As células do cumulus oophorus são firmemente conectadas umas às outras e ao oócito e participam ativamente do amadurecimento oocitário. Essas células apresentam abundância de receptores para o FSH. Os carboidratos da molécula do FSH podem terminar em resíduos de ácido siálico, permitindo que ele exista em numerosas isoformas. As isoformas mais ácidas do FSH contêm mais resíduos de ácido siálico. Os níveis dessas isoformas variam na fase folicular do ciclo menstrual e possuem diferentes capacidades de estímulo ovariano, sendo as menos ácidas mais potentes e de curta meia-vida plasmática. Objetivo: padronizar a técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas e verificar a ação de uma isoforma menos ácida do FSH, a Folitropina Beta (Puregon®) sobre o potencial de membrana dessas células, comparando com a ação de um FSH com menor grau de pureza e mais ácido (FSH ovino). Metodologia: o potencial de membrana foi registrado utilizando células de cumulus oophorus de pacientes encaminhadas ao procedimento de injeção intracitoplasmática de espermatozóides (ICSI). As células do cumulus foram cultivadas em placas de cultura com meio HTF (Life Global® - Guilford, CT USA) acrescido de 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) por 24 a 48 horas. Durante o experimento eletrofisiológico, a placa foi perfundida com o fluxo de 1 mL/min de meio de Hank´s acrescido de Hepes com 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) mantido a 37º C. O registro intracelular foi realizado com microeletrodos preenchidos com KCl 3 M com uma resistência de 15 a 25 MΩ e acoplados a um eletrômetro. A resistência da membrana ao fluxo de cargas foi avaliada com a aplicação de pulsos quadrados de corrente (0,5 nA, 0,5 Hz e 200 ms) através do eletrodo de registro. Os hormônios testados (FSH ovino e Folitropina Beta) foram aplicados topicamente na placa. Para a análise estatística foi utilizado o teste de Anova de duas vias (seguido do pós-teste de Bonferroni) ou o teste exato de Fischer. Resultados: Foi realizada a padronização da técnica de registro eletrofisiológico para as células do cumulus e o potencial de membrana médio em repouso obtido foi de -34,02 mV, com desvio-padrão de ± 6,46 e erro-padrão da média de ± 2,04. A resistência média das membranas ao fluxo de íons foi de 16,5 MΩ, com desvio-padrão de ± 4,03 e erro-padrão da média de ± 1,8. A aplicação do FSH Ovino (1 μM) causou uma lenta despolarização, estatisticamente significativa 180 segundos após a aplicação do hormônio (P<0,01). A aplicação da Folitropina Beta (Puregon® 1 μM) causou uma lenta despolarização, estatisticamente significativa 120 e 180 segundos após a aplicação do hormônio (P<0,001). O padrão de despolarização foi semelhante entre as duas isoformas, sendo o efeito da Folitropina Beta mais imediato que o do FSH Ovino aos 120 segundos. Conclusões: A partir da padronização da técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas, registrou-se o potencial médio de membrana em repouso (-34,02 mV) e a resistência média da membrana ao fluxo de íons (16,5 MΩ). O FSH Ovino (1 μM) e a Folitropina Beta (Puregon®) (1 μM) possuem uma ação despolarizante sobre o potencial de membrana das células do cumulus. Para o FSH Ovino, essa despolarização foi significativa 180 segundos após a aplicação do hormônio e para a Folitropina Beta, a despolarização foi significativa já aos 120 segundos após a aplicação. Há uma semelhança no padrão de despolarização produzida pelo FSH Ovino e pela Folitropina Beta. Um melhor entendimento das diferenças no funcionamento das isoformas do FSH pode proporcionar o desenvolvimento de melhores protocolos de tratamento para as pacientes, com menos efeitos colaterais. Esse conhecimento também pode ser utilizado para o aprimoramento das técnicas de maturação in vitro dos oócitos, que podem poupar as pacientes dos efeitos colaterais e dos altos custos da estimulação exógena com gonadotrofinas. / The cumulus oophorus cells are firmly connected to each other and to the oocyte and they take an important part on the oocyte maturation. These cells present numerous FSH receptors. The carbohydrates present in the FSH molecule may end in sialic acid residues, allowing FSH to exist in various isoforms. The more acidic isoforms present more sialic acid residues in their carbohydrate moieties. These isoforms exhibit fluctuations in the follicular phase of the menstrual cycle and possess different capabilities of ovarian stimulation, with the least acidic isoform having higher potency and lower plasmatic half-life. Objective: The aim of this study was to standardize the intracellular electrophysiology register technique to the human cumulus oophorus cells and to investigate the membrane potential action of a less acidic FSH isoform (Puregon™), comparing with the membrane action using a less purified, more acidic FSH isoform (sheep FSH). Methods: The membrane potential was registered in cumulus oophorus cells from patients assigned to the intracytoplasmatic sperm injection (ICSI) technique. The cumulus cells were cultured in plates with HTF medium for 24 to 48 hours. During the electrophysiological experiment, the plate was superfused with 1 mL/min of Hank`s medium supplemented with Hepes, maintained at 37º C. The intracellular register was performed with microcapillaries filled with 3 M KCl with resistance ranging between 15 and 25 MΩ, and coupled to an electrometer. The membrane resistance to the ion flow was assessed by the application of square current pulses (0,5 nA, 0,5 Hz and 200 ms) through the register electrode. The tested hormones (sheep FSH and Beta Follitropin Puregon™) were applied topically onto the plate. Statistical analysis was performed using the ANOVA test for repeated measures (with Bonferroni post-test) or Fischer´s exact test. Results: the standardization of the electrophysiological register technique for the cumulus cells was successfully achieved and the mean resting membrane potential obtained was -34,02 ± 2,04 mV (SEM). The mean resistance of the membrane to the ion flow was 16,5 ± 1,8 MΩ (SEM). The sheep FSH application (1 μM) led to a slow depolarization, statistically significant 180 seconds after the hormone application (P<0,01). Beta Follitropin administration (1 μM) led to a slow depolarization, statistically significant 120 and 180 seconds after the hormone application (P<0,001). The depolarization pattern was similar between both isoforms, being that Beta Follitropin`s effect was more immediate than sheep FSH`s. Conclusion: With the electrophysiological intracellular register technique standardization to the human cumulus cells, the mean resting membrane potential was registered (-34,02 mV) and the mean membrane resistance to the ion flow was obtained (16,5 MΩ). Sheep FSH (1 μM) and Beta Follitropin (Puregon™) (1 μM) prompted a depolarizing action in the membrane potential of cumulus cells. Sheep FSH depolarization was significant at 180 seconds after the hormone application and Beta Follitropin depolarization was significant at 120 seconds after its application. Both isohormones have similar depolarization patterns. A better understanding of the differences in the action of both FSH isoforms shall make it possible to develop better hormone induction protocols in fertility treatments, with less side effects for the patients. This knowledge will potencially also make it possible to improve the in vitro oocyte maturation techniques, sparing patients of the hormone treatment side effects and of the high financial costs of the exogenous gonadotrophins administration.

Made available in DSpace on 2014-06-11T19:32:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-24Bitstream added on 2014-06-13T20:42:39Z : No. of bitstreams: 1 macharelli_ca_dr_botfm.pdf: 2403268 bytes, checksum: a44fffeaff80c6818630f8724462477f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O tracoma ainda é um importante problema de saúde pública, causando morbidade, deficiência visual e cegueira em diversos países subdesenvolvidos ou em desenvolvimento. Em 2006 foi realizado um estudo transversal nas escolas estaduais na de Bauru, estado de São Paulo, que revelou uma prevalência de 3,7% de tracoma no município. O presente estudo tem como objetivo analisar o comportamento espacial da ocorrência dos casos de tracoma detectados na cidade de Bauru pelo referido estudo, a fim de utilizar as informações apuradas para definir áreas prioritárias para a otimização dos recursos do setor Saúde. Para atingirmos nossos objetivos lançamos mão de uma ferramenta que é o geoprocessamento em saúde ‐ uma das maneiras de se conhecer mais detalhadamente as condições de saúde da população através de mapas que permitem observar a distribuição espacial de situações de risco e dos problemas de saúde. A quantificação dos fenômenos observados pode ser feita de várias formas, sendo a estimativa de Kernel uma delas. Esse procedimento permite filtrar a variabilidade de um conjunto de dados, sem alterar as suas características locais. Os casos de tracoma foram georreferenciados com uso do GPS e depois as coordenadas transformadas em latitudes e longitudes decimais, com o auxílio do TCGeo. As análises espaciais foram feitas com o TerraView, utilizando‐se os pontos dos casos, das escolas, e dados do censo IBGE 2000, contidos nos CD‐Rom: Base de informações por setor censitário, Censo demográfico 2000, Resultados do Universo. Bauru. IBGE, 2002. O protocolo empregado possibilitou que todas as crianças detectadas pelo estudo de Ferraz (2006) fossem visitadas pela equipe executora do presente e georeferenciadas usando aparelho de GPS. As crianças que fizeram parte da amostra apresentavam média de idade de 8.5 anos, eram na sua maioria do sexo masculino e... / Trachoma still is an important issue for public health; it increases morbidity, causing visual deficiency and blindness in several underdeveloped or developing countries. In 2006, a transversal study was carried out among public schools in Bauru, São Paulo state. The study revealed a prevalence of 3.7% of trachoma in the town. This study aims at analyzing the spatial behavior of the cases detected in Bauru by the 2006 research, in order to define areas that should have priority when improving the municipal health system. We used health geoprocessing – which is done by maps that show the spatial distribution of risk situations and health problems, allowing to know details about the population health. The phenomena observed can be quantified in different manners – Kernel estimation is one of them, and the one we used, because it permits to filter the variability of a set of data without altering its local features. Trachoma cases were georeferenced with a GPS and their coordinates were converted with TCGeo into decimal latitudes and longitudes. Spatial analyses were carried out with TerraView, using points of cases and schools, and data from IBGE 2000 census found in the CD‐Rom: Base de informações por setor censitário, Censo demográfico 2000, Resultados do Universo. Bauru. IBGE, 2002 (Information database on each censual sector. Demographic census 2000, Universal Results. Bauru. IBGE, 2002). The procedures allowed our team to visit and georeference all the children detected by Ferraz (2006). The children are mostly males, with a mean age of 8.5 years and presenting uni or bilateral trachoma. Geoprocessing was important for it facilitates the process of spatial localization through Google Earth (2007) – which allowed us to notice a concentration of cases in regions with low income. We could also observe that individuals with low income are not concentrated on the ...(Complete abstract click electronic access below)

As células do cumulus oophorus são firmemente conectadas umas às outras e ao oócito e participam ativamente do amadurecimento oocitário. Essas células apresentam abundância de receptores para o FSH. Os carboidratos da molécula do FSH podem terminar em resíduos de ácido siálico, permitindo que ele exista em numerosas isoformas. As isoformas mais ácidas do FSH contêm mais resíduos de ácido siálico. Os níveis dessas isoformas variam na fase folicular do ciclo menstrual e possuem diferentes capacidades de estímulo ovariano, sendo as menos ácidas mais potentes e de curta meia-vida plasmática. Objetivo: padronizar a técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas e verificar a ação de uma isoforma menos ácida do FSH, a Folitropina Beta (Puregon®) sobre o potencial de membrana dessas células, comparando com a ação de um FSH com menor grau de pureza e mais ácido (FSH ovino). Metodologia: o potencial de membrana foi registrado utilizando células de cumulus oophorus de pacientes encaminhadas ao procedimento de injeção intracitoplasmática de espermatozóides (ICSI). As células do cumulus foram cultivadas em placas de cultura com meio HTF (Life Global® - Guilford, CT USA) acrescido de 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) por 24 a 48 horas. Durante o experimento eletrofisiológico, a placa foi perfundida com o fluxo de 1 mL/min de meio de Hank´s acrescido de Hepes com 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) mantido a 37º C. O registro intracelular foi realizado com microeletrodos preenchidos com KCl 3 M com uma resistência de 15 a 25 MΩ e acoplados a um eletrômetro. A resistência da membrana ao fluxo de cargas foi avaliada com a aplicação de pulsos quadrados de corrente (0,5 nA, 0,5 Hz e 200 ms) através do eletrodo de registro. Os hormônios testados (FSH ovino e Folitropina Beta) foram aplicados topicamente na placa. Para a análise estatística foi utilizado o teste de Anova de duas vias (seguido do pós-teste de Bonferroni) ou o teste exato de Fischer. Resultados: Foi realizada a padronização da técnica de registro eletrofisiológico para as células do cumulus e o potencial de membrana médio em repouso obtido foi de -34,02 mV, com desvio-padrão de ± 6,46 e erro-padrão da média de ± 2,04. A resistência média das membranas ao fluxo de íons foi de 16,5 MΩ, com desvio-padrão de ± 4,03 e erro-padrão da média de ± 1,8. A aplicação do FSH Ovino (1 μM) causou uma lenta despolarização, estatisticamente significativa 180 segundos após a aplicação do hormônio (P<0,01). A aplicação da Folitropina Beta (Puregon® 1 μM) causou uma lenta despolarização, estatisticamente significativa 120 e 180 segundos após a aplicação do hormônio (P<0,001). O padrão de despolarização foi semelhante entre as duas isoformas, sendo o efeito da Folitropina Beta mais imediato que o do FSH Ovino aos 120 segundos. Conclusões: A partir da padronização da técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas, registrou-se o potencial médio de membrana em repouso (-34,02 mV) e a resistência média da membrana ao fluxo de íons (16,5 MΩ). O FSH Ovino (1 μM) e a Folitropina Beta (Puregon®) (1 μM) possuem uma ação despolarizante sobre o potencial de membrana das células do cumulus. Para o FSH Ovino, essa despolarização foi significativa 180 segundos após a aplicação do hormônio e para a Folitropina Beta, a despolarização foi significativa já aos 120 segundos após a aplicação. Há uma semelhança no padrão de despolarização produzida pelo FSH Ovino e pela Folitropina Beta. Um melhor entendimento das diferenças no funcionamento das isoformas do FSH pode proporcionar o desenvolvimento de melhores protocolos de tratamento para as pacientes, com menos efeitos colaterais. Esse conhecimento também pode ser utilizado para o aprimoramento das técnicas de maturação in vitro dos oócitos, que podem poupar as pacientes dos efeitos colaterais e dos altos custos da estimulação exógena com gonadotrofinas. / The cumulus oophorus cells are firmly connected to each other and to the oocyte and they take an important part on the oocyte maturation. These cells present numerous FSH receptors. The carbohydrates present in the FSH molecule may end in sialic acid residues, allowing FSH to exist in various isoforms. The more acidic isoforms present more sialic acid residues in their carbohydrate moieties. These isoforms exhibit fluctuations in the follicular phase of the menstrual cycle and possess different capabilities of ovarian stimulation, with the least acidic isoform having higher potency and lower plasmatic half-life. Objective: The aim of this study was to standardize the intracellular electrophysiology register technique to the human cumulus oophorus cells and to investigate the membrane potential action of a less acidic FSH isoform (Puregon™), comparing with the membrane action using a less purified, more acidic FSH isoform (sheep FSH). Methods: The membrane potential was registered in cumulus oophorus cells from patients assigned to the intracytoplasmatic sperm injection (ICSI) technique. The cumulus cells were cultured in plates with HTF medium for 24 to 48 hours. During the electrophysiological experiment, the plate was superfused with 1 mL/min of Hank`s medium supplemented with Hepes, maintained at 37º C. The intracellular register was performed with microcapillaries filled with 3 M KCl with resistance ranging between 15 and 25 MΩ, and coupled to an electrometer. The membrane resistance to the ion flow was assessed by the application of square current pulses (0,5 nA, 0,5 Hz and 200 ms) through the register electrode. The tested hormones (sheep FSH and Beta Follitropin Puregon™) were applied topically onto the plate. Statistical analysis was performed using the ANOVA test for repeated measures (with Bonferroni post-test) or Fischer´s exact test. Results: the standardization of the electrophysiological register technique for the cumulus cells was successfully achieved and the mean resting membrane potential obtained was -34,02 ± 2,04 mV (SEM). The mean resistance of the membrane to the ion flow was 16,5 ± 1,8 MΩ (SEM). The sheep FSH application (1 μM) led to a slow depolarization, statistically significant 180 seconds after the hormone application (P<0,01). Beta Follitropin administration (1 μM) led to a slow depolarization, statistically significant 120 and 180 seconds after the hormone application (P<0,001). The depolarization pattern was similar between both isoforms, being that Beta Follitropin`s effect was more immediate than sheep FSH`s. Conclusion: With the electrophysiological intracellular register technique standardization to the human cumulus cells, the mean resting membrane potential was registered (-34,02 mV) and the mean membrane resistance to the ion flow was obtained (16,5 MΩ). Sheep FSH (1 μM) and Beta Follitropin (Puregon™) (1 μM) prompted a depolarizing action in the membrane potential of cumulus cells. Sheep FSH depolarization was significant at 180 seconds after the hormone application and Beta Follitropin depolarization was significant at 120 seconds after its application. Both isohormones have similar depolarization patterns. A better understanding of the differences in the action of both FSH isoforms shall make it possible to develop better hormone induction protocols in fertility treatments, with less side effects for the patients. This knowledge will potencially also make it possible to improve the in vitro oocyte maturation techniques, sparing patients of the hormone treatment side effects and of the high financial costs of the exogenous gonadotrophins administration.

As células do cumulus oophorus são firmemente conectadas umas às outras e ao oócito e participam ativamente do amadurecimento oocitário. Essas células apresentam abundância de receptores para o FSH. Os carboidratos da molécula do FSH podem terminar em resíduos de ácido siálico, permitindo que ele exista em numerosas isoformas. As isoformas mais ácidas do FSH contêm mais resíduos de ácido siálico. Os níveis dessas isoformas variam na fase folicular do ciclo menstrual e possuem diferentes capacidades de estímulo ovariano, sendo as menos ácidas mais potentes e de curta meia-vida plasmática. Objetivo: padronizar a técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas e verificar a ação de uma isoforma menos ácida do FSH, a Folitropina Beta (Puregon®) sobre o potencial de membrana dessas células, comparando com a ação de um FSH com menor grau de pureza e mais ácido (FSH ovino). Metodologia: o potencial de membrana foi registrado utilizando células de cumulus oophorus de pacientes encaminhadas ao procedimento de injeção intracitoplasmática de espermatozóides (ICSI). As células do cumulus foram cultivadas em placas de cultura com meio HTF (Life Global® - Guilford, CT USA) acrescido de 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) por 24 a 48 horas. Durante o experimento eletrofisiológico, a placa foi perfundida com o fluxo de 1 mL/min de meio de Hank´s acrescido de Hepes com 10% de Soro Sintético (SSS- Life Global® - Guilford, CT USA) mantido a 37º C. O registro intracelular foi realizado com microeletrodos preenchidos com KCl 3 M com uma resistência de 15 a 25 MΩ e acoplados a um eletrômetro. A resistência da membrana ao fluxo de cargas foi avaliada com a aplicação de pulsos quadrados de corrente (0,5 nA, 0,5 Hz e 200 ms) através do eletrodo de registro. Os hormônios testados (FSH ovino e Folitropina Beta) foram aplicados topicamente na placa. Para a análise estatística foi utilizado o teste de Anova de duas vias (seguido do pós-teste de Bonferroni) ou o teste exato de Fischer. Resultados: Foi realizada a padronização da técnica de registro eletrofisiológico para as células do cumulus e o potencial de membrana médio em repouso obtido foi de -34,02 mV, com desvio-padrão de ± 6,46 e erro-padrão da média de ± 2,04. A resistência média das membranas ao fluxo de íons foi de 16,5 MΩ, com desvio-padrão de ± 4,03 e erro-padrão da média de ± 1,8. A aplicação do FSH Ovino (1 μM) causou uma lenta despolarização, estatisticamente significativa 180 segundos após a aplicação do hormônio (P<0,01). A aplicação da Folitropina Beta (Puregon® 1 μM) causou uma lenta despolarização, estatisticamente significativa 120 e 180 segundos após a aplicação do hormônio (P<0,001). O padrão de despolarização foi semelhante entre as duas isoformas, sendo o efeito da Folitropina Beta mais imediato que o do FSH Ovino aos 120 segundos. Conclusões: A partir da padronização da técnica de registro eletrofisiológico intracelular para as células do cumulus oophorus humanas, registrou-se o potencial médio de membrana em repouso (-34,02 mV) e a resistência média da membrana ao fluxo de íons (16,5 MΩ). O FSH Ovino (1 μM) e a Folitropina Beta (Puregon®) (1 μM) possuem uma ação despolarizante sobre o potencial de membrana das células do cumulus. Para o FSH Ovino, essa despolarização foi significativa 180 segundos após a aplicação do hormônio e para a Folitropina Beta, a despolarização foi significativa já aos 120 segundos após a aplicação. Há uma semelhança no padrão de despolarização produzida pelo FSH Ovino e pela Folitropina Beta. Um melhor entendimento das diferenças no funcionamento das isoformas do FSH pode proporcionar o desenvolvimento de melhores protocolos de tratamento para as pacientes, com menos efeitos colaterais. Esse conhecimento também pode ser utilizado para o aprimoramento das técnicas de maturação in vitro dos oócitos, que podem poupar as pacientes dos efeitos colaterais e dos altos custos da estimulação exógena com gonadotrofinas. / The cumulus oophorus cells are firmly connected to each other and to the oocyte and they take an important part on the oocyte maturation. These cells present numerous FSH receptors. The carbohydrates present in the FSH molecule may end in sialic acid residues, allowing FSH to exist in various isoforms. The more acidic isoforms present more sialic acid residues in their carbohydrate moieties. These isoforms exhibit fluctuations in the follicular phase of the menstrual cycle and possess different capabilities of ovarian stimulation, with the least acidic isoform having higher potency and lower plasmatic half-life. Objective: The aim of this study was to standardize the intracellular electrophysiology register technique to the human cumulus oophorus cells and to investigate the membrane potential action of a less acidic FSH isoform (Puregon™), comparing with the membrane action using a less purified, more acidic FSH isoform (sheep FSH). Methods: The membrane potential was registered in cumulus oophorus cells from patients assigned to the intracytoplasmatic sperm injection (ICSI) technique. The cumulus cells were cultured in plates with HTF medium for 24 to 48 hours. During the electrophysiological experiment, the plate was superfused with 1 mL/min of Hank`s medium supplemented with Hepes, maintained at 37º C. The intracellular register was performed with microcapillaries filled with 3 M KCl with resistance ranging between 15 and 25 MΩ, and coupled to an electrometer. The membrane resistance to the ion flow was assessed by the application of square current pulses (0,5 nA, 0,5 Hz and 200 ms) through the register electrode. The tested hormones (sheep FSH and Beta Follitropin Puregon™) were applied topically onto the plate. Statistical analysis was performed using the ANOVA test for repeated measures (with Bonferroni post-test) or Fischer´s exact test. Results: the standardization of the electrophysiological register technique for the cumulus cells was successfully achieved and the mean resting membrane potential obtained was -34,02 ± 2,04 mV (SEM). The mean resistance of the membrane to the ion flow was 16,5 ± 1,8 MΩ (SEM). The sheep FSH application (1 μM) led to a slow depolarization, statistically significant 180 seconds after the hormone application (P<0,01). Beta Follitropin administration (1 μM) led to a slow depolarization, statistically significant 120 and 180 seconds after the hormone application (P<0,001). The depolarization pattern was similar between both isoforms, being that Beta Follitropin`s effect was more immediate than sheep FSH`s. Conclusion: With the electrophysiological intracellular register technique standardization to the human cumulus cells, the mean resting membrane potential was registered (-34,02 mV) and the mean membrane resistance to the ion flow was obtained (16,5 MΩ). Sheep FSH (1 μM) and Beta Follitropin (Puregon™) (1 μM) prompted a depolarizing action in the membrane potential of cumulus cells. Sheep FSH depolarization was significant at 180 seconds after the hormone application and Beta Follitropin depolarization was significant at 120 seconds after its application. Both isohormones have similar depolarization patterns. A better understanding of the differences in the action of both FSH isoforms shall make it possible to develop better hormone induction protocols in fertility treatments, with less side effects for the patients. This knowledge will potencially also make it possible to improve the in vitro oocyte maturation techniques, sparing patients of the hormone treatment side effects and of the high financial costs of the exogenous gonadotrophins administration.

In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well‐known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin‐stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P4) and estradiol (E2) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P4, but not E2, secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin‐releasing hormone agonist (GnRH‐a)‐treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH‐a groups. EtOH at 20 mM or more inhibited EGF stimulated P4 secretion in human GC from all three patient groups. EtOH inhibited P4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH‐a groups, but not in human GC from the hMG‐treated patients. These results suggest that basal and EGF or LH‐stimulated P4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.

The function of light in the reproductive cycle in amphibians has not been extensively studied. However, Farner (1965) feels that the small amount of experimental evidence suggests that photoperiod may play a role in the testicular cycle of Rana esculenta. The purpose of this paper is to study the effect of light on the gonads and the gonadotropic hormone forming cells of the adenohypophysis in the newt Taricha granulosa granulosa.

Ce mémoire porte sur l’analyse de la croissance folliculaire dans l’ovaire chez la vache et l’humain. La transcription des gènes dans les cellules folliculaires indique quels mécanismes moléculaires sont modulés dans le follicule. Le premier volet de ce projet, chez le bovin, avait pour but de déterminer le profil transcriptomique des follicules de taille moyenne, entre 6 et 9 mm, appartenant à la phase de développement portant les ovocytes les plus compétents. Dans le deuxième volet, réalisé avec des tissus humains, l’impact de l’administration de hCG, une hormone pré-ovulatoire, a été évalué au niveau transcriptomique dans un cycle stimulé. Pour la première fois, des indicateurs géniques sont associés spécifiquement à trois phases clés du développement folliculaire, mais plus particulièrement à la phase plateau, ou atrésie précoce, correspondant à une compétence accrue des ovocytes à produire un embryon. Ces résultats apportent une contribution importante dans la définition des mécanismes moléculaires agissant dans le développement folliculaire. / This master’s thesis focused on the analysis of follicle growth in the ovary of both cows and humans. Genes’ transcription in follicular granulosa cells indicates which molecular mechanisms are modulated in the follicle. The first part of this project, in bovine, aimed to determine the transcriptomic profile of medium-sized follicles, between 6-9 mm, belonging to the follicular phase containing the most competent oocytes. In the human facet, the impact of hCG administration, a pre-ovulatory hormone, was evaluated at the transcriptomic level in a stimulated cycle. For the first time, genic indicators are specifically associated to three key phases of follicular development, especially the plateau phase, or early atresia, which correspond to an enhanced competence of the oocyte to produce an embryo. These results are an important step towards further defining the molecular mechanisms at work during follicular development, or folliculome.

Le développement des follicules, des ovocytes et des embryons sont des processus biologiques qui contrôlent la possibilité de devenir un organisme vivant. La maturation des ovocytes est un événement tout à fait unique avec deux divisions asymétriques, la méiose, mais également caractérisée par l'arrêt de la transcription et le stockage des transcrits et des protéines nécessaires au développement embryonnaire précoce. Comme nous le savons, le liquide folliculaire forme partie du micro environnement autour de l'ovocyte et influence la compétence de l'ovocyte. Cependant, la composition du liquide folliculaire peut changer sous divers statuts nutritionnels, tels que l'obésité chez l'homme et un bilan énergétique négatif chez les vaches et les truies. Ces modifications moléculaires du liquide folliculaire induites par les conditions métaboliques maternelles peuvent affecter la qualité des ovocytes, le développement embryonnaire et peuvent par la suite provoquer des troubles métaboliques et autres maladies chez les prochaines générations. Ces dernières années, de nouvelles études se sont concentrées sur les effets de contextes nutritionnels anormaux, p.ex., niveaux élevés d'acides gras non estérifiés (AGNE), sur les cellules de la granulosa, les cellules du cumulus, les ovocytes et le développement précoce de l'embryon chez diverses espèces, notamment chez l'homme, la souris et le bétail. Dans cette thèse, le porc a été utilisé comme modèle expérimental. Le porc est un excellent modèle pour étudier l'impact de la nutrition sur la fonction ovarienne chez l'homme, puisqu'il existe de nombreuses similitudes entre les porcs et l'homme telles que la physiologie digestive et la durée approximative de la maturation in vitro (IVM). En outre, les niveaux élevés d'AGNE induits par la nutrition existent également pendant la lactation chez les porcs. Enfin, les embryons de porc peuvent être générés in vitro et leur étude n'est pas soumise aux mêmes limitations éthiques que les humains. Pour mieux comprendre les effets de niveaux élevés d'AGNE au cours de la maturation des ovocytes chez le porc, des ovocytes porcins ont été exposés au milieu de maturation in vitro contenant une combinaison de niveaux élevés d'AGNE. Des puces à ADN ont été utilisées pour obtenir des informations globales sur les schémas transcriptomiques et de méthylation de l'ADN des blastocystes provenant des ovocytes exposés aux AGNE. Afin de mieux mimer un environnement folliculaire fonctionnel lors de la maturation, des ovocytes exposés aux AGNE ont été co-cultivés ou non avec des cellules de la granulosa porcine issues de follicules de taille moyenne (diamètre entre 3 et 5 mm). Pour capturer la réponse des cellules de la granulosa aux AGNE, les informations transcriptomiques des cellules de la granulosa co-cultivées ont également été étudiées. Le travail réalisé dans cette thèse nous a permis de mettre en évidence et d'expliquer les impacts de niveaux élevés d'AGNE folliculaires sur les blastocystes issus d'ovocytes exposés aux AGNE et les cellules de la granulosa co-cultivées. Les ovocytes exposés aux AGNE ont moins de potentiel de se développer en blastocystes, et les blastocystes dérivés ont montré une inflammation, plus d'apoptose, et des changements métaboliques des schémas transcriptomiques et de méthylation de l'ADN. La méthylation différente de l'ADN ouvre la possibilité de transmettre les impacts induits par les AGNE aux générations suivantes. Lorsque les voies affectées des blastocystes provenant d'ovocytes avec coculture de cellules de la granulosa ont été comparées à celles sans coculture, davantage de voies anti-inflammatoires ont été observées dans des conditions de coculture, suggérant le rôle protecteur des cellules de la granulosa dans la diminution des effets néfastes des AGNE sur les ovocytes. En examinant le transcriptome des cellules de la granulosa avec ou sans AGNE, nous avons observé que les cellules de la granulosa étaient capables d'atténuer les effets néfastes des AGNE sur les ovocytes par la régulation du métabolisme, des facteurs anti-inflammatoires et l'inhibition de la transition épithéliale-mésenchymateuse. Le dernier chapitre résume le métabolisme des acides gras dans les ovocytes, les cellules folliculaires et les embryons, et traite également des effets néfastes de l'exposition aux AGNE pendant la maturation in vitro sur les ovocytes, les cellules folliculaires et les embryons. Les informations dérivées des résultats mettent en évidence les effets néfastes des niveaux élevés d'AGNE dans le liquide folliculaire induits par l'état nutritionnel maternelle avec un aperçu global des schémas transcriptomiques et de méthylation de l'ADN chez les porcs. Ensuite, le rôle des cellules de la granulosa dans la protection des ovocytes envers l'environnement défavorable des acides gras en excès est proposé. Les connaissances acquises donnent une idée du métabolisme du niveau élevé d'acides gras dans les follicules et suggèrent l'importance d'une concentration appropriée d'acide gras pendant la folliculogenèse et la maturation des ovocytes. / The development of follicles, oocytes, and embryos are the biological processes that control the possibility to become a living organism. The oocyte maturation is a quite unique event with two asymmetric cleavages, the meiosis, but also characterized by the arrest of transcription and the storage of transcripts and proteins required for early embryonic development. As we know, the follicular fluid is part of the microenvironment surrounding the oocyte and influences the oocyte competence. However, the composition of follicular fluid may change under various nutrition statuses, such as obesity in humans and negative energy balance in cows and sows. These maternal metabolic conditions induce follicular fluid changes may affect oocyte quality, embryonic development, and subsequently cause metabolic problems and diseases in the next generations. In recent years, new studies focused on the effects of abnormal nutrition contexts, e.g., elevated levels of non-esterified fatty acids (NEFAs), on granulosa cells, cumulus cells, oocytes, and early embryo development in various species including humans, mice, and cattle. In this thesis, the pig was used as the experimental model. The pig is an excellent model to study the impacts of nutrition on human ovarian function since there are many similarities between pigs and humans, such as digestive physiology and approximate length of in vitro maturation (IVM). Besides, the nutrition-induced elevated levels of NEFA also exist during the lactation in pigs. Finally, porcine embryos can be generated in vitro and are not subjected to the same ethical limitations as human embryos. To better understand the effects of elevated levels of NEFAs during oocyte maturation in pigs, porcine oocytes were exposed to an in vitro maturation medium containing a combination of high level of NEFAs. Microarrays were used to draw global information of the transcriptomic and DNA methylation patterns of blastocysts originated from NEFAs' exposed oocytes. To better mimic a functional follicular environment during maturation, oocytes exposed to NEFAs were co-cultured or not with porcine granulosa cells from medium size follicles (diameter between 3 and 5 mm). To capture the response of granulosa cells to NEFAs, the transcriptomic information of the cocultured granulosa cells was also investigated. The work completed in this thesis allowed us to highlight and explain the impacts of elevated levels of follicular NEFAs on the blastocysts originated from NEFAs-exposed oocytes and the cocultured granulosa cells. NEFAs-exposed oocytes have less potential to develop into blastocysts, and the derived blastocysts showed inflammation, more apoptosis, and metabolic changes both in transcriptomic and DNA methylation patterns. The altered DNA methylation opens the possibility of transmitting the NEFAs-induced impacts to subsequent generations. When the affected pathways of blastocysts originated from oocytes cocultured with granulosa cells were compared to those without coculture, more anti-inflammatory pathways were observed in coculture condition, suggesting the protective role of granulosa cells in decreasing the detrimental effects of NEFAs on oocytes. Looking at the granulosa cell transcriptome with or without NEFAs, we observed that the granulosa cells were able to attenuate the adverse effects of NEFAs on oocytes through regulation of metabolism, anti-inflammatory factors, and inhibition of the epithelial-mesenchymal transition. The last chapter summarizes the metabolism of fatty acids in oocytes, follicular cells, and embryos, and also discusses the adverse impacts of exposure to NEFAs during IVM on oocytes, follicular cells, and embryos. The information derived from the results highlights the detrimental impacts of maternal nutrition-induced elevated NEFAs levels in follicular fluid with a global overview of the transcriptomic and DNA methylation patterns in pigs. Then the role of granulosa cells in protecting the oocytes from the adverse environment of excess fatty acids is proposed. The acquired knowledge provides the idea about the metabolism of the high level of fatty acids in follicles and suggests the importance of a suitable concentration of fatty acids during folliculogenesis and oocyte maturation.

La membrane est aujourd'hui perçue comme une mosaïque de micro-domaines membranaires, ou « lipid rafts ». Ces domaines sont moins fluides que le reste de la membrane, car ils sont enrichis en cholestérol et en sphingolipides à longues chaînes saturées. Ils comportent peu de protéines, mais plusieurs de celles-ci sont impliquées dans la signalisation cellulaire. C'est pour cette raison que les « rafts » sont considérés comme des plates-formes de signalisation cellulaire. Les investigations réalisées sur les « rafts » au niveau des gamètes femelles ont essentiellement été pratiquées sur les œufs de Xenopus, mais chez les mammifères presque rien n'a encore été démontré quant à la présence et surtout à la fonctionnalité des « rafts » dans les ovocytes et autres cellules du follicule ovarien. Ce mémoire présente une étude des « lipid rafts » des ovocytes, des cellules de la granulosa et des complexes ovocyte-cumulus porcins. / Nowadays, plasma membrane is perceived as a mosaic of micro-domains called lipid rafts. Because of their enrichment in cholesterol and sphingolipids, these domains are less fluid than the bulk membrane. Only a few proteins partition into lipid rafts, but most of these are implicated in cell signalling. For this reason, lipid rafts are considered as platforms for cell signalling. Most of the investigations performed on female gametes were realised on Xenopus eggs, but not much as been done yet to assess the presence and especially the functionality of lipid rafts of the oocytes and other cell types of the mammalian ovarian follicle. The present study investigates the presence and the functionality of lipid rafts in porcine oocytes, granulosa cells and cumulus-oocyte complex.

Thèse ou mémoire avec insertion d'articles. / Thèse en cotutelle : « Université Laval, Québec, Canada, Philosophiæ doctor (Ph. D.) et Université de Carthage, Tunis, Tunisie » / Depuis des décennies, l'hormone synthétisée et sécrétée naturellement par la glande pinéale appelée « mélatonine » a fait son entrée dans le monde scientifique. Les études ont montré que le rythme de sécrétion de la mélatonine dépend de la photopériode, cette dernière étant influencée par la saison. Chez les petits ruminants, la mise en veilleuse de la fonction de reproduction pour une période de l'année est désignée par la reproduction saisonnée. L'information photopériodique est traduite en termes de variation de la durée de sécrétion de la mélatonine. Chez l'espèce ovine, des études ont montré que la mélatonine régule indirectement l'axe gonadotrope en modulant la sécrétion de GnRH. Cette modulation se fait à travers les récepteurs spécifiques à la mélatonine MT1 et MT2. Cependant, il existe des variations considérables dans la densité et l'emplacement de l'expression des récepteurs de la mélatonine entre les espèces. Chez les animaux saisonniers, la mélatonine est impliquée dans la fonction ovarienne en activant des récepteurs multiples et des voies de signalisation de différents types de cellules cibles, en particulier les cellules de la thèque et de granulosa. La confirmation de la présence de cette hormone avec des quantités plus élevées au niveau du liquide folliculaire que dans le plasma a encouragé les chercheurs à tester son rôle spécifiquement au niveau du follicule ovarien. Dans un premier temps, cette thèse s'intéresse à comprendre l'effet de la mélatonine sur une culture de cellules de granulosa à travers une prédiction des principales voies de signalisation impliquées par la mélatonine dans la manipulation des signaux métaboliques au sein des follicules ovariens au stade antrale. L'objectif étant d'étudier la réponse folliculaire à la mélatonine au niveau de l'expression des gènes en utilisant l'outil de séquençage à haut débit. Nous avons constaté une image transcriptomique différente pour la culture des cellules de granulosa traitées avec de la mélatonine comparée à la culture non traitée. De plus, l'analyse des gènes différentiellement exprimés et des principales voies biologiques y étant associée nous ont démontré que cette hormone agit à travers la voie PKB/mTOR pour reprogrammer le métabolisme des cellules de granulosa afin de maintenir une lente croissance et différenciation ainsi que de prévenir l'atrésie folliculaire. Dans un second temps, cette thèse s'intéresse à la caractérisation des voies de signalisation activées, via les récepteurs spécifiques MT1 et MT2, dans la médiation de l'effet de la mélatonine au niveau des cellules de granulosa humaines. En utilisant des agonistes et des antagonistes spécifiques aux deux récepteurs, cette étude a permis de mettre en évidence un rôle clair du récepteur MT1 dans la médiation de l'effet antiproliférative de la mélatonine, via la voie PKB. Dans un troisième temps, cette thèse s'intéresse aussi à comprendre l'effet de la mélatonine à travers son récepteur MT1 sur le cycle reproductif chez les petits ruminants. Cette étude consiste à établir le lien entre les polymorphismes existants au niveau du récepteur MT1 et la saisonnalité de reproduction chez 77 brebis de deux races ovines locales en Tunisie, la Barbarine (B) et la Queue Fine de l'Ouest (QFO). Cette étude nous a permis de trouver certaines mutations au niveau du gène de récepteur à la mélatonine MT1 chez ces brebis, spécifiquement les SNPs liés au caractère saisonnier de la reproduction chez d'autres races ovines dans le monde. Les analyses statistiques nous ont prouvé que les brebis de la race B de génotype G/G et G/A qui sont les moins sensibles à la photopériode et répondent rapidement à l'effet bélier que les brebis de génotype A/A, ce qui nous laisse conclure que les brebis de la race (B) ont un anœstrus moins profond que les brebis de la race QFO. Globalement, les résultats de ces études suggèrent un aperçu global de l'action de la mélatonine au niveau des cellules de granulosa humaine tout en améliorant les connaissances sur le rôle de ses récepteurs spécifiques MT1 et MT2 ainsi que les principaux réseaux de signalisation impliqués à la suite de leur activation. Cette thèse permet de mettre en lumière les différents polymorphismes au niveau du gène de récepteur MT1 chez deux races ovines et révèle également une certaine sensibilité d'une race plus que l'autre à la photopériode. Ces résultats servent à optimiser les programmes d'amélioration génétique de reproduction et à éclaircir la réponse génomique des cellules de la granulosa humaine sur l'échelle transcriptomique. / For decades, the hormone synthesized and secreted naturally by the pineal gland called melatonin has entered the scientific world. This sleep hormone, which was discovered in 1960, has attracted the attention of researchers who have begun to explore more and more about its effects on animals. Studies have shown that the melatonin secretion rate depends on the photoperiod, which is influenced by the season. In nature, the seasonal variation in climate and food availability are important parameters in which, different animal species must adapt to survive well. In small ruminants, the dormancy of the reproductive function for a period of the year is referred to as the seasonal breeding. The photoperiod information is translated in terms of variation during the secretion of melatonin. In sheep, melatonin indirectly regulates the gonadotropic axis by modulating GnRH secretion. This modulation is done through melatonin-specific receptors MT1 and MT2. However, there are considerable variations in the density and location of melatonin receptor expression among species. In seasonal animals, melatonin is involved in ovarian function by activating multiple receptors and signaling pathways of different types of target cells, especially theca and granulosa cells. Confirmation of the higher amount of this hormone in the follicular fluid than in the plasma encouraged the researchers to test its role specifically on the ovarian follicle. First, this thesis is interested in understanding the effect of melatonin on a culture of human granulosa cells. It is also intended to predict the main signaling pathways involved with melatonin in the manipulation of metabolic signals within the antral ovarian follicles. The objective of this study is to understand the follicular response to melatonin in terms of gene expression and epigenetic adaptation while using the high-throughput sequencing tool. This relatively simple system could allow exploring the molecular mechanism that acts across generations, it allows the production of a global image of the activated pathways in the presence of melatonin. The foundation of different transcriptomic images for the culture of granulosa cells treated with melatonin than untreated culture. In addition, the analysis of differentially expressed genes and the main biological pathways associated with them showed that this hormone may act through the PKB/mTOR pathway to reprogram granulosa cell metabolism to maintain slow growth and differentiation as well as prevent follicular atresia. In the second stage, this thesis focuses on the characterization of activated signaling pathways, via specific receptors MT1 and MT2, in the mediation of the effect of melatonin in human granulosa cells. The aim was to identify which receptor is most involved in the effect of melatonin using specific agonists and antagonists to the two receptors. This study demonstrated a clear role of the MT1 receptor in mediating the antiproliferative effect of melatonin via PKB pathway by reprogramming the metabolism of human granulosa cells. To understand the effect of melatonin through its MT1 receptor on the reproductive cycle in small ruminants, the third chapter consists in establishing the link between the existing polymorphisms on MT1 receptor gene and the seasonal reproduction of 77 ewes of two local sheep breeds in Tunisia, the Barbarine and the Queue Fine de l'Ouest (QFO). This study allowed us to find certain mutations in the MT1 melatonin receptor gene in ewes of both breeds, specifically, SNPs related to the seasonality of reproduction in other sheep breeds worldwide. Statistical analyses have shown us that the Barbarine ewes with G/G and G/A genotypes are less sensitive to the photoperiod and spread rapidly to the ram effect than ewes with A/A genotype. This leads us to conclude that the Barbarine breed showed to be ready to resume reproductive activity earlier than the QFO ewes at the ram introduction. Overall, the results of these studies suggest a global overview of the melatonin action on human granulosa cells while improving our knowledge on the role of its specific MT1 and MT2 receptors as well as the main signaling networks involved following their activation. This thesis allows us to highlight the different polymorphisms at the level of the MT1 receptor gene in two Tunisian sheep breeds and reveals a certain sensitivity of one breed more than the other to the photoperiod. These results serve to optimize genetic breeding improvement programs and to clarify the genomic response of human granulosa cells on the transcriptomic scale.