Šio darbo tikslas buvo nustatyti ovuliacijos pasireiškimo laiko priklausomybę nuo kalės veislės dydžio ir amžiaus bei metų laiko, bei palyginti GnRH agonisto Suprelorin implanto sukeltos ovuliacijos pasireiškimo laiką su natūraliai pasireiškusios rujos ovuliacijos laiku. Šiam tikslui pasiekti pirmiausiai buvo ištirta 194 kalių (51 skirtingos veislės) progesterono koncentracija kraujo serume. Iš šių kalių buvo atrinktos tos kalės, kurioms pagal progesterono koncentraciją pasireiškė ovuliacija (53 kalės). Nustatyta, kad progesterono koncentracija kraujo serume siekė 20-30 nmol/l. Kalės buvo suskirstytos į grupes: pagal amžių (2 grupės – iki 5 metų (n=41); 5 metų ir vyresnės (n=12) ), pagal veislės dydį (3 grupės – mažos (n=22), vidutinės (n=14) ir didelės (n=17) veislės), pagal sezoną (4 grupės – žiema (n=3), pavasaris (n=16), ruduo (n=11), vasara (n=23) ). Pagal kiekvieną požymį statistiškai buvo įvertinti ovuliacijos pasireiškimo laiko skirtumai tarp grupių, bei įvertinta ovuliacijos priklausomybė nuo kalės amžiaus, dydžio, metų laiko. Statistiškai patikimų skirtumų tarp grupių ir koreliacinių ryšių tarp ovuliacijos pasireiškimo laiko ir kalės amžiaus ir dydžio, bei metų laiko nebuvo nustatyta. Panašių tyrimų iki šiol buvo atlikta labai mažai, todėl informacija yra vertinga. Paskutiniame tyrimųetape4 kalėms buvo panaudoti GnRH agonisto Suprelorin implatai rujai sukelti. Implantas buvo įvedamas po oda, bambos srityje. Po įvedimo buvo imamas kraujas kelis kartus kas 3-5 dienas... [toliau žr. visą tekstą] / The main goal of this paper was to determine whether or not there is a significant influence of GnRH agonist, seasonality, age and size of the bitch on ovulation time, and to compare ovulation time induced by GnRH agonist Suprelorin implant and the ovulation time that occured naturally. To achieve this goal we tested the blood of 194 bitches (51 breeds) for progesterone levels in blood serum. Out of these 194 bitches we selected the ones (53 bitches) that ovulated. Meaning, the progesterone levels for these 53 bitches varied between 20-30 nmol/l. These 53 bitches were divided into groups: by age (2 groups – younger than 5 years (n=41); 5 years old and older (n=12),, by size (3 groups – small (n=22), medium (n=14), large (n=17), and by season (4 groups – winter (n=3), spring (n=16), summer (n=11), autumn (n=23). All the groups were statistically analyzed to determine whether or not there is a significant difference between the groups, and to determine if there are correlations between the ovulation time and the size and age of the bitch and time of year (season). No significant difference or correlations were determined. We have found a little information about influence of seasonality, and age and size of the bitch on ovulation time, so the information in this paper is valuable. For the last step to achieving the goal of the paper, GnRH agonist Suprelorin implant was used on 4 bitches. The agonist was implanted subcutaneously around the navel area. After the implantation... [to full text]

Chez les mammifères, le contrôle de la reproduction est médié par un réseau hypothalamique qui intègre divers stimuli pour réguler la sécrétion de la Gonadotropin Releasing Hormone (GnRH). Ces neurones à GnRH naissent dans la placode olfactive et migrent vers le cerveau le long des axones vomeronasaux et terminaux au cours du développement embryonnaire. Bien que ce processus a bien été étudié chez les rongeurs, sa caractérisation complète chez l'homme reste inachevée. Il est largement admis que des perturbations dans le développement ou dans la sécrétion de GnRH sont associées chez l’homme à un hypogonadisme hypogonadotrope congénital (CHH), qui est un trouble caractérisé par un retard ou une absence de la puberté conduisant à l'infertilité.Les systèmes GnRH et olfactif ont des liens complexes au cours du développement, le syndrome de Kallmann (KS) représente un trouble qui associe l'hypogonadisme dû à une déficience en GnRH et l'anosmie. Le CHH et le KS sont des troubles oligogéniques, les mutations génétiques sous-jacentes n’expliquent que 50% des cas cliniques.Dans cette étude, nous avons entrepris une caractérisation complète du processus migratoire des neurones à GnRH au cours du premier trimestre de gestation sur une grande série d'embryons et de fœtus humains, ce qui nous a permis d’élaborer le premier atlas chronologique et quantitatif de la distribution de GnRH. En effet, l'utilisation d’une nouvelle approche de transparisation des tissus embryologiques humains par de solvants organiques, a permis d’établir pour la première fois, une véritable représentation des neurones dans leur contexte natal in vivo.De plus, les résultats de cette étude ont non seulement révélé que le nombre de neurones GnRH chez l'homme était significativement plus élevé que prévu, mais aussi que ces derniers migrent vers plusieurs régions du cerveau extra-hypothalamique, en plus de l'hypothalamus. Leur présence dans ces régions soulève l'hypothèse qu’ils pourraient exercer des rôles non reproductifs, créant de nouvelles pistes pour la recherche sur les fonctions du système