Comprendre les événements paracriniens qui régulent la fertilité chez la vache est nécessaire non seulement en raison de l'importance agricole de cette espèce, mais aussi pour son utilisation potentielle comme modèle chez l’humain. L'oxyde nitrique (NO), un gaz de radicaux libres, a été impliqué dans la croissance folliculaire et l'ovulation chez les rongeurs et d'autres espèces, mais chez la vache c’est une énigme fascinante : le NO est produit par les cellules de la granulosa bovine et est régulé par la FSH, mais la présence et le profil d'expression des enzymes responsables de la synthèse de NO (NOS) dans les cellules de la granulosa tout au long de la croissance folliculaire ne sont pas claires. Les objectifs de cette thèse sont: (1) élucider le mécanisme de contrôle des NOS et les conséquences de la production d'oxyde nitrique pour le fonctionnement des cellules de la granulosa au cours du développement folliculaire chez la vache et (2) déterminer la régulation des NOS pendant la cascade ovulatoire induite par LH chez les cellules de la granulosa bovine et si l'activité des NOS pour l’expression des gènes critiques dans la cascade ovulatoire chez cette espèce. Les résultats sont séparés en 2 articles. Dans le premier article, la régulation de NOS2 dans les cellules de la granulosa bovine a été explorée. L'abondance des ARNm codant pour NOS2 a été stimulée par la FSH et l’IGF1 en augmentant l’estradiol, et un blocage de l'action de l’estradiol a conséquemment réduit les niveaux d'ARNm codant pour NOS2. De plus, l'inhibition de l'activité des NOS a augmenté l'apoptose dans les cellules de la granulosa in vitro. Dans le second article, il a été démontré que le pic de LH induit une activation des NOS dans les cellules de la granulosa, et que l'activité de NOS induit la production de NO, ce qui est essentiel pour l’expression des gènes critiques dans la cascade ovulatoire induite par LH comme EREG/AREG/PTGS2. Ensemble, les résultats présentés dans ces 2 articles suggèrent que les niveaux physiologiques d'activité des NOS peuvent contribuer à la croissance et la survie des cellules de la granulosa et indiquent également que NO peut être essentiel pour l'ovulation chez les bovins. / Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth and ovulation in rodents and other species, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the presence and the expression pattern in granulosa cells of the enzymes responsible for NO synthesis (NOS) throughout follicular growth are unclear. The objectives of the present thesis were (1) to elucidate the mechanism of control of NOS and the consequences of nitric oxide production for granulosa cell function during follicle development in cattle; and (2) to determine the regulation of NOS during the LH-induced ovulatory cascade in bovine granulosa cells and whether NOS activity is critical for the ovulatory cascade in this species. The results are separated in 2 articles. In the first article, the regulation of NOS2 in bovine granulosa cells was explored. Abundance of mRNA encoding NOS2 was stimulated by FSH and IGF1 through increased estradiol, and a blockade of estradiol action consequently lowered NOS2 mRNA levels. Further, inhibition of NOS activity increased apoptosis in granulosa cells in vitro. In the second article, it was demonstrated that the LH surge induces NOS activation in granulosa cells, and that NOS activity induces the production of NO, which is essential for EREG/AREG/PTGS2 expression, critical genes in the LH-induced ovulatory cascade. Together, the results presented in these 2 articles suggest that physiological levels of NOS activity may contribute to growth and survival of granulosa cells, and also indicate that NO may be essential for ovulation in cattle.

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.

