The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues.

There has been a global rise in obesity in the last three decades, and at present one in five women are obese at antenatal booking. Maternal obesity is associated with an increased risk of adverse pregnancy outcomes, including increased fetal size and prolonged pregnancy. In the longer-term, offspring of obese are at increased risk of premature death from a cardiovascular event in their adulthood. One mechanism that has been linked to these outcomes is fetal exposure to glucocorticoids in utero. During normal pregnancy, the maternal hypothalamic pituitary adrenal (HPA) axis undergoes major changes, resulting in exponentially increasing levels of the major circulating glucocorticoid cortisol, and other HPA axis hormones, such as corticotrophin releasing hormone (CRH). Cortisol and CRH are vital for normal fetal growth and length of gestation, but in excess they are associated with fetal growth restriction and preterm labour. In non-pregnant obesity, it is thought that the HPA axis is dysregulated, although evidence is inconclusive. Little is known about the effects of maternal obesity in pregnancy on the HPA axis. The work in this Thesis used clinical studies to test the hypothesis that the HPA axis is dysregulated in obese pregnant women with altered release, clearance and placental metabolism of cortisol. Associations with clinical outcomes related to fetal size and length of gestation were also studied. The HPA axis activity during pregnancy was investigated in a prospective case-control study cohort. Fasting serum cortisol levels were measured at 16, 28 and 36 weeks of gestation (obese n=276, lean n=135). In a subset (obese n=20, lean n=20), corticosteroid binding globulin (CBG), CRH, estrogens and progesterone were measured. Salivary cortisol was measured in samples collected at bedtime, waking and 30 minutes after waking at 16 weeks. Urinary glucocorticoid metabolites were measured at 19 weeks and 36 weeks (obese n=6, lean n=5) and non-pregnant (obese n=7, lean n=7) subjects. All circulating hormone levels rose similarly in obese and lean during pregnancy, but were significantly lower in obese women. The diurnal rhythm of cortisol was maintained. Urinary glucocorticoids increased with gestation in lean, but not in obese, indicating a lesser activation of the HPA axis in obese compared with lean pregnancy. These findings associated with increased birthweight and longer gestation in obese pregnancy, suggesting that decreased HPA axis activity may underlie these obese related adverse pregnancy outcomes. Whether or not lower glucocorticoids in obese pregnancies are maintained at delivery was investigated by measuring active glucocorticoids (cortisol and corticosterone) and their inactive versions (cortisone and 11- dehydrocorticosterone, respectively) from matched maternal and cord plasma samples (n=259, BMI 18 – 55 kg/m2). Active glucocorticoids were significantly higher in maternal than cord blood, and inactive versions were significantly higher in cord than maternal blood. Increased maternal BMI associated with lower maternal cortisol, corticosterone and 11-dehydrocorticosterone. Despite significant correlations between maternal and cord blood glucocorticoid levels, increased maternal BMI did not associate with lower cord blood glucocorticoids. This suggests that conditions at delivery may overcome any potential negative effects of low maternal glucocorticoids on the fetus in the short-term. However, it may not preclude the longer-term effects of fetal exposure to lower glucocorticoid levels during obese pregnancy, and offspring follow-up studies are required. Potential mechanisms leading to altered HPA axis activity in obese pregnancy were explored by studying the pulsatile release and placental metabolism of glucocorticoid hormones. Glucocorticoid pulsatility is thought to be important for transcriptional regulation of glucocorticoid responsive genes, and disruptions to pulsatility have been reported in some disease processes. Glucocorticoids