Yuen Chi Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 84-106). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.2 --- Gonadotropins (GTHs) --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Function --- p.5 / Chapter 1.2.3 --- Regulation --- p.7 / Chapter 1.2.3.1 --- Neuroendocrine hypothalamic regulators --- p.9 / Chapter 1.2.3.1.1 --- Gonadotropin-releasing hormone (GnRH) --- p.9 / Chapter 1.2.3.1.2 --- Dopamine (DA) --- p.11 / Chapter 1.2.3.2 --- Endocrine regulators from the gonads --- p.12 / Chapter 1.2.3.2.1 --- Gonadal steroids (T and E2) --- p.12 / Chapter 1.2.3.2.2 --- Negative steroid effect on pituitary GTH regulation --- p.12 / Chapter 1.2.3.2.3 --- Positive steroid effect on pituitary GTH regulation --- p.13 / Chapter 1.2.3.3 --- Paracrine regulators from within the pituitary --- p.14 / Chapter 1.3 --- Activin --- p.14 / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.4 --- Follistatin (FS) --- p.17 / Chapter 1.4.1 --- Structure --- p.17 / Chapter 1.4.2 --- Function --- p.19 / Chapter 1.5 --- Temporal Variations in the GTH Expressional and Releasing Profile and Sex Steroid Level in the Goldfish --- p.19 / Chapter 1.5.1 --- Hormone changes during annual cycle --- p.20 / Chapter 1.5.2 --- Hormone changes during ovulatory cycle --- p.21 / Chapter 1.6 --- Objectives of the Present Study --- p.23 / Chapter Chapter 2 --- "Effects of Dopamine on the Expression of Gonadotropin (GTH) Subunits in the Dispersed Pituitary Cells of the Goldfish, Carassius auratus" --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Materials --- p.27 / Chapter 2.2.2 --- Primary culture of dispersed goldfish pituitary cells --- p.28 / Chapter 2.2.3 --- mRNA analysis --- p.29 / Chapter 2.2.4 --- Data analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Effects of DA on GTH-Iβ and GTH-IIβ expression --- p.30 / Chapter 2.3.2 --- Effects of DA D1 and D2 agonists on GTH-Iβ expression --- p.33 / Chapter 2.3.3 --- Effects of DA D1 and D2 antagonists on DA- inhibited GTH-Iβ expression --- p.33 / Chapter 2.3.4 --- Effects of α-adrenergic agonists on GTH-Iβ expression --- p.33 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Seasonal Variation of Activin-regulated Goldfish Pituitary GTH-Ip and GTH-IIβ Expression and Evidence for the Involvement of Gonadal Steroids --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Materials --- p.42 / Chapter 3.2.2 --- Gonadectomy of the goldfish --- p.42 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.43 / Chapter 3.2.4 --- mRNA analysis --- p.43 / Chapter 3.2.5 --- Data analysis --- p.44 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1 --- Effects of goldfish activin B on the expression of GTH-Iβ and GTH-IIβ --- p.44 / Chapter 3.3.2 --- Seasonal variation of activin-regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.3 --- Effects of gonadectomy on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.4 --- Effects of sex steroids on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ in vitro --- p.50 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Evidence for the Autocrine/Paracrine Regulation of Gonadotropin Expression by Activin in the Goldfish Pituitary --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.63 / Chapter 4.2.1 --- RNA isolation --- p.63 / Chapter 4.2.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.63 / Chapter 4.2.3 --- Primary culture of goldfish pituitary cells --- p.63 / Chapter 4.2.4 --- Slot-blot analysis --- p.64 / Chapter 4.2.5 --- Data analysis --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Expression of activin βB subunit and activin type IEB receptor in the goldfish pituitary --- p.65 / Chapter 4.3.2 --- Effects of human activin A on goldfish GTH-Iβ and GTH-IIβ expression --- p.65 / Chapter 4.3.3 --- Effects of follistatin on basal and activin- regulated GTH-Iβ and GTH-IIβ expression --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter Chapter 5 --- General Discussion --- p.77 / Chapter 5.1 --- Overview --- p.77 / Chapter 5.2 --- Contribution of the Present Study --- p.78 / Chapter 5.2.1 --- Dopamine as a potential neuroendocrine regulator in the differential regulation of GTH-Iβ and GTH-IIβ expression --- p.78 / Chapter 5.2.2 --- Seasonal variation of the effects of activin on GTH-Iβ and GTH-IIβ expression --- p.79 / Chapter 5.2.3 --- Autocrine/paracrine regulation of GTH expression by activin --- p.79 / Chapter 5.3 --- Future Prospects --- p.81 / References --- p.84

