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.

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Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-25Bitstream added on 2014-06-13T21:00:19Z : No. of bitstreams: 1 portocarrerosanchez_c_me_botfm.pdf: 410152 bytes, checksum: 7c21ba36fbce5cc498fe42900b430f52 (MD5) / Fundação de Ensino e Pesquisa em Ciências da Saúde (FEPECS) / A taxa de sucesso de ovulação com uso do citrato de clomifene é excelente, alcançando valores entre 80% e 85%, porém a taxa de gravidez é de apenas 20% a 40%. Quando o citrato de clomifene é usado como indutor de ovulação, em mulheres portadoras de síndrome dos ovários policísticos, a literatura não define qual o momento ideal de ruptura folicular que se correlaciona com sucesso de gravidez. Identificar o diâmetro de ruptura folicular de pacientes com síndrome de ovários policísticos (SOP) estimuladas com citrato de clomifeno que melhor se correlacione com ocorrência de gravidez. Estudo corte transversal realizado entre janeiro a dezembro de 2010, que incluiu 104 pacientes com ciclos ovulatórios após uso de citrato de clomifeno, acompanhadas com ultrassonografia até determinar-se o diâmetro de ruptura folicular, que foi posteriormente correlacionado com a ocorrência ou não de gravidez. Para o estudo da associação entre as variáveis aplicou-se o teste do qui-quadrado e para as variáveis quantitativas a análise de variância seguida do método de Tukey ou teste t de Student, com valor de p <0,05. No grupo de pacientes com ruptura folicular com menos de 25 mm (n: 54) a taxa de gravidez foi de 35,2% e no grupo com ruptura folicular com mais de 25 mm (50) essa taxa foi de 34%, sem diferencia estatística significativa. Quando relacionamos aleatoriamente os diferentes diâmetros de ruptura folicular com a ocorrência de gravidez também não houve diferença significativa. O presente estudo permitiu concluir que, a taxa de gravidez de mulheres portadoras de síndrome dos ovários policísticos estimuladas com citrato de clomifene, não tem correlação com o diâmetro de ruptura folicular / The success rate of ovulation using clomiphene citrate is excellent, reaching values between 80% and 85%, but the pregnancy rate is only 20% to 40%. When clomiphene citrate is used to induce ovulation in women with polycystic ovary syndrome (PCOS), the literature does not define what the optimal timing of follicular rupture that correlates with successful pregnancy is. To identify the diameter of follicular rupture in patients with PCOS stimulated with clomiphene citrate that best correlates with the occurrence of pregnancy. Cross-sectional study conducted between January and December 2010, which included 104 patients with ovulatory cycles after the use of clomiphene citrate, followed by ultrasound to determine the diameter of follicular rupture, which was later correlated with the occurrence of pregnancy or not. To the study of the association between variables the chi-square test for quantitative variables and the analysis of variance followed by Tukey’s method or Student’s t test, with p <0.05 was applied. In patients with follicular rupture less than 25mm (n = 54) the pregnancy rate was 35.2%, in the group with follicular rupture with more than 25mm (50) this rate was 34%, no statistically significant difference was found. When relating the different diameters of randomly follicular rupture with the occurrence of pregnancy there was no significant difference. This study concluded that the pregnancy rate of women with PCOS stimulated with clomiphene citrate does not correlate with the diameter of follicular rupture