were measured in 10-minute serum sampling between 08.00h-11.00h and 16.00h- 19.00h. Peripheral tissue cortisol was measured from 20-minute sampling of interstitial fluid, over 24-hours, at 16-24 weeks and 30-36 weeks (obese n=7, lean n=8), and non-pregnant controls (obese n=4, lean n=3). Total circulating serum cortisol levels were higher in pregnancy than non-pregnancy in lean and obese, and increased significantly with advancing gestation in lean but not in obese. Pulsatility of cortisol was demonstrated in interstitial fluid in both non-pregnancy and pregnancy. In obese pregnancy, interstitial fluid pulse frequency was lower with advancing gestation. This may be a novel mechanism underlying the observed decreased HPA axis activity in obese pregnancy. Placental cortisol metabolism and transport was studied using an ex vivo placental perfusion model, perfused with a deuterium-labelled cortisol tracer combined with computational modeling. The findings challenge the concept that maternal cortisol diffuses freely across the placenta, but confirmed that 11β- HSD2 acts as major ‘barrier’ to cortisol transfer to the fetus, protecting the fetus from the high maternal circulating cortisol levels. In addition we showed preliminary evidence of local cortisol production within the placenta. The model is able to predict maternal-fetal cortisol transfer and can now be used in future experimental design. In conclusion, in obese pregnancy, lower maternal cortisol and urinary clearance suggested reduced HPA axis activity. Altered glucocorticoid pulsatility may underlie this change. Future studies of placental cortisol metabolism in maternal obesity could be conducted using an ex vivo perfusion model. The lower HPA axis activity in obese pregnancy represents a novel pathway underlying increased fetal growth.

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Made available in DSpace on 2014-10-09T12:52:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:55Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-06-11T19:29:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-25Bitstream added on 2014-06-13T19:38:27Z : No. of bitstreams: 1 bonin_bf_me_botfmvz.pdf: 208253 bytes, checksum: b815ba940b43478c6f605909f134aaef (MD5) / Universidade Estadual Paulista (UNESP) / O presente trabalho objetivou avaliar os efeitos de doses baixas do extrato de pituitária eqüina (EPE) no crescimento folicular, taxa de ovulação e de recuperação embrionária em éguas idosas. Doadoras de embrião (n=20) da raça Quarto de Milha de um programa comercial com idade de 15 a 20 anos de idade, foram utilizadas em 3 ciclos consecutivos. O primeiro e o terceiro ciclo foi utilizado como grupo controle e entre eles o grupo tratado com EPE no segundo ciclo. Em todos os ciclos, a ultrassonografia transretal dos ovários foi realizada diariamente após aplicação de prostaglandina e monitorando os dois maiores folículos. Durante os três ciclos as éguas foram inseminadas com sêmen a fresco ou refrigerado do mesmo garanhão e receberam uma aplicação de acetato de deslorelina após a detecção de pelo menos um folículo de 35 a 40 mm de diâmetro. A recuperação embrionária foi realizada por lavado uterino 8 dias pós ovulação seguidas da administração de prostaglandina. No ciclo tratado, injeções de EPE (7mg/IM/duas vezes ao dia) se iniciaram no dia 8 após a ovulação até que um folículo pré-ovulatório fosse observado. A porcentagem de éguas com mais de uma ovulação foi comparada pelo teste de Fisher e a média do número de ovulações e embriões, assim como o diâmetro folicular, entre os ciclos foram comparados usando teste de Student’s. A porcentagem de éguas com mais de uma ovulação e o número de ovulações foram maiores (p<0,05) no ciclo tratado com EPE (65% das éguas e 1,8 ovulações) do que o grupo controle antes (5% e 1,0) e depois do tratamento (5% e 1,0). O número de embriões recuperados por ciclo também foi maior (p<0,05) no grupo tratado com EPE (1,0/ciclo) comparado com os ciclos controle (0,42/ciclo). Entretanto a taxa de embrião recuperado por ovulação foi