Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / The aim of this study were to evaluate the effect of different concentrations of Concanavalin A (Con A) and FSH on activation, and survival in vitro growth of goat primordial follicles, as well as investigating the effects of Con A and FSH on the expression of mRNA for PCNA, KL, c-Kit, BMP-15 and GDF-9. For this, ovarian cortex fragments were in vitro cultured for 1 to 6 days in &#945;-MEM+ supplemented with 0, 5, 10, 20 or 40 &#956;g/mL of Con A lectin (experiment 1). At the end of the growing period, the fragments of ovarian cortex were fixed for histology. Next, we evaluated the percentage of primordial follicles or under development. After determination of the concentration of Con A more efficient (10 &#956;g/mL), fragments of ovarian cortex were cultured in situ for 6 days in &#945;-MEM+ alone or supplemented with 50 ng/mL of FSH, 10 &#956;g/mL of Con A or both (experiment 2). Tissues from the control uncultured or after culture in different treatments were fixed for histology or stored at -80ÂC to assess the expression profile of mRNA for PCNA, KL, c-Kit, BMP-15 and GDF-9. The results of Experiment 1 demonstrated that after 6 days of culture, the percentage of developing follicles was significantly higher in samples cultured with &#945;-MEM+ supplemented with 10 and 40 &#956;g/mL Con A, when compared to the other treatments (P <0.05). In experiment 2, the presence of FSH, or both Con A promoted a reduction in the percentage of primordial follicles and increase developing follicles compared to control medium (&#945;-MEM+). After 6 days of culture, the presence of FSH promoted an increase in mRNA levels for PCNA, but this effect was blocked by Con A. Moreover, the Con A increased mRNA levels for c-KIT compared to the control not grown, but Con A or Con A and FSH both reduced the levels of mRNA for KL. Furthermore, a reduction of BMP-15 mRNA was observed in follicles cultured in control medium, but the presence of FSH inhibited this reduction. Furthermore, Con A promoted an increase in levels of GDF-9 mRNA after in vitro culture. In conclusion, the Con A alone or in combination with FSH promote activation of primordial follicles goat after 6 days of in vitro culture and regulate the expression of mRNA for PCNA, KL, c-Kit, BMP-15 and GDF-9 / Os objetivos deste estudo foram avaliar o efeito de diferentes concentraÃÃes de Concanavalina A (Con A) e do FSH sobre a ativaÃÃo, sobrevivÃncia e crescimento in vitro de folÃculos primordiais caprinos, bem como investigar os efeitos da Con A e do FSH sobre a expressÃo de RNAm para PCNA, KL, c-Kit, BMP-15 e GDF-9. Para isto, fragmentos de cÃrtex ovariano foram cultivados in vitro por 1 ou 6 dias em &#945;-MEM+ suplementado com 0, 5, 10, 20 ou 40 &#956;g/mL da lectina Con A (experimento 1). No final do perÃodo de cultivo, os fragmentos de cÃrtex ovariano foram fixados para histologia clÃssica. Em seguida, avaliou-se a percentagem de folÃculos primordiais ou em desenvolvimento. ApÃs a determinaÃÃo da concentraÃÃo de Con A mais eficiente (10 &#956;g/mL), os fragmentos do cÃrtex ovariano foram cultivados in situ por 6 dias em &#945;-MEM+ sozinho ou suplementado com 50 ng/mL de FSH, 10 &#956;g/mL de Con A ou ambos (experimento 2). Os tecidos provenientes do controle nÃo cultivado ou apÃs cultivo nos diferentes tratamentos foram fixados para histologia clÃssica ou armazenados a -800C para avaliar o perfil de expressÃo de RNAs mensageiros para PCNA, KL, c-Kit, BMP-15 e GDF-9. Os resultados do experimento 1 demonstraram que apÃs 6 dias de cultivo, a percentagem de folÃculos em desenvolvimento foi significativamente maior nos fragmentos cultivados com &#945;-MEM+ suplementado com 10 e 40 &#956;g/mL de Con A, quando comparado aos demais tratamentos (P <0,05). No experimento 2, a presenÃa de FSH, Con A ou ambos promoveram uma reduÃÃo na percentagem de folÃculos primordiais e aumento de folÃculos em desenvolvimento em relaÃÃo ao meio controle (&#945;-MEM+). ApÃs 6 dias de cultivo, a presenÃa de FSH promoveu um aumento nos nÃveis de RNAm para PCNA, mas este efeito foi bloqueado pela Con A. Por outro lado, a Con A aumentou os nÃveis de RNAm para c-kit em comparaÃÃo ao controle nÃo cultivado, mas Con-A ou ambos Con A e FSH reduziram os nÃveis de RNAm para KL. AlÃm disso, uma reduÃÃo dos RNAm para BMP-15 foi observado em folÃculos cultivados no meio controle, mas a presenÃa de FSH inibiu esta reduÃÃo. AlÃm disso, a Con A promoveu um aumento nos nÃveis de RNAm para GDF-9 apÃs cultivo in vitro. Em conclusÃo, a Con A sozinha ou em combinaÃÃo com FSH promovem a ativaÃÃo de folÃculos primordiais caprinos apÃs 6 dias de cultivo in vitro e regulam a expressÃo de RNAm para PCNA, KL, c-Kit, BMP-15 e GDF-9.

