novilhas que não manifestaram estro nesse período, receberam uma dose de 100μg de GnRH por via IM e IATF nas 16 a 24h seguintes. As fêmeas do grupo eGPG receberam o protocolo similar ao anterior porém, sem o MAP. A percentagem de estro dos grupos eMGPG e eGPG após a aplicação da prostaglandina foram de 23,5% (20/85) e 22,3% (19/85) respectivamente (p>0,05). Igualmente, os índices de concepção e prenhez nos grupos eMGPG (65,0%; 13/20 e 37,6%; 32/85) e eGPG (68,4%; 13/19 e 28,2%; 24/85) não diferiram estatisticamente. Com intuito de avaliar a resposta de folículos de diferentes tamanhos após administração de eCG, novilhas de 12-14 meses foram aleatoriamente distribuídas para receber 400UI de eCG (IM) na presença de folículos de 5,0, 8,5 e >10,0mm, durante o diestro. Os diâmetros médios dos folículos no dia 3 da dinâmica foram, de 13mm, de 10mm e de 8mm nos grupos de folículos grandes, de folículos médios e de folículos pequenos respectivamente. Conclui-se que a aplicação prévia de eCG para aumentar o índice de folículos responsivos ao GnRH não substitui uma fonte de gestágeno na sincronização/indução de estro em novilhas de corte com 12 a 14 meses de idade. Pode-se concluir também que algumas fêmeas, apesar da aplicação prévia de eCG, dependendo do momento do crescimento folicular em que se encontram, não atingem um diâmetro folicular compatível com resposta ao GnRH

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Made available in DSpace on 2016-05-02T13:55:02Z (GMT). No. of bitstreams: 1 Leandro Francisco Gofert- Dissertacao.pdf: 644155 bytes, checksum: c324806273039e5418f42b4f7ed29fa2 (MD5) Previous issue date: 2015-02-27 / To test a new follicular growth inducer on the increase of follicular growth, the rate of ovulation and pregnancy in cows submitted to fixed time artificial insemination (FTAI). Was associated with a GnRH analogue (gonadorelin, Fertagyl®, MSD, São Paulo, Brazil) diluted in 20% sodium hyaluronate solution. It was used 27 Nellore cows, lactating, body condition score (BCS) of 2.5 (range 1-5) in anoestrus (fol. <8 mm and absence of corpus luteum). On day 0 all cows received a progesterone device (Primer® - Tecnopec, São Paulo, Brazil) and 2 mg of estradiol benzoate (RIC-BE® - Tecnopec, São Paulo, Brazil). Eight days later were evaluated by ultrasound and divided into 3 groups, balancing groups by follicular condition, that day all animals had their devices removed and received 500 g of sodium Cloprostenol (Ciosin® - MSD, São Paulo, Brazil). In the G100 group was administered intramuscularly the priming solution of growth with 100 &#956;g of gonadorelin. In the G200 was applied to priming solution of growth with 200 &#956;g of gonadorelin. In the control group (CG) was applied 2 mL of saline solution. On day 9 the animals received 1 mg of estradiol benzoate as ovulation inducer. All animals were inseminated on day 10, 54 h after removal of the devices. In day 9 and 10 the animals were evaluated by ultrasound for follicular measurement, on day 19 for corpus luteum ID (ovulation rate) and day 44 for pregnancy diagnosis. Follicular diameter did not differ between groups at D0: 5.2 ± 1.16 mm vs. 5.8 ± 1.45 mm vs. 5.2 ± 1.55 mm; at D8: 7.3 ± 1.83 mm vs. 8.2 ± 1.67 mm vs. 7.4 ± 1.60 mm; in D9: 9.2 ± 1.69 mm vs. 9.6 ± 1.55 mm vs. 9.2 ± 1.18 mm, respectively, for G100, G200 and GC. Follicular growth between day 8 and 9 also did not differ between groups: 1.9 mm vs. 1.4 mm vs. 1.8 mm, for G100, G200 and GC respectively. The cows treated with GnRH, evaluated in the D10, had anticipation of ovulation in relation to GC: 100% vs. 77.8% vs. 0%, respectively, for G100, G200 and GC. The ovulation rate determined by the presence of CL in the D19 did not differ between the groups: 100% vs. 77.8% vs. 77.8% respectively for G100, G200 and GC, the diameters of corpora lutea did not differ, when evaluated in D19: 1.56 ± 0.24 mm vs. 1.68 ± 0.40 vs. 1.51 ± 0.30 mm, respectively, for G100, G200 and GC. The pregnancy rate was higher in GC than those treated: 33.4% vs. 0 vs. 0, respectively for GC, G100 and G200. The data suggest that the association between the GnRH analogue and hyaluronate 20% (at the doses tested) did not increase follicular growth or ovulation rate. Probably this association induced a premature LH surge, leading to early ovulation and harming the pregnancy rates of the treated animals. / Para testar um novo indutor de crescimento folicular sobre o aumento do crescimento folicular, a taxa de ovulação e prenhez em vacas submetidas à inseminação artificial em tempo fixo (IATF). Associou-se um análogo de GnRH (gonadorelina, Fertagyl®, MSD, São Paulo, Brasil) diluído em solução de hialuronato de sódio a 20%. Foram utilizadas 27 vacas Nelore, lactantes, com escore de condição corporal (ECC) de 2,5 (escala de 1 a 5), em anestro (fol. < 8 mm e ausência de corpo lúteo). No dia 0 todas as vacas receberam um dispositivo de progesterona (Primer® - Tecnopec, São Paulo, Brasil) e 2 mg de benzoato de estradiol (RIC-BE® - Tecnopec, São Paulo, Brasil). Oito dias depois foram avaliadas por ultrassom e divididas em 3 grupos, equilibrando-se os grupos pela condição folicular. Nesse dia todos os animais tiveram seus dispositivos removidos e receberam 500 µg de Cloprostenol sódico (Ciosin® - MSD, São Paulo, Brasil). No grupo G100 foi aplicada por via IM a solução indutora de crescimento com 100 µg de gonadorelina. No G200 foi aplicada a solução indutora de crescimento com 200 µg de gonadorelina. No grupo controle (GC) foi aplicado 2 ml de solução fisiológica. No dia 9 os animais receberam 1 mg de benzoato de estradiol como indutor de ovulação. Todos os animais foram inseminados no dia 10, 54 h após a retirada dos dispositivos. Nos dias 9 e 10 os animais foram avaliados por ultrassonografia para mensuração folicular, no dia 19 para identificação de corpo lúteo (taxa de ovulação) e no dia 44 para diagnóstico de gestação. O diâmetro folicular não diferiu entre os grupos no D0: 5,2±1,16 mm vs. 5,8±1,45 mm vs. 5,2±1,55 mm; no D8: 7,3±1,83 mm vs. 8,2±1,67 mm vs. 7,4±1,60 mm; no D9: 9,2±1,69 mm vs. 9,6±1,55 mm vs. 9,2±1,18 mm, respectivamente para G100, G200 e GC. O crescimento folicular entre o dia 8 e 9 também não diferiu entre os grupos: 1,9 mm vs. 1,4 mm vs. 1,8 mm, para G100, G200 e GC respectivamente. As vacas tratadas com GnRH, avaliadas no D10, tiveram antecipação da ovulação em relação ao GC: 100% vs. 77,8% vs. 0%, respectivamente para G100, G200 e GC. A taxa de ovulação avaliada pela presença de CL no D19 não diferiu entre os grupos: 100% vs. 77,8% vs. 77,8% respectivamente para G100, G200 e GC. Os diâmetros dos corpos lúteos também não diferiram quando avaliados no D19: 1,56±0,24 mm vs. 1,68±0,40 mm vs. 1,51±0,30 mm, respectivamente para G100, G200 e GC. A taxa de prenhez foi superior no GC em relação aos tratados: 33,4% vs. 0 vs. 0, respectivamente para GC, G100 e G200. Os dados sugerem que, a associação entre análogo de GnRH e hialuronato a 20% (nas doses testadas) não aumentou o crescimento folicular, nem a taxa de ovulação. Provavelmente essa associação induziu um pico precoce de LH, levando à ovulação antecipada e prejudicando as taxas de prenhez dos animais tratados.

Während der letzten Jahre stellten GnRH-II-Antagonisten einen Interessenschwerpunkt bei der endokrinen Therapie gynäkologischer und auch anderer Karzinome wie des Prostatakarzinoms dar. Es konnte dargestellt werden, dass GnRH-II-Antagonisten schon in nanomolaren Konzentrationen in vitro und in vivo zu Apoptose von Mamma- Endometriums- und Ovarialkarzinomen führen. In dieser Studie sollte untersucht werden, ob eine Wirkungssteigerung des Antitumoreffekts durch die Kombination des GnRH-II-Antagonisten [(AcD-2-Nal1), (D-4Cpa2), (D-3Pal3), (D-3Pal6), (D-Leu8), (D-Ala10)] mit dem selektiven Estrogenrezeptormodulator (SERM) Tamoxifen beziehungsweise mit dem selektiven Estrogenrezeptordestabilisator (SERD) Fulvestrant erzielt werden kann. Hierzu wurden das Proliferationsverhalten der Ovarialkarzinomzelllinien OVCAR-3, SKOV-3, EFO-21 und EFO-27 sowie Veränderungen der Rezeptorexpression des ER-α, ER-β, GPR-30 und des GnRH-II-Rezeptors bei Behandlung der Zellen mit den einzelnen Substanzen und der Kombination von Tamoxifen und GnRH-II-Antagonist untersucht. Auch die Apoptosewege wurden anhand des Nachweises der Phosphorylierung von p38 und JNK und der Spaltung von Caspase-3 bei Behandlung mit der Kombination von Tamoxifen und GnRH-II-Antagonist untersucht. Die Behandlung der Zelllinien mit der Kombination von Tamoxifen beziehungsweise Fulvestrant und GnRH-II-Antagonist führte bei drei von vier Zelllinien zu einer stärkeren Verminderung der Zellzahl als die Behandlung der Zellen mit den einzelnen Substanzen. Auch auf Rezeptorbasis zeigte die Kombinationstherapie Vorteile gegenüber der Monotherapie mit Tamoxifen und GnRH-II-Antagonist. Apoptose fand bei den untersuchten Zelllinien SKOV-3 und EFO-27 vor allem durch den Weg der Phosphorylierung von JNK statt. Die Phosphorylierung von JNK erfolgte bei der Kombinationstherapie in kürzerer Zeit und fiel intensiver aus als bei einzelner Anwendung der Substanzen Tamoxifen und GnRH-II-Antagonist. Insgesamt konnte gezeigt werden, dass die Kombinationstherapie von Tamoxifen und GnRH-II-Antagonist sowohl bezüglich der Zellproliferation als auch der Rezeptorexpression und der Induktion von Apoptose einer Monotherapie mit den Substanzen überlegen war.

Die GnRH-Agonisten-Applikation zur Downregulation vor IvF ist "gold standard", überwiegend im sogenannten langen Protokoll. Die Behandlung soll den vorzeitigen LH-Anstieg mit vorzeitiger Ovulation verhindern. Die unerwünschten Wirkungen sind dosisabhängig und rechtfertigen die Suche nach der optimal niedrigen Dosis des GnRH-Agonisten. Mit dieser Fragestellung wurde eine prospektive randomisierte Dosisfindungsstudie durchgeführt. 200 sterile Frauen zwischen 18 und 38 Jahren erhielten vor der IvF-Behandlung im langen Protokoll die Standarddosis von 4,12 mg Triptorelinazetat-Depot (1 Amp. i.m. = Gruppe B: n = 100) versus 2,1 mg Triptorelinazetat Depot (1/2 Amp. i.m = Gruppe A. n = 100) zur Downregulation. Folgende Parameter wurden bestimmt: E2, LH, Progesteron. Die Behandlungsergebnisse wurden korreliert mittels der Anzahl der gewonnenen Oocyten, der fertilisierten Oocyten, der transferierten Embryonen und der Schwangerschaftsraten pro Embryotransfer. Abgebrochene IvF-Zyklen wurden einzeln analysiert. Bezüglich der Hormonwerte waren beide Gruppen ohne signifikanten Unterschied. In der Gruppe der Patientinnen mit der halbierten Dosis (A) kam es nur in einem Fall zu einer vorzeitigen Luteinisierung, in der Standartdosisgruppe (B) in keinem Fall. Wegen low response wurde in Gruppe A in 5 Fällen die Therapie abgebrochen, versus 3 Fälle in Gruppe B (ns). Ebenfalls vergleichbar war das IvF-outcome, nur die ET-Rate pro begonnener Stimulation zeigte einen signifikanten Unterschied: 88 % (A) versus 96 % (B), p / The GnRH agonist application for the downregulation prior to IVF is 'gold standard', mainly in the so-called long protocol. This should avoid premature ovulations. The dose-dependent, undesired effects justify the search for the optimal low dose of the GnRH agonist. A prospective randomised dose-finding study was carried out in this respect. Among 200 sterile women (18 and 38 years) for the planned IVF and/or IVF / ICSI treatment in the long protocol, n = 100 in group A received 2.1 mg Triptorelinacetate depot (1/2 amp., i.m.) and n = 100 in group B the standard dose of 4.12 mg (1 amp., i.m.) for the downregulation. The hormone values E2, LH, progesterone were determined. The treatment results were compared by means of the number and quality of the oocoytes, the embryo transfers and the pregnancy rates. Cancelled IVF cycles were analysed. With respect to the hormone values, neither of the two groups showed significant differences. A premature luteinization occurred in group A (reduced dose) in only one case; in the standard dose of group B, none occurred. Due to the low response, the therapy was cancelled in 5 cases in group A, in comparison to 3 cases in group B (ns). The IVF outcome showed a comparable result. The only significant difference was the ET rate per started stimulation (p