Magister Scientiae - MSc / The main thesis hub was on development of two electrochemical biosensors for the determination of 17β-estradiol-estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17β-estradiol-estradiol endocrine disrupting compound; consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide. / South Africa

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Depuis plusieurs années il est devenu essentiel de comprendre le déterminisme sexuel des espèces à fort intérêt économique afin d’optimiser leur production au sein d’écloseries émergentes.L’objectif principal de cette thèse était de mettre en évidence les mécanismes impliqués dans la détermination et la différenciation sexuelle, et notamment du sexe femelle, chez l’huître perlière P. margaritifera, espèce hermaphrodite protandre et espèce clé de la perliculture, la seconde ressource économique pour la Polynésie française. Pour atteindre cet objectif, deux approches ont été menées : une approche transcriptomique visant à étudier les mécanismes moléculaires du déterminisme et de la différenciation sexuelle, et une approche expérimentale visant à comprendre le phénomène de la sexualisation par des forçages environnementaux et hormonaux en s’intéressant plus particulièrement au déterminisme et à la différenciation sexuelle femelle.Dans l’approche transcriptomique, le transcriptome de la gonade de P. margaritifera a été séquencé à partir de plusieurs échantillons gonadiques d’huîtres de sexe mâle et femelle à différents stades de développement. Après le séquençage Illumina et l'assemblage du transcriptome, 70 147 contigs ont été obtenus. L’analyse fonctionnelle de ces 70 147contigs, a permis d’identifier des gènes d’intérêt et ainsi de constituer un catalogue de 87 ARNm codant pour 67 protéines impliquées dans la détermination, la différenciation sexuelle et/ou la gamétogenèse. Ensuite une analyse stricte des données de quantification RNAseq a révélé 1 937 contigs exprimés de manière différentielle entre les catégories histologiques des gonades. À partir de l’analyse de leurs profils d’expression au sein de chaque échantillon, un nouveau modèle de la reproduction de P. margaritifera, basé sur une double approche analytique, eg. histo-moléculaire, a été proposé. Ce modèle révèle notamment que le déterminisme sexuel de P. margaritifera chez l’adulte se produirait durant une phase de régression de la gonade. Considérant ainsi les nouveaux stades définis par ce modèle, 9 gènes biomarqueurs de la voie sexuelle femelle ont pu être identifiés révélant un modèle prédictif de la voie sexuelle basé sur 3 rapports d’expressions de gènes impliquant 2 gènes inconnus pmarg-c43476 et pmarg-c54338 et 2 gènes connus pmarg-foxl2 et pmarg-fem1-like. Ce deuxième modèle suggère fortement l'implication de pmarg-foxl2 et pmarg-fem1-like dans le déterminisme du sexe de P. margaritifera. Dans l’approche expérimentale, deux expérimentations séparées ont été réalisées pour mettre en évidence l’effet i) de plusieurs combinaisons de température et de niveau trophique, et ii) de l’œstradiol-17β administré par injection directe dans la gonade ; sur le sexe, la gamétogenèse et l’expression des neuf gènes biomarqueurs de la voie sexuelle femelle identifiés précédemment. Les résultats ont montré que la condition combinant la température de 28°C et la concentration en algues de 40 000 cellules mL-1 était la plus favorable non seulement à la maturation des gonades mâles et femelles mais aussi au maintien du sexe femelle. Ce serait dans cette condition environnementale qu’il serait possible d’induire un changement de sexe de mâle vers femelle. Dans la seconde expérimentation, il a été clairement démontré que la reproduction de P. margaritifera pouvait être régulée par les hormones œstrogènes. Les résultats montrent un effet négatif de l’œstradiol sur le développement et la différenciation mâle. Enfin les résultats du modèle prédictif de la voie sexuelle de P. margaritifera, suggèrent une programmation génétique du sexe femelle qui toutefois resterait soumise aux conditions environnementales validant ainsi l’hypothèse d’un mode de détermination mixte du sexe chez P. margaritifera. / For several years it has become essential to understand sex determination of species with high economic interest to maximize their production in emerging hatcheries.The main objective of this thesis was to identify the mechanisms involved in sex determination and sex differentiation, and particularly in female sex, in the pearl oyster P. margaritifera, a protandrous hermaphrodite species and the key species of the pearl farming, the second economic resource for French Polynesia. To achieve this