Tse, Chung Kwan Anna. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 86-105). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgement --- p.v / Table of content --- p.vii / List of figures and tables --- p.xi / Symbols and abbreviation --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Folliculogenesis --- p.1 / Chapter 1.1.1 --- In Mammals --- p.1 / Chapter 1.1.2 --- In Teleosts --- p.2 / Chapter 1.1.2.1 --- Vitellogenesis --- p.3 / Chapter 1.1.2.2 --- Oocyte Maturation --- p.4 / Chapter 1.1.3 --- Oocyte Control of Folliculogenesis --- p.5 / Chapter 1.2 --- Epidermal Growth Factor Family --- p.7 / Chapter 1.2.1 --- Epidermal Growth Factor Ligand Family --- p.7 / Chapter 1.2.1.1 --- Discovery --- p.7 / Chapter 1.2.1.2 --- Structure --- p.8 / Chapter 1.2.1.3 --- Shedding --- p.8 / Chapter 1.2.1.4 --- Functions --- p.9 / Chapter 1.2.2 --- Epidermal Growth Factor Receptor Family --- p.10 / Chapter 1.2.2.1 --- Structure --- p.10 / Chapter 1.2.2.2 --- Ligand Binding and Dimerization --- p.11 / Chapter 1.2.2.3 --- Signaling and Internalization --- p.12 / Chapter 1.2.3 --- EGF Family in Reproduction --- p.13 / Chapter 1.2.3.1 --- Reproductive Function of EGF Family --- p.13 / Chapter 1.2.3.2 --- EGF Network as Downstream Mediator of Gonadotropins --- p.15 / Chapter 1.3 --- Objectives of the Present Study --- p.18 / Chapter Chapter 2 --- Spatiotemporal Expression of the EGF Family in Zebrafish Ovary / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Chemicals --- p.22 / Chapter 2.2.2 --- Animals --- p.22 / Chapter 2.2.3 --- Isolation of Different Tissues and Ovarian Follicles --- p.22 / Chapter 2.2.4 --- Separation of Oocyte and Follicle layers --- p.23 / Chapter 2.2.5 --- Embryo Collection --- p.23 / Chapter 2.2.6 --- Tyrphostin AG 1478 Treatment of Embryos --- p.23 / Chapter 2.2.7 --- Total RNA Isolation and Reverse Transcription --- p.24 / Chapter 2.2.8 --- Semi-quantitative and Real-time Polymerase Chain Reaction (PCR) --- p.24 / Chapter 2.2.9 --- Data Analysis --- p.25 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Validation of Semi-quantitative PCR Quantification --- p.27 / Chapter 2.3.2 --- Tissue Distribution of the EGF Family --- p.27 / Chapter 2.3.3 --- Spatial Expression of the EGF and the Activin Families within the Follicles --- p.27 / Chapter 2.3.4 --- Temporal Expression of the EGF Family in Folliculogenesis --- p.29 / Chapter 2.3.5 --- Temporal Expression of the EGF Family in Embryogenesis --- p.29 / Chapter 2.3.6 --- Effect of Blocking EGFR on Embryonic Development --- p.30 / Chapter 2.4 --- Discussion --- p.31 / Chapter Chapter 3 --- Regulation of the EGF Family in Cultured Follicle Cells and Their Effects on the Expression of Activin Subunits / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Chemicals --- p.58 / Chapter 3.2.2 --- Primary Follicle Culture --- p.58 / Chapter 3.2.3 --- Ovarian Fragment Incubation --- p.58 / Chapter 3.2.4 --- Total RNA Isolation and Reverse Transcription --- p.58 / Chapter 3.2.5 --- Semi-quantitative and Real-time Polymerase Chain Reaction (PCR) --- p.59 / Chapter 3.2.6 --- Microinjection --- p.59 / Chapter 3.2.7 --- Data analysis --- p.59 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Expression of the EGF Family in Cultured Follicle Cells --- p.61 / Chapter 3.3.2 --- Effects of EGF on the EGF Family Expression in Cultured Follicle Cells --- p.61 / Chapter 3.3.3 --- Effects of BTC and HB-EGF on the Expression of EGF Ligands in Cultured Follicle Cells --- p.61 / Chapter 3.3.4 --- Effect of EGF Ligands on the Expression of Activin Subunits in Cultured Follicle Cells --- p.62 / Chapter 3.3.5 --- Effects of Actinomycin D on Ovarian Fragments --- p.62 / Chapter 3.3.6 --- Effects of Microinjecting Anti-EGF Morpholino on Full Grown Follicles --- p.62 / Chapter 3.4 --- Discussion --- p.64 / Chapter Chapter 4 --- General Discussion --- p.81 / References --- p.86

published_or_final_version / Paediatrics / Master / Master of Philosophy

No description available.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 220-222) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 220-222) Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 79-99).

published_or_final_version / Paediatrics / Master / Master of Philosophy

Epidermal growth factor (EGF) markedly stimulates cell proliferation in a variety of mammalian systems. For this reason, EGF and factors related to EGF were examined for a possible role in the promotion and maintenance of the uncontrolled growth state that is a characteristic of malignant neoplasias. Phorbol ester tumor promoters, compounds that are capable of promoting tumors in the mouse skin carcinogenesis assay, act synergistically with EGF to stimulate DNA synthesis in cultured fibroblasts despite an inhibitory effect on the binding of EGF to its cellular receptor. Sparing of EGF degradation in combination with recovery of EGF binding was postulated to be responsible for the increased role of DNA synthesis in cells exposed to the phorbol esters. The hypothesis is presented that the hyperplasiogenic component of tumor promotion may be mediated, at least in part, by local alterations in growth factor levels. Recent evidence suggests the existance of a family of molecules related to EGF as defined by the ability to bind to the EGF receptor. These factors (transforming growth factors) appear to be responsible for the phenotypic alterations characteristic of transformed cells. Using a radioreceptor assay, two EGF-like factors were isolated from mouse submaxillary gland. These factors were not transforming growth factors and appeared to be modified EGF molecules. Two EGF-like factors, molecular weight 27,000 and 13,000, were identified in medium conditioned by Rous sarcoma virus (RSV)-transformed cells and were shown to possess the characteristics of a transforming growth factor. In addition, rat fetus extracts contained a 55,000 molecular weight EGF-like factor with the properties of a transforming growth factor. The EGF-effector system may therefore play an important role in embryonic development and in the maintenance of the neoplastic phenotype. The difference in the molecular weights of the RSV-factor and the fetus factor indicates that there are numerous members of the class of EGF-like molecules, and that RSV-transformation probably does not induce the re-expression of a fetal growth stimulatory factor. The results of these experiments suggest that EGF and EGF-like factors are biologically important growth-stimulating molecules that must be tightly regulated to maintain normal physiological conditions.

Modifications of cellular responses to epidermal growth factor (EGF) induced by tumor promoters and anti-promoters were examined. The effect of the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) EGF binding was studied using mouse epidermal cells. Initially, TPA decreased EGF binding. However, when cells were incubated continuously in TPA plus a low concentration of EGF, more EGF bound to TPA-treated cells than to control cells. It was shown that the effects of TPA were partially reversible and that the greater amount of EGF bound to TPA-treated cells over controls after long-term incubation was due to larger amounts of whole EGF in the media of TPA-treated cells when cells have regained their ability to bind EGF. The ability of TPA to induce DNA synthesis synergistically with EGF may depend on the transient sparing of EGF from degradation and subsequent binding of the spared EGF. Fluocinolone acetonide (FA) and retinoic acid (RA) are potent anti-promoters able to induce increased EGF binding. The possibility that these compounds exerted their anti-promoting activities through offsetting TPA-induced EGF binding alterations was studied. Rat-1 fibroblasts were used to examine the effect of FA on TPA-mediated changes in EGF binding and EGF-induced ornithine decarboxylase (ODC) activity and DNA synthesis. Pretreatment with FA caused increased EGF binding and decreased ODC activity and DNA synthesis stimulated by high or low EGF concentrations. The glucocorticoid lowered ODC and DNA synthesis induced by EGF in combination with TPA to levels closer to control (EGF alone) levels. These data indicated that the anti-hyperplasiagenic effect of FA may be partially mediated through the EGF receptor. The effects of RA on EGF binding and EGF-induced cellular responses were examined in Rat-1 and Swiss 3T3 fibroblasts. Pretreatment with RA resulted in increased EGF binding to 3T3 cells only. However, RA treatment was able to enhance ODC activity in both cell lines. Retinoic acid binding protein was detected only in Rat-1 cells. It was therefore unlikely that the effects of RA on ODC induction were mediated by either altered EGF binding or the presence of CRABP. Experiments with 3T3 cells demonstrated that TPA alone was able to induce ODC activity. It is therefore possible that TPA exerts part of its tumor promoting action through the EGF receptor, but other sites of action also contribute to its promoting properties.