Follicular dendritic cells (FDCs) are key elements of secondary lymphoid organs where they form the stromal component of B-cell follicles. FDCs possess extensive dendritic process that trap intact antigen via Fc and complement receptors on the cell surface. The antigen is displayed to B-cells, providing a basis for selection of high affinity B cells. FDC also have important roles in facilitating the clearance of apoptotic B cells by the secretion of the opsonising factor MFGE8. It is well established that lymphotoxin signalling is required for FDC maturation but the specific details of the molecular mechanisms that regulate FDC development and differentiation are not fully understood. MicroRNAs (miRNAs) are non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level. MiRNAs bind to their target gene transcripts as part of the RNA induced silencing complex and repress translation of the target gene product. The objective of this study was to identify miRNAs that play a role in the development and function of FDCs. An in vivo murine model of FDC de-differentiation was used to provide material for miRNA analysis. By comparison of miRNA profiles from spleen tissue with FDC at different stages of de-differentiation, we would be able to obtain a miRNA signature for mature FDC. Spleens were collected at various time points over a 28 day period following transient blockade of lymphotoxin signalling. A variety of methods were used to profile the miRNAs expressed at different time points during the suppression and recovery of the FDC network. Comparison of the miRNA profiles of spleens containing mature, partially de-differentiated, and fully de-differentiated FDC identified a number of miRNAs that were differentially expressed during FDC de-differentiation. To assess the role of specific miRNAs in FDC development, the mouse FDC-like cell line, FL-YB, was used as an in vitro model system. FL-YB cells were used to perform gain-offunction and loss-of-function studies on selected miRNAs and to assess the effects of various stimuli/conditions on miRNA expression. The effects of different treatments on cell proliferation, morphology and adhesion, and on gene expression by FL-YB, were monitored. Loss-of-function studies for one of the selected miRNA (miR-100-5p) revealed a significant effect on a number of gene transcripts involved in mediation of the germinal centre response (Il-6, Tlr4, Ptgs1/2). These data indicate that miR-100-5p has a role in regulating Il-6, Tlr4 and Ptgs1/2 transcripts. None of these transcripts contain predicted target sites for miR-100-5p and so the effect of miR-100-5p on these transcripts is likely to be indirect. Further studies on these miRNA: target interactions are required to elucidate the mechanisms and biological consequences of miRNA regulation in FDCs.

<p>A series of experiments were designed to study alternative estrus synchronization and resynchronization protocols to facilitate the use of artificial insemination in cattle. </p><p>Studies were conducted to study the effects of estradiol cypionate (ECP) on follicular dynamics, time of ovulation, and pregnancy rate to timed-AI (TAI) in CIDR-based protocols. Although administration of 1 mg ECP did not result in synchronous follicular wave emergence, a dose of 0.5 mg ECP synchronized LH release and ovulation. Administration of ECP 24 h after CIDR removal resulted in acceptable pregnancy rate. However, treatment with ECP at CIDR removal resulted in acceptable pregnancy rate only if follicular wave emergence was synchronized with estradiol-17â (E-17â). </p><p>The efficacy of two estradiol preparations (5 mg of E-17â or estadiol valerate; EV) and reduced doses of EV on CL and ovarian follicular dynamics and superovulatory response were examined. When doses of 5 mg were compared, EV treatment resulted in a more variable interval to follicular wave emergence and a lower superovulatory response than E-17â. However, EV at a dose of 1 or 2 mg was efficacious in synchronizing follicle wave emergence in CIDR-treated cattle.</p><p>Pregnancy rates were compared following TAI in cattle given a new or previously used CIDR and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. Pregnancy rate following TAI did not differ between cattle treated with a new or once-used CIDR, but pregnancy rate was lower in cattle treated with one or two twice-used CIDR. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate. </p><p>The efficacy of progestins (used CIDR and MGA), and E-17â, ECP, GnRH, or progesterone treatment for resynchronization of estrus in cattle not pregnant following TAI were investigated. Progestin treatment resulted in the majority of nonpregnant heifers detected in estrus over a 4-d interval. Conception rates were higher in heifers resynchronized with a once-used CIDR than with MGA. GnRH at CIDR insertion synchronized follicular wave emergence in cows, but did not increase conception rate in heifers. E-17â at CIDR insertion (1.5 mg) and removal (0.5 mg) resulted in decreased pregnancy rate following TAI. In summary, protocols described in this thesis resulted in acceptable pregnancy rates following TAI and resynchronization of previously inseminated heifers with progestins resulted in variable estrus and pregnancy rates.</p>