goal, two approaches were undertaken: a transcriptomic approach to investigate the molecular mechanisms of sex determinism and sex differentiation, and an experimental approach to understand the phenomenon of sexualization by environmental and hormonal forcing focusing especially on female sex determinism and female sex differentiation.In the transcriptomic approach, the gonad transcriptome of P. margaritifera was sequenced from several samples of male and female oyster gonads at different stages of development. After Illumina sequencing and assembly of the transcriptome, 70,147 contigs were obtained. Functional analysis of these 70,147 contigs identified genes of interest and allowed the constitution of a catalog of 87 mRNAs encoding 67 proteins involved in sex determination, sex differentiation and/or gametogenesis. Then a strict analysis of RNAseq quantification data revealed 1,937 contigs differentially expressed between the histological categories of gonad. From the analysis of their expression profiles in each sample, a new model of reproduction of P. margaritifera, based on dual analytical approach, i.e. histo-molecular, has been proposed. This model shows that sex determination of adult P. margaritifera pearl oysters occur during a regression phase of the gonad. And considering the new stages defined on this model, 9 biomarkers genes of the female sexual pathway have been identified revealing a 3-gene-pair expression ratio based model, which makes it possible to predict the sexual pathway in this hermaphrodite species. This predictive model involves two unknown genes pmarg-c43476 and pmarg-c54338 and 2 known genes pmarg-foxl2 and pmarg-fem1-like, and strongly suggests the involvement of pmarg-foxl2 and pmarg-fem1-like in sex determinism in P. margaritifera.In the experimental approach, two separated experiments were conducted to demonstrate the effect of i) various combinations of temperature and trophic level, and ii) 17β-estradiol administered by direct injection into the gonad; on sex, gametogenesis and expression of the nine biomarkers genes of the female sexual pathway previously identified. The results showed that the condition combining a temperature of 28 °C and a concentration of 40 000 cells of algae mL-1 was the most favorable not only for the maturation of the male and female gonads but also for the maintenance of the female sex. It would be in this environmental condition that it would be possible to induce a sex change from male to female. In the second experiment, it was clearly demonstrated that the reproduction of P. margaritifera could be regulated by estrogen hormones. The results show a negative effect of estradiol on male development and differentiation. Finally the results of the predictive model of the sexual pathway of P. margaritifera, suggest a genetic programming of the female sex, which however remain subject to environmental conditions, thus validating the hypothesis of a mixed sex determinism mode in P. margaritifera.

Sex determination among reptiles is a very variable matter across it's taxa. We meet there temperature sex determination and genotypic sex determination with many independent transitions between them. It is a group suitable to study evolution of sex determination, sex chromosomes and sex determination genes. Rare cases of sex reversal caused by extreme incubation temperature or exogenous hormones have been reported in recent years. In case of Acrodont lizard, Pogona vitticeps, was reported sex reversal caused by high incubation temperatures. Our purpose was to repeat the experiment, mainly due to insufficient conclusiveness of used methods. We wanted to expand the experiment by hormonal reversal, studying persistence of sex reversal to maturity and fertility of reversed individuals. We managed successfully to demonstrate sex reversal in both treatments by histological examination. Individuals with discordant phenotypic and genotypic sex were breed till one year of life, which demonstrate persistence of reversal. Our outcomes are concordant with most recent work on this species and show full functional phenomenon of sex reversal with reptiles, which studying could contribute to our understanding of evolution of sex determination.

Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...

Contexte : Les parabènes sont des substances antibactériennes et antifongiques utilisées comme conservateurs dans de nombreux produits d’usage courant (cosmétiques, soins personnels, pharmaceutiques et alimentaires). Des études in vitro et in vivo ont signalé les effets de perturbation endocrinienne des parabènes, soulevant des inquiétudes quant à leurs risques potentiels pour la santé humaine. Objectif : Évaluer l’association entre les concentrations urinaires de parabènes et les concentrations sériques d’hormones de la reproduction (estradiol, progestérone, hormone folliculostimulante et hormone lutéinisante) chez les jeunes filles de la population générale canadienne. Méthodes: Les données de l'Enquête canadienne sur les mesures de la santé (2014 – 2015) concernant les filles âgées entre 6 et 17 ans ont été utilisées pour cette étude. L’association entre les concentrations urinaires de parabènes et des hormones a été analysée par régression linéaire multivariée, en ajustant pour les covariables suivantes : âge, indice de masse corporelle, ethnicité, revenu, et saison lors de la collecte des échantillons urinaires et sanguins. Résultats : Les 382 participantes incluses dans cette étude étaient majoritairement blanches (76%), avaient un indice de masse corporelle normal (73%) et des niveaux détectables d’au moins un parabène (92 %). Des concentrations de parabènes plus élevées étaient associées à des concentrations d'hormones de la reproduction significativement inférieures pour l’estradiol, et les hormones folliculostimulante et lutéinisante; il n’y avait pas d’association pour la progestérone. Un doublement des concentrations des parabènes urinaires était associé à des concentrations inférieures d'estradiol de 5,8% (IC à 95% -9,3; -2,1), d’hormone folliculostimulante inférieure de 4,2% (IC à 95% -7,9; -0,3) et d’hormone lutéinisante inférieure de 10,8% (IC à 95% -17,4; -3,7). Discussion : Cette étude montre que l'exposition aux parabènes était associée à des concentrations circulantes plus faibles d'hormones de la reproduction chez des jeunes filles de la population générale. Le devis transversal ne permet pas de réaliser d’inférences causales. Néanmoins, ces résultats concordent avec les études animales et suggèrent que l’exposition aux parabènes pendant le développement puisse altérer le fonctionnement du système reproducteur. Des études longitudinales permettraient de confirmer, ou non, ces résultats. / Background: Parabens are chemical substances used as preservatives for their antibacterial and antifungal properties in many everyday products (personal care, cosmetics, pharmaceutical and food). Several in vitro and in vivo studies have shown their endocrine disrupting potential, raising some concerns for potential adverse human health effects. Objective: To assess the cross-sectional association between urinary concentration of parabens and serum reproductive hormones (estradiol, progesterone, follicle stimulating hormone and luteinizing hormone) in girls in the general Canadian population. Methods: Data from the Canadian Health Measures Survey (2014 – 2015) on girls aged 6-17 were used for this study. Associations between hormones and parabens were analyzed with multivariable linear regressions, adjusting for potential confounders (i.e., age, body mass index, ethnicity, household income, and sampling season). Results: The girls and teens included in the study (n=382) were mostly white (76%), had a normal body mass index (73%), and detectable levels of at least one paraben (92%). We observed significantly lower concentrations of reproductive hormones with higher paraben concentrations; there was no association with progesterone concentrations. A doubling in urinary parabens was associated with lower estradiol by 5.8% (95% CI -9.3, -2.1), lower FSH by 4.2% (95% CI -7.9, -0.3), and lower LH by 10.8% (95% CI -17.4, -3.7). Discussion: This study shows that exposure to parabens is associated with lower circulating levels of reproductive hormones in young girls in the general population. The cross-sectional design does not allow to make causal inferences. However, these results are consistent with animal studies and suggest that exposure to parabens during development may affect the functioning of the reproductive system. Future studies should employ a longitudinal design verify these results.

Einfluss des Wachstumsfaktors Insulin-like growth factor-I (IGF-I) auf das Follikelwachstum beim Weißbüschelaffen (Callithrix jacchus) Aus dem Veterinär-Physiologisch-Chemischen Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im September 2010 (86 S., 16 Abb., 9 Tab., 225 Lit., 4 S. Anhang) Schlüsselwörter: Insulin-like Growth Factor-I (IGF-I), Granulosazellen, Steroidhormone (Östradiol, Progesteron), Gonadotropine (FSH, hCG) In der vorliegenden Studie wurde die Rolle von IGF-I und das Zusammenwirken mit den Gonadotropinen (FSH, hCG) auf die Sekretion der Steroidhormone (Progesteron, Östradiol) kultivierter Granulosazellen von 13 Weißbüschelaffen untersucht, um zu prüfen, ob und wie weit IGF-I die Sekretion und Reifung der Granulosazellen beeinflusst. Für die in vitro-Experimente wurden Zellkulturen mit Granulosazellen kleiner ( 0,5 - 1 mm) und präovulatorischer Follikel ( > 2 mm) von Ovarien am 7. Tag der Follikelphase verwendet. Vor jedem Versuch wurde das Wachstum der Follikel durch zwei Ultraschalluntersuchungen kontrolliert. Während der Kultur wurden drei Inkubationsintervalle von je 48 h durchgeführt. Die Zellen wurden mit IGF-I allein oder in Kombination mit FSH bzw. hCG stimuliert. Zum Teil wurden die Gonadotropine auch zur Prästimulation verwendet. Das Signifikanzniveau der Hormoneffekte lag bei p<0,05. Bei den Granulosazellen kleiner Follikel lässt sich durch die alleinige Gabe von IGF-I nur am Ende der Kultur (144 h) eine signifikante Erhöhung der Progesteronsekretion feststellen. Bei einer Kombinationsgabe von IGF-I und FSH findet sich schon am Anfang (48 h) ein signifikanter Einfluss auf die Sekretion von Progesteron und Östradiol. Bei der Progesteronsekretion ist der Effekt der Kombination signifikant höher als bei Einzelgabe beider Hormone. Dagegen ist bei der Östradiolsekretion der Effekt der Kombination zwar nicht höher als bei einer alleinigen Gabe von FSH, aber die Zellen reagieren wesentlich schneller auf IGF-I, wenn sie zusammen mit FSH stimuliert werden. Keine signifikante Wirkung in der Steroidhormonsekretion ruft die Hormonkombination IGF-I und hCG im Vergleich zur alleinigen Gabe der beiden Hormone hervor. Bei dem Vergleich beider Gonadotropine ist eine signifikante Erhöhung der Steroidhormonsekretion nur bei alleiniger Gabe von FSH zu beobachten. Bei den Experimenten mit Prästimulationen (FSH oder hCG) lässt sich nur bei der FSH-Prästimulation mit einer nachfolgenden Kombinationsgabe von hCG und IGF-I eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Dies bedeutet, dass FSH die kleinen Granulosazellen auf die Wirkung von hCG sensibilisiert, wobei IGF-I diesen Vorgang unterstützt. Im Gegensatz zu den kleinen Follikeln lässt sich bei den Granulosazellen der präovulatorischen Follikel ein signifikanter Effekt von verschiedenen Hormonstimulationen schon früh beobachten. Durch alleinige IGF-I-Gabe lässt sich bereits am Anfang der Kultur (48 h) eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Eine Kombinationsgabe von IGF-I und der Gonadotropine (FSH oder hCG) zeigt, dass die Kombination mit FSH zu einer signifikanten Erhöhung beider Steroide im Vergleich zur Kontrolle führt. Dagegen zeigt sich bei einer Kombination von IGF-I und hCG eine signifikante Erhöhung der Steroidhormonsekretion schon ab 48 h sowohl im Vergleich zur Kontrolle als auch zur alleinigen Gabe dieser Hormone. Bei der Untersuchung des Effekts beider Gonadotropine (FSH oder hCG) ist schon ab 48 h ein signifikanter Effekt auf beide Steroidhormone zu erkennen. Beide Gonadotropinprästimulationen (FSH oder hCG) mit nachfolgender Hormonkombination (hCG und IGF-I) führen bei den Granulosazellen der präovulatorischen Follikel zu einer signifikant geringeren Steroidhormonsekretion im Vergleich zur Gabe von hCG und IGF-I ohne Prästimulation. Die Zellen reagieren offenbar in dieser Art und Weise, um eine mögliche übermäßige Steroidgenese, und somit eine pathologische Situation, zu verhindern. Die vorliegende Arbeit zeigt, dass IGF-I bei den kleinen und präovulatorischen Follikeln unterschiedliche Wirkungen hervorruft. Es scheint, dass IGF-I die Sekretion von Progesteron und Östradiol auf unterschiedliche Art und Weise beeinflusst, und dass die Granulosazellen der Weißbüschelaffen erst während der Follikelentwicklung die Fähigkeit erwerben, auf IGFI entsprechend zu reagieren. Die Ergebnisse zeigen weiterhin, dass IGF-I bei den Granulosazellen der kleinen Follikel eine eher unterstützende Rolle für die Gonadotropine spielt, und dass IGF-I mit den Gonadotropinen bei der Reifung und der Differenzierung der Follikel mitwirkt. Möglicherweise spielt IGF-I auch während der Entwicklung und des Wachstums des präovulatorischen Follikels sowie bei der Regulierung der Progesteronsekretion eine Rolle. Die vorliegenden Ergebnisse unterstützen die Hypothese, dass IGF-I zusammen mit hCG die Zelldifferenzierung bei den Granulosazellen der präovulatorischen Follikel fördert. Außerdem kann vermutet werden, dass bei den Granulosazellen der präovulatorischen Follikel IGF-I zusammen mit FSH in unabhängiger Weise wirkt. Abschließend kann gesagt werden, dass ein Zusammenwirken zwischen den Gonadotropinen und IGF-I in Bezug auf die Bildung des präovulatorischen Follikels und die darauffolgende Ovulation existiert, dies gilt es auch bei pathologischen Situationen der Follikelreifung und Ovulation zu berücksichtigen.

Die Wirkung von zwei endogen neuroprotektiven Substanzen, Melatonin und 17 beta-Estradiol wurde an drei Caspase-abhängigen, apoptotischen, aber Exzitotoxin-unabhängigen Schadensmodellen an neuronalen Primärkulturen untersucht und mit der bei vorwiegend nekrotischen Schadensmodellen verglichen. Es zeigten sich eine Abhängigkeit des neuroprotektiven Potentials von der Art des Zelluntergangs sowie unterschiedliche Mechanismen der Neuroprotektion. Melatonin wirkte in allen drei apoptischen Modellen nicht neuroprotektiv, sondern verstärkte die Schädigung der Neurone noch, während partiell gegen die OGD-induzierte Nekrose (OGD, engl. Oxygen glucose deprivation, kombinierter Sauerstoff- und Glukoseentzug) kortikaler Neurone Schutz erzielt wurde. Der Einsatz des endogenen neuroprotektiven Faktors Melatonin als Therapeutikum ist möglicherweise nur bei neurodegenerativen Erkrankungen mit exzitotoxischer Schädigung durch Glutamat oder oxidativem Stress wie bei Epilepsie oder dem Schlaganfall durch Ischämie sinnvoll. Die fehlende bzw. potenzierenden Wirkung von Melatonin bei neuronaler Apoptose in vitro, stellt jedoch einen therapeutischen Erfolg bei der Behandlung der mit apoptotischer Schädigung einhergehenden Alzheimer'schen Erkrankung in Frage. Bei klinischer Anwendung ist auch der von uns erhobene Befund zu beachten, dass in vitro native neuronale Zellen durch Melatonin geschädigt werden. 17 beta-Estradiol wirkte sowohl bei nekrotischer als auch bei apoptotischer Zellschädigung. Dabei zeigten sich wesentliche Unterschiede in den Mechanismen der Neuroprotektion und in der Ansprechbarkeit verschiedener Regionen des Gehirns. Schutz vor Apoptose konnte nur durch eine Langzeitvorbehandlung (20 h) in septalen und hippokampalen Kulturen, nicht jedoch in kortikalen Kulturen beobachtet werden. Dieser Effekt liess sich durch Rezeptorantagonisten, Proteinsynthesehemmung sowie durch Hemmung der Phosphoinositol-3-Kinase blockieren. Eine Kurzzeitbehandlung war gegen Apoptose nicht wirksam, zeigte gegen OGD und Glutamattoxizität jedoch neuroprotektives Potential. Dieser Effekt liess sich nicht antagonisieren, so dass hier ein direkter antioxidativer Mechanismus wahrscheinlich erscheint. Die antiapoptotische Wirkung in septalen und hippokampalen Kulturen korrelierte mit einer höheren Dichte des Estrogenrezeptors-alpha und einer erhöhten Expression antiapoptotischer Proteine in diesen Regionen. Da bei der Alzheimer'schen Erkrankung der Kortex betroffen ist, könnte der fehlende Effekt von 17 beta-Estradiol in kortikalen Neuronen sowohl auf die neuronale Apoptose als auch auf die Proteinexpression von Bcl-2 und Bcl-xL möglicherweise auf experimenteller Basis erklären, warum eine langfristige Estrogentherapie bei Frauen mit milder bis moderater Alzeimer'scher Erkrankung den Progress der Erkrankung nicht aufhalten konnte (Mulnard et al. 2000). / The neuroprotective effect of melatonin and 17 beta-estradiol has been evaluated in several in vitro models of neuronal apoptosis and necrosis. Melatonin was not neuroprotective in three models of apoptosis but showed a pro-apoptotic effect in primary cortical neurons. Melatonin revealed to damage naïve neurons, too. Partial protection was observed against necrotic neurodegeneration after oxygen-glucose deprivation (OGD). The use of melatonin as a therapeutic agent might be of interest in neurodegenerative diseases with excitotoxic damage like epilepsia or ischemia, but is questioned in case of apoptotic neurodegeneration. 17 beta-estradiol was neuroprotectiv in both necrotic and apoptotic neurodegeneration. Differences in the mechanism of neuroprotetion and in the efficacy in different regions of the brain were observed. A neuroprotective effect was visible only in hippocampal and septal cultures if 17 beta-estradiol was applied 20 h prior (long term pre-treatment) but not in cortical neurons. This effect correlates with an increased density of estrogen receptor-alpha and an increased expression of anti-apoptotic proteins like Bcl-2 and Bcl-xL in these regions. These effect could be blocked with receptor antagonists, protein synthesis inhibitors and an inhibitor of the phosphatidylinositol 3-kinase. A short term pre-treatment revealed a receptor independent neuroprotective potential against OGD and glutamate toxicity. The failure of 17 beta-estradiol to protect cortical neurons against apoptosis could be an experimental basis to understand, why a long lasting treatment with estrogens of women with mild to moderate Alzheimer´s disease failed to inhibit the progress of the illness (Mulnard et al., 2000)