Made available in DSpace on 2014-10-09T12:52:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:55Z (GMT). No. of bitstreams: 0 / 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. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:26:59Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:49Z (GMT). No. of bitstreams: 0 / Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

FSH é caracterizada por uma grande variabilidade clínica inter- e intrafamilial. Aproximadamente 10-20% dos pacientes ficam em cadeira de rodas, enquanto que 20-30% dos portadores do alelo com a contração permanecem assintomáticos ou minimamente afetados. Interessantemente, estes casos parecem estar concentrados em determinadas famílias, sugerindo que algum mecanismo deve estar agindo nestes indivíduos, protegendo-os dos efeitos da doença. Para tentar explicar esta variabilidade clínica em FSH, nós comparamos o perfil de expressão gênica a partir do músculo de três membros (afetado, portador assintomático e controle normal) de cinco famílias diferentes através do microarray de expressão e de exons. Nossos resultados sugerem que a expressão dos genes no cromossomo 4q está alterada nos afetados e nos assintomáticos. Interessantemente, as alterações observadas nas amostras dos assintomáticos estão relacionadas aos genes de quemocinas, enquanto que as alterações vistas nas amostras dos afetados estão relacionadas com os genes envolvidos nos processos de acetilação de histonas e da modificação pós-traducional âncora-GPI. Além disto, os pacientes afetados e os assintomáticos compartilham o haplótipo 4qA161 e, desta forma, estes polimorfismos sozinhos não explicam a patogenicidade do alelo com a contração. Nossos resultados corroboram com as observações anteriores de FSH deve ser causada pela desregulação transcricional de vários genes, tanto in cis como in trans, e sugerem alguns fatores potencialmente importantes na patogênese de FSH. O estudo do perfil da expressão gênica dos portadores assintomáticos é uma abordagem nova que está revelando resultados novos e bem interessantes. Entender tal mecanismo é um grande desafio, mas que certamente levará ao desenvolvimento de novas ferramentas para o prognóstico e um possível tratamento. / FSHD is characterized by a great clinical inter and intrafamilial variability. Approximately 10-20% of patients eventually becoming wheelchair-bound while 20-30% with a shortened D4Z4 array, remains asymptomatic or minimally affected. Interestingly, these cases seem to be concentrated in some particular families, suggesting that some mechanism might be acting in these individuals, protecting them form the effects of the disease. In order to try to explain this clinical variability observed in FSHD, we compared the expression profiles of muscle tissue from three members (affected, asymptomatic carrier and normal control) from five unrelated FSHD families through expression and exon microarrays. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype, thus these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Together, our results support the previous evidences that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of gene expression profiles from asymptomatic carriers is a novel approach that is revealing new and interesting results. Understanding such mechanisms is a great challenge, but will certainly lead to the development of new tools for prognosis and also for future treatment.