GnRH dans les processus cognitifs, comportementaux et physiologiques.Le second objectif de ce travail a visé à caractériser un nouveau gène candidat impliqué dans le développement du système GnRH: L'hormone Anti-Müllerienne (AMH), connue pour son rôle dans la différenciation de la gonade bipotentielle chez les mâles. Néanmoins, une récente étude menée par notre équipe a mis en évidence son rôle extragonadique sur les neurones à GnRH en période post-natale.Le séquençage complet d'une large cohorte de patients européens a révélé plusieurs nouvelles mutations faux-sens dans le gène de l’AMH chez les patients atteints de CHH et KS, non retrouvés dans la cohorte des témoins. L’évaluation de la pertinence fonctionnelle de ces mutations a ensuite été effectuée par diverses analyses biochimiques in vitro de la bioactivité des mutations, ainsi que par la caractérisation d'une lignée de souris transgénique. Ce qui a entraîné une diminution de la sécrétion de l'AMH et une diminution de la bioactivité de la protéine sécrétée dans les études in vitro; Conduisant à une éventuelle réduction de la capacité migratoire. Cela suggère fortement que ces mutations pourraient avoir un effet pathogène.En outre,nous montrons que le récepteur AMHR2 est exprimé le long des fibres olfactives et par les neurones à GnRH pendant le processus migratoire GnRH. L'analyse pathohistologique des souris Amhr2 -/- a révélé une altération de la migration embryonnaire des neurones à GnRH vers le cerveau antérieur basal, entraînant une réduction significative du nombre total de neurones GnRH dans les cerveaux adultes de ces animaux, conduisant à une fertilité réduite. L’ensemble de ces travaux indiquent que l'insuffisance de signalisation AMH contribuerait à la pathogenèse des troubles de CHH chez l’homme, et met en évidence un nouveau rôle de l'AMH dans développement et la fonction des neurones GnRH. / The control of mammalian reproduction is mediated by a hypothalamic network that integrates various stimuli to regulate the periodic secretion of gonadotropin-releasing hormone (GnRH). GnRH neurons, originate in the olfactory placode and enter the brain along vomeronasal and terminal axons during embryonic development. This process has been well studied in rodents, however, a full characterisation in humans was lacking. Alterations either in the development of this system or in the secretion of GnRH are associated with congenital hypogonadotropic hypogonadism (CHH) in humans, a condition characterized by failure of sexual competence. Due to the inextricable links in the development of the olfactory and GnRH systems, there also exists the developmental condition, Kallmann syndrome (KS), associating hypogonadism due to GnRH deficiency and anosmia. Both CHH and KS are oligogenic disorders, with the underlying genetic mutations only explained in 50% of the clinical cases.At the heart of this study, we have undertaken a full characterisation of the GnRH migratory process during the first trimester of gestation in a large series of human embryos and foetuses and provide the first chronological and quantitative atlas of GnRH distribution. This has been aided by the novel application of organic solvent based tissue clearing techniques to human embryological tissue. This allows, for the first time, a true representation and appreciation of cells in their native, in vivo context. The results of this study have revealed not only that the number of GnRH neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioural and physiological processes.The second aim of this work has been to characterise a novel candidate gene responsible for the development of the GnRH system. Anti-Müllerian hormone (AMH), best known for its role in facilitating the differentiation of the bipotential gonad in males, has recently been shown by our lab to exert significant effects on GnRH neurons postnatally. Here we have undertaken whole exome sequencing of a large cohort of European CHH and KS patients, identifying several novel missense mutations in the Amh gene that are not present in the control cohort. Various in vitro and biochemical analyses of mutations bioactivity as well as analysis of a transgenic mouse line have been used to assess the functional relevance of these mutations.Mutations in Amh resulted in impaired secretion of AMH and reduced bioactivity of the secreted protein in in vitro studies; eventually leading to reduced migratory capacity. This strongly suggests that these mutations could have a pathogenic effect. We also show that its receptor AMHR2 is expressed along the olfactory fibres and by GnRH neurons during the GnRH migratory process. Pathohistological analysis of Amhr2-/- mice revealed defective embryonic migration of GnRH cells to the basal forebrain, leading to a significant reduction in the total number of GnRH neurons in the adult brains of these animals resulting in reduced fertility. We therefore propose that AMH signalling insufficiency contributes to the pathogenesis of human CHH conditions and highlights a novel role for AMH in the correct development and function of GnRH neurons.