As técnicas de cultivo de folículos e oócitos in vitro, com o objetivo de se obter oócitos maduros para procedimentos de Reprodução Assistida (RA), têm sido aplicadas em diferentes contextos. O sucesso destes procedimentos está diretamente relacionado ao sistema de cultivo utilizado. A utilização de células da granulosa (CG) humanas cultivadas in vitro como um suporte para o co-cultivo destes oócitos imaturos e folículos tem sido descrita por alguns autores. A criopreservação destas células, considerando-se o contexto de sua obtenção em procedimentos de RA, permitiria a viabilização da aplicação destas células na prática clínica diária. Sendo assim, o objetivo deste estudo foi padronizar o congelamento de células da granulosa (CG) humanas para aplicação em sistemas de co-cultivos de folículos e oócitos imaturos. Foram obtidas CG de 20 voluntárias em tratamento de reprodução assistida, células de 10 voluntárias foram cultivadas em meio ?-MEM suplementado para interrupção da luteinização e congeladas após 48 horas em container \"Cryostep\" (grupo 2C- 2 cultivos) (etapa 2) e células de 10 voluntárias foram congeladas em container \"Cryostep\" sem cultivo prévio (grupo CD- congelamento direto) (etapa 3). Após o descongelamento estas células foram (re)cultivadas por 144 horas, com troca de meio em 48, 96 e 144 horas para avaliações da produção de estradiol (E2) e progesterona (P4) (ng/mL). Verificamos redução na contagem celular e na viabilidade celular tanto no método de congelamento direto (CD) quanto no método com dois cultivos (2C) após o descongelamento (p<0,05), e isso se refletiu na produção de estradiol e progesterona que foi maior nas culturas de células frescas em relação às células criopreservadas (p<0,05). Porém, a relação de E2/célula foi mantida após o descongelamento, sugerindo que esta redução na produção se deve à redução no número de células, as que sobrevivem se mantém normofuncionantes (p=0,23).O CD foi mais eficiente pois permitiu uma maior recuperação celular e uma melhor viabilidade quando comparado ao grupo 2C. A relação estradiol/progesterona foi mantida em todos os tempos de cultivo, fresco, CD e 2C (p>0,05), indicando que a característica funcional destas células foi preservada após o descongelamento. Concluímos que a criopreservação de CG humanas obtidas durante a captação de oócitos compromete a contagem celular e a viabilidade geral da cultura, entretanto, a capacidade funcional e a característica destas células se mantêm preservadas (manutenção das relações E2/célula e E2/P4) / Follicle and oocyte in vitro culture techniques, aiming to obtain mature oocytes for Assisted Reproductive Treatments (ART), have been applied to different contexts. The success of these procedures depends on the culture system used. The use of human granulosa cells (GC) in co-culture systems for follicle and oocyte maturation have been described by some authors. The cryopreservation of these cells, considering the context in which they are obtained during ART, would enable the usage of these cells in such procedures in daily clinical practice. Thus, the objective of this study was to standardize the freezing protocol for human granulosa cells (GC) for future applications in co-culture systems for follicle and oocyte maturation. Twenty volunteers submitted to ART donated their granulosa cells after oocyte retrieval, 10 were cultivated previously in order to interrupt the luteinization process and then frozen \"Cryostep\" container (group 2C- two cultures) (step 2) and 10 were directly frozen with no previous culture in the \"Cryostep\" container (group DF- direct freeze) (step 3). After thawing these cells were (re)cultured for 144 hours, with medium exchange at 48, 96 and 144 hours to evaluate the estradiol (E2) and progesterone (P4) production (ng/mL). After thawing, there was a reduction in the cell number (p<0,05) and cell viability in both methods, the direct freezing (DF) and the two cultures (2C) (p<0,05); this had an impact in the production of estradiol and progesterone, which were higher in fresh cultures than in the frozen ones (p<0,05). However, the E2/cell ratio was maintained after thawing (p=0.23), suggesting that this impairment in steroid production was probably due to the reduction in the cell count. The cells that survive remain functionally normal. The DF was more efficient since it allowed greater cell recovery and better viability when compared to 2C. The estradiol/progesterone ratio was maintained in all culture times, in the fresh, DF or 2C groups (p>0.05), indicating that the functional characteristic of these cells was preserved post-thawing. We conclude that cryopreservation of human GC obtained during oocyte retrieval compromises the cell count and the overall viability of the culture; however, the functional capacity and the characteristic of these cells are preserved (maintenance of E2/cell and E2/P4 relations)