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

<p>A series of experiments were designed to study alternative estrus synchronization and resynchronization protocols to facilitate the use of artificial insemination in cattle. </p><p>Studies were conducted to study the effects of estradiol cypionate (ECP) on follicular dynamics, time of ovulation, and pregnancy rate to timed-AI (TAI) in CIDR-based protocols. Although administration of 1 mg ECP did not result in synchronous follicular wave emergence, a dose of 0.5 mg ECP synchronized LH release and ovulation. Administration of ECP 24 h after CIDR removal resulted in acceptable pregnancy rate. However, treatment with ECP at CIDR removal resulted in acceptable pregnancy rate only if follicular wave emergence was synchronized with estradiol-17â (E-17â). </p><p>The efficacy of two estradiol preparations (5 mg of E-17â or estadiol valerate; EV) and reduced doses of EV on CL and ovarian follicular dynamics and superovulatory response were examined. When doses of 5 mg were compared, EV treatment resulted in a more variable interval to follicular wave emergence and a lower superovulatory response than E-17â. However, EV at a dose of 1 or 2 mg was efficacious in synchronizing follicle wave emergence in CIDR-treated cattle.</p><p>Pregnancy rates were compared following TAI in cattle given a new or previously used CIDR and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. Pregnancy rate following TAI did not differ between cattle treated with a new or once-used CIDR, but pregnancy rate was lower in cattle treated with one or two twice-used CIDR. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate. </p><p>The efficacy of progestins (used CIDR and MGA), and E-17â, ECP, GnRH, or progesterone treatment for resynchronization of estrus in cattle not pregnant following TAI were investigated. Progestin treatment resulted in the majority of nonpregnant heifers detected in estrus over a 4-d interval. Conception rates were higher in heifers resynchronized with a once-used CIDR than with MGA. GnRH at CIDR insertion synchronized follicular wave emergence in cows, but did not increase conception rate in heifers. E-17â at CIDR insertion (1.5 mg) and removal (0.5 mg) resulted in decreased pregnancy rate following TAI. In summary, protocols described in this thesis resulted in acceptable pregnancy rates following TAI and resynchronization of previously inseminated heifers with progestins resulted in variable estrus and pregnancy rates.</p>

In the ewe, one to three antral follicles emerge or grow from a pool of small antral follicles (1 to 3 mm in diameter) every 3 to 5 days and reach diameters of ¡Ý5 mm before regression or ovulation. Each follicular wave is triggered by a peak in serum concentrations of FSH. It is not clear what characteristics of an FSH peak cause follicular wave emergence and what aspects of development of a follicular wave are regulated by its preceding FSH peak.<p> In Experiment 1, we found that the amplitude of FSH peaks decreased, while basal serum FSH concentrations increased across the inter-ovulatory interval (P < 0.05). However, there were no associated changes in the growth, static or regression phases of follicular waves or the number and size of follicles in a wave. In Experiment 2, using computer-assisted quantitative echotextural analysis, we found that the numerical pixel value (NPV) for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than for waves 1 and 2 at the time of wave emergence but it decreased as follicles reached maximum follicular diameter (P < 0.05). A tendency for a similar pattern for the wall of follicles in the last wave of the cycle was also observed (P = 0.07).<p> In Experiment 3, treatment with ovine FSH (oFSH) increased the amplitude of an FSH peak by 5 to 6 fold. This treatment increased estradiol production (P < 0.05) but had little effect on other characteristics of the subsequent follicular wave. Daily injections of oFSH (Experiment 4) for four days, resulted in the occurrence of 4 discrete peaks in serum FSH concentrations. Each injection of oFSH resulted in the emergence of a new follicular wave.<p> In Experiment 5, six cyclic ewes received oFSH (0.1 ¦Ìg/kg, sc) every 6 h for 42 h, to try to give a gradual increase in the leading slope of an FSH peak. Serum FSH concentrations increased in oFSH treated ewes (P < 0.05) resulting in an additional peak between two endogenously driven FSH peaks and therefore, did not give the planned gradual leading slope to an FSH peak. Ovine FSH treatment occurred in the early growth phase of wave 1 of the inter-ovulatory interval and increased the growth rate of growing follicles in that wave, compared to control ewes (P < 0.05). This apparently induced dominance in follicles in wave 1, causing them to suppress wave emergence in response to the injected FSH. In Experiment 6, oFSH was infused constantly (1.98 ¦Ìg/ewe/h, iv, n = 6) for 60 h. Infusion of oFSH maintained serum FSH concentrations at a level similar to the zenith of a peak. This resulted in a superstimulatory effect with a peak in the mean number of large follicles on Day 2 after the start of FSH infusion (P < 0.001).<p> A hormonal milieu similar to low serum progesterone concentrations was created by treatment of ewes with prostaglandin and medroxyprogesterone acetate (MAP) sponges (Experiment 7). This treatment delayed regression of the penultimate follicular wave of a cycle. However, the delayed follicular atresia was accompanied by a greater degree of apoptosis in somatic cells of follicles growing in the penultimate wave compared to those in the final wave of the cycle, when collected one day before expected ovulation.<p> In conclusion, trends in basal serum concentrations of FSH and peaks in serum FSH concentrations, across the estrous cycle, are associated with changes in the image attributes of follicles emerging later in the estrous cycle, perhaps reflecting a greater readiness of those follicles for ovulation and formation of CL. The ovine ovary can respond to discrete peaks in serum FSH concentrations with the emergence of new follicular waves on a daily basis. This led us to conclude that follicular dominance is not evident in the ewe and peaks in serum FSH concentrations are likely to be driven by some endogenous rhythm that is unrelated to ovarian follicular secretory products. However, direct dominance can be induced by giving supplemented FSH during the growth phase of a follicle. Extended exposure of ovine ovaries to the serum concentrations of FSH found at the zenith of a peak overrides the mechanisms that recruit follicles into a wave and induces a superovulatory response in cyclic ewes. Finally, an increase in the incidence of apoptosis occurs in antral follicles in sheep that have an extended lifespan, prior to any morphological changes detectable by ultrasonography. This would seem to cause decreased follicular viability and lowered fertility of the oocytes that the follicles contain.

Follicular lymphoma (FL) is an attractive model for discovering biomarkers and elucidating mechanisms of tumour progression. We hypothesized that alterations in the expression of proteins with known roles in cancer biology and hematological cells might correlate with clinical outcome and thereby shed light on biological mechanisms. Sections from a tissue microarray (TMA) containing FL samples from 67 patients were immunostained for candidate biomarkers, including p53, p16INK4a, Bcl-2, Bcl-6, MUM1, PML, phospho-ERK, and p27Kip1. The Kaplan-Meier method and log-rank test were used to identify markers that correlate significantly (p<0.05) with overall survival (OS). The chi-squared or Fisher exact test were used to examine associations between histological markers and baseline clinical features, including the Follicular Lymphoma International Prognostic Index (FLIPI) score. Expression of p16INK4a or p53, or absent CD10 expression correlated with poor survival. Patients with p16INK4a-negative tumours had a median OS of 13.4 years compared to 8.3 years for those with p16INK4a-positive tumours (p=0.006). Expression of p16INK4a was significantly associated with low hemoglobin, elevated serum lactate dehydrogenase (LDH), high histological grade, high cell proliferation index, presence of associated diffuse large B-cell lymphoma (DLBCL) and high-risk FLIPI classification. Our observation of a positive association between p16INK4a expression and indicators of tumour aggressiveness is novel and perhaps surprising since loss of the INK4a tumour suppressor gene is one of the most frequently observed lesions in human cancers, including lymphoma. Expression of p16INK4a may be part of a cellular response to unidentified pro-mitotic mutations, such as deleterious mutations of the RB tumour suppressor gene, associated with more aggressive instances of FL. Immunostaining FL diagnostic biopsies for expression of p16INK4a may serve as an informative prognostic biomarker to aid clinicians managing FL patients. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-07-04 15:55:16.121