Made available in DSpace on 2015-03-26T13:54:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 259691 bytes, checksum: 3eb4834dd0179533658dbe83bf931de6 (MD5) Previous issue date: 2008-07-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The experiment was done with the following objectives: a) to evaluate the effect of GnRH administered at different times of the reproductive management on the pregnancy rate and serum P4 concentration; and b) to evaluate the effect of gonadotropins releasing hormone (GnRH) given twelve days after FTAI on serum levels of progesterone and above the rate of pregnancy. For the first objective 82 crossbred cows (Bos taurus indicus x Bos taurus Taurus) were allocated into three treatments: TControl (n = 28): the animals were observed for estrus detection and were inseminated after 8 to 12 hours; TGnRH0 (n = 27): similar to TControl plus the administration of 25 μg of GnRH at the AI time; and TGnRH12 (n = 27): similar to TControl plus the administration of 25 μg of GnRH at the AI time and also 12 days after AI. The pregnancy diagnostic was done on day 35 after the AI, by ultrasound scans through trans-rectal ultrasound. The pregnancy rate for TControl cows was 57.15% (16/28) for the first service, and for TGnRH0 and TGnRH12 cows, 63.00% (17/27) in each treatment. The protocol used did not affect the pregnancy rate of crossbred cows (P > 0.05). Also, there was no difference between the serum P4 concentrations between treatments, (P > 0.05). In conclusion, the administration of GnRH analogue, at the time of AI or 12 days after AI, did not improve the reproductive performance in crossbred cows. For the second objective 59 beef crossbred cows (Bos taurus indicus x Bos taurus taurus) were allocated into two treatments. TBE (n = 30): the day 0 - insertion of progesterone intravaginal device (Primer®) plus 2 mg of BE (Estrogin®), im; on 8, Remove the Primer® and applied 300 IU of eCG (Novormon ®) and 0.15 mg of PGF2 α (Prolise®), im, on 9, was applied 1 mg BE, im, and was do FTAI 48-56 hours after the withdrawal of PRIMER; and TBEGnRH12 (n = 29): the protocol was similar to TBE, but with administration of 25 μg of GnRH (Gestran Plus®, Lecirelin) twelve days after the FTAI. The pregnancy diagnostic was done on day 35 after the AI, by ultrasound scans through trans-rectal ultrasound. The pregnancy rate for TBE cows was 53.33% (16/30) for the first service, and for TBEGnRH12 cows, 37.93% (11/29). The protocol used did not affect the pregnancy rate of crossbred cows (P> 0.05). Also, there was no difference between the serum P4 concentrations between treatments (P> 0.05). In conclusion, the administration of the analogue of GnRH, lecirelina, at twelve days after the FTAI did not affect the pregnancy rates, neither affect the serum P4 concentration, between treatments, on days zero, five, 12 and 20 after the FTAI. / Este trabalho foi realizado com os seguintes objetivos: a) avaliar o efeito do hormônio liberador de gonadotropina (GnRH), administrado em diferentes momentos do manejo reprodutivo, na taxa de prenhez e concentração sérica de P4 em gado de corte mestiço; e b) avaliar o efeito do hormônio liberador de gonadotropinas (GnRH), administrado 12 dias após IATF, sobre os níveis séricos de progesterona e, principalmente, na taxa de prenhez em vacas de corte mestiças. Para o primeiro objetivo, utilizaram-se 82 fêmeas bovinas mestiças (Bos taurus indicus x Bos taurus taurus), alocadas ao acaso, em três tratamentos: Tcontrole (n = 28): os animais foram observados para detecção de estros e inseminados após 8 a 12 horas; TGnRH0 (n = 27): similar ao Tcontrole, com administração de 25 μg de GnRH (Gestran Plus®, Lecirelina) no momento da IA; e TGnRH0-12 (n = 27): similar ao Tcontrole, com administração de 25 μg de GnRH no momento da IA e no dia 12 após a IA. No dia 35, após IA, foi feito o diagnóstico de gestação