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this study was to investigate the factors controling the expression of angiotensin II (AngII) receptors and to determine the physiological role of AngII in granulosa cells. The AGTR2 receptor was localized in granulosa (and theca) cells from follicles of different sizes. Bovine ovaries were collected at a local abattoir and small follicles (2-5mm) were isolated for harvesting granulosa cells. The cells were cultured in free medium serum in non-luteinizing conditions without FSH (control group) or with graded doses of FSH or IGF1. In other cultures, cells were cultured with or without IGF1 and bone morphogenetic protein-7 (BMP-7), and fibroblast growth factor-2 (FGF-2). Treatment with FSH, IGF1 and BMP-7 increased (P<0.05) estradiol secretion and AGTR2 mRNA expression relative to control cultures. In contrast, none of these treatments affected AGTR1 receptor expression. Addition of FGF-2 significantly decreased estradiol secretion but did not affect AGTR1 or AGTR2 expression. Cells were cultured with FSH plus graded doses of FGF-7 or FGF-10, and the effects of these factors on AGTR2 protein levels were measured by Western blot. AGTR2 protein levels decreased in the groups treated with FGF-7 (10 and 100ng/ml) and FGF-10 (all concentrations; P<0.05), and estradiol secretion was significantly inhibited by the highest dose of each FGF (P<0.05). Bovine follicles greater than 5 mm diameter were dissected and granulosa and theca cells were separated for RNA extraction, and follicle fluid assayed for estradiol (E2) and progesterone (P) content. Non-atretic follicles (P less than 100ng/ml) were classed as estrogenic (E2 greater than 100ng/ml) or non-estrogenic (E2 less than 40ng/ml). There were no differences in AGTR1 receptor expression in theca and granulosa cells between estrogenic and nonestrogenic follicles. Likewise, there were no changes in AGTR2 receptor expression in theca cells with follicle state. However, AGTR2 receptor mRNA levels were significantly higher in granulosa cells of estrogenic compared to non-estrogenic follicles (P<0.01), and AGTR2 receptor mRNA was correlated with E2 concentrations in follicular fluid. To determine the physiological consequences of AT activation in granulosa cells, cells from small (2-5mm) bovine follicles were cultured in serum-free medium with FSH ± AngII. The addition of AngII had no effect on estradiol or progesterone secretion, but significantly inhibited protease nexin-1 (PN-1) mRNA levels and protein secretion (P<0.05). PN-1 is an inhibitor of proteases involved in extracellular matrix remodeling and follicle rupture. Bovine granulosa cells from large (>10 mm) follicles were cultured for 6h, 12h and 24h with LH (100ng/ml) or AngII, with or without angiotensin receptor blocker (losartan for AGTR1 and PD123,319 for AGTR2). These cells expressed Ptgs2 under basal culture conditions, which was not upregulated by either LH or AngII alone. However, LH and AngII in combination significantly enhanced Ptgs2 (P<0.05) mRNA and protein accumulation. Similarly, expression of the proteolytic enzymes uPA and tPA, and their inhibitor, PN-1, were upregulated by the combination of LH and AngII but not by either factor alone. The addition of AGTR blockers inhibited the effect of AngII. In conclusion, AGTR2 receptor is present in granulosa bovine cells, and mRNA and protein are regulated by FSH, IGF-1, BMP-7, FGF-7 and FGF-10 in bovine granulosa cells in vitro, AGTR2 but not AGTR1 receptor mRNA levels are regulated during follicular growth in cattle, and that AngII regulates granulosa PN-1 secretion. These