Orientador: Silvana Artioli Schellini / Banca: Ivete Dalben Soares / Banca: Luciene Cristina Barbarini Ferraz / Banca: Paula Araújo Opromolla / Banca: Norma Helen Medina / Resumo: 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 ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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) / Doutor

O objetivo deste estudo foi analisar as concentrações do fator de crescimento semelhante à insulina tipo 1 (IGF1) e insulina (INS) no fluido folicular durante foliculogênese em éguas, e localizar o receptor tipo 1 de IGF (IGF1R) nas paredes foliculares. Durante a estação reprodutiva, 40 éguas mestiças enviadas para abate em matadouro, com idades variando entre 6 e 16 anos foram utilizadas. As éguas foram abatidas de forma humanitária. Os tratos reprodutivos internos foram recuperados dentro de 10 min após o abate, ovários de éguas cíclicas foram separados do resto do aparelho. As éguas foram classificadas em quatro grupos: G1 – Folículo ≤ 13,5 mm e CL identificável; G2 – Folículo de 13,6 - 22,5 mm e CL identificável; G3 – Folículo de 22,6 - 31,5 mm e CL identificável; G4 – Folículo ≥ 31,6 mm e CL difícil de identificar. O fluido folicular (FF) foi aspirado e armazenado a -80 ° C até a sua utilização. Após a punção do FF, um fragmento da porção ventral da parede folicular foi removido para realizar a imunohistoquímica para localização de receptores IGF1R. O IGF1 livre foi determinado por ensaio imunoradiométrico. A INS foi determinada por um radioimunoensaio de fase líquida. A concentração de IGF1 livre e INS no FF aumentou com o crescimento folicular (P < 0,05). O escore imunohistoquímico de IGF1R nas células da granulosa e da teca interna da parede folicular equina em diferentes grupos aumentou (P < 0,05) com aumento folicular. Concluímos que as concentrações de IGF1 livre e INS no FF e de IGF1R em paredes foliculares de éguas aumentam com o crescimento folicular durante a foliculogênese, dando evidência do papel importante do IGF1 e INS no desenvolvimento folicular, e uma interação entre ambos hormônios na fisiologia reprodutiva ovariana. Sendo a égua um modelo de pesquisa para estudos comparativos na dinâmica folicular para consideração em mulheres, a conclusão deste estudo pode ser aplicada, também, a humanos. / The aim of this study was to analyze free type 1 insulin-like growth factor (IGF1) and insulin (INS) concentrations in follicular fluid during folliculogenesis in mares, and to localize type 1 IGF receptor (IGF1R) on follicular walls. During the breeding season, 40 mixed-breed mares sent to slaughter at an abattoir, with ages ranging between 6 to 16 years were used. Mares were humanely slaughtered. Internal reproductive tracts were recovered within 10 min after slaughter; ovaries of cyclic mares were separated from the rest of the tract. Mares were categorized into four groups: G1 – Follicle ≤ 13.5 mm and CL identifiable; G2 – Follicle of 13.6 to 22.5 mm and CL identifiable; G3 – Follicle of 22.6 to 31.5 mm and CL identifiable; G4 – Follicle ≥ 31.6 mm and CL difficult to identify. The follicular fluid (FF) was aspirated and stored at -80°C until use. After puncture of the FF, a fragment of the ventral portion of the follicular wall was removed to perform immunohistochemistry to localize IGF1R receptors. Free IGF1 was determined by immunoradiometric assay. INS was determined by a liquid phase radioimmunoassay. The concentration of free IGF1 and INS in FF increased with follicle growth (P < 0.05). Immunohistochemical score of IGF1R on granulosa and internal theca cells from equine follicular wall on different groups increase (P < 0.05) with follicular raise. We conclude that free IGF1 and INS concentrations in FF and IGF1R in follicular walls of mares increase with follicular growth during folliculogenesis, giving evidence of important role of IGF1 and INS in follicular development, and an interaction between both hormones in ovarian reproductive physiology. As the mare is a model research for comparative studies in follicular dynamics for consideration in women, the conclusion of this study can be applied to humans also.

Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation versus apoptosis of granulosa cells (GCs), and can influence whether small antral follicles continue their growth or undergo atresia. Transforming Growth Factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function, yet has been largely overshadowed by current interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMPs) and anti-Mullerian hormone (AMH). In the current study, effects of TGFα on bovine GC proliferation, intracellular signaling and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5mm) from bovine ovaries were aspirated and the cells were initially plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 degrees Celsius in 5% CO2 for 3-4 days. Once confluent, the cells were sub-cultured to 96-, 12- or 6-well plates in serum-free conditions (insulin 100 ng/mL; transferrin 55 ng/mL; sodium selenite 6.7 pg/mL). 24-hour treatment of bovine GCs with TGFα (10 and 100ng/mL) stimulated cell proliferation compared to control (p<0.05; n = 7 ovary pairs). Cell proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 hours of treatment, as measured by phosphorylated ERK1/2 expression (p<0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, p<0.05). Additionally, pre-exposure of the bovine GCs to TGFα (100ng/mL) failed to prevent Fas Ligand (100ng/mL)-induced apoptosis, as determined by caspase 3/7 activity (P<0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bovine GCs from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting TGFα may be incapable of promoting the persistence of follicles during the process of follicular selection and deviation.

Pig follicle development is a complex but poorly understood process involving both gonadotrophins and local ovarian factors. A series of studies sought to investigate these intrafollicular cell-to-cell interactions. Microarray analysis combined with gene ontology revealed that the oocyte, granulosa and theca cells each expressed more than 650 potential secreted factors and receptors, including members of the TGF-β, IGF1, EGF and FGF families. Using a well-defined in vivo experimental paradigm that generates follicles and oocytes of different quality, the temporal expression of several growth factors in the oocyte, granulosa and theca cells collected from sows during the recruitment and mid-selection phases, as well as the final selection of the preovulatory follicle population before and after the LH surge was studied. IGF1 expression patterns indicated its potential for modulating granulosa and theca cell function during the selection stages, and an involvement in creating differences in follicular quality between the first and second preovulatory wave post-weaning. Transient up-regulation of AREG and EREG mRNA around the LH surge, suggested their involvement in ovulation. Results of a second study investigating TGF-β superfamily expression, suggested a role for GDF9 in follicle selection through the up-regulation of TGFBR1 expression, while BMP15 could be involved in ovulation through the up-regulation of BMPR1B. Expression of angiogenesis-related genes during follicle development was also investigated. During mid-selection, ANGPT2 may allow VEGFA and similar factors to stimulate vascular development or destabilize the vasculature and cause follicular atresia, while ANGPT1 may be required in the preovulatory follicle population. Associations among the transcription factor HIF1A, VEGFA and ANGPT1, suggested interactions between the ligands in regulation of angiogenesis. Finally, the effects of the pig oocyte on cumulus cell function was assessed by co-culturing cumulus cell complexes with or without denuded oocytes isolated from large oestrogenic follicles. Presence of oocytes decreased FSHR and increased HSD3B expression, potentially stimulating progesterone while attenuating oestradiol production. In conclusion, oocytes were shown to control cumulus cell function in a way that reflects their specific environment and further evidence was obtained for a complex network of growth factors and receptors in the follicle and their involvement in regulation of pig follicle development. / Animal Science