por exames ultrassonográficos pela via transretal. Foi observado que 57,14% das vacas (16/28) no Tcontrole e no TGnRH0 e TGnRH12, 62,96% (17/27) ficaram gestantes após o primeiro serviço. Não foi observada diferença entre os animais dos tratamentos (P > 0,05). Também não se observou diferença entre as concentrações de P4 séricas entre os animais dos tratamentos. Concluiu-se que a administração de análogo de GnRH no momento da IA ou 12 dias após a IA não melhorou o desempenho reprodutivo em vacas mestiças, nem a sintese de P4 pelo corpo lúteo. Para o segundo objetivo, foram utilizadas 59 fêmeas bovinas mestiças (Bos taurus indicus x Bos taurus taurus), alocadas ao acaso, em dois tratamentos: TBE (n = 30): no dia 0, inseriu-se o dispositivo intravaginal de progesterona (Primer®) mais 2,0 mg de BE (Estrogin®), im; no dia 8, retirou-se o PRIMER e aplicaram-se 300 UI de eCG (Novormon®) e 0,15 mg de PGF2α (Prolise®), im; no dia 9, aplicou-se 1 mg de BE, im, e realizou-se a IATF 48-56 horas após a retirada do PRIMER; e TBEGnRH12 (n = 29): o protocolo foi similar ao do TBE, porém com administração de 25 μg de GnRH (Gestran Plus®, Lecirelina) no dia 12 após a IA. No dia 35, após IA, foi feito o diagnóstico de gestação por exames ultrassonográficos pela via transretal. Neste estudo foi observado que 53,33% das vacas (16/30) no TBE e 37,93% (11/29) no TBEGnRH12 ficaram gestantes após o primeiro serviço. Não foi observada diferença entre os animais dos tratamentos (P > 0,05). Também não se observou diferença entre as concentrações de P4 entre os animais dos tratamentos (P > 0,05). Concluiu-se que a administração do análogo de GnRH, lecirelina, no dia 12, após a IATF, não afetou as taxas de prenhez e nem a concentração de progesterona dos animais dos tratamentos, nos dias 0, 5, 12 e 20 após a IATF.

Neurônios GnRH e kisspeptina representam as populações neuronais de maior importância no controle da reprodução. Estradiol liga-se ao seu receptor expresso pelos neurônios kisspeptina para regular a libertação de GnRH. No modelo animal OVX+E a atividade do neurônio GnRH e pico de LH é depende do estradiol e hora do dia. Nesse estudo, embora a taxa de disparo dos neurônios GnRH seja similar entre os grupos, o padrão dos potenciais revelou uma mudança para maior duração do estouro em camundongos no proestrous, além do fato de uma maior resposta da hipófise. A prolactina tem grande impacto na modulação do eixo HPG e kisspeptina são mediadores dos efeitos da prolactina sobre a reprodução. Uma pequena porcentagem de neurônios de kisspeptina do AVPV foi indiretamente despolarizada pela prolactina. Este efeito requeria a via de sinalização PI3K. Camundongos portadores de inativação de Stat5a/b em células kisspeptina exibiram um início precoce de ciclicidade estro, indicando que os fatores de transcrição STAT5 exercem um efeito inibitório sobre o momento da puberdade. / GnRH and kisspeptina neurons represent the most important neuronal populations in the control of reproduction. Estradiol binds to its receptor expressed by the kisspeptina neurons to regulate the release of GnRH. In the animal model OVX+E the activity of the GnRH neuron and LH surge is dependent of estradiol and time of day. In this study, although the firing rate of GnRH neurons was similar between groups, the pattern of potentials revealed a change to longer burst duration in mice in proestrous, and the pituitary response was greater in this group. Prolactin has impact on HPG axis modulation and kisspeptin is a mediator of the effects of prolactin on reproduction. A small percentage of AVPV kisspeptin neurons were indirectly depolarized by prolactin. This effect required the PI3K signaling pathway. Mice bearing Stat5a/b inactivation on kisspeptin cells exhibited an early onset of estrus cyclicity, indicating that STAT5 transcription factors exert an inhibitory effect on the time of puberty.