data suggest that AngII is a physiological co-factor necessary for the expression of genes in granulosa cells that are critical for ovulation. / O objetivo deste trabalho foi estabelecer o controle de expressão dos receptores de angiotencina II (AngII) e determinar a ação fisiológica da AngII em células da granulosa (CG) cultivadas in vitro. Os receptores de AngII tipo 1 (AGTR1) e tipo 2 (AGTR2) foram localizados em folículos de bovinos de diferentes tamanhos. Verificouse que as CG de bovinos provenientes de folículos entre 2- 5 mm e cultivadas com FSH, IGF-1, BMP-7 apresentaram aumento na expressão do receptor AGTR2 (P<0,05) em relação ao grupo controle (GC), bem como aumento da secreção de estradiol (E2; P<0,05). Em contraste, as CG tratadas com 10 ng/ml de FGF-2 ou 10 e 100 ng/ml de FGF-7 e FGF-10 apresentaram uma redução na secreção de E2 (P<0,05), porém somente os grupos FGF-7 e 10 nas doses 10 e 100 ng/ml reduziram (P<0,05) a expressão do receptor AGTR2. Para os dois experimentos, não houve diferença na expressão do receptor AGTR1 entre o GC e os grupos tratados. As CG e células da teca (CT) foram coletadas de ovários provenientes de abatedouro para extração de RNA e o fluido folicular para dosagem de E2 e progesterona P4. Esses folículos foram classificados como dominantes (FD; P4<100ng/ml e E2>100ng/ml) e atrésicos (FA; P4>40ng/ml). A expressão dos receptores AGTR1 e AGTR2 foi mensurada por RTPCR. Não houve diferença na expressão do receptor AGTR1 entre FD e FA. No entanto, o receptor AGTR2 apresentou um aumento (P<0,05) na expressão em CG de FD em relação a FA. A expressão do receptor AGTR1 se manteve constante em CG e CT de FD e FA. Para determinar os afeitos da AngII através da ativação de seus receptores CG foram cultivadas em meio livre de soro com FSH e/ou AngII. A AngII não apresentou efeito na secreção de E2 ou P4, mas inibiu (P<0.05) mRNA e proteína para a protease nexin-1 (PN-1). Considerando a redução de expressão da PN-1 envolvida no controle do remodelamento da matriz extracelular (RME), é possível especular um efeito de AngII sobre RME durante o desenvolvimento folicular. Em um terceiro experimento, as CG de folículos grandes (>10mm) foram cultivadas durante 6h, 12h e 24h na presença de Ang (10-5) com ou sem LH (100ng/ml). A combinação de AngII e LH aumentou significativamente (P<0,05) a expressão de mRNA e proteína para COX-2, ativador do plasminogênio tipo U e T, bem como para PN-1. Entretanto, AngII ou LH não aumentaram a expressão de COX-2. O aumento da expressão destes gene indica uma função de AngII no processo de ovulação através das CG. Em um segundo momento, verificou-se através de qual receptor a AngII atua para controlar a expressão desses genes. As CG foram cultivadas por 6h com LH e/ou AngII com ou sem inibidores específicos para o receptor AGTR1 (losartan) e AGTR2 (PD123,319). Os resultados demonstraram que a presença do inibidor de AGTR2 bloqueou o efeito da associação de LH e AngII em relação ao grupo controle (P<0.05), demonstrado que a ação da AngII é mediada pelo receptor AGTR2 em CG. Em conclusão, o receptor AGTR2 está presente nas células da granulosa de bovinos e o mRNA para o receptor AGTR2 é regulado durante o crescimento folicular. Além disso, a expressão do mRNA e a tradução da proteína para o AGTR2 são reguladas por FSH, IGF-1, BMP-7, FGF-7 e FGF-10 em CG de bovinos cultivadas in vitro. Os dados também sugerem que AngII regula a proteína PN-1 em CG e age como um co-fator fisiológico necessário para a ovulação.

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.

L'efficacité de la reproduction bovine a considérablement diminué dans les dernières décennies et cette diminution constitue un problème économique majeur. Pour mieux contrer ce problème, la physiologie des cellules stéroïdogéniques ovariennes dont les cellules de granulosa (CG) doit être mieux comprise au cours des dernières étapes de la croissance folliculaire, de l'ovulation et de la lutéinisation. En ce sens, nous avons précédemment identifié divers gènes induits dans les CG des follicules ovulatoires bovins par la LH/hCG incluant Ankyrin-repeat and SOCS-box protein 9 (ASB9). Cependant, les mécanismes d’action d’ASB9 dans les CG étaient encore indéfinis. Les objectifs de cette étude étaient d'élucider