Made available in DSpace on 2014-06-11T19:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-06Bitstream added on 2014-06-13T20:42:44Z : No. of bitstreams: 1 caixeta_es_dr_botib.pdf: 1185802 bytes, checksum: ef647e66df4e5f5d7f8ca9a758116412 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O oócito participa ativamente dos mecanismos reguladores da maturação do complexo cumulus-oócito (COC) via secreção de fatores parácrinos. A proteína morfogênica óssea 15 (BMP15) e o fator de crescimento e diferenciação 9 (GDF9) têm concentrado a maior parte da atenção direcionada aos fatores secretados pelo oócito (FSO) e têm sido associados com a melhora na competência para o desenvolvimento do COC. Em adição, recentemente, detectamos a expressão de fatores de crescimento fibroblástico (FGFs) no oócito e seus receptores nas células do cumulus (FGF10 e seus receptores FGFR1B e 2B; FGF8 e 17 e seus receptores FGFR2C e 3C), sugerindo o envolvimento do sistema FGF na regulação da diferenciação das células do cumulus. O presente trabalho investigou a regulação da expressão do RNAm de FSOs (BMP15, GDF9, FGF8, FGF10 e FGF17) e seus receptores, bem como de membros da família de fatores de crescimento epidermal (EGF)-like [ampiregulina (AREG), epiregulina (EREG) e betacelulina (BTC)] durante a maturação in vitro (MIV) bovina estimulada pelo FSH. O FSH estimulou a expressão do FGFR2C, FGFR3C, FGFR1B, ALK6, AREG e EREG nas células do cumulus durante a MIV. A expressão do RNAm do FGF8 e FGF17, mas não da BMP15, GDF9 e FGF10 diminuiu no oócito durante a MIV. Em adição foram investigadas especificamente as ações da BMP15 e do FGF10 sobre a expansão do cumulus e expressão gênica de membros da família desintegrina e metaloproteinases (ADAM10 e ADAM17), membros da família dos fatores EGF-like (AREG, EREG e BTC) e de genes sabidamente envolvidos no controle da expansão do cumulus [ciclooxigenase 2 (COX2), hialurona sintetase 2 (HAS2), proteína indutora do fator de necrose tumoral 6 (TSG6) e pentraxina 3 (PTX3)]. A BMP15 e o FGF10 aumentaram a porcentagem de COCs completamente... / The oocyte actively participates in the regulatory mechanisms of cumulus-oocyte complex (COC) maturation via secretion of paracrine factors. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) have concentrated most of the attention directed to oocyte secreted factors (OSFs) and have been shown to enhance developmental competence of the COC. In addition, fibroblast growth factors (FGFs) have also been recognized as important OSFs. Recently, we detected the expression of FGFs in the oocytes and their receptors in cumulus cells (FGF10 and its receptors FGFR1B and 2B; FGF8 and 17 and their receptors FGFR2C and 3C), suggesting the involvement of the FGF system in the regulation of cumulus cells differentiation. The present study investigated the mRNA expression pattern for FSOs (BMP15, GDF9, FGF8, FGF10 and FGF17) and their receptors, as well as of epidermal growth factor (EGF)-like family members [ampiregulina (AREG), epiregulina (EREG) and betacelulina (BTC)] during bovine COC in vitro maturation (IVM) stimulated by FSH. The FSH stimulated mRNA expression of FGFR2C, FGFR3C, FGFR1B, ALK6, AREG and EREG in cumulus cells during IVM. Messenger RNA expression of FGF8 and FGF17, but not of BMP15, GDF9 and FGF10, decreased in the oocyte during IVM. In addition were specifically investigated the actions of BMP15 and FGF10 on cumulus expansion and gene expression of disintegrin and metalloproteinase family members (ADAM10 and ADAM17), of EGF-like family members (AREG, EREG and BTC) and the major expansion-inducing genes genes [cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3), tumor necrosis factor-stimulated gene-6 protein (TSG6)]. BMP15 and FGF10 increased the percentage of fully expanded cumulus-oocyte... (Complete abstract click electronic access below)

Orientador: José Buratini Junior / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: José Antonio Visintin / Banca: Mario Binelli / Banca: Marcelo Marcondes Seneda / Resumo: O oócito participa ativamente dos mecanismos reguladores da maturação do complexo cumulus-oócito (COC) via secreção de fatores parácrinos. A proteína morfogênica óssea 15 (BMP15) e o fator de crescimento e diferenciação 9 (GDF9) têm concentrado a maior parte da atenção direcionada aos fatores secretados pelo oócito (FSO) e têm sido associados com a melhora na competência para o desenvolvimento do COC. Em adição, recentemente, detectamos a expressão de fatores de crescimento fibroblástico (FGFs) no oócito e seus receptores nas células do cumulus (FGF10 e seus receptores FGFR1B e 2B; FGF8 e 17 e seus receptores FGFR2C e 3C), sugerindo o envolvimento do sistema FGF na regulação da diferenciação das células do cumulus. O presente trabalho investigou a regulação da expressão do RNAm de FSOs (BMP15, GDF9, FGF8, FGF10 e FGF17) e seus receptores, bem como de membros da família de fatores de crescimento epidermal (EGF)-like [ampiregulina (AREG), epiregulina (EREG) e betacelulina (BTC)] durante a maturação in vitro (MIV) bovina estimulada pelo FSH. O FSH estimulou a expressão do FGFR2C, FGFR3C, FGFR1B, ALK6, AREG e EREG nas células do cumulus durante a MIV. A expressão do RNAm do FGF8 e FGF17, mas não da BMP15, GDF9 e FGF10 diminuiu no oócito durante a MIV. Em adição foram investigadas especificamente as ações da BMP15 e do FGF10 sobre a expansão do cumulus e expressão gênica de membros da família desintegrina e metaloproteinases (ADAM10 e ADAM17), membros da família dos fatores EGF-like (AREG, EREG e BTC) e de genes sabidamente envolvidos no controle da expansão do cumulus [ciclooxigenase 2 (COX2), hialurona sintetase 2 (HAS2), proteína indutora do fator de necrose tumoral 6 (TSG6) e pentraxina 3 (PTX3)]. A BMP15 e o FGF10 aumentaram a porcentagem de COCs completamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The oocyte actively participates in the regulatory mechanisms of cumulus-oocyte complex (COC) maturation via secretion of paracrine factors. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) have concentrated most of the attention directed to oocyte secreted factors (OSFs) and have been shown to enhance developmental competence of the COC. In addition, fibroblast growth factors (FGFs) have also been recognized as important OSFs. Recently, we detected the expression of FGFs in the oocytes and their receptors in cumulus cells (FGF10 and its receptors FGFR1B and 2B; FGF8 and 17 and their receptors FGFR2C and 3C), suggesting the involvement of the FGF system in the regulation of cumulus cells differentiation. The present study investigated the mRNA expression pattern for FSOs (BMP15, GDF9, FGF8, FGF10 and FGF17) and their receptors, as well as of epidermal growth factor (EGF)-like family members [ampiregulina (AREG), epiregulina (EREG) and betacelulina (BTC)] during bovine COC in vitro maturation (IVM) stimulated by FSH. The FSH stimulated mRNA expression of FGFR2C, FGFR3C, FGFR1B, ALK6, AREG and EREG in cumulus cells during IVM. Messenger RNA expression of FGF8 and FGF17, but not of BMP15, GDF9 and FGF10, decreased in the oocyte during IVM. In addition were specifically investigated the actions of BMP15 and FGF10 on cumulus expansion and gene expression of disintegrin and metalloproteinase family members (ADAM10 and ADAM17), of EGF-like family members (AREG, EREG and BTC) and the major expansion-inducing genes genes [cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3), tumor necrosis factor-stimulated gene-6 protein (TSG6)]. BMP15 and FGF10 increased the percentage of fully expanded cumulus-oocyte... (Complete abstract click electronic access below) / Doutor

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

The gonadotropin-induced resumption of meiosis and cumulus expansion in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like factors, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. In vitro, the EGF-like peptides are also produced in cumulus cells upon stimulation by FSH. Both FSH and the EGF-like peptides stimulate resumption of meiosis and cumulus expansion in vitro via activation of a broad signaling network in cumulus cells. To define signaling pathways that drive FSH- and AREG-induced cumulus expansion and meiotic resumption, in vitro cultured pig cumulus-oocyte complexes (COCs) were treated with specific protein kinase inhibitors. The results document that FSH-stimulated, but not the AREG-stimulated resumption of meiosis, depends on the PKA and MAPK14 activities; both modes of stimulation require activation of EGFR and MAPK3/1. To characterize the effects of FSH and EGF-like peptides on gene expression in cumulus cells, transcriptomes of cumulus cells were analysed using microarray approach. Both FSH and AREG+EREG increased the expression of genes associated with regulation of cell proliferation, blood coagulation and extracellular matrix remodeling. In contrast to AREG+EREG, FSH also increased the expression of genes coding...

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.

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.