Ko Nga Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-131). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiv / List of Tables --- p.xvii / Symbols and Abbreviations --- p.xviii / Scientific Names --- p.xxi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.5 / Chapter 1.1.3.1 --- Hypothalamic regulators (GnRH) --- p.5 / Chapter 1.1.3.2 --- Endocrine regulators from gonads (steroids) --- p.7 / Chapter 1.1.3.3 --- Paracrine regulators (activin) --- p.9 / Chapter 1.1.4 --- Promoter analysis --- p.9 / Chapter 1.2 --- Activin Family of Growth Factors --- p.12 / Chapter 1.2.1 --- Activin --- p.12 / Chapter 1.2.1.1 --- Structure --- p.12 / Chapter 1.2.1.2 --- Function --- p.13 / Chapter 1.2.1.3 --- Signaling --- p.15 / Chapter 1.2.2 --- Follistatin --- p.16 / Chapter 1.2.2.1 --- Structure --- p.16 / Chapter 1.2.2.2 --- Function --- p.17 / Chapter 1.3 --- Objectives --- p.18 / Chapter Chapter 2 --- Establishment and Characterization of Stable LβT2 Cell Lines Containing and Expressing SEAP Driven by the Goldfish FSHβ Promoter / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.31 / Chapter 2.2.1 --- Construction of expression plasmid --- p.31 / Chapter 2.2.2 --- Cell culture --- p.32 / Chapter 2.2.3 --- Cotransfection of LβT2 cells --- p.32 / Chapter 2.2.4 --- G418 selection of transfected LpT2 cells --- p.33 / Chapter 2.2.5 --- SEAP reporter gene assay --- p.33 / Chapter 2.2.6 --- Cloning of pSEAP/gfFSHβ promoter and pBK- CMV-transfected LβT2 cells by limited dilution --- p.34 / Chapter 2.2.7 --- Extraction of genomic DNA --- p.34 / Chapter 2.2.8 --- Isolation of total RNA --- p.35 / Chapter 2.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Optimization of G418 concentration for selection --- p.36 / Chapter 2.3.2 --- Expression of SEAP reporter gene by pSEAP/gfFSHβ promoter and pBK-CMV-transfected LβT2 cells --- p.37 / Chapter 2.3.3 --- Establishment of LβT2 cell lines that contain a functional gfFSHp promoter --- p.37 / Chapter 2.3.4 --- Characterization of LβT2#23 that contains a functional gfFSHβ promoter --- p.38 / Chapter 2.4 --- Discussion --- p.39 / Chapter Chapter 3 --- Hormonal Regulation of Goldfish Follicle-Stimulating Hormone β (FSHβ) Promoter Activity in LpT2#23 Cells / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Cell culture --- p.55 / Chapter 3.2.2 --- Drug treatment --- p.56 / Chapter 3.2.3 --- SEAP reporter gene assay --- p.56 / Chapter 3.2.4 --- Isolation of total RNA --- p.57 / Chapter 3.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.57 / Chapter 3.2.6 --- Data analysis --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Effects of goldfish activin on FSHβ promoter --- p.59 / Chapter 3.3.2 --- Blockade of activin effects by follistatin --- p.59 / Chapter 3.3.3 --- Effects of different hormones and steroids on FSHβ promoter --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- Promoter Analysis for the Activin Responsive Element (ARE) in the Goldfish Follicle-Stimulating Hormone β (FSHβ) Gene / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Materials and Methods --- p.74 / Chapter 4.2.1 --- Generation of SEAP reporter plasmids containing the gfFSHβ promoter of different lengths --- p.74 / Chapter 4.2.2 --- PCR screening and restriction analysis --- p.75 / Chapter 4.2.3 --- Midiprep --- p.76 / Chapter 4.2.4 --- Cell culture --- p.77 / Chapter 4.2.5 --- Transfection of the pSEAP/gfFSHβ promoter constructs into LβT2 cells --- p.77 / Chapter 4.2.6 --- Activin treatment --- p.77 / Chapter 4.2.7 --- SEAP assay --- p.78 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- Subcloning of the gfFSHβ promoter of decreasing length into SEAP reporter vector --- p.78 / Chapter 4.3.2 --- Activin stimulation of the pSEAP/gfFSHβ promoter constucts in LβT2 cells --- p.79 / Chapter 4.4 --- Discussion --- p.80 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- Overview --- p.92 / Chapter 5.2 --- Contribution of the present research --- p.95 / Chapter 5.2.1 --- Establishment of stable LβT2 cell lines containing and expressing SEAP driven by gfFSHβ promoter --- p.95 / Chapter 5.2.2 --- Hormonal regulation of the gfFSHβ promoterin LβT2#23 cells --- p.95 / Chapter 5.2.3 --- Identification of the activin responsive element (ARE) on the gfFSHβ promoter --- p.96 / Chapter 5.3 --- Future research direction --- p.96 / References --- p.98

Cheng Fu Yip Gheorghe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 97-116). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract (in English) --- p.III / Abstract (in Chinese) --- p.V / Table of Content --- p.VII / Symbols and Abbreviations --- p.XII / Scientific Names --- p.XIV / List of Tables --- p.XV / List of Figures --- p.XVI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropin / Chapter 1.1.1 --- Structure --- p.2 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.4 / Chapter 1.1.3.1 --- Neuroendocrine and endocrine regulation of GTHs --- p.4 / Chapter 1.1.3.1.1 --- Hypothalamic neuropeptides and neurotransmitters --- p.6 / Chapter 1.1.3.1.2 --- Gonadal steroids --- p.7 / Chapter 1.1.3.2 --- Paracrine regulation of GTH --- p.8 / Chapter 1.2 --- Activin / Chapter 1.2.1 --- Structure --- p.8 / Chapter 1.2.2 --- Function --- p.9 / Chapter 1.2.3 --- Regulation of activin activity --- p.12 / Chapter 1.2.3.1 --- Intracellular blockade of activin signaling by Smad7 --- p.12 / Chapter 1.2.3.2 --- Extracellular control of activin access --- p.13 / Chapter 1.2.3.2.1 --- Inhibin --- p.13 / Chapter 1.2.3.2.2 --- Activin-binding protein --- p.14 / Chapter 1.3 --- Follistatin / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.3.3 --- Regulation in the pituitary --- p.19 / Chapter 1.4 --- Objectives of the Present Study --- p.20 / Chapter Chapter 2 --- Cloning and Recombinant Production of Goldfish Follistatin / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Reagents --- p.26 / Chapter 2.2.2 --- Animal --- p.26 / Chapter 2.2.3 --- Extraction of total RNA and reverse transcription --- p.27 / Chapter 2.2.4 --- Cloning of full-length cDNA encoding goldfish follistatin --- p.27 / Chapter 2.2.5 --- Sequencing of the cDNA --- p.29 / Chapter 2.2.6 --- Distribution of follistatin mRNA in different tissues --- p.29 / Chapter 2.2.7 --- Production of rgFS --- p.30 / Chapter 2.2.8 --- RT-PCR of the rgFS-positive clones --- p.34 / Chapter 2.2.9 --- Extraction of genomic DNA from rgFS-positive clones --- p.34 / Chapter 2.2.10 --- Functional analysis of rgFS --- p.35 / Chapter 2.2.11 --- Data Analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Cloning and sequence analysis of goldfish follistatin --- p.37 / Chapter 2.3.2 --- Tissue distribution of follistatin mRNA in the goldfish --- p.39 / Chapter 2.3.3 --- Production and bioassay of rgFS --- p.43 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Function and Regulation of Follistatin in the Goldfish Pituitary; Evidence for an Intrinsic Activin/Follistatin Regulatory Feedback Loop / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Reagents --- p.57 / Chapter 3.2.2 --- Animals --- p.57 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.57 / Chapter 3.2.4 --- RNA extraction and reverse transcription --- p.58 / Chapter 3.2.5 --- Ovariectomy on pituitary follistatin expression --- p.5 9 / Chapter 3.2.6 --- Seasonal expression profile of follistatin --- p.59 / Chapter 3.2.7 --- Validation of semi-quantitative RT-PCR assays --- p.61 / Chapter 3.2.8 --- Real-time PCR for assay on follistatin and β-actin expression --- p.61 / Chapter 3.2.9 --- Data analysis --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Expression of follistatin in the goldfish pituitary --- p.64 / Chapter 3.3.2 --- Validation of semi-quantitative RT-PCR assay --- p.64 / Chapter 3.3.3 --- Activin regulation of pituitary follistatin --- p.64 / Chapter 3.3.4 --- Effects of sex steroids on pituitary follistatin expression --- p.69 / Chapter 3.3.5 --- Effect of GnRH on follistatin expression in the pituitary --- p.74 / Chapter 3.3.6 --- Effect of intracellular cAMP level on pituitary follistatin expression --- p.74 / Chapter 3.3.7 --- Seasonal variation profile of goldfish pituitary follistatin --- p.78 / Chapter 3.4 --- Discussion --- p.78 / Chapter Chapter 4 --- General Discussion / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- Contribution of the Present Study / Chapter 4.2.1 --- Cloning of full-length goldfish follistatin cDNA --- p.91 / Chapter 4.2.2 --- Establishment of stable cell line for expression of rgFS --- p.92 / Chapter 4.2.3 --- Evidence for the presence of intrinsic feedback loop of activin in the goldfish pituitary --- p.92 / Chapter 4.2.4 --- Modulation of follistatin expression in the pituitary by sex steroids --- p.93 / Chapter 4.2.5 --- Conclusions --- p.93 / Chapter 4.3 --- Future Prospects / Chapter 4.3.1 --- Production of rgFS --- p.95 / Chapter 4.3.2 --- Regulation of activin-follistatin system in the pituitary --- p.95 / Reference --- p.96