le rôle d'ASB9 dans les CG ainsi que ses effets sur ses partenaires spécifiques PAR1, TSG6 et TAOK1. Un modèle in vivo de CG provenant de follicules à différentes phases de développement: petits follicules (SF), follicules dominants (DF) et follicules ovulatoires (OF), et un modèle in vitro de CG en culture ont été utilisées. L'inhibition d’ASB9 dans les CG via CRISPR/Cas9 a montré une augmentation significative de PAR1, PCNA, CCND2 et CCNE2 et une diminution significative de TAOK1, TSG6 et CASP3. Dans le modèle in vivo, PAR1 a été différentiellement exprimé dans DF et TSG6 et TAOK1 ont été induits dans OF. L'inhibition de l'ASB9 a aussi entraîné une diminution de l'apoptose des CG et de l'activité caspase3/7. Des analyses Western blot ont démontré que l'induction d'ASB9 dans OF, après l'injection d'hCG, était concomitante avec une diminution significative des niveaux de phosphorylation de MAPK3/1 tandis que pMAPK3/1 augmentait après l'inhibition d'ASB9. Ces résultats supportent qu'ASB9 pourrait être un régulateur de l'activité et de la fonction des CG en ciblant des protéines spécifiques qui affectent la signalisation MAPK, limitant la prolifération des CG. Ces résultats contribuent à une meilleure compréhension de l’activité ovarienne et de la reproduction bovine. / The efficiency of bovine reproduction has considerably decreased in recent decades and this decrease constitutes a major economic problem. To better counter this problem, the physiology of ovarian steroidogenic cells including granulosa (CG) cells needs to be better understood during the later stages of follicular growth, ovulation and luteinization. In this sense, we have previously identified various genes induced in the CGs of bovine ovulatory follicles by LH / hCG including Ankyrin-repeat and SOCS-box protein 9 (ASB9). However, ASB9 mechanisms of action in GC were still undefined. The objectives of this study were to elucidate the role of ASB9 in CG as well as its effects on target partners PAR1, TSG6 and TAOK1, and on MAPK signaling. An in vivo model of GC from follicles at different developmental stages: small follicles (SF), dominant follicles (DF), and ovulatory follicles (OF) and an in vitro model of cultured GC along with the CRISPR/Cas9 approach to inhibit ASB9 were used. Inhibition of ASB9 in GC resulted in significant increase in PAR1, PCNA, CCND2, and CCNE2 and significant decrease in TAOK1, TNFAIP6, and CASP3 expression. From in vivo samples, PAR1 was differentially expressed in DF as compared to OF while TSG6 and TAOK1 were induced in OF. Further analyses showed an increase in GC number and a decrease in apoptosis and caspase3/7 activity following ASB9 inhibition. Western blot analyses demonstrated that ASB9 induction in OF by hCG was concomitant with a significant decrease in MAPK3/1 phosphorylation levels while pMAPK3/1 increased following ASB9 inhibition. These results provide strong evidence that ASB9 is a regulator of GC activity and function by modulating MAPK signaling pathway likely through specific binding partners such as PAR1, therefore controlling GC proliferation. These results contribute to a better understanding of ovarian activity and bovine reproduction.

Invasiv äggstockscancer anses vara en av de mest dödligaste gynekologiska maligniteterna. Granulosacelltumör i äggstocken är en sällsynt subtyp av äggstockstumör, som utgör 1–5% av alla äggstockstumörer. De kan kännetecknas av den långsamma tillväxthastigheten och produktionen av höga östrogennivåer. Återfallsfrekvensen efter den första behandlingen är låg, men dödligheten på ett återfall är så hög som 80%. Det är därför av intresse att undersöka nya terapeutiska mål som kan användas för att behandla granulosacelltumör i framtiden. Granulosacelltumörer tros uppstå från de sena preovulatoriska granulosacellerna eftersom de delar liknande egenskaper; de uttrycker follikelstimulerande hormonreceptorer och producerar östrogen som respons på follikelstimulerandehormoner. Östrogen och dess motsvarande kärnreceptorer, östrogenreceptor α och β spelar en avgörande roll i utvecklingen av äggstocksfolliklarna under äggstockscykeln. Uttrycket av östrogenreceptorn α är ganska lågt i granulosacellerna, istället tros östrogenreceptorn β (ERβ/ESR2) spela den övervägande rollen