Chez la vache Prim’Holstein, un QTL de fertilité a été localisé sur le chromosome 3, et deux haplotypes ont été déterminés : « Fertil+ » et « Fertil- ». Les « Fertil- » ont un plus fort taux d’échec de gestation précoce. Le 1er objectif de cette thèse était de déterminer si un dysfonctionnement des complexes ovocyte-cumulus (COC) pouvait expliquer l’échec précoce de gestation chez les « Fertil- ». L’analyse révèle un retard de maturation, une dérégulation de gènes appartenant aux voies des prostaglandines et des MAPK, et à la famille des Tribbles dans les CC ou l’ovocyte des « Fertil- ». Trois gènes composent la famille des Tribbles chez le bovin: TRIB1, TRIB2 et TRIB3, leur fonction est indéterminée dans le COC. Le 2nd objectif était de caractériser le patron d’expression des Tribbles et leur(s) fonction(s) au sein du COC bovin. Les Tribbles joueraient un rôle dans le métabolisme lipidique et l’inflammation au niveau folliculaire. Le métabolisme lipidique au sein du COC bovin étant peu caractérisé, le 3ème objectif était d’appréhender les profils des gènes du métabolisme des acides gras (AG) dans les CC au cours de la maturation. Ainsi, les CC expriment les gènes lipotytiques et lipogéniques. Enfin, la β-oxydation des AG est une fonction primordiale pour la maturation ovocytaire. / In Prim’Holstein cow, a fertility QTL was localized on chromosome 3 and two haplotypes were determined: « Fertil+ » and « Fertil- ». « Fertil- » have a higher early pregnancy failure rate. The 1st objective of the thesis was to define if complex oocyte-cumulus COC dysfunction could explain the early pregnancy failure in « Fertil- ». Analysis highlighted a maturation delay, a dysregulation of genes involved in prostaglandin and MAPK pathway along with one member of the Tribbles family in « Fertil- » CC or oocyte. In bovine, the Tribbles family is composed of three genes: TRIB1, TRIB2 and TRIB3, their function is unknown within the COC. The 2nd objective was to characterize the Tribbles expression pattern and their function in the bovine COC. The tribbles might play a role in lipid metabolism and in inflammation at the follicular level. Lipid metabolism within bovine COC is poorly understood, thus the third objective was to apprehend fatty acid (FA) genes pattern in CC during maturation. Thus, CC express lipolytic and lipogenic genes. Lastly, FA β oxidation is found to be important for oocyte maturation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The process of meiotic resumption in oocytes, arrested since fetal life, and the expansion of compact layers of cumulus cells is triggered by intrafollicular mediators stimulated by LH. These events are extremely complex. In mice, among system components of natriuretic peptides (NP), only the C-type NP (CNP) has a role to inhibit the resumption of meiosis. However, little is know about the function of NPs on resumption of meiosis, nuclear maturation and cumulus expansion in monovular species. The aim of this study was to characterize the natriuretic peptide system, studing its role in the resumption of meiosis and cumulus expansion. We also proposed a new model to study cumulus expansion. Initially, we detected the presence of mRNA for the ANP, CNP, natriuretic peptide receptor 1 (NPR-1), NPR-2 and NPR-3 in the cumulus cells and NPR-2 mRNA in the oocyte. Using an in vitro model, in which the oocytes are arrested in germinal vesicle (VG) by the action of forskolin (100 μM), we demonstrated that ANP, BNP and CNP, alone or in combination, induce resumption of meiosis after 12 h of maturation. In another experiments, we observed that the concentration of 100 μM forskolin inhibited cumulus expansion stimulated by FSH for12 h, which was reversed by adding ANP, BNP and CNP in the COC culture system. Thus, we demonstrated for the first time the localization of mRNA for the NP system in COCs. Furthermore, we found that the ANP, BNP and CNP are likely mediators of LH to induce meiotic resumption and cumulus expansion in monovuluar species, using the bovine as the animal model. / O processo de retomada da meiose no oócito, bloqueada desde a vida fetal, e a expansão de compactas camadas de células do cumulus que o envolvem é desencadeado por mediadores intrafoliculares estimulados pelo LH, sendo eventos extremamente complexos. Em camundongos, dentre os componentes do sistema peptídeos natriuréticos (NP) somente o NP tipo C (CNP) apresenta função, bloqueando a retomada da meiose. Entretanto, em espécie monovular, o conhecimento sobre a ação dos NP, na maturação nuclear de oócitos e expansão do cumulus, é extremamente escasso. O objetivo do presente estudo foi caracterizar o sistema peptídeo natriurético, demonstrar sua função na retomada da meiose e expansão do cumulus, além de propor