similar (p>0,05) entre os grupos controles... / The present study aimed to evaluate the effect of the use of low doses of equine pituitary extract (EPE) on follicular growth, ovulation rate and embryo recovery rates in old mares. Embryo donors (n=20) of Quarter Horse breed from a commercial embryo center aging 15 to 20 years old , were used during 3 consecutive cycles. The first and third cycles were use as control being the EPE treatment performed on the second cycle. In all cycles transrectal ultrasonography of the uterus and ovaries was performed daily after PG injection to follow the development of the two largest follicles. During the 3 estrus mares were inseminated with cooled or fresh semen from the same stallion and received an injection of deslorelin (1 mg i.m.) upon detection of at least one follicle of 35 to 40 mm in diameter. The embryo flushing’s were performed on day 8 post-ovulation and were always followed by administration of PG. On the treated cycle, EPE injections (7mg twice daily i.m.) were started on day 8 after ovulation until a pre-ovulatory follicle was observed. The percentage of mares with more than one ovulation was compared by means using Fischer’s test and the mean number of ovulations and embryos, as well as follicular diameters, between cycles were compared using Student’s t test. The percentage of mares with more than one ovulation and number of ovulations were higher (p<0.05) in the EPE-treated cycle (65% of mares and 1.8 ovulations) than in the control cycle before (5% and 1.0) and after treatment (5% and 1.0). Also the mean number of embryos recovered per cycle were higher (p<0.05) on EPE treated cycle (1.0 /cycle) than in no treated cycles (0.42/cycle). However the embryo rate per ovulation was similar (p>0.05) between no treated cycles (0.4 embryo/ovulation) and treated cycle (0.6 embryo/ovulation). The treatment with EPE allowed the recovery of embryos in ix stage of development more... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-17Bitstream added on 2014-06-13T19:17:40Z : No. of bitstreams: 1 gimenes_am_me_botfmvz.pdf: 692315 bytes, checksum: 7da5bd34c5afb8b570d2c0e11f7e30c9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo visou avaliar a resposta ovariana e a taxa de recuperação embrionária em éguas tratadas com EPE nas doses de 6, 8 e 12,5 mg sendo o tratamento iniciado após a aplicação de prostaglandina F2 no oitavo ou sexto dia após a ovulação com a finalidade de reduzir o tempo e custo do tratamento. Foram realizados dois experimentos, para o experimento 1, 40 ciclos estrais de éguas Mangalarga Marchador, entre cinco e 24 anos, e para o segundo experimento 30 ciclos estrais de éguas mestiças entre quatro e 12 anos de idade, foram estudados. Foi aplicada 7,5 mg i.m.de prostaglandina F2 (Dinoprost Trometamina) no oitavo (experimento 1) ou sexto (experimento 2) dia após a ovulação. Nesse momento as éguas foram distribuídas aleatoriamente em cinco grupos para o experimento 1: (n=8 ciclos estrais/grupo): Grupo F20-23mm 6mg e F20-23mm 8mg receberam 6 e 8mg, respectivamente, de EPE por via i.m. a cada 12 horas, a partir da detecção de folículo(s) entre 20-23mm de diâmetro. Nos Grupos D8 - 6mg e D8 - 8mg foi aplicado 6 ou 8 mg de EPE, respectivamente, a cada 12 horas por via i.m. a partir do D8 (concomitante a PGF2 ). No Grupo F20-23mm Salina (Controle): as éguas receberam solução salina respeitando os mesmos intervalos que os grupos tratados com EPE a partir da detecção de folículo(s) entre 20-23mm de diâmetro. No experimento 2, a metodologia foi similiar ao experimento 1, contudo, as éguas receberam 12,5 mg de EPE, e o tratamento foi iniciado no sexto dia após a ovulação para o Grupo D6-12,5 mg (n=9), ou quando detectou-se folículo(s) entre 20-23mm, Grupo F20-23mm 12,5 mg (n=10) e F20-23mm Salina (Controle, n=10). Em todos os grupos (Exp. 1 e 2) o tratamento (EPE ou salina) foi mantido até 12 horas anterior a detecção de folículo(s) com diâmetro 35 mm, nesse momento a ovulação foi induzida com única dose de 2500 U.I. de hCG, i.v. (Vetecor®)... / The present