att aktivera intracellulära signalvägar som främjar cellproliferation och överlevnad i äggstocken. Det finns också många varianter av östrogenreceptorn β, främst ERβ1, ERβ2 ibland kallad för ERβcx, ERβ3, ERβ4, och ERβ5. Deras roll i äggstocken är fortfarande okända och har varit svårt att studera eftersom de saknar ligand-bindningsdomänen. DNA-bindningsdomänen är fortfarande bevarad, så de kan fortfarande vara viktiga transkriptionsfaktorer i äggstocken. Nya ChIP-sekvenseringsresultat från en studie visade att ERβ och leverreceptorn homolog 1 (LRH-1/NR5A2) delar många av kromatinbindningsställena i en normal musäggstock. Detta resultat tyder på att dessa två transkriptionsfaktorer troligtvis interagerar med varandra fysiskt eller indirekt. ChIP-qPCR användes för att bekräfta ERβ-varianternas kromatinbindning i den mänskliga granulosatumörcellinjen COV434. Dubbel luciferasanalys användes för att undersöka om ERβ påverkar LRH-1s transaktivering aktivitet. Co-IP utfördes för att undersöka om ERβ och LRH-1 fysiskt interagerar med varandra. Vi kunde bekräfta att ERβ-varianterna ERβcx, ERβ5 och ERβ4 binder till LRP6 genen och att varianterna kan binda direkt till DNA. För att ytterligare bekräfta flera målgener för ERβ-varianterna måste DNA-proverna skickas för sekvensering. Resultaten från denna studie indikerar att ERβ undertrycker LRH-1s transaktiverings aktivitet med eller utan liganden östradiol, hur ERβ fungerar som repressor är fortfarande okänt. Vi kunde inte visa om ERβ fysiskt interagerar med LRH-1 i denna studie. / Invasive ovarian cancer is considered to be one of the most fatal gynecological malignancies. Granulosa cell tumor in the ovary is a rare subtype of ovarian tumor that makes up almost 1-5% of all ovarian tumors. It can be characterized by the slow growth rate and production of high estrogen levels. The recurrence rate after the first treatment is fairly low, but the mortality rate for the recurrence is as high as 80%. Therefore, it is interesting to investigate new therapeutic targets that can be used to treat granulosa cell tumors in the future. Granulosa cell tumors are thought to originate from the late preovulatory granulosa cells because they share similar features; they express follicle-stimulating hormone receptors and produce estrogen in response to follicle-stimulating hormone. Estrogen and its corresponding nuclear receptors, estrogen receptors α and β, play a crucial role in the development of the ovarian follicles during the ovarian cycle. The expression of the estrogen receptor α is relatively low in the nucleus of the granulosa cells; instead, estrogen receptor β (ERβ/ESR2) is thought to play the predominant role of activating intracellular signaling pathways that promote cell proliferation and survival in the ovary. There are also many splice variants of the estrogen receptor β, mainly ERβ1, ERβ2 or also sometimes called ERβcx, ERβ3, ERβ4, and ERβ5. Their role in the ovary is still unknown. Most of the splice variants lack the ligand-binding domain. However, the DNA-binding domain is still preserved, so they could be crucial transcriptional factors of different pathways in the ovary. Recent ChIP-sequencing results from a study showed that two nuclear receptors, ERβ and the liver receptor homolog 1 (LRH-1/NR5A2), share many chromatin binding sites in normal mouse ovary. This finding suggests that these two transcriptional factors may interact with each other physically or indirectly. ChIP-qPCR was used to confirm chromatin binding of ERβ’s splice variants in the human granulosa tumor cell line COV434. Dual luciferase assay was used to investigate if ERβ affects the transactivation activity of LRH-1. Co-IP was performed to investigate if ERβ and LRH-1 physically interact. We could confirm that LRP6 is a target gene of ERβ splice variants ERβcx, ERβ4 and ERβ5. We also demonstrated that these ERβ splice variants bind directly to DNA which has not been shown before. The DNA samples must be sent for further sequencing to confirm more target genes of the ERβ splice variants. The results from this study indicate that ERβ represses the transactivation activity of LRH-1, how ERβ acts as a repressor is still unknown. We could not show whether ERβ and LRH-1 physically interact in this study.

L'ovulation est le processus de rupture du follicule ovarien qui conduit à la libération d'un ovocyte dans l'oviducte pour la fécondation. Le processus ovulatoire est initié par un pic de LH qui conduit à l'activation du récepteur EGF (EGFR) dans les cellules de la granulosa (CG) qui stimule par conséquent l'expression de plusieurs gènes pré-ovulatoires essentiels. Environ 15% de l'infertilité chez les vaches est due à la présence de kystes ovariens qui se développent normalement à partir de follicules pré-ovulatoires qui n'ovulent pas. Chez les femmes et les rongeurs, alors que l'activité physiologique des effecteurs de signalisation Hippo semble essentielle pour la signalisation de l'EGF dans les CG, leur dérégulation peut jouer un rôle important dans les troubles kystiques anovulatoires. À notre connaissance, aucune étude n'a rapporté les rôles physiologiques ou physiopathologiques des effecteurs de la voie Hippo liés à l'ovulation ou à la formation de kystes ovariens chez les bovins. Les principaux objectifs de la présente thèse étaient donc (1) d'élucider l'importance de l'activité physiologique des effecteurs de signalisation Hippo dans la cascade pré-ovulatoire et dans l'ovulation chez les ruminants, et (2) de déterminer si la dérégulation de YAP dans le CG bovin est impliquée dans la pathogenèse de la maladie kystique des ovaires (MKO). Les résultats sont séparés en 2 articles. Dans le premier article, nous avons d'abord utilisé un système de culture de CG préovulatoire bien défini pour montrer que la signalisation YAP est essentielle pour l'expression de l'EGFR et des gènes cibles en aval EREG, EGR1 et TNFAIP6. Plus important encore, en utilisant un système d'injection folliculaire guidée par ultrasons, nous avons également montré que l'inhibiteur classique de la signalisation Hippo, la vertéporfine, inhibe l'ovulation induite par la GnRH in vivo chez les bovins. Dans le deuxième article, nous avons utilisé un modèle bovin de MKO spontanée pour montrer que l'abondance de l'ARNm et des protéines de YAP est significativement plus élevée dans les cellules de la granulosa et de la thèque (CT) isolées des follicules kystiques (structures folliculaires d'au moins 20 mm de diamètre) par rapport aux types de cellules respectifs isolés à partir de grands follicules non kystiques (≥12 mm). De plus, le schéma de phosphorylation de YAP et l'abondance accrue d'ARNm codant pour les gènes cibles YAP-TEAD classiques suggèrent que l'activité transcriptionnelle de YAP est augmentée dans les CG des kystes folliculaires par rapport aux grands follicules non kystiques. Ensemble, ces résultats montrent pour la première fois que la voie Hippo est essentielle à la cascade ovulatoire chez les bovins et que sa dérégulation peut jouer un rôle clé dans la pathogenèse de la maladie kystique des ovaires chez cette espèce. / Ovulation is the process of rupture of the ovarian follicle, which leads to the release of an oocyte into the oviduct for fertilization. The ovulatory process is initiated by a surge of LH that leads to the activation of the EGF receptor (EGFR) in granulosa cells (GC) which consequently stimulates the expression of several essential ovulatory genes. About 15% of infertility in cows is due to the presence of ovarian cysts that generally develop from pre-ovulatory follicles that fail to ovulate. In women and rodents, in addition to the essential physiological activity of Hippo signalling effectors for the EGF signalling in GC, their deregulation also plays an important role in anovulatory cystic disorders. To the best of our knowledge, no studies report physiological or pathophysiological roles of Hippo pathway effectors related to ovulation or ovarian cyst formation in cattle. The main objectives of the present thesis were, therefore, (1) to elucidate the importance of the physiological activity of Hippo signaling effectors in the pre-ovulatory cascade and in ovulation in ruminants and (2) to determine whether dysregulation of YAP in bovine GC is implicated in the pathogenesis of the cystic ovarian disease (COD). The results are separated into 2 articles. In the first article, we employed a well-defined preovulatory GC culture system to show that YAP signaling is essential for expressing EGFR and the downstream target genes EREG, EGR1 and TNFAIP6. Most importantly, using an ultrasound-guided follicular injection system, we have also shown that the classic Hippo signaling inhibitor verteporfin inhibits GnRH-induced ovulation in vivo in cattle. In the second article, we employed a bovine model of spontaneous COD to show that YAP mRNA and protein abundance are significantly higher in granulosa and theca (TC) cells isolated from cystic follicles (follicular structures of at least 20 mm in diameter) compared to the respective cell types isolated from large non-cystic follicles (≥12 mm). Moreover, the phosphorylation pattern of YAP and the increased abundance of mRNA encoding classical YAP-TEAD target genes suggest that the transcriptional activity of YAP is increased in GC of follicular cysts compared to large non-cystic follicles. Together, these results show for the first time that the Hippo pathway is critical to the ovulatory cascade in cattle and its dysregulation may play a key role in the pathogenesis of cystic ovarian disease in this species.

Au cours des dernières années, une sélection génétique importante a été faite pour améliorer la production de lait des bovins, ceci au détriment des performances reproductives. Cette diminution de performance n’a cependant pas été rapportée chez la génisse présentant un même potentiel génétique. Cette immense production de lait et les changements métaboliques qui l’accompagnent ont donc un impact négatif sur l’efficacité reproductive des vaches laitières qui subissent un stress métabolique supérieur à celui des génisses. Le but de l’étude était d’acquérir une meilleure connaissance des différences moléculaires et métaboliques entre ces deux groupes d’animaux pour amener à une meilleure compréhension de la pathogenèse de l’infertilité chez la vache laitière. Pour ce faire, les vagues folliculaires de vaches en lactation (30-50 jours en lait; N = 12) et de génisses (N = 10) ont été synchronisées par ablation écho guidée des follicules et par traitement hormonal avec injection de prostaglandine et insertion d’un implant de progestérone. L’aspiration du liquide folliculaire et des cellules de la granulosa du follicule dominant a été faite au jour 6. Les paramètres métaboliques mesurés chez les animaux à partir de prises de sang, faites au jour 6, confirment un plus grand stress métabolique chez la vache, les niveaux de BHBA, acides biliaires et cholestérol étant plus élevés et le niveau de glucose plus bas chez celles-ci. Un total de six échantillons a été utilisé pour le séquençage d’ARN et des analyses bio-informatiques ont été effectuées. Plusieurs gènes et voies de signalisation ont présenté des différences entre les deux groupes d’animaux incluant le cycle cellulaire et la production d’hormones. Une confirmation des résultats par PCR en temps réel a été faite, mais la grande variation intragroupe a nui à l’obtention de résultats significatifs. Conjointement, une culture primaire de cellules de la granulosa a été réalisée pour évaluer l’effet des acides biliaires sur la stéroïdogenèse suite à la détection d’une plus grande quantité de ceux-ci chez la vache laitière. La présence d’acide biliaire dans la culture cellulaire cause une diminution de l’accumulation d’estradiol ainsi que de l’expression des gènes CYP19A1 et CYP11A1. Les résultats présentés dans ce mémoire indiquent une différence potentielle au niveau métabolique et moléculaire des follicules dominants entre la vache laitière et la génisse pouvant avoir une responsabilité dans la diminution de l’efficacité reproductive observée chez la vache laitière. / Over the last fifty or more years, genetic selection has been employed to improve milk production in dairy cattle. This selection was made at the expense of reproductive performance. The observed decrease in fertility does not occur in heifers with the same genetic merit. The enormous milk production and the metabolic challenge that accompany it have a negative impact on the reproductive efficiency due to the metabolic stress of lactation. The purpose of the study was to gain a better knowledge of the molecular and metabolic difference between the two groups of animals in order to better understand the pathogenesis of infertility in dairy cows. To do this, the follicular wave of twelve lactating cows (30-50 days in milk; N = 12) and ten heifers (N = 10) were synchronized by ultrasound guided follicle ablation and by hormonal treatment with injection of prostaglandin-F2α and insertion of a progesterone implant. Follicular fluid and granulosa cells of the dominant follicle were aspirated on day 6. The metabolic indicators BHBA, total bile acids, cholesterol and glucose, were measured in the animals from the blood samples also taken on day 6 confirming greater metabolic stress in the cows when compared to the heifers. A total of six samples were used for RNA sequencing and bioinformatics analyses were performed. Several genes and signaling and cellular function pathways were shown to differ between the two groups of animals, including the cell cycle signaling pathway and hormone production pathway. A confirmation of the results by real-time PCR was undertaken, but the great intragroup variation obviated significant results. In the second set of experiments, primary culture of granulosa cells was conducted to evaluate the effect of bile acids on steroidogenesis to further explore the larger amount of the bile acids in the dairy cows when compared to heifers. The results demonstrate a difference in the metabolic status of the animals; BHBA, total bile acids and cholesterol being higher and glucose being lower in the dairy cow relative to the heifer. Presence of bile acids in the granulosa cell culture caused a decrease in expression of CYP19A1, CYP11A1 and estradiol accumulation. The differences at the metabolic and molecular level of the dominant follicles between dairy cows and heifers may be implicated in the reduced reproductive efficiency of the dairy cows.

No description available.