um novo modelo para estudo da expansão do cumulus. Inicialmente, demonstramos a presença de RNAm para ANP, CNP, receptor peptídeo natriurético 1 (NPR-1), NPR-2 e NPR-3 nas células do cumulus, sendo que no oócito somente foi detectado RNAm do NPR-2. Utilizando um modelo in vitro, no qual os oócitos permanecem bloqueados em vesícula germitava (VG) por ação do forskolin (100 μM), demonstramos que os ANP, BNP e CNP, isoladamente ou em associação, induzem o reinício da meiose após 12 h de maturação. Em outros experimentos, observamos que a concentração de 100 μM de forskolin inibiu, por 12 h, a expansão das células do cumulus estimulada por FSH e que o ANP, BNP e CNP revertem o efeito inibitório do forskolin sobre a expansão do cumulus. Dessa forma, demonstramos pela primeira vez a localização de RNAm para o sistema NP em CCOs. Além disso, foi demonstrado que em espécie monovuluar, utilizando o bovino como modelo animal, os peptídeos natriuréticos (ANP, BNP e CNP) apresentam função de mediadores intrafoliculares do LH, na qual estimulam a retomada da meiose e expansão do cumulus.

Made available in DSpace on 2015-03-26T13:47:14Z (GMT). No. of bitstreams: 1 texto completo.pdf: 929339 bytes, checksum: fddfad690cd5f53dd8bcd64c3c0e07a5 (MD5) Previous issue date: 2012-12-20 / In vitro production of pig embryos has yet taken unsatisfactory results due to low production blastocyst, the percentage of embryos produced from oocytes matured in vitro is lower than those matured in vivo. Several factors affect the success of maturation, however, the oocyte competence is one of the most important factors. Only competent oocytes are able to undergo fertilization and have normal embryonic development. The oocyte competence can be evaluated from various parameters such as morphology of the oocyte, follicle size, stained with cresyl brilliant blue (BCB), the time of cleavage and age of the donor. In recent years these parameters have been used for studies of gene expression associated with the task. This study aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 and using the constitutive gene GAPDH as endogenous control in immature oocytes of different morphological classes which are: 1, 2, 3 and 4 to elucidate the mechanisms required for oocyte acquires competence and provide important molecular markers linked to the development capability. Oocytes total RNA was extracted and used as a template for synthesis of cDNA first tape. The results of gene expression were analyzed using a mixed model, considering the gene expression data dependent and independent variables oocyte classes. Genes BMP15, RYBP ZAR1 and showed similar expression oocyte in categories 1, 2 and 3, only 4 categories differ in their expression (P <0.05). The MATER gene was expressed similarly in all categories oocyte studied (P> 0.05). The qPCR technique, in this work, was efficient to detect these transcripts in oocytes of different categories. However, to better understand the involvement of these transcripts in the acquisition of oocyte competence are needed most studies evaluating different morphological classes of oocytes at different stages of development and implication of other genes involved in oocyte competence. / A produção in vitro de embriões suínos ainda tem tidos resultados insatisfatórios devido a baixa de produção de blastocisto, a porcentagem de embriões produzidos de ovócitos maturados in vitro é inferior àqueles maturados in vivo. Diversos fatores interferem no sucesso da maturação, entretanto, a competência ovocitária é um dos fatores mais importantes. Somente ovócitos competentes são capazes de sofrer a fecundação e ter desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada a partir de vários parâmetros, tais como morfologia do ovócito, tamanho do folículo, coloração com brilliant cresyl blue (BCB), momento da clivagem e idade da doadora. Nos últimos anos esses parâmetros têm sido utilizados para os estudos da expressão dos genes associados com a competência. Este trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER, ZAR1 e como controle endógeno o gene constitutivo GAPDH em ovócitos imaturos de diferentes classes morfológicas sendo elas: 1, 2, 3 e 4. Como finalidade elucidar os mecanismos necessários para que o ovócito adquira competência e proporcionar importantes marcadores moleculares relacionados com a capacidade de desenvolvimento. O RNA total ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando um modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3, somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de qPCR, no presente trabalho, foi eficiente para detecção destes transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento e implicação de outros genes envolvidos com a competência ovocitária.

Orientador: Anthony César de Souza Castilho / Resumo: A fim de mimetizar melhores condições do sistema in vivo, um meio de cultura mais proximo à composição do fluido do oviduto foi criado, denominado fluido ovidutal sintético (SOF), este foi formulado com base na análise bioquímica do oviduto das ovelhas. In vivo os momentos finais de maturação oocitária ocorrem em contato com o FO, já in vitro a maturação oocitária é realizada com protocolos de maturação 24h sem nenhuma alteração no meio de maturação. Portanto, o presente estudo investigou o impacto do meio de cultivo SOF durante as últimas quatro horas da maturação in vitro (MIV) e seus efeitos sobre a progressão meiótica e taxa de produção embrionária. Para tanto, os CCOs foram recuperados de ovário de vacas abatidas e maturados in vitro, nos seguintes grupos: SFB/ SFB; SFB/ SFB+SOF; BSA/BSA; BSA/BSA+SOF, todos submetidos há uma troca total de meio nas últimas quatro horas de maturação, adicionando o meio SOF de acordo com os grupos citados anteriormante. Os CCOs maturados foram fertilizados e cultivados em atmosfera e umidade controlada até o estágio de blastocisto (7 e 8 dias após a fertilização). A análise estatística foi realizada transformando os dados em arcoseno e as médias foram comparadas pelo teste T usando JMP (software JMP, SAS Institute Cary, NC). Diferenças significativas foram consideradas quando p≤ 0,05. O resultado obtido mostrou que o tratamento com SOF não tem efeito na progressão da meiose (p= 0,5944). Entretanto, quando analisamos as taxas embrionárias e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The oviduct and its secretion, the ovidutal fluid (OF), promote an adequate environment for the final maturation of the oocyte, sperm training, fertilization, transport and initial development of the embryo. In this context, in order to mimic better conditions of in vivo mechanisms, a culture medium similar to the composition of the oviduct fluid, called synthetic ovidutal fluid (SOF), was formulated based on biochemical analysis of the oviduct of sheep. Specifically, regarding the importance of OF for the final maturation of the oocyte in cattle, only the last four hours of oocyte maturation occur in the oviduct, which therefore pemits a new strategy of oocyte maturation in vitro (MIV) in the bovine species. Therefore, the present study aimed to investigate the impact of SOF culture medium during the last four hours of IVM and its effects on the meiotic progression and embryonic production rate. Therefore, the COCs were recovered from ovaries of slaughtered and matured cows in vitro, in base medium (composed of 199 MCT with Earles salts supplemented with pyruvate, amicacin, BSA, follicle stimulating hormone and luteinizing hormone, and another group in 199 MCT medium with Earles salts supplemented with 10% fetal bovine serum (FBS), pyruvate, both in moist atmosphere at 5% CO2 for 20 hours (4 replicates with 20 COCs/group). In the last four hours of maturation, the medium was completely changed by adding the SOF medium. Matured COCs were fertilized and cultivated in a control... (Complete abstract click electronic access below) / Mestre

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux. / The nuclear receptor Nr5a2 is expressed in the ovary, exclusively in granulosa and luteal cells. Conditional disruption of Nr5a2 in granulosa cells beginning with primary follicles by means of Nr5a2-floxed and Amhr2-Cre mice (Nr5a2f/fAmhr2Cre/+) results in failure in cumulus expansion, ovulation and luteinization. We hypothesize that Nr5a2 regulates intercellular connections in ovarian follicles through connexin 43 (Cx43), a gap-junctional protein related to cumulus expansion. The first objective of this study was to determine whether the lack of cumulus expansion in Amhr2Cre-cKO mice is related to the absence of normal cell-to-cell communication in cumulus/granulosa cells of preovulatory follicles. To address this, immature ovaries of Amhr2Cre-cKO and non-transgenic littermates mice were collected (n=3) after superstimulation to induce follicle development and ovulation. Using RT-PCR, the Cx43 mRNA levels were shown to be downregulated prior to and at the time of the ovulatory stimulus (0 h and 2 h) in the Amhr2Cre-cKO group (P<0.01), which may lead to the failure in the acquisition of oocyte developmental competence. On the other hand, by the time of ovulation (12 h), mRNA levels of Cx43 are upregulated in Amhr2Cre-cKO group, which may prevent the cumulus cells to detach one from another. We conclude that Cx43 is one of the downstream genes under Nr5a2 control and its dysregulation can be one reason for the defect in cumulus expansion in Amhr2Cre-cKO females. To examine the role of Nr5a2 in ovulation and luteinization in different stages of the follicle maturation, we hypothesized that Nr5a2 modulates the events leading to ovulation in a temporal sequence. By crossing Nr5a2-floxed and Cyp19-Cre mice (Nr5a2f/fCyp19Cre/+), Nr5a2 was disrupted in granulosa cells of antral and preovulatory follicles. No litters were born to Cyp19Cre-cKO females (n=4) during a 6 months breeding trial. Cyp19Cre-cKO enabled the development of a luteal-like structure, and 1-year-old females presented hemorrhagic follicular cysts and hypertrophic ovarian surface epithelium. Both knockout models display lack of cumulus expansion and ovulation. We conclude that Nr5a2 differentially regulates folliculogenesis and ovulation in granulosa cells of small and antral follicles.

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune. / The nuclear receptor Nr5a2, also known as liver receptor homolog-1 (Lrh-1), is expressed in the mouse ovary, exclusively in granulosa and luteal cells. Granulosa-specific disruption of Nr5a2 in mice from primary follicles onward in the follicle development trajectory has shown that Nr5a2 is a key regulator of ovulation and female fertility. We hypothesized that Nr5a2 modulates peri- and post-ovulatory events in a temporal sequence during folliculogenesis. To examine the role of Nr5a2 in ovulation and luteinization at different stages of the follicular development, we generated two Nr5a2 granulosa-specific knockout mice models: 1) Nr5a2Amhr2-/-, with Nr5a2 depletion from primary follicles forward; and 2) Nr5a2Cyp19-/-, with Nr5a2 depletion from the antral stage of development forward. The lack of Nr5a2 beginning in antral follicles resulted in infertility in Nr5a2Cyp19-/- females, with ovaries displaying non-functional luteal-like structures, synthesizing reduced progesterone levels and failing in supporting pseudopregnancy. Progesterone synthesis was affected by the lack of Nr5a2 through the downregulation of the cholesterol transport-related genes, Scarb1, StAR and Ldlr, as shown by qPCR. The cumulus-oocyte complexes of superstimulated Nr5a2Cyp19-/- immature females underwent expansion in vivo, but ovulation was disrupted, likely due to the downregulation of the progesterone receptor (Pgr) gene. An in vitro cumulus expansion assay showed defective cumulus expansion in Nr5a2Amhr2-/- associated with a dysregulation in the gap junction alpha-1 (Gja1; Cx43). In vitro cumulus expansion in Nr5a2Cyp19-/- was less affected than in Nr5a2Amhr2-/- cumulus-oocyte complexes. Data from qPCR showed a downregulation in the gene expression of Areg, Ereg, Btc and Tnfaip6 in both knockout ovarian cells at 2 h and 4 h post hCG. We found that 85% of the oocytes in both mutant genotypes can undergo germinal vesicle breakdown, confirming their capability to mature in vivo. Intracytoplasmic sperm injection (ICSI) showed the oocytes in both mutant models to be fertilizable and 70% of the resulting embryos proceeded to a blastocyst stage, independent of the genotype. In conclusion, Nr5a2 regulates female fertility along the entire process of the follicular development. Nr5a2 is shown to be essential for luteinization and its disruption in ovarian somatic cells does not compromise oocyte fertilizability. In overview, we provided a novel and comprehensive investigation, using multiple models and techniques to determine the mechanisms by which Nr5a2 regulates the important processes of cumulus expansion, ovulation and formation of the corpus luteum.

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux. / The nuclear receptor Nr5a2 is expressed in the ovary, exclusively in granulosa and luteal cells. Conditional disruption of Nr5a2 in granulosa cells beginning with primary follicles by means of Nr5a2-floxed and Amhr2-Cre mice (Nr5a2f/fAmhr2Cre/+) results in failure in cumulus expansion, ovulation and luteinization. We hypothesize that Nr5a2 regulates intercellular connections in ovarian follicles through connexin 43 (Cx43), a gap-junctional protein related to cumulus expansion. The first objective of this study was to determine whether the lack of cumulus expansion in Amhr2Cre-cKO mice is related to the absence of normal cell-to-cell communication in cumulus/granulosa cells of preovulatory follicles. To address this, immature ovaries of Amhr2Cre-cKO and non-transgenic littermates mice were collected (n=3) after superstimulation to induce follicle development and ovulation. Using RT-PCR, the Cx43 mRNA levels were shown to be downregulated prior to and at the time of the ovulatory stimulus (0 h and 2 h) in the Amhr2Cre-cKO group (P<0.01), which may lead to the failure in the acquisition of oocyte developmental competence. On the other hand, by the time of ovulation (12 h), mRNA levels of Cx43 are upregulated in Amhr2Cre-cKO group, which may prevent the cumulus cells to detach one from another. We conclude that Cx43 is one of the downstream genes under Nr5a2 control and its dysregulation can be one reason for the defect in cumulus expansion in Amhr2Cre-cKO females. To examine the role of Nr5a2 in ovulation and luteinization in different stages of the follicle maturation, we hypothesized that Nr5a2 modulates the events leading to ovulation in a temporal sequence. By crossing Nr5a2-floxed and Cyp19-Cre mice (Nr5a2f/fCyp19Cre/+), Nr5a2 was disrupted in granulosa cells of antral and preovulatory follicles. No litters were born to Cyp19Cre-cKO females (n=4) during a 6 months breeding trial. Cyp19Cre-cKO enabled the development of a luteal-like structure, and 1-year-old females presented hemorrhagic follicular cysts and hypertrophic ovarian surface epithelium. Both knockout models display lack of cumulus expansion and ovulation. We conclude that Nr5a2 differentially regulates folliculogenesis and ovulation in granulosa cells of small and antral follicles.

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune. / The nuclear receptor Nr5a2, also known as liver receptor homolog-1 (Lrh-1), is expressed in the mouse ovary, exclusively in granulosa and luteal cells. Granulosa-specific disruption of Nr5a2 in mice from primary follicles onward in the follicle development trajectory has shown that Nr5a2 is a key regulator of ovulation and female fertility. We hypothesized that Nr5a2 modulates peri- and post-ovulatory events in a temporal sequence during folliculogenesis. To examine the role of Nr5a2 in ovulation and luteinization at different stages of the follicular development, we generated two Nr5a2 granulosa-specific knockout mice models: 1) Nr5a2Amhr2-/-, with Nr5a2 depletion from primary follicles forward; and 2) Nr5a2Cyp19-/-, with Nr5a2 depletion from the antral stage of development forward. The lack of Nr5a2 beginning in antral follicles resulted in infertility in Nr5a2Cyp19-/- females, with ovaries displaying non-functional luteal-like structures, synthesizing reduced progesterone levels and failing in supporting pseudopregnancy. Progesterone synthesis was affected by the lack of Nr5a2 through the downregulation of the cholesterol transport-related genes, Scarb1, StAR and Ldlr, as shown by qPCR. The cumulus-oocyte complexes of superstimulated Nr5a2Cyp19-/- immature females underwent expansion in vivo, but ovulation was disrupted, likely due to the downregulation of the progesterone receptor (Pgr) gene. An in vitro cumulus expansion assay showed defective cumulus expansion in Nr5a2Amhr2-/- associated with a dysregulation in the gap junction alpha-1 (Gja1; Cx43). In vitro cumulus expansion in Nr5a2Cyp19-/- was less affected than in Nr5a2Amhr2-/- cumulus-oocyte complexes. Data from qPCR showed a downregulation in the gene expression of Areg, Ereg, Btc and Tnfaip6 in both knockout ovarian cells at 2 h and 4 h post hCG. We found that 85% of the oocytes in both mutant genotypes can undergo germinal vesicle breakdown, confirming their capability to mature in vivo. Intracytoplasmic sperm injection (ICSI) showed the oocytes in both mutant models to be fertilizable and 70% of the resulting embryos proceeded to a blastocyst stage, independent of the genotype. In conclusion, Nr5a2 regulates female fertility along the entire process of the follicular development. Nr5a2 is shown to be essential for luteinization and its disruption in ovarian somatic cells does not compromise oocyte fertilizability. In overview, we provided a novel and comprehensive investigation, using multiple models and techniques to determine the mechanisms by which Nr5a2 regulates the important processes of cumulus expansion, ovulation and formation of the corpus luteum.

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

A Síndrome dos Ovários Policísticos (SOP) representa um dos distúrbios endócrinos reprodutivos mais comuns em mulheres em idade reprodutiva. As mulheres com SOP normalmente respondem bem ao tratamento de reprodução assistida (TRAs), mas frequentemente apresentam oócitos de baixa qualidade e capacidade reprodutiva, a qual está correlacionado com a interação do oócito com as células do cumulus. A baixa qualidade oocitária pode estar relacionada a perda de estabilidade genômica do oócito, ou até mesmo das células do cumulus, o que pode levar a uma redução gradativa da fertilidade feminina. Os telômeros e a atividade da telomerase possuem um papel fundamental na manutenção da estabilidade genômica e são considerados importantes marcadores de viabilidade celular, podendo ser um indicativo da qualidade oocitária. O objetivo do estudo foi avaliar o comprimento do telômero e atividade da telomerase nas células do cumulus de mulheres com SOP. Neste estudo prospectivo caso-controle foram incluídas 110 voluntárias, sendo 43 mulheres com SOP e 67 controles no período de Setembro de 2015 a Junho de 2017. Foram avaliados os dados como idade, Índice de massa corporal (IMC), hormônio luteinizante (LH), hormônio folículo estimulante (FSH), globulina de ligação de hormônios sexuais (SHBG), prolactina, estradiol, insulina, testosterona total, androstenediona, índice de androgênio livre (FAI), homocisteína e proteína c-reativa. Foi avaliado o comprimento do telômero nas células do cumulus de oócitos imaturos (CCI), nas células do cumulus de oócitos maduros (CCM), nos oócitos imaturos no estágio de vesícula germinativa (VG), nos oócitos imaturos em metáfase I (MI) e nos leucócitos pelo método quantitativo da reação em cadeia da polimerase (qPCR). A atividade da telomerase das CCI, CCM, dos oócitos VG e MI foram avaliadas pelo Kit TRAPeze® XL. A análise estatística foi determinada pelo teste Mann-Whitney, regressão linear múltipla e correlação de Spearman. Os resultados foram que as variáveis IMC (p=0,001), LH (p=0,015), estradiol (p=0,004), insulina (p=0,002), testosterona (p<0,0001), androstenediona (p=0,001), FAI (p<0,0001) e proteína c-reativa (p=0,003) foram maiores no grupo SOP. FSH (p=0,0002) foi menor no grupo SOP. A prolactina e a homocisteína não diferiram entre os grupos. O comprimento do telômero nas CCI não diferiu entre os grupos SOP e controle (1,60±0,56 vs 1,58±0,33; p=0,649, respectivamente), bem como o comprimento do telômero nas CCM não diferiu entre os grupos SOP e controle (1,61±0,47 vs 1,70±0,43; p=0,378, respectivamente). Entretanto, nos leucócitos o comprimento do telômero foi menor no grupo SOP (p=0,025). A atividade da telomerase nas CCI foi maior no grupo SOP do que no grupo controle (1,62±1,49 vs 0,30±0,42; p=0,003, respectivamente) e a atividade da telomerase nas CCM também foi maior no grupo SOP do que no grupo controle (1,39±1,63 vs 0,55±0,84; p=0,022, respectivamente). O comprimento do telômero e a atividade da telomerase nos oócitos VG e MI não diferiu entre os grupos. Uma correlação positiva foi observada entre a atividade da telomerase e o comprimento do telômero nas CCI no grupo controle (p=0,051). O grupo SOP apresentou uma correlação positiva entre a atividade da telomerase e o comprimento do telômero, porém nas CCM (p=0,048). Foi observada uma correlação positiva do comprimento do telômero entre as células (leucócitos, CCI e CCM) em ambos os grupos. Os dados sugerem que a SOP parece não afetar o comprimento do telômero nas CCI e CCM, apenas nos leucócitos. Por outro lado, uma maior atividade da telomerase nas CCI e CCM pode ser necessária para a manutenção do comprimento telomérico à nível reprodutivo nas mulheres com SOP. / Polycystic Ovarian Syndrome (PCOS) represents one of the most common reproductive endocrine disorders in women of reproductive age. Women with PCOS, despite responding well to Assisted Reproduction Treatments (ART), the oocytes usually have low quality and reproductive capacity, which is correlated to oocyte interaction with cumulus cells. The low oocyte quality may be related to loss of genomic stability of oocytes, or even cumulus cells, and may lead to a reduction of female fertility. The telomere length and telomerase activity play a fundamental role in maintaining genomic stability, which is considered an important molecular marker of cell viability, whose alterations are related to apoptosis and/or senescence, maybe also an indicative of oocyte quality. The aim of the study was to evaluate the telomere length and telomerase activity in cumulus cells of women with PCOS. In this prospective case-control study, 110 volunteers were included, 43 women with PCOS and 67 controls from September 2015 to June 2017. Data were evaluated as age, body mass index (BMI), luteinizing hormone (LH), follicle stimulating hormone (FSH), sex hormone binding globulin (SHBG), prolactin, estradiol, insulin, total testosterone, androstenedione, index of free androgen (FAI), homocysteine and c-reactive protein. Telomere length in cumulus cells from immature (ICC) and mature (MCC) oocytes, leukocytes and immature oocytes in the germinal vesicle stage (VG) and in metaphase I (MI) were evaluated by quantitative real-time polymerase chain reaction (qPCR). Telomerase activity of ICC, MCC, VG and IM oocytes were evaluated by TRAPeze® XL Kit. Statistical analyses were determined by the MannWhitney test, multiple linear regression and Spearman\'s correlation. The results were that the variables BMI (p=.001), LH (p=.015), estradiol (p=.004), insulin (p=.002), testosterone (p<.0001), androstenedione (p=.001), FAI (p<.0001) and c-reactive protein (p=.003) was increased in PCOS group. FSH (p=.0002) was smaller in PCOS group. Prolactin and homocysteine were not different between the groups. The telomeres length in ICC did not differ between PCOS and control groups (1.60±0.56 vs 1.58±0.33; p=.649, respectively). The telomeres length in MCC did not differ between PCOS and control groups (1.61±0.47 vs 1.70±0.43; p=.378, respectively). However, in leukocytes reduced telomeres were observed in the PCOS (p=.025), respectively. The telomerase activity in ICC was higher in the PCOS group than in the control group (1.62±1.49 vs 0.30±0.42; p=.003, respectively) and the telomerase activity in MCC was higher in the PCOS group than in the control group (1.39±1.63 vs 0.55±0.84; p=.022, respectively). The telomere length and telomerase activity in VG and MI oocytes did not differ between groups. A positive correlation between telomerase activity and telomere length in the ICC was observed in control group (p=.051). PCOS also presented a positive correlation between telomerase activity and telomere length in MCC (p=.048). Telomere length of leukocytes, ICC and MCC were a positive correlated in both groups. The data suggest that PCOS does not appear to affect telomere length in ICC and MCC, only in leukocytes. On the other hand, a greater activity of telomerase in CCI and CCM may be necessary for the maintenance of telomere length at the reproductive level in women with PCOS.