study aimed to evaluate the ovarian response and embrionary recovery in mares treated with doses of 6, 8 and 12.5 mg of EPE. The treatment was started after application of prostaglandin F2 for reduce the period and cost of treatment. Two experiments were conducted, for the experiment one, 40 estrous cycles of Mangalarga Marchador mares and ranged in age from 5 to 24 yrs, and for the second experiment 30 estrous cycles of crossbreed mares ranged in age from four to 12 yrs was used. In the eighth (experiment 1) or sixth (experiment 2) after ovulation was applied 7.5 mg i.m. of prostaglandin F2 (Dinoprost Trometamina). In this moment, all mares in experiment 1 was randomly assigned to treatment and control groups as follows: Groups 1 and 2 (n=8 cycles/group) received 6 mg of EPE, i.m. twice daily, beginning when largest follicle (s) was (were) 20-23 mm (Group F20-23mm 6mg) or regardless of follicle size on Day 8 (Group D8 – 6 mg); Groups 3 and 4 (n=8 cycles/group), received 8 mg of EPE, i.m. twice daily, beginning when largest follicle was 20-23 mm (Group F20-23 mm 8 mg) or regardless of follicle size on Day 8 (Group D8 – 8mg); Group F20-23mm Saline (Control, n=8 cycles) received saline, i.m. beginning when largest follicle (s) was (were) 20-23 mm in the same range of groups treated with EPE. In the second experiment was used the same methodology of the experiment 1, however, the mares received 12.5mg of EPE, and the treatment began in the sixth day after ovulation for the Group D6- 12.5mg (n=9), or when the largest follicle (s) was (were) 20-23 mm, Group F20-23mm 12,5 mg (n=10) e F20-23mm Saline (Control, n=10). Treatments with EPE or saline continued until 12 hours before detection of follicle (s) reached 35 mm, at which time a single dose of hCG (2500 U.I., i.v., Vetecor®) was given. Groups were compared using ANOVA and mean differences among groups were... (Complete abstract click electronic access below)

Orientador: Marco Antonio Alvarenga / Banca: Julio Cesar Ferraz Jacob / Banca: José Antonio Dellaqua Junior / Resumo: O presente trabalho objetivou avaliar os efeitos de doses baixas do extrato de pituitária eqüina (EPE) no crescimento folicular, taxa de ovulação e de recuperação embrionária em éguas idosas. Doadoras de embrião (n=20) da raça Quarto de Milha de um programa comercial com idade de 15 a 20 anos de idade, foram utilizadas em 3 ciclos consecutivos. O primeiro e o terceiro ciclo foi utilizado como grupo controle e entre eles o grupo tratado com EPE no segundo ciclo. Em todos os ciclos, a ultrassonografia transretal dos ovários foi realizada diariamente após aplicação de prostaglandina e monitorando os dois maiores folículos. Durante os três ciclos as éguas foram inseminadas com sêmen a fresco ou refrigerado do mesmo garanhão e receberam uma aplicação de acetato de deslorelina após a detecção de pelo menos um folículo de 35 a 40 mm de diâmetro. A recuperação embrionária foi realizada por lavado uterino 8 dias pós ovulação seguidas da administração de prostaglandina. No ciclo tratado, injeções de EPE (7mg/IM/duas vezes ao dia) se iniciaram no dia 8 após a ovulação até que um folículo pré-ovulatório fosse observado. A porcentagem de éguas com mais de uma ovulação foi comparada pelo teste de Fisher e a média do número de ovulações e embriões, assim como o diâmetro folicular, entre os ciclos foram comparados usando teste de Student's. A porcentagem de éguas com mais de uma ovulação e o número de ovulações foram maiores (p<0,05) no ciclo tratado com EPE (65% das éguas e 1,8 ovulações) do que o grupo controle antes (5% e 1,0) e depois do tratamento (5% e 1,0). O número de embriões recuperados por ciclo também foi maior (p<0,05) no grupo tratado com EPE (1,0/ciclo) comparado com os ciclos controle (0,42/ciclo). Entretanto a taxa de embrião recuperado por ovulação foi similar (p>0,05) entre os grupos controles... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study aimed to evaluate the effect of the use of low doses of equine pituitary extract (EPE) on follicular growth, ovulation rate and embryo recovery rates in old mares. Embryo donors (n=20) of Quarter Horse breed from a commercial embryo center aging 15 to 20 years old , were used during 3 consecutive cycles. The first and third cycles were use as control being the EPE treatment performed on the second cycle. In all cycles transrectal ultrasonography of the uterus and ovaries was performed daily after PG injection to follow the development of the two largest follicles. During the 3 estrus mares were inseminated with cooled or fresh semen from the same stallion and received an injection of deslorelin (1 mg i.m.) upon detection of at least one follicle of 35 to 40 mm in diameter. The embryo flushing's were performed on day 8 post-ovulation and were always followed by administration of PG. On the treated cycle, EPE injections (7mg twice daily i.m.) were started on day 8 after ovulation until a pre-ovulatory follicle was observed. The percentage of mares with more than one ovulation was compared by means using Fischer's test and the mean number of ovulations and embryos, as well as follicular diameters, between cycles were compared using Student's t test. The percentage of mares with more than one ovulation and number of ovulations were higher (p<0.05) in the EPE-treated cycle (65% of mares and 1.8 ovulations) than in the control cycle before (5% and 1.0) and after treatment (5% and 1.0). Also the mean number of embryos recovered per cycle were higher (p<0.05) on EPE treated cycle (1.0 /cycle) than in no treated cycles (0.42/cycle). However the embryo rate per ovulation was similar (p>0.05) between no treated cycles (0.4 embryo/ovulation) and treated cycle (0.6 embryo/ovulation). The treatment with EPE allowed the recovery of embryos in ix stage of development more... (Complete abstract click electronic access below) / Mestre

Orientador: Marco Antonio Alvarenga / Resumo: Vários estudos têm relatado que a égua é refratária a todas as drogas rotineiramente utilizadas, visando superovulação em outras espécies, como o FSH e o eCG (Mc Cue, 1996). O extrato de pituitária eqüina (EPE) é um preparado parcial de gonadotrofina eqüina, e o único composto que consistentemente induz ovulações múltiplas em éguas (Squires et al, 1999); contudo, a resposta superovulatória tem sido baixa (1-3 ovulações/égua) (Mc Cue, 1996). Estudos mais recentes demonstram uma melhora no percentual de ovulações múltiplas em éguas superovuladas com a administração do EPE duas vezes ao dia (4-7ovulações/égua), entretanto com baixa taxa de recuperação embrionária (Scoggin et al., 2002; Alvarenga et al., 2001). Este fato pode estar relacionado a produção de oócitos de baixa qualidade (anormalidade na maturação folicular e oocitária) (Dippert et al., 1994; Palmer et al., 1993), a falha do folículo em liberar o oócito e a dificuldade na captação do mesmo para o interior do oviduto, assim como o trânsito deste ou do embrião pelo oviduto (Dippert et al., 1994). O extrato de pituitária eqüina (EPE) foi preparado no laboratório do Departamento de Reprodução Animal e Radiologia Veterinária da Universidade Estadual Paulista (UNESP), campus de Botucatu - SP, através do método proposto por Guillon & Combarnous, (1983). Seis éguas em bom estado nutricional, com idade variando entre quatro e 15 anos e massa corpórea de 400 a 500 Kg, além de um bom histórico reprodutivo, foram monitoradas diariamente durante os períodos compreendidos entre os meses de novembro (2001) a março (2002). Foram utilizados dois ciclos estrais de cada égua subdivididos em dois grupos: doses constantes e doses decrescentes; porém entre os tratamentos esperou-se um intervalo de dois ciclos estrais. O experimento foi conduzido no posto de monta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Several studies have reported the is unresponsive to all the routinely used drugs aiming the superovulation in other species, such as the FSH and the eCG. The equine pituitary extract (EPE) is a partial equine gonadotropin preparation, and the only compound which consintenty induces multiple ovulation in mares; however, the superovulatory