Graduation date: 1992

A prolonged growth phase of the ovulatory follicle results in follicular aging. Whether follicular aging is detrimental or beneficial to oocyte competence is not fully known. The objective of this thesis is to investigate the effects of follicular aging on oocyte competence and granulosa cell gene expression in cattle. Four sets of experiments were designed to address the objective. The following hypotheses were tested during the course of these studies: 1) oocyte competence will improve by the longer growing phase but will be adversely affected by FSH starvation, 2) follicles that undergo superstimulation will have different gene expression than dominant follicles from a natural cycle, 3) extending the superstimulation protocol by 3 days will allow follicles to mature better and 4) markers of maturity, cellular health and survival will be turned off by FSH starvation. The objective of the first study (Chapter 3) was to determine the effects of extending the length of superstimulation and follicular aging on oocyte competence by in vitro embryo production. Multiple follicles were allowed to grow for 4 (Short FSH) or 7 days (Long FSH) under the treatment of 8 or 14 injections of FSH (at 12-hour intervals), respectively. Multiple follicles in the FSH starvation group were allowed to grow for 7 days but FSH was provided for only the first 4 days of superstimulation. Extending the duration of follicular growth by superstimulation resulted in a greater number of ≥9 mm follicles and in 2.5 more transferable embryos per animal (morulae+blastocysts) at Day 9 of in vitro embryo culture. The FSH starvation resulted in a greater proportion of poor quality oocytes lower cleavage rate and lower embryonic development. Microarray analysis was used to assess the effect of superstimulation (Chapter 4), follicular aging (Chapter 5) and FSH starvation (Chapter 6) on the gene expression profile of superstimulated granulosa cells. Gene expression of granulosa cells from the post-LH preovulatory dominant follicle was compared (Chapter 4) with those from follicles of the same status after a standard 4-day superstimulation (same protocol as Short FSH group from Chapter 3). A total of 190 genes were down-regulated and 280 genes were upregulated in the superstimulated group when compared with the reference (non-superstimulated control). Data analysis showed that superstimulated follicles are still in a growing phase compared to untreated dominant follicles (most of the upregulated genes are related to matrix remodeling due to tissue proliferation) and did not respond to LH properly (down regulation of LH gene markers). Four-day superstimulation also disturbed genes related to angiogenesis and activated oxidative stress response genes. Extending the superstimulation protocol (7 days; same protocol as Long FSH from Chapter 3) allowed more time for follicles to leave the growing stage and properly respond to LH surge (most of the upregulated genes in the Long FSH group are markers of post LH surge) when compared to the standard 4 day superstimulation protocol (Short FSH; reference group) (Chapter 5). Moreover, the follicles from Long FSH show proximity to ovulation. The continuous FSH support during the extended superstimulation protocol is crucial for follicular health since FSH starvation disturbed genes markers of oocyte quality and embryo development (Chapter 6). Granulosa cells that underwent FSH starvation do not respond to LH surge, which could be detrimental to ovulation (Chapter 6). Therefore, follicles from Short FSH are delayed in maturation and differentiation but the oocyte competence is not compromised. Extending superstimulation protocol by 3 d enhanced the ovarian response to FSH treatment and allowed more time for follicles to mature and properly respond to the LH stimulus. A period of FSH starvation after superstimulatory treatment compromised follicular health, ability to respond to LH and ovulate, oocyte quality and the fertilization process.

No description available.

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Chez la vache le développement des techniques d’induction d’ovulations multiples et de production d’embryons est limité par la variabilité inter-individuelle de la réponse ovarienne au traitement de superovulation. Chez la femme, l’Hormone anti-Müllérienne (AMH) est le meilleur marqueur endocrinien de la réponse folliculaire au traitement de stimulation ovarienne. Les objectifs de mon travail étaient d’étudier l’AMH comme marqueur endocrinien de la réponse ovarienne à un traitement gonadotrope chez la vache et d’étudier la régulation de la production d’AMH par les cellules de granulosa. Nos résultats montrent que les concentrations plasmatiques d’AMH sont prédictives de la réponse ovulatoire à un traitement gonadotrope. Au cours du cycle, les concentrations d’AMH ont un profil en 2 temps : elles diminuent entre les chaleurs et J5 à J8, puis augmentent jusqu’aux chaleurs suivantes. Pour prédire efficacement la réponse ovulatoire, les concentrations d’AMH doivent être mesurées aux chaleurs ou après J12 du cycle. L’étude de la régulation de la production in vitro d’AMH par les cellules de granulosa montre que BMP stimule tandis que FSH inhibe la production d’AMH. / In the cow, the development of multiple ovulation and embryo transfer technologies is limited by the variability between animals in ovarian responses to superovulatory treatments. In the women, the anti-Mullerian Hormone (AMH) is the best marker of follicular response to a stimulatory treatment. The objectives of my work were to study AMH as an endocrine marker of the ovulatory response to a stimulatory treatment in the cow and to study the regulation of AMH production by granulosa cells. Our results show that plasma AMH concentrations can predict the ovulatory responses to a stimulatory treatment. During the estrous cycle, AMH concentrations have a dynamic profile in 2 steps: AMH concentrations decrease between estrus and D5 to D8 and then they increase until the next estrus. To predict in optimal conditions, AMH has to be measured at the estrus time or after D12 of the estrous cycle. In vitro, the study about regulation of AMH production by granulosa cells indicates that BMP enhances whereas FSH decreases AMH production.