In vitro, meotic maturation of porcine oocytes and cumulus expansion are induced by FSH and EGF-like peptides AREG and EREG. FSH and EGF-like peptides induce expression of cumulus expansion-related genes (HAS2, PTGS2 and TNFAIP6). To define signaling pathways that control FSH- and AREG-induced cumulus expansion, porcine cumulus-oocyte complexes were treated with specific protein kinase inhibitors. Inhibitors of MAPK3/1, MAPK14 and ERBB1 significantly reduced both FSH- and AREG-induced expression of HAS2, PTGS2 and TNFAIP6. These inhibitors decreased FSH/LH-induced expression of AREG and EREG in mural granulosa cells. Surprisingly, inhibitor of PKA had no effect on AREG expression in cumulus-oocyte complexes but the inhibitor decreased expression of TNFAIP6 induced by AREG. Inhibitor of PI3K increased expression levels of AREG and PTGS2 but EREG, HAS2 and TNFAIP6 were reduced. Expression levels of the cumulus expansion-related genes were not affected by an analog of cGMP (8-CPT-cGMP). However, 8-CPT-cGMP blocked spontaneous in vitro meiotic maturation of porcine oocytes and its effect was abolished by FSH. Key words: cumulus expansion, cumulus expansion-related genes, meotic maturation, FSH, amphiregulin, cGMP

Contexte : Le récepteur hormone folliculo-stimulante (FSHR) est exprimé par les cellules de l'endothélium vasculaire dans un large éventail de tumeurs humaines primaires. Notre but était d'évaluer l'intérêt de FSHR comme marqueur des vaisseaux sanguins tumoraux associés aux sarcomes, les sous-types moléculaires du cancer du sein et des métastases ainsi que comme biomarqueur prédictif de la réponse au traitement anti-angiogénique. Méthodes : Nous avons utilisé l'immunohistochimie comme technique de révélation. Ceci implique la production d'un anticorps monoclonal hautement spécifique anti-FSHR (produit chez la souris) et l'hybridation in situ pour détecter FSHR dans des échantillons de tissus provenant de patients atteints de sarcomes (308 patients), les sous-types moléculaires du cancer du sein (84 patients), et des métastases (203 patients). Pour évaluer FSHR comme marqueur prédictif du traitement anti-angiogénique du cancer du rein métastatique avec le sunitinib, nous avons utilisé la microscopie confocale à immunofluorescence. Nous avons également co-localiser FSHR avec le facteur von Willebrand, un marqueur des cellules endothéliales vasculaires (50 patients).RésultatsFSHR est exprimé dans les 11 sous-types de patients atteints de sarcomes ont analysés et dans 75% des tumeurs métastatiques examinées, ainsi que dans tous les sous-types moléculaires des cancers du sein. Dans cadre de l'étude du cancer du rein métastatique, le pourcentage de vaisseaux marqués FSHR était en moyenne cinq fois plus élevé pour les patients qui ont répondu au traitement par rapport au groupe stable et presque huit fois plus élevé que dans le groupe non-réponse (57%, 11% et 7 %, respectivement).ConclusionsNos résultats montrent que, en plus des cancers signalés précédemment, FSHR peut être considéré comme marqueur tumoral pour les sarcomes et les métastases. En outre, FSHR peut être utilisé, avec une sensibilité et une spécificité élevée, en tant que biomarqueur prédictif de la réponse au traitement par sunitinib des patients atteints de cancer du rein métastatique. / Background : Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human primary tumors. Our purpose was to further evaluate FSHR as marker of tumor blood vessels associated with sarcomas, breast cancer molecular subtypes, and metastases as well as predictive biomarker of response to antiangiogenic treatment.MethodsWe used immunohistochemistry involving a highly specific mouse monoclonal anti-FSHR antibody and in situ hybridization to detect FSHR in tissue samples from patients with sarcomas (308 patients), breast cancer molecular subtypes (84 patients), and metastases (203 patients). To evaluate FSHR as predictive marker of antiangiogenic treatment of metastatic kidney cancer with sunitinib, we used immunofluorescence confocal microscopy to co-localize FSHR with von Willebrand factor, a marker of vascular endothelial cells (50 patients).ResultsFSHR is expressed in all 11 subtypes of sarcoma patients analysed, in 75% of metastatic tumors examined as well as in all different molecular subtypes of breast cancers. In metastatic kidney cancer patients the percentage of FSHR stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non-responsive group (57%, 11%, and 7%, respectively).ConclusionsOur results suggest that, in addition to the cancers previously reported, FSHR can be considered as tumor marker for sarcomas and metastasis. Moreover, FSHR can be used, with high sensitivity and specificity, as predictive biomarker for the response to sunitinib treatment of patients with metastatic kidney cancer.

Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as it is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in tR between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) for the pituitary and recombinant preparations. The linearity of the dose-response curve (r = 0.9965, n = 15) for this RP-HPLC methodology, as well as an inter-assay precision with relative standard deviation less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behavior and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the accurate molecular mass of the individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for characterization and quality control of this hormone that is not yet described in the main pharmacopoeias.

Submitted by Kelson Anthony de Menezes (kelson@ucb.br) on 2016-12-19T19:37:39Z No. of bitstreams: 1 FabiodeBarrosAmaralDissertacao2014.pdf: 2938215 bytes, checksum: 3d899bf568eeae36d21979ba7aafab3c (MD5) / Made available in DSpace on 2016-12-19T19:37:39Z (GMT). No. of bitstreams: 1 FabiodeBarrosAmaralDissertacao2014.pdf: 2938215 bytes, checksum: 3d899bf568eeae36d21979ba7aafab3c (MD5) Previous issue date: 2014-03-31 / The equine chorionic gonadotropin (eCG) has been used as an important resource in the processes of reproduction in animals of economic interest, especially horses and cattle. The eCG molecule consists of a glycoprotein composed of two subunits called alpha and beta which together perform the function of both LH (luteinizing hormone) and FSH (follicle stimulating hormone) and due to this characteristic has great potential to induce ovulation in household species. Based on the aforementioned characteristics, the present work was was aimed at the study and characterization of the structure of eCG using molecular modeling techniques as well as testing its production in cells of the microorganism Pichia pastoris. Furthermore, chimeric models have been proposed linking the eCG and immunoglobulin G (IgG) molecule based on studies that reveal the great potential of it in raising the half-life of other molecules circulating in the organism. Four chimeric models were prepared so that the eCG alpha and beta chains are connected by a hinge, short and flexible regions, while the terminal region of the alpha chain was connected to the N-terminal portion of the CH2 domain of the IgG by another hinge region, thus resulting in a model in which two different molecules are linked. / A gonadotrofina cori??nica equina (eCG) tem sido um importante recurso usado nos processos de reprodu????o em animais de interesse econ??mico, principalmente equinos e bovinos. A mol??cula de eCG consiste em uma glicoprote??na composta de duas subunidades denominadas alfa e beta que juntas desempenham a fun????o tanto de LH (horm??nio luteinizante) como FSH (horm??nio fol??culo estimulante) e devido a essa caracter??stica possui um grande potencial para induzir a ovula????o em esp??cies dom??sticas. Baseando-se nas caracter??sticas citadas, o presente trabalho teve como objetivo estudar e caracterizar a estrutura do eCG utilizando-se t??cnicas de modelagem molecular assim como testar a sua produ????o em c??lulas do microrganismo Pichia pastoris. Al??m disso, foram propostos modelos quim??ricos ligando eCG ?? mol??cula de imunoglobulina G (IgG) tendo como base estudos que revelam o grande potencial da mesma em elevar a meia vida de outras mol??culas em circula????o no organismo. Quatro modelos quim??ricos foram apresentados de forma que as cadeias beta e alfa do eCG s??o conectadas por regi??es hinge que s??o sequ??ncias curtas e flex??veis da mol??cula enquanto que a regi??o terminal da cadeia alfa foi conectada por meio de outra regi??o hinge, ?? por????o N-terminal do dom??nio CH2 da IgG, portanto, resultando em um modelo em que est??o ligadas duas mol??culas diferentes.

Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / A reversed-phase high-performance liquid chromatography (RP-HPLC) method for the qualitative and quantitative analysis of intact human follicle-stimulating hormone (hFSH) was established and validated for accuracy, precision and sensitivity. Human FSH is a dimeric glycoprotein hormone widely used as a diagnostic analyte and as therapeutic product in reproductive medicine. The technique developed preserves the protein integrity, allowing the analysis of the intact heterodimeric form rather than just of its subunits, as it is the case for the majority of the conditions currently employed. This methodology has also been employed for comparing the relative hydrophobicity of pituitary, urinary and two Chinese hamster ovary (CHO)-derived hFSH preparations, as well as of two other related glycoprotein hormones of the anterior pituitary: human thyroid-stimulating hormone (hTSH) and human luteinizing hormone (hLH). The least hydrophobic of the three glycohormones analyzed was hFSH, followed by hTSH and hLH. A significant difference (p<0.005) was observed in tR between the pituitary and recombinant hFSH preparations, reflecting structural differences in their carbohydrate moieties. Two main isoforms were detected in urinary hFSH, including a form which was significantly different (p<0.005) for the pituitary and recombinant preparations. The linearity of the dose-response curve (r = 0.9965, n = 15) for this RP-HPLC methodology, as well as an inter-assay precision with relative standard deviation less than 4% for the quantification of different hFSH preparations and a sensitivity of the order of 40 ng, were demonstrated. The chromatographic behavior and relative hydrophobicity of the individual subunits of the pituitary and recombinant preparations were also analyzed. Furthermore, the accurate molecular mass of the individual hFSH subunits and of the heterodimer were simultaneously determined by matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS). The present methodology represents, in our opinion, an essential tool for characterization and quality control of this hormone that is not yet described in the main pharmacopoeias.

Chez la brebis, les BMPs inhibent la synthèse et la libération de FSH. La détection des ARNm de certaines BMPs dans l’hypophyse et de leurs récepteurs sur les cellules gonadotropes suggère une action paracrine/autocrine des BMPs sur la production de FSH. L’objectif de cette thèse a visé à déterminer l’importance des BMPs hypophysaires, en particulier BMP-4, dans la régulation de la sécrétion de FSH chez la brebis. Nous montrons que l’expression des ARNm du système BMP-4,analysée par RT-PCR en temps réel, ne varie pas au cours du cycle oestrien ou in vitro quelque soit le temps d’incubation (6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires.Afin de déterminer si les cellules hypophysaires ovines sécrètent des BMPs, des milieux conditionnés(MC) hypophysaires ont été soumis à un test d’activité biologique reposant sur des cellules embryonnaires transfectées avec un élément de réponse BMPs couplé au gène rapporteur luciférase.Aucun effet des MC n’a été observé sur l’activité luciférase comparé au milieu non conditionnésuggérant l’absence ou la très faible activité BMP des MC, et cela quelque soit le temps d’incubation(6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires. Toutefois, nous détectons une activité inhibitrice de BMPs qui est augmentée par la GnRH et l’oestradiol suggérant l’implication d’inhibiteurs de BMPs dans la régulation de la production de FSH. En conclusion,l’ensemble de nos résultats n’est pas en faveur d’un rôle de BMP-4 hypophysaire dans la synthèse de FSH chez l’adulte. Un rôle des BMPs au niveau hypophysaire via la voie endocrine peut s’envisager puisque nous avons mis en évidence une bioactivité de type BMP dans le sérum ovin. Les inhibiteurs BMPs produits par l’hypophyse, qui restent à identifier, pourraient moduler la biodisponibilité des BMPs atteignant l’hypophyse par la voie sanguine. / BMPs inhibit FSH synthesis and release in ewe. The detection of BMP mRNAs in the pituitary as well as the colocalisation of the two types of BMP receptors on gonadotrope cells suggest that these BMPs can exert paracrine/autocrine actions on FSH production. The aim of this thesis work was to determine the importance of pituitary BMPs in the regulation of FSH production in the ewe. We showed that the level of mRNAs for BMP-4 system, analyzed by real-time RT-PCR, did not vary across the oestrous cycle or in vitro whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of pituitary cells. By using a bioactivity test based on embryonic mesenchymal cells transfected with an expression construct containing a BMP-responsive element fused to firefly luciferase reporter gene,we detected no BMP activity within conditioned media from pituitary cells whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of the pituitary cells. However, we detected a BMP inhibitory activity which is increased by GnRH and oestradiol suggesting the implication of BMP inhibitor(s) in FSH regulation. In conclusion, the results are not in favor of a role for pituitary BMP-4 in the regulation of FSH synthesis in adult ewe. An endocrine action of BMPs at pituitary level can be evoked since BMP bioactivity was detected within ovine serum. BMP inhibitors that remain to be identified can modulate the bioavailability of BMPs reaching the pituitary by the blood way.

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La FSH est une des hormones clés qui régule la reproduction chez les mammifères. Chez le mâle, elle cible les cellules de Sertoli, qui expriment le RFSH. La cellule de Sertoli a un rôle trophique important pour le bon développement de la spermatogenèse. Dans cette thèse, nous avons établi le premier traductome, c’est-à-dire l’ensemble des ARNm en cours de traduction, dépendant du RFSH. La traduction de certains ARNm significativement modulés par la FSH exercerait un rétrocontrôle sur la signalisation FSH-dépendante. L’analyse du protéome nous a permis de valider ce traductome au niveau systémique. Nous avons également démontré l’implication des β-arrestines dans la traduction d’ARNm dépendante de la FSH. Les β-arrestines forment un assemblage moléculaire avec le module de traduction p70S6K/rpS6. Cet assemblage est impliqué dans la traduction des ARNm 5’TOP, qui encodent la machinerie traductionnelle. C’est l’activation FSHdépendante des protéines G qui promeut l’activation de p70S6K au sein du module β-arrestines/ p70S6K/ rpS6. Ce travail constitue une nouvelle avancée sur les mécanismes grâce auxquels la FSH exerce sa fonction biologique de dans ses cellules-cibles naturelles de la gonade mâle. / FSH is one of the key hormones that regulate the reproductive function in mammals. In the male, FSH targets Sertoli cells, which express the FSHR. Sertoli cells play an important trophic role in the development of spermatogenesis. Here, we have provided the first FSHR-induced translatome, that encompasses all the mRNA being actively translated. The translation of some mRNAs significantly modulated by FSH may exert a feedback control on FSH-dependent signaling. The analysis of the proteome has validated the FSHR translatome at the systems level. We also demonstrated the involvement of β-arrestins in the FSH-stimulated translation of mRNA. β-arrestins form a molecular assembly with the p70S6K / rpS6 translation module. This molecular assembly is involved in the translation of 5'TOP mRNA, which encode proteins of the translational machinery. FSH-activated G proteins leads to p70S6K activation within the β-arrestins/ p70S6K/ rpS6 module. This work provides new advance on the mechanisms whereby FSH exerts its biological function in its natural target cells of the male gonad.

Die GABA-ergen Neurone als Teil des GnRH-Netzwerkes spielen eine Rolle bei den Veränderungen der altersabhängigen Prozesse der Reproduktionsfunktion. Um die Regulation der gonadalen Steroide auf die Expression von GAD in reproduktionsabhängigen Regionen zu untersuchen, wurde im ersten Teil der vorliegenden Arbeit das männliche Rattenmodell gewählt. Nach der Untersuchung des endokrinen Zustands der Tiere anhand der Radioimmunoassay-Methode (RIA) wurden die zellulären Gentranskripte der beiden Isoformen von GAD, GAD65 und GAD67, mittels der Methode der in situ Hybridisierung in der POA, im Nukleus suprachiasmaticus (SCN), im mediobasalen Hypothalamus (MBH) und im Gyrus dentatus bestimmt. In allen untersuchten Regionen konnte nach der Kastration und einer anschließenden dreiwöchigen Erholungszeit kein Effekt beobachtet werden. Die Administration von Estradiol bewirkt in der POA eine signifikante Erhöhung der Expression von GAD65 und GAD67 um nahezu 40%. In den restlichen Regionen konnte dagegen kein Effekt gemessen werden. Die Testosteronbehandlung zeigte eine negative Wirkung auf die Regulation nur von GAD67: Eine 30%-ige Abnahme in der POA und eine 15%-ige im SCN. Im Gegensatz dazu trat im MBH und im Gyrus dentatus eine Verminderung der Expression nur bei GAD65 auf. Aus den hier vorgestellten Ergebnissen kann folgendes abgeleitet werden: Testosteron und Estradiol regulieren in unterschiedlicher Weise die Expression von GAD und so wiederum die inhibitorische Funktion von GABA. Da in der SCN, im MBH und im Gyrus dentatus im Gegensatz zu Estradiol eine Testosteron-Wirkung gemessen wurde, existiert eine eigene androgene Regulation von GAD. Weil die Estradiol-Zugabe eine Zunahme der Expression von GAD bewirkte und dieser Effekt von einer Abnahme der LH-Konzentration im Serum der betroffenen Tiergruppe begleitet wurde, ist die These bestätigt, dass GABA mit ihren inhibitorischen Funktionen zur Übermittlung der positiven Rückkopplung von Estradiol auf die LH-Freisetzung auf der Ebene der POA und nicht auf der Ebene der Axone agiert. Im Gegensatz zu Estradiol kann eine Progesteronbehandlung bei persistent östrischen Ratten einen LH-Peak auslösen und somit den Östrus-Zyklus wieder in Gang bringen. Aufgrund dieser Tatsache wurde im zweiten Teil der vorliegenden Arbeit ein Tiermodell zur Untersuchung der molekularbiologischen altersabhängigen Veränderungen entwickelt. Dabei wurden drei Monate alte proöstrische Ratten (Y) und 12 Monate alte persistent östrische Ratten (MA) benutzt. Die MA-Ratten wurden mit Progesteron behandelt. Sowohl die MA-Ratten als auch die Y-Ratten wurden um 13 Uhr und um 17 Uhr getötet. Eine unbehandelte MA-Gruppe, deren Tiere um 10 Uhr getötet wurden, diente hier als Kontrollgruppe. Anhand der LH-Messung der untersuchten Gruppen wurde ein Kontrollwert (5 ng/ml LH) für die positive Reaktion der Tiere auf Progesteron (responding animals) festgestellt. Es konnte bei 44% der persistent östrischen Ratten ein erhöhter LH-Spiegel erfolgreich wieder erreicht werden. In den Gruppen dieses Modells entstand eine Analogie zwischen den Gruppen der behandelten MA-13-Uhr und Y-13-Uhr Tiere sowie zwischen den responding animals und den Y-17 Uhr-Tieren. Um aussagekräftige statistische Veränderungen entlang der hypothalamo-hypophysio-ovariellen Achse in individuellen Tieren zu erhalten, wurde die Taqman®-PCR und die quantitative, kompetitive RT-PCR eingesetzt. Dabei wurden die folgenden Gene untersucht: ER α und ER β, GnRH, GnRH-R, GAD65 und GAD67, sowie FSH-β. In der POA, Hypophyse und im Ovar wurde altersabhängigen Genexpression beobachtet: Eine signifikante Abnahme der Expression von ER β sowohl in der Gruppe responding animals als auch in deren analoger Gruppe wurde in der POA (34 %), Hypophyse (44 %) und im Ovar (um die 30 %) gemessen. In der Hypophyse verzeichneten die mRNA-Transkripte von ER α bei der Gruppe der behandelten mittelalten Ratten der 13 Uhr-Gruppe eine Zunahme von 55% und bei der 13-Uhr-Gruppe der jungen Ratten einen Anstieg von 153 %. Ebenso nehmen die mRNA-Konzentrationen von FSH-β sowohl bei den responding animals als auch bei deren analoger Gruppe in gleichem Masse (ungefähr 300 %) zu. Da die Veränderungen der Expression von ER β, ER α und FSH-β bei den zwei analogen Gruppen auftritt, ist zu vermuten, dass diese Gene altersabhängig expremiert und an der Zyklusregulation ursächlich beteiligt sind. Die restlichen Gene zeigten entlang der Achse keine altersrelevanten Veränderungen. Da ER β-Expressionsveränderungen in der POA, in der Hypophyse und im Ovar gemessen wurden, konnte der wichtigste Schluss der hier vorgestellten Untersuchungen gezogen werden, dass nämlich ER β für den Erhalt des Zyklus essentiell sein kann. In diesem Teil der vorliegenden Arbeit wurde ein Tiermodell zur molekular biologischen Untersuchung der altersabhängigen Veränderungen mit sehr zufriedenstellender Ausbeute zur Wiederherstellung des Östrus-Zyklus (44%) erfolgreich entwickelt. Dieses Modell ermöglichte darüber hinaus die Untersuchung einer relativ hohen Anzahl an Genen entlang der hypothalamo-hypophysio-ovariellen Achse.