response has been low (1-3 ovulations/mare). More recent studies demonstrate some improvement in the percentage of multiple ovulation in superovulated mares with the EPE administration twice a day (4-7 ovulations/mare), nevertheless with a low rate of embryo recovery. This fact can be related to the low quality oocyte production (abnormality in the follicular and oocytary maturation), the follicular failure in releasing the oocyte and its collection towards the inner part of the oviduct, as its or the embryo traffic through the oviduct. The improvement on embryo recovery rate has been shownin cows with the utilization of decreasing doses of FSH. The present experiment aimmed to compare the ovulatory answer and embryo production of mares treated twice daily with constant and decreasing doses of EPE. Six mares in good nutrition status, with ages ranging from four and 15 years old and weighing from 400 to 500 Kg, together with a good reproductive history, were monitored daily through the preiods between the months of november/2001 to march/2002. Two estrous cytrous cycles were used from each mare and subdivided into two groups: constant doses and decresing doses;however between the treatments na interval of two estrous cycles was given. The experiment was carried out in the riding station (UNESP), placed in Lageado, a farm in the city of Botucatu - SP. For both groups, the treatment was started in the seventh post-ovuatory day, presenting a very evident corpora lutea and follicles equal or under 25 mm of diameter in the... (Complete abstract, click electronic address below) / Mestre

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Made available in DSpace on 2014-10-09T12:52:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:55Z (GMT). No. of bitstreams: 0 / Um método de cromatografia líquida de alta eficiência por fase reversa (RP-HPLC) para análise qualitativa e quantitativa do hormônio folículo estimulante humano íntegro (hFSH), foi estabelecido e validado quanto à exatidão, precisão e sensibilidade. O FSH humano é um hormônio glicoprotéico dimérico largamente utilizado em medicina reprodutiva tanto para diagnóstico quanto para terapia. A metodologia desenvolvida preserva a integridade da proteína, permitindo a análise da forma heterodimérica intacta, e não somente de suas subunidades, como é normalmente obtida na maioria das condições geralmente empregadas. Esta técnica foi também utilizada para a comparação da hidrofobicidade relativa de preparações de hFSH hipofisária, urinária e derivadas de células de ovário de hamster chinês (CHO) bem como de outros dois hormônios glicoprotéicos, sintetizados na hipófise anterior: hormônio humano estimulante da tireóide (hTSH) e hormônio luteinizante humano (hLH). O menos hidrofóbico dos três hormônios analisados foi o hFSH, seguido do hTSH e do hLH. Uma diferença significativa (p<0,005) foi observada entre o tempo de retenção (tR) das preparações hipofisária e recombinante de hFSH, refletindo diferenças estruturais nas suas cadeias de carboidratos. Duas isoformas principais foram detectadas no hFSH urinário, incluindo uma forma que foi significativamente diferente (p<0,005) das preparações hipofisária e recombinante. Foram demonstradas linearidade da curva dose-resposta (r=0,9965, n=15) para esta metodologia de RP-HPLC, bem como uma precisão inter-ensaio, cujo coeficiente de variação é menor que 4%, para a quantificação de diferentes preparações de hFSH e uma sensibilidade da ordem de 40 ng. Foram também analisados o comportamento cromatográfico e a hidrofobicidade relativa das subunidades individuais das preparações recombinantes e hipofisária de hFSH. Além disso, a exata massa molecular das subunidades individuais de hFSH e do heterodímero foram simultaneamente determinadas por espectrometria de massa MALDI-TOF. A presente metodologia representa, em nossa opinião, uma ferramenta essencial para a caracterização e controle de qualidade deste hormônio, que ainda não consta das principais farmacopéias. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP