The intra-oocyte signaling pathways that control oocyte growth and early follicular development are largely unknown. The aim of this thesis was to investigate the regulation and functions of phosphatidylinositol 3 kinase (PI3K) pathway in the oocyte, focusing in the roles of Foxo3a, p27, and Pten (phosphatase and tensin homolog deleted on chromosome ten). The physiological significance of Foxo3a in oocytes had been investigated by generating a transgenic mouse, whereby constitutively active Foxo3a is maintained in oocytes using the oocyte-specific ZP3 (Zona pellucida) promoter. The expression of the constantly active “negative” molecule Foxo3a in mouse oocytes was found to cause retardation of oocyte growth, resulting in a significant reduction in oocyte volume in secondary follicles. The transgenic mice also showed arrested follicular development and were infertile. In addition, when Foxo3a was overexpressed in oocytes of primary follicles, oocyte growth and follicular development were retarded. One of the causes of this phenotype may be the retained expression of the cyclin-dependent kinase (Cdk) inhibitor 1B (Cdkn1b), commonly known as p27kip1 or p27, in the nuclei of oocytes. The role and related mechanisms of p27 in controlling early follicular development and oocyte growth were then investigated using wild-type and p27-deficient (p27-/-) mice, and we demonstrated that (i) p27 suppresses follicle endowment/formation and activation, (ii) p27 induces follicle atresia that occurs prior to sexual maturity, and (iii) the overactivated follicles in p27-/- ovaries are depleted in early adulthood, causing premature ovarian failure (POF). In this thesis, we also provide genetic evidence that in mice with conditional deletion of Pten a major negative regulator of PI3K in oocytes, the entire pool of primordial follicles becomes activated, and subsequently all activated follicles are depleted in young adulthood, causing POF. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling, which led to upregulation of both expression and activation of ribosomal protein S6 (rpS6) in oocytes. The results thus show that the mammalian oocyte serves as the headquarters of programming of the occurrence of follicle activation, and that the PI3K pathway of the oocyte governs follicle activation through control of initiation of oocyte growth.

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

No description available.

Orientador: Eunice Oba / Resumo: As substâncias desreguladores endócrinas de diversas origens e propriedades químicas acomentem uma diversidade de mamíferos, animais silvestres e seres humanos, que podem interagir com o sistema reprodutivo feminino e prejudicar a homeostase hormonal. Dentro dessa classificação, os parabenos podem ser utilizados como conservantes em diversos produtos comerciais. Seus efeitos se tornam cada vez mais complexos em relação à idade, história reprodutiva, ambiente endócrino da espécime no momento da exposição, como também não é sabido todos os reais mecanismos desses compostos quando associados. Sendo assim, o objetivo deste estudo foi avaliar os efeitos dos parabenos em ratas Wistar durante um período de 90 dias consecutivos de exposição. Assim, as ratas foram distribuídas em quatro grupos experimentais: controle, que recebeu somente óleo de milho como veículo, e tratados com propil e buti-parabeno nas doses de 10 mg/kg, 100 mg/kg, ou 200 mg/kg e expostas via subcutânea. Foi utilizada a técnica de citologia vaginal para investigar a ciclicidade das fêmeas, bem como a caracterização das fases do ciclo estral pelo método Shorr. Além desses parâmetros, foram analisados aspectos morfológicos de ovários e útero, quantificação das estruturas ovarianas e avaliação ultraestutural de folículos pré-ovulatórios. Foram avaliados a concentração plasmática de estrógeno, peso corporal bem como o peso dos órgãos reprodutivos. Os resultados revelaram que o método Shorr, foi eficaz para identificar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The endocrine disruption substances of many origins and chemistry properties affect a diversity of mammals, wild animals and human beings. These substances can interact with the female reproductive system and damage the hormonal homeostasis. In this classification, the paraben could be used as preservatives in different commercial products. Its effects get even worst and complex with the age, reproductive history, and endocrine environment of the specie in the moment of exposure of the drug. In complement, it is not known the actual mechanisms of those compounds when they are associated. Thus, the goal of this experiment was evaluate the effects of parabens in females Wistar during 90 consecutive days of exposure. Therefore, the females were divided in four experimental groups: Control group – Corn oil; and a mixture of propil and butyl paraben with 10 mg/kg, 100 mg/kg and 200 mg/kg and exposed through the subcutaneous pathway. The vaginal cytology was performed to investigate the cyclicality as well as the characterization of the estrous phases by the Shor method. Besides that, morphological aspects of the ovary and uterus, counting of ovaries structure and ultrastructural of pre-ovulatory follicle. To complete the evaluation, the plasmatic estrogen, body weight and reproductive organ weight were assessed. The Shor method was effective to characterize and identify the cells types of the vaginal epithelium and identify the estrous cycle phase. Moreover, the relative and absol... (Complete abstract click electronic access below) / Mestre

Resumo: O objetivo deste estudo foi verificar os efeitos da suplementação energética e com diferentes fontes de gordura na produção de embriões FIV e no perfil metabólico em novilhas nelore. Foram realizados dois experimentos, no primeiro, vinte novilhas Nelore (Bos taurus indicus) com idade média de 30 meses, com peso médio de 417,0 ± 42,4 kg foram distribuidas aleatoriamente em 3 tratamentos: T1 (n= 7), animais receberam 100% das exigências de manutença de energia, calculados conforme NRC (1997), T2 onde os animais receberam 170% das exigências de manutenção de energia (1,7 X M), e T3, onde os animais receberam 170 % das exigências de manutenção de energia com adição de 200 g de gordura protegida-AGCL (Megalac®). No segundo experimento 12 novilhas Nelore (Bos taurus indicus) com idade variando de 14 a 18 meses, com peso médio inicial de 309,6 kg, foram distribuidas em 4 tratamentos, de acordo com peso e população folicular avaliada por ultrasonografia: T1, animais receberam 100% dos requerimentos de mantença de energia, calculados conforme NRC (1997), T2- animais receberam 170% das exigências de mantença de energia, T3- idem ao T2, com adição de 4% de óleo de soja na MS, e T4- idem ao T2, com adição de 200 g de AGCL (Megalac ®). O período de adaptação foi quinze dias, onde os animais receberam feno de capim Brachiaria e ração concentrada... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Two experiments were carried to evaluate the effect of the energy and fat in the metabolic profile, follicular dynamic, oocyte and in vitro embryo production in Nelore heifers (Bos taurus indicus). In the first, twenty Nelore heifers with 30 months old, and average weight 417,0 ± 42,4 were allocated in 3 treatments, in accordance with weight and follicular population evaluated by ultrasound: T1, animals received 100% of energy maintenance, calculated as NRC (1997), T2- animals received 170% of energy maintenance; T3: animals received 170% of energy maintenance with addition of 200 g Megalac ®. In the second experiment, 12 Nelore heifers with 14-18 months and 309,6 kg average weight, were allocated in 3 treatments, in tree periods: T1, animals received 100% of energy maintenance, calculated as NRC (1997), T2- animals received 170% of energy maintenance; T3: animals received 170% of energy maintenance with addition of 4% soybean oil in Dry Matter and T4: animals received 170% of energy maintenance with addition of 200 g Megalac ®. After adaptation period, the treatment initiated, animals had synchronized ovulation, blood samples were collected, and follicular aspiration was carried to evaluate the oocytes and in vitro embryo production. Follicular development was monitored daily by ultrasonography until day post heat. The consumption of MS and the weight gain were lower in group T1 (P< 0.05), in relation to the others treatments. The FSH, LH, and albumin concentrations were not different (P > 0.05) among treatments. The cholesterol concentrations were higher in the T3 and T4 groups (P< 0.05) only in the second experiment... (Complete abstract, click electronic access below) / Orientadora: Gisele Zoccal Mingoti / Coorientador: Roberto Sartori Filho / Banca: Ciro Moraes Barros / Banca: César Roberto Esper / Banca: Paulo Henrique Franceschini / Doutor

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-08Bitstream added on 2014-06-13T19:45:13Z : No. of bitstreams: 1 nogueira_e_dr_jabo.pdf: 663672 bytes, checksum: f044bd71ec8b6a869bb1626ab1b3b25b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi verificar os efeitos da suplementação energética e com diferentes fontes de gordura na produção de embriões FIV e no perfil metabólico em novilhas nelore. Foram realizados dois experimentos, no primeiro, vinte novilhas Nelore (Bos taurus indicus) com idade média de 30 meses, com peso médio de 417,0 ± 42,4 kg foram distribuidas aleatoriamente em 3 tratamentos: T1 (n= 7), animais receberam 100% das exigências de manutença de energia, calculados conforme NRC (1997), T2 onde os animais receberam 170% das exigências de manutenção de energia (1,7 X M), e T3, onde os animais receberam 170 % das exigências de manutenção de energia com adição de 200 g de gordura protegida-AGCL (Megalac®). No segundo experimento 12 novilhas Nelore (Bos taurus indicus) com idade variando de 14 a 18 meses, com peso médio inicial de 309,6 kg, foram distribuidas em 4 tratamentos, de acordo com peso e população folicular avaliada por ultrasonografia: T1, animais receberam 100% dos requerimentos de mantença de energia, calculados conforme NRC (1997), T2- animais receberam 170% das exigências de mantença de energia, T3- idem ao T2, com adição de 4% de óleo de soja na MS, e T4- idem ao T2, com adição de 200 g de AGCL (Megalac ®). O período de adaptação foi quinze dias, onde os animais receberam feno de capim Brachiaria e ração concentrada... / Two experiments were carried to evaluate the effect of the energy and fat in the metabolic profile, follicular dynamic, oocyte and in vitro embryo production in Nelore heifers (Bos taurus indicus). In the first, twenty Nelore heifers with 30 months old, and average weight 417,0 ± 42,4 were allocated in 3 treatments, in accordance with weight and follicular population evaluated by ultrasound: T1, animals received 100% of energy maintenance, calculated as NRC (1997), T2- animals received 170% of energy maintenance; T3: animals received 170% of energy maintenance with addition of 200 g Megalac ®. In the second experiment, 12 Nelore heifers with 14-18 months and 309,6 kg average weight, were allocated in 3 treatments, in tree periods: T1, animals received 100% of energy maintenance, calculated as NRC (1997), T2- animals received 170% of energy maintenance; T3: animals received 170% of energy maintenance with addition of 4% soybean oil in Dry Matter and T4: animals received 170% of energy maintenance with addition of 200 g Megalac ®. After adaptation period, the treatment initiated, animals had synchronized ovulation, blood samples were collected, and follicular aspiration was carried to evaluate the oocytes and in vitro embryo production. Follicular development was monitored daily by ultrasonography until day post heat. The consumption of MS and the weight gain were lower in group T1 (P< 0.05), in relation to the others treatments. The FSH, LH, and albumin concentrations were not different (P > 0.05) among treatments. The cholesterol concentrations were higher in the T3 and T4 groups (P< 0.05) only in the second experiment... (Complete abstract, click electronic access below)

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