A falta de conhecimento sobre a reprodução do avestruz dificulta o desenvolvimento da sua criação comercial devido a uma baixa eficiência reprodutiva. O objetivo deste estudo foi avaliar a ação da aplicação de um análogo de GnRH (lecirelina - Gestran Plus®) na indução da ovulação em fêmeas adultas de avestruz. Primeiramente, um grupo de 10 fêmeas foi avaliado para determinação de seu status reprodutivo, por meio da mensuração das concentrações séricas de estradiol e progesterona (coletas de sangue 3x/semana); da observação do comportamento reprodutivo; da determinação do desenvolvimento ovariano por exame ultrassonográfico (a cada 21 dias); e pela postura de ovos. Após o término desta primeira fase, as fêmeas foram separadas em grupo controle (n = 3) e grupo tratado (n = 6). A cada semana, 2 fêmeas do grupo tratado eram selecionadas para a aplicação intramuscular de 1ml (25μl) de lecirelina, e os seus efeitos avaliados pela análise das concentrações séricas de estradiol, progesterona e corticosterona (0h, 6h, 24h, 48h e 96h pós-aplicação); pela verificação do desenvolvimento ovariano e ocorrência de ovulação através de exames ultrassonográficos (24h, 48h e 96h); pela observação de comportamento reprodutivo (0h, 48h e 96h) e pela postura de ovos. O tratamento foi repetido no mesmo animal a cada 21 dias e em cada tratamento realizado, 2 fêmeas do grupo controle eram submetidas às mesmas análises. Os resultados indicam que a aplicação de lecirelina foi capaz de induzir a ovulação em 77% dos tratamentos realizados, com postura de ovos normais em 50% das induções. Assim, o tratamento proposto foi considerado eficiente na indução da ovulação em fêmeas de avestruz desde que seja aplicado em fêmeas que apresentem ovário bem desenvolvido. / The lack of knowledge about the ostrich reproduction delays the development of the ostrich commercial breeding due to a low reproductive performance. The aim of this study was to evaluate the effect of the application of an analog of GnRH (Lecirelin - Gestran Plus®) for induction of ovulation in adult female ostriches. Initially, a group of 10 adult female ostriches were evaluated in order to determine their reproductive status, by measuring serum levels of estradiol and progesterone (blood sampling - 3 times a week), by the observation of reproductive behavior, by the evaluation of the ovarian development through ultrasound examination (repeated every 21 days) and by the egg laying data. After the end of the first phase, female ostriches were separated into a control group (n = 3) and a treated group (n = 6). Every week, two females from the treated group were selected to receive the treatment: an intramuscular application of 1 ml (25μl) of Lecirelin. The effect of the application was evaluated by the analysis of serum concentrations of estradiol, progesterone and corticosterone (0h, 6h, 24h, 48h and 96h post-application), by the verification of the ovarian development and the occurrence of ovulation through ultrasound examinations (24h, 48h and 96h), by the observation of reproductive behavior (0h, 48h and 96h) and by the egg laying data. The intramuscular application was repeated in the same animal every 21 days and two females from the control group were subjected to the same analysis during each treatment. The results indicate that the application of Lecirelin was able to induce ovulation in 77% of the treatments with normal egg laying in 50% of inductions. Thus, the treatment was found effective in inducing ovulation in female ostrich provided that it is applied in females who exhibit well developed ovary.

Les hypogonadismes hypogonadotropes congénitaux (CHH) sont des maladies héréditaires caractérisées par un déficit de sécrétion des gonadotrophines par l’hypophyse, à l’origine d’une infertilité ou d’une absence complète de puberté. On distingue les formes isolées avec olfaction normale (nCHH) et les formes syndromiques associant au déficit gonadotrope d’autres signes, tel qu’un défaut d’olfaction dans le cas du syndrome de Kallmann (SK), la forme plus fréquente de CHH. Les gènes identifiés dans le SK participent au développement embryonnaire et les gènes des nCHH sont impliqués dans la régulation de la sécrétion de la GnRH ou de son action. A ce stade, deux populations de neurones hypothalamiques gonadotropes sont connues, le neurone à GnRH et le neurone KNDy, sécrétant les Kisspeptines et la Neurokinine B. On estimait que l’ensemble des gènes identifiés couvraient moins de 20% des étiologies génétiques. L’objectif de ce doctorat était d’étudier prévalences et mécanismes physiopathologiques des gènes connus et d’identifier de nouvelles étiologies génétiques de CHH. Dans la première partie, nous avons caractérisé la fonctionnalité de tous les variants identifiés sur les gènes KISS1R, TACR3 et TAC3. Cela a permis de préciser les prévalences chez 600 patients, d’identifier un profil neuroendocrinien propre à l’altération de la signalisation Neurokinine B et de démontrer l’implication des Kisspeptines au cours de la vie embryonnaire. Enfin, nous proposons un modèle d’interaction entre le neurone à GnRH et le neurone KNDy. Dans la seconde partie, nous avons identifié deux nouveaux gènes, SEMA3A dans une forme familiale de SK et PNPLA6 dans une forme familiale rare de CHH syndromique. En conclusion, notre connaissance accrue des formes génétiques de CHH, a permis de développer un panel d’exome ciblé dédié au diagnostic par séquençage nouvelle génération permettant l’analyse simultanée de gènes candidats et de gènes connus. / Congenital hypogonadotropic hypogonadism (CHH) is characterized by deficient or absent pubertal development due to deficient or absent secretion of the pituitary gonadotropins. The many known genetic causes are generally classified into distinct nosological groups. One comprises abnormalities that affect the pre-natal development or migration of GnRH neurons, the paradigm of which is Kallmann syndrome. The other encompasses molecular abnormalities that only affect hypothalamic GnRH synthesis, GnRH release or GnRH signaling at pituitary level. At this stage, two populations of hypothalamic neurons implicated in a gonadotrop function are identified, GnRH neurons and KNDy neurons secreting kisspeptins and neurokinin B. All of the identified genes would represent less than 20% of genetic etiologies.The aim of this PhD was to study the prevalence and pathophysiology mechanisms of known genes and to identify new genetic etiologies of CHH.In the first part, we characterized the function of all molecular events identified on KISS1R, TACR3 and TAC3 genes. Prevalences were estimated in 600 patients. A particular neuroendocrine profile was identified in patients presenting an alteration of neurokinin B signaling. Importance of Kisspeptins during embryonic life was validated. According to these data, a model of interaction between GnRH and KNDy neurons was proposed.In the second part, we identified two new CHH genes using various molecular genetics approaches. SEMA3A was identified in a familial form of Kallmann syndrome and PNPLA6 in a rare familial form of CHH.Finally, our increased knowledge of the various genetic forms of CHH allows proposing a new genetic approach based on next generation sequencing to test together all known and several candidate genes.

Untersuchungen zu Schlachtkörperqualität und Ebergeruchsstoffen bei mit einem GnRH-Analogon geimpften, chirurgisch kastrierten und intakten männlichen Mastschweinen Franziska Sauer Universität Leipzig, Medizinische Tierklinik, Leipzig, Deutschland Zielstellung Ziel der vorliegenden Studie war, Auswirkungen der Impfung gegen Ebergeruch bei männlichen Mastschweinen in konventioneller Haltung zu untersuchen und mit intakten Mastebern und Kastraten zu vergleichen. Tiere und Methode Insgesamt 348 männliche Mastschweine wurden in vier Gruppen wie folgt unterteilt: Zwei Gruppen enthielten mit Improvac® geimpfte Schweine (1. Impfung 11. Lebenswoche (LW)): Gruppe 1: n=84, 2. Impfung 18. LW, Gruppe 2: n=83, 2. Impfung 21. LW. Gruppe 3 bestand aus 90 Kastraten und Gruppe 4 aus 91 unkastrierten männlichen Mastschweinen. Die Schweine wurden im Alter von 26 bzw. 27 Wochen geschlachtet. Mast- und Schlachtleistung, das Fettsäuremuster im Rückenfett sowie Hodengewicht, Hodenhistologie und Ebergeruchsstoffe im Rückenfett wurden untersucht. Ergebnisse GnRH-geimpfte Schweine wiesen eine bessere Futterverwertung als chirurgisch kastrierte auf. Die Schlachtkörper der Geimpften hatten einen signifikant höheren Magerfleischanteil und weniger Rückenfett als die der Kastraten und erzielten dadurch einen höheren Schlachterlös. Das Fettsäuremuster der geimpften Schweine gleicht im Hinblick auf die Menge an PUFA eher den intakten als den chirurgisch kastrierten. In früherem Alter geimpfte Schweine zeigen histologisch eher eine Hodenhypoplasie, später geimpfte eher eine Hodenatrophie. Ebergeruchsstoffe im Rückenfett waren in beiden Impfgruppen und bei den Kastraten signifikant niedriger als bei intakten Mastebern. Schlussfolgerung Die Impfung männlicher Schweine mit Improvac® ist eine tierschutzgerechte, praktikable und wirtschaftliche Alternative zur Vermeidung von Ebergeruch. Literatur Sattler et al: BMTW 2014; 127:10-16 Sauer et al: WTM 2014;101:103-109 franziska.sauer@vetmed.uni-leipzig.de:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 2 2.1 Ebergeruch 2 2.2 Methoden zur Vermeidung von Ebergeruch 3 2.2.1 Chirurgische Kastration 3 2.2.2 Ebermast 3 2.2.3 Impfung gegen Ebergeruch 4 2.2.3.1 Wirkungsweise 4 2.2.3.2 Rückstände 5 2.2.3.3 Wirkung auf Kryptorchiden 6 2.2.3.4 Frühe Impfung 6 2.2.3.5 Langzeiteffekte und Regeneration 7 2.2.3.6 Wirkung auf das Verhalten 8 2.2.3.7 Wirkung auf die Mastleistung 9 2.2.3.8 Wirkung auf die Schlachtkörper- und Fleischqualität 9 3 Publikation 1: Effect of time of second GnRH vaccination on feed intake, carcass quality and fatty acid composition of male fatteners compared to entire boars and barrows 11 4 Publikation 2: Einfluss des Alters bei der zweiten Improvac®-Vakzination auf Hodengewicht, Hodenhistologie und Ebergeruchsstoffe von männlichen Mastschweinen im Vergleich zu intakten Mastebern und Kastraten 31 5 Diskussion 39 5.1 Futterverwertung 40 5.2 Körpermasseentwicklung 41 5.3 Schlachtkörperqualität 41 5.4 Fettsäuremuster 42 5.5 Hodenhistologie 43 5.5.1 Atrophie-Degenerations-Score 44 5.6 Ebergeruchsstoffe 45 5.6.1 Androstenon 46 5.6.2 Skatol und Indol 47 5.7 Ökonomische Aspekte 48

In der vorliegenden Arbeit werden Beiträge zur Entwicklung neuer qualitativer und quantitativer analytischer Methoden für die Untersuchung von Cetrorelix und anderen GnRH-Antagonisten während des Aggregationsprozesses vorgestellt. Für die qualitative Untersuchung von GnRH-Antagonisten wurden am Modellpeptid Cetrorelix massenspektrometrische Methoden entwickelt und auf weitere GnRH-Antagonisten übertragen. Eingesetzt wurde sowohl die ESI-TOF-MS als auch die MALDI-TOF-MS. Beide massenspektrometrische Verfahren zeigten für die untersuchten GnRH-Antagonisten polydisperse Verteilungen oberhalb der fluoreszenzspektroskopisch bestimmten kritischen Aggregatbildungskonzentration (cac). Bei den ESI-TOF-MS-Messungen konnte ein stark von Cetrorelix abweichendes Aggregationsverhalten für Ozarelix beobachtet werden. Für die Quantifizierung des Aggregatanteils in Cetrorelix- bzw. Ozarelixproben wurde eine Methode mit der MALDI-TOF-MS etabliert, die Untersuchungen an pharmazeutisch relevanten Peptidhormon-Formulierungen ermöglicht. Systematische Untersuchungen zum Einfluss verschiedener An- und Kationen sowie deren Konzentrationen auf die Cetrorelixaggregation erfolgten mit der Fluoreszenzspektroskopie. Die erhaltenen Ergebnisse ermöglichen eine bessere Charakterisierung der Aggregation und lassen Abschätzungen über das Aggregationsverhalten von Cetrorelix in verschiedenen Medien mit unterschiedlichen Elektrolyten zu. Mit dem Einsatz der Transmissionselektronenmikroskopie war es erstmals möglich, Aggregate von GnRH-Antagonisten visuell darzustellen.

La mucoviscidose est la maladie héréditaire autosomique récessive, rare, létale, la plus fréquente dans la population caucasienne. Cette maladie est causée par des mutations du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) codant la protéine CFTR. Cette protéine est principalement un canal chlorure (Cl-) AMPc-dépendant localisé dans la membrane apicale des cellules épithéliales. La mutation F508del entraîne un défaut de maturation de la protéine qui est retenue dans le réticulum endoplasmique avant d’être dégradée. Cependant, une faible quantité de protéines malformées échappe à ce système de contrôle et parvient à la membrane plasmique.Des travaux de notre équipe ont montré une augmentation des efflux ioniques dépendants du CFTR dans des lignées cellulaires épithéliales bronchiques (CFBE41o-), exprimant le CFTR sauvage ou le CFTR muté F508del, après un traitement par une hormone: la gonadolibérine (GnRH, Gonadotropin releasing hormone, 1h, 10-9M). Cette augmentation est vraisemblablement due à un nombre plus important de canaux CFTR à la membrane plasmique.L’objectif de cette thèse a été de tester un analogue de la GnRH comme modulateur de l’exportation membranaire et/ou de l’activité canal du CFTR muté, sur des cultures primaires de cellules épithéliales nasales humaines homozygotes pour la mutation F508del. Dans un premier temps, nous avons vérifié la présence du récepteur à la GnRH (R-GnRH) dans notre modèle cellulaire. Puis, nous avons étudié l’effet de l’analogue sur la fonction du CFTR par des techniques d’électrophysiologie. Nous avons observé une augmentation des efflux d’ions Cl- médiés par le canal CFTR après un traitement à l’analogue (2h, 10-12M). Enfin, une étude protéomique nous a permis d’identifier des protéines différentiellement exprimées après traitement. Certaines protéines mises en évidence pourraient appartenir à des voies de signalisation intracellulaires ayant un rôle dans la régulation de la protéine CFTR et être des cibles thérapeutiques.Par ailleurs, le canal CFTR est régulé par le Mg2+ intracellulaire ([Mg2+]i). Le canal TRPM7 est le principal régulateur du [Mg2+]i. La [Mg2+]i a été mesurée et l’expression de TRPM7 vérifiée dans des cellules Hela transfectées avec le CFTR sauvage (wt) ou mutés (G551D et F508del). Nous avons étudié la localisation, la fonction et la régulation de TRPM7 dans nos modèles cellulaires avant de rechercher un possible lien fonctionnel entre le CFTR et TRPM7. Dans les modèles CF, l’expression, la fonction et la localisation du canal TRPM7 sont altérées. Il existerait un lien fonctionnel entre TRPM7 et le CFTR par l’intermédiaire de la diminution du [Mg2+]i impliquant TRPM7 dans la physiopathologie de la mucoviscidose. / Cystic fibrosis is the most common lethal autosomal recessive disease in the Caucasian population. This disease is caused by mutations in the gene encoding the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) protein. This protein is a cAMP-regulated chloride channel expressed at the apical membrane of epithelial cells. The F508del mutation causes a defect in CFTR protein folding preventing its maturation. Some misfolded proteins escape the control system and reach the plasma membrane.We previously showed a rise of CFTR-dependent ion efflux in wt- and F508del-CFTR human bronchial epithelial expressing cells (CFBE41o-) after incubation with GnRH (Gonadotropin releasing hormone; 1h, 10-9M). This increase was probably due to an increased cell surface expression of CFTR.The aim of the present study was to test a GnRH analog as a modulator of CFTR delivery to the plasma membrane and/or activity of CFTR on primary culture of human nasal epithelial cells (F508del/F508del).We checked the GnRH receptor expression in our model. Then, we studied the GnRH effect on CFTR’s function by electrophysiology. We found a significant increase of CFTR dependent chloride efflux in cells pretreated with analogue (2h, 10-12M). Proteomic study enabled us to identify differentially expressed proteins after treatment. Some highlighted proteins could be part of signalling pathways regulating CFTR and could be therapeutic targets.Moreover, CFTR is regulated by intracellular Mg2+ ([Mg2+]i). TRPM7 (Transient Receptor Potential Melastatin 7) is the main channel regulating [Mg2+]i. [Mg2+]i and TRPM7 expression were measured in Hela cells stably expressing wildtype and two CFTR mutants (F508del-CFTR and G551D-CFTR). We studied TRPM7 expression, function and regulation in our cell models before examining a functional link between TRPM7 and CFTR. In CF models, TRPM7 expression, localization and function are altered. There should be a functional link between TRPM7 and CFTR through the decreased [Mg2+]i involving TRPM7 in cystic fibrosis physiopathology.

Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA. Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NFκB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro. An NFκB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NFκB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin.