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