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

Abstract Anti-Müllerian hormone (AMH) was identified originally in connection with its role in male sexual differentiation. In females, AMH is secreted by ovarian granulosa cells of growing follicles and its serum levels correlate well with the remaining number of follicles, thus reflecting ovarian reserve. The aim of this study was to explore the expression and secretion of AMH in human ovarian development and in various disorders resulting in decreased reproductive function. In fetal ovaries, AMH expression was found to be initiated at midgestation and it increased gradually towards term. In serum samples from infant girls, transient postnatal activation of the pituitary-ovarian axis was found and it occurred later in premature infants, reflected in lower serum AMH levels and lower numbers of growing follicles. This immaturity resulted in insufficient feedback from ovary to pituitary and may explain the higher levels of follicle-stimulating hormone (FSH) in these girls. Ovarian follicle reserve is typically diminished in women with primary ovarian insufficiency (POI). Assay of serum AMH may be used to identify women with POI and existing follicles, as women with FSH receptor mutation who were known to have follicles had serum AMH levels in low normal range and girls/women with Turner syndrome mosaicism had higher serum AMH levels compared with those with other karyotypes. The diagnostics and follow-up of ovarian granulosa cell tumors (GCTs) are challenging as a result of the rarity of the disease and late possible recurrences. Assay of serum AMH combined with assay of the currently used marker inhibin B was shown to improve follow-up of GCTs in individual patients, compared with serum inhibin B measurement alone. The analyses revealed that AMH and inhibin B assays perform similarly in detecting macroscopic tumors. Continuous use of combined hormonal contraceptives inhibited ovarian activity independently of the administration route, as serum AMH levels decreased significantly and similarly during oral, transdermal or vaginal administration. The decrease of serum AMH levels indicates that AMH is secreted partially from FSH-dependent follicles. The study provides novel information on AMH secretion in ovarian development, and the use of AMH assay in assessing ovarian reserve and detecting ovarian disorders such as ovarian insufficiency and GCTs. / Tiivistelmä Naisen munasarjassa munarakkuloiden granuloosasolut erittävät anti-Müllerian hormonia (AMH), ja sen pitoisuudet seerumissa heijastavat jäljellä olevien munarakkuloiden määrää. Tässä tutkimuksessa tarkasteltiin AMH:n eritystä ja ilmentymistä munasarjan kehityksen aikana ja erilaisissa munasarjan toiminnan häiriötiloissa. Tuloksina havaittiin, että AMH:a ilmentyy sikiökaudella munasarjassa jo keskiraskaudesta eteenpäin. Syntymän jälkeen otetuissa seeruminäytteissä todettiin ohimenevä aivolisäkkeen ja munasarjan aktivaatio, joka ilmeni myöhemmin ennenaikaisesti syntyneillä tytöillä. Heillä havaittiin vähemmän kasvavia munarakkuloita ja matalammat seerumin AMH-pitoisuudet syntymän jälkeen kuin täysiaikaisilla tytöillä. Tämä kypsymättömyys johtui todennäköisesti munasarjan riittämättömästä palautejärjestelmästä aivolisäkkeeseen, mikä voi selittää korkeammat follikkelia stimuloivan hormonin (FSH) pitoisuudet ennenaikaisesti syntyneillä tytöillä. Ennenaikainen munasarjojen toiminnan hiipuminen voi johtua esim. kromosomi- tai geenivirheestä. Naisilla, joilla on virheellinen FSH-reseptori, tiedetään olevan munarakkuloita jäljellä, ja tulokset osoittivat seerumin AMH-pitoisuuksien olevan lähes normaali näillä naisilla. Myös tytöillä ja naisilla, joilla on todettu Turnerin oireyhtymän mosaikismi, AMH-pitoisuudet olivat merkittävästi korkeammat verrattuna muihin karyotyyppeihin. Täten AMH-määrityksen avulla voidaan mahdollisesti löytää ennenaikaisesta munasarjojen toiminnan hiipumisesta kärsivien joukosta ne naiset, joilla on vielä jäljellä munarakkuloita. Munasarjan granuloosasolukasvaimen diagnostiikka ja seuranta ovat haastavia kasvaimen myöhäisen uusiutumistaipumuksen ja harvinaisuuden vuoksi. Tulokset osoittivat, että seerumin AMH-määrityksen yhdistäminen tällä hetkellä käytössä olevaan inhibiini B -määritykseen voisi parantaa yksittäisten potilaiden seurantaa makroskooppisen kasvaimen toteamisen osalta. Tutkimus osoitti hormonaalisten yhdistelmäehkäisyvalmisteiden jatkuvan käytön vähentävän munasarjan aktiivisuutta merkittävästi annostelureitistä riippumatta, koska seerumin AMH-pitoisuudet laskivat samalla tavalla ehkäisypilleriä, -rengasta ja -laastaria käyttävillä naisilla. Tutkimus toi uutta tietoa AMH:n erityksestä munasarjan kehityksen aikana sekä AMH-määrityksen käytön mahdollisuuksista munarakkuloiden määrän arvioinnissa ja eri häiriötilojen tunnistamisessa.

Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...

Recent studies have shown that postmenopausal follicle stimulating hormone (FSH) levels may be predictive of future cardiovascular disease risk. However, little is known about postmenopausal FSH levels, including the level of variation between women and factors associated with this variation. We assessed the relationship of multiple reproductive factors with FSH levels among 588 postmenopausal women in the Kuopio Ischemic Heart Disease Risk Factor Study. Participants were aged 53 to 73 years and not using hormone therapy at baseline (1998-2001). Reproductive factors were assessed at baseline, along with FSH levels. After adjustment for sex steroids, adiposity measures, plasma lipids, blood pressure, and behavioral factors, we observed that women with 3 or more births and an age at first birth of 25 or later had mean FSH levels that were significantly 7.6 IU/L lower than those of women with a 1 to 2 births and an age at first birth of 24 or less. Number of miscarriages was inversely correlated with FSH levels. Women reporting a 7 or more years of OC use and 4-6 or 7 or more years of HT use each had significantly higher mean FSH levels than women who had never used OCs or HT. In summary, multiple reproductive factors were associated with postmenopausal FSH levels, independent of estradiol, adiposity, and other factors. These findings warrant replication and further exploration of potential underlying mechanism.

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluídos os do sistema reprodutor e parece estar envolvido na maturação oocitária. No entanto, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar nos níveis de outro nucleotídeo (AMPc) via fosfodiesterases (PDE). O AMPc é um importante elemento da via de sinalização do FSH nos complexos cumulus-oócitos e no controle da maturação oocitária. O objetivo do presente projeto foi investigar a influência do FSH na via do NOS/NO e seus componentes em oócitos bovinos maturados in vitro e o envolvimento das células do cumulus (CC) na via de sinalização. Para tanto, complexos cumulus-oócito (CCO) e oócitos desnudos (OD - maturados sem células do cumulus) foram maturados in vitro por 24h na presença ou ausência de FSH. As amostras foram avaliadas quanto a: 1) taxa da maturação nuclear; 2) níveis de produção de NO; 3) níveis de AMPc e GMPc; 4) abundância relativa de RNAm de NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A e PKA1. O FSH, na concentração de 0,05UI/mL, estimulou positivamente a maturação nuclear em CCO e OD, com 80,6 e 89% de oócitos maturados, respectivamente. Quando comparados diretamente os grupos CCO e OD, o FSH não influenciou as taxas de maturação (71 e 71,3%, p>0,05), nem os níveis de produção de NO (12,8 e 7,4 µM/mL, p>0,05). Os níveis de GMPc em CCO aumentaram após 1 e 3 h de MIV na presença de FSH (266,3 e 187,2 pmol/pool com FSH e 240,5 e 168,5 pmol/pool sem FSH, respectivamente, p<0,05). Após 6 h os níveis de GMPc declinaram de forma mais acentuada no grupo sem FSH (46,3 e 106,9 pmol/pool, com e sem FSH, respectivamente, p<0,05). Os níveis de AMPc em CCO também foram mais elevados na presença de FSH à 1 e 3 h de MIV (7,60 e 7,81 pmol/pool, respectivamente) em comparação com CCO maturados sem FSH (0.30 e 0,76 pmol/pool, respectivamente, p<0,05). Após 6h, os níveis declinaram e foram similares para ambos os grupos (0,43 pmol/pool, p>0,05). Em relação à expressão dos genes selecionados, todos foram detectados nos oócitos (CCO e OD), porém, em células do cumulus, foram detectados apenas PDE5A, ADCY6, ADCY9 e PKA1. Quando observados os resultados do grupo CCO, apenas os genes PKG1, ADCY6 e PDE3A sofreram influência do FSH (p<0,05), apresentando um aumento destes transcritos. No grupo OD, apenas o gene PKG1 sofreu influência do FSH, também apresentando um aumento destes transcritos (p<0,05). Em células do cumulus, os genes ADCY6 e ADCY9 sofreram influência do FSH, sendo que para a ADCY6 provocou um aumentos destes transcritos, e para a ADCY9 provocou uma queda dos mesmos (p<0,05). Em conclusão, o FSH pode exercer influência positiva na maturação nuclear de oócitos bovinos, agindo sobre os níveis de GMPc e AMPc, mas não sobre o NO. O FSH pode influenciar a expressão gênica em oócitos e em células do cumulus de bovinos. / Nitric oxide (NO) is a chemical messenger generated by the nitric oxide synthase (NOS) enzyme, which was detected in several organs including the reproductive system and appears too involved in oocyte maturation. However, there are few studies on the role of this system in bovine oocytes. NO is known to act via guanylate cyclase (GC) stimulating the production of the nucleotide cGMP, which in turn is capable of influencing the levels of another nucleotide, cAMP via phosphodiesterases (PDE). cAMP is an important factor in FSH signaling in cumulus-oocyte complexes (COC) for the control of maturation. The aim of the present work was to investigate the influence of FSH on the NOS/NO pathway and its components in bovine oocytes matured in vitro and the involvement of cumulus cells (CC) in the signaling pathway. COC and denuded oocytes (DO - matured without cumulus cells) were matured in vitro for 24 h with or without FSH. Samples were assessed for: 1) maturation rate; 2) levels of NO production; 3) levels of cGMP and cAMP; 4) relative abundance for mRNA of NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A and PKA1. FSH positively stimulated oocyte maturation at 0.05UI/mL concentration for both COC and OD (80.6 and 89% maturation rates, respectively). When COC and OD were compared directly, FSH did not affect maturation rates (71 and 71.3%, p>0.05) nor NO production levels (12,8 and 7,4 µM/mL), p>0.05). cGMP levels increased after 1 and 3 h in vitro maturation (IVM) with FSH (266.3 and 187.2 pmol/pool with FSH and 240.5 and 168.5 pmol/pool without FSH, respectively, p<0.05). After 6 h IVM, cGMP levels in COC declined more in the group cultured with FSH (46.3 and 106.9 pmol/pool, with and without FSH, respectively, p<0.05). cAMP levels in COC were also increased in the presence of FSH at 1 and 3 h IVM (7.60 and 7.81 pmol/pool, respectively) in comparison to COC cultured without the hormone (0.30 and 0.76 pmol/pool, respectively, p<0.05). After 6 h, the levels declined and were similar for both groups (0.43 and 0.02 pmol/pool, p>0.05). Regarding mRNA expression for the selected genes, all of them were detected in oocytes, but only four of them were detected in cumulus cells: PDE5A, ADCY6, ADCY9 and PKA1. For COC only PKG1, ADCY6 and PDE3A were influenced by FSH (p<0.05), with an increase in transcript relative abundance, For DO, only PKG1 was influenced by FSH and also showed an increase in these transcripts (p<0.05). In cumulus cells, ADCY6 and ADCY9 were affected by FSH, with an increase for ADCY6 and a decrease in ADCY9 transcripts (p<0.05). In conclusion, FSH may positively influence nuclear maturation, acting on cGMP and cAMP levels, but not on NO. FSH may also influence gene expression in bovine oocytes and cumulus cells.

L’hormone anti-Müllerienne (AMH), une glycopréotéine exclusivement produite par les cellules de la granulosa (CG) des follicules ovariens de la femme, est un marqueur unique de du statut folliculaire ovarien. Contrairement à l’inhibine B, l’estradiol et la FSH, l’AMH est produite par un large éventail de follicules allant des follicules primaires aux follicules à petit antrum. Cependant, les mécanismes précis régulant la production d’AMH par les CG restent mal connus. Nous avons montré que la sélection folliculaire précoce au cours de la phase de transition lutéo-folliculaire, un phénomène fréquemment retrouvé chez les femmes ayant un vieillissement ovarien, caractérisé par la présence d’au moins un follicule surdéveloppé au cours de la phase folliculaire précoce, n’altérait pas la puissance de la relation entre le compte folliculaire antral et les concentrations sériques d’AMH. En revanche, cette situation perturbait significativement celle entre le nombre de follicules antraux et les taux sériques de FSH, d’inhibine B et d’estradiol. Nous avons par la suite mis en évidence, en utilisant un nouvel outil, nommé Follicular Output RaTe (FORT), que le pourcentage de follicules qui répondent effectivement à la FSH exogène en atteignant la maturation pré-ovulatoire, était négativement et indépendamment lié aux taux sériques d’AMH, ce qui va dans le sens de l’hypothèse d’un effet inhibiteur de l’AMH sur la sensibilité des follicules à la FSH. Ensuite, nous avons regardé si la production d’AMH par ovaire et par follicule était altérée chez les femmes n’ayant plus qu’un seul ovaire suite à une ovariecomie unilatérale. En effet, tout indique que chez ces femmes, des réarrangements majeurs de la folliculogenèse sont mis en place pour maintenir une fonction ovarienne malgré la perte brutale d’une partie du pool folliculaire. Ainsi, par une analyse extensive et comparative de la folliculogenèse utilisant des marqueurs hormono-folliculiares, nous n’avons pu mettre aucune modification significative chez les femmes avec un ovaire unique, comparativement aux contrôles. A l’aide du modèle précédemment utilisé, nous avons constaté une augmentation de la sensibilité des follicules antraux à la FSH exogène, évaluée par le FORT, chez des femmes avec un seul ovaire, comparativement aux femmes avec 2 ovaires. Ces résultats supportent l’hypothèse d’une augmentation de la sensibilité folliculaire à la FSH, qui pourrait faire partie des possibles mécanismes compensatoires en jeu dans le maintien d’une folliculogenèse efficace chez les femmes ayant eu une ovariectomie unilatérale.Finalement, à l’aide de 2 approches complémentaires, in vitro and in vivo, nous avons montré que la FSH et l’AMPc stimulaient la transcription de l’AMH, et que la LH avait un effet additif. Nous avons montré que les gonadotrophines et l’AMPc agissaient à travers la protéine kinase A et la P38 MAP Kinase, impliquant notamment les facteurs de transcription GATA binding factor-4 et le steroidogenic factor-1. Par ailleurs, nous avons également mis en évidence que l’expression d’AMH pouvait être régulée de manière différentielle par l’estradiol, en fonction du type de récepteur aux estrogènes exprimés par les CG. Ainsi, la chute d’expression de l’AMH au sein des CG des follicules matures, qui expriment essentiellement ERβ, est probablement liée à un effet de l’estradiol. En résumé, ces travaux de thèse ont permis d’apporter de nouvelles données sur la régulation de la croissance folliculaire et sur la production d’AMH chez la femme. / Anti-Müllerian hormone (AMH), a glycoprotein that is exclusively produced by the granulosa cells (GC) of ovarian follicles in the adult female, is a unique biomarker of ovarian follicular status. In contrast with inhibin B, estradiol and FSH, AMH is produced in a wide range of follicles that goes from the primary to the small antral stages of folliculogenesis. However, the precise mechanisms that drive AMH expression by GC remain poorly understood.We showed that untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase does not alter the strength of the relationship between antral follicle count and serum AMH levels but does affect the relationship between serum FSH, inhibin B and estradiol levels and the number of antral follicles. The heftiness of AMH in relation to advanced antral follicle growth provides a further explanation for the reported stronger association between serum AMH levels and antral follicle counts as compared with the other hormonal markers of the ovarian fertility status. We subsequently demonstrated, using an innovative tool, the Follicular Output RaTe (FORT), that the percentage of follicles that effectively respond to exogenous FSH by reaching pre-ovulatory maturation is negatively and independently related to serum AMH levels, which is in keeping with the hypothesis that AMH inhibits follicle sensitivity to FSH. Given this hypothesis, we wondered if per-ovary and per-follicle AMH production could be altered in patients having a single ovary as a result of unilateral oophorectomy. Indeed, all indicate that major rearrangements of folliculogenesis occur to preserve and maintain ovarian function despite the abrupt halving of follicular stockpile in these patients. We performed an extensive and comparative evaluation of the folliculogenesis using homono-follicular markers failing to show major changes in unilaterally oophorectomized when compared with control women. Using the same model, we demonstrated an increased antral follicle responsiveness to exogenous FSH, as assessed by FORT, in normo-ovulating unilaterally oophorectomized women undergoing controlled ovarian hyperstimulation. These results support the hypothesis that increased FSH sensitivity ranks among the possible compensating mechanisms at stake in the maintenance of successful folliculogenesis after unilateral oophorectomy.Finally, using complementary approaches, in vitro and in vivo, we showed that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by GC. We also demonstrated that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors by GC. Therefore the decrease in AMH expression by GC of mature follicles, which mainly express ERβ, is likely due to the effect of estradiol.In short, this Ph.D. work offers new insight into the regulation of the follicular growth and AMH production in woman.