Lin Sze-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 111-127). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / Abbreviations --- p.x / Scientific Names --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.1 / Chapter 1.1.3 --- Regulation --- p.2 / Chapter 1.1.3.1 --- Gonadotropin-releasing hormone (GnRH) --- p.3 / Chapter 1.1.3.2 --- Dopamine --- p.4 / Chapter 1.1.3.3 --- Sex steroids --- p.5 / Chapter 1.1.3.3.1 --- Functions --- p.5 / Chapter 1.1.3.3.2 --- Working mechanism´ؤEstrogen signaling pathway --- p.7 / Chapter 1.1.3.4 --- Gonadal peptides --- p.9 / Chapter 1.1.3.4.1 --- Functions --- p.9 / Chapter 1.1.3.4.2 --- Working mechanism一Activin signaling pathway --- p.11 / Chapter 1.2 --- Transcriptional regulation of pituitary gonadotropin subunit genes at the promoter level --- p.13 / Chapter 1.2.1 --- Transcriptional regulation of mammalian glycoprotein a subunits --- p.13 / Chapter 1.2.1.1 --- GnRH --- p.14 / Chapter 1.2.1.2 --- Activin --- p.15 / Chapter 1.2.1.3 --- Steroids --- p.15 / Chapter 1.2.2 --- Transcriptional regulation of mammalian FSHβ and LHβ subunits --- p.16 / Chapter 1.2.2.1 --- Regulation of LHβ expression by GnRH --- p.17 / Chapter 1.2.2.1.1 --- Roles of SP-1 binding sites on LHβ promoter --- p.17 / Chapter 1.2.2.1.2 --- Effect of SF-1 on LHp expression --- p.17 / Chapter 1.2.2.1.3 --- Effect of Egr-1 on LHp expression --- p.18 / Chapter 1.2.2.1.4 --- "Synergistic effect ofSP-1, SF-1 and Egr-1 on LHp expression." --- p.18 / Chapter 1.2.2.1.5 --- Effect of Pitx-1 on LHβ expression --- p.19 / Chapter 1.2.2.1.6 --- "Effect of SF-1, Egr-1 and Pitx-1 on LHβ expression of other mammalian counterparts" --- p.19 / Chapter 1.2.2.1.7 --- Effect of other transcription factors on mammalian LHβ expression --- p.19 / Chapter 1.2.2.2 --- Regulation of LHβ expression by steroids and activin --- p.20 / Chapter 1.2.2.3 --- Regulation of FSHβ expression by activin and GnRH --- p.20 / Chapter 1.2.2.4 --- Regulation of FSHβ expression by steroids --- p.21 / Chapter 1.2.2.5 --- Regulation of FSHβ expression by other transcription factors --- p.22 / Chapter 1.2.3 --- Transcriptional regulation of fish FSHβ and LHβ subunits --- p.22 / Chapter 1.3 --- The project objectives and long-term significance --- p.24 / Chapter CHAPTER 2 --- CLONING OF ZEBRAFISH FSHB AND LHB PROMOTERS. --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Animals --- p.27 / Chapter 2.2.3 --- Isolation of genomic DNA --- p.28 / Chapter 2.2.4 --- Cloning of promoters of zebrafish FSHβ and LHβ from the genomic DNA --- p.28 / Chapter 2.2.5 --- Construction of the reporter plasmids containing zebrafish FSHβ and LHβ promoters --- p.30 / Chapter 2.2.6 --- Cell culture and transient transfection --- p.31 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.32 / Chapter 2.2.8 --- β-galactosidase reporter gene assay --- p.32 / Chapter 2.2.9 --- Data analysis --- p.33 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Cloning of zebrafish FSHβ and LHβ promoters --- p.33 / Chapter 2.3.2 --- Sequence characterization of zebrafish FSHβ and LHβ promoters --- p.34 / Chapter 2.3.3 --- Basal FSHp and LHβ promoter activities in LβT2 cells --- p.35 / Chapter 2.4 --- Discussion --- p.36 / Chapter CHAPTER 3 --- ROLES OF ACTIVIN/SMADS AND ESTROGEN/ERS IN THE REGULATION OF ZEBRAFISH FSHB AND LHB PROMOTER ACTIVITY --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Chemicals --- p.56 / Chapter 3.2.2 --- Animals --- p.56 / Chapter 3.2.3 --- Isolation of total RNA --- p.57 / Chapter 3.2.4 --- Rapid amplification of full-length cDNA (RACE) --- p.57 / Chapter 3.2.5 --- Construction of expression plasmids --- p.57 / Chapter 3.2.6 --- cell culture and transient transfection --- p.59 / Chapter 3.2.7 --- SEAP reporter gene assay --- p.59 / Chapter 3.2.8 --- p-galactosidase reporter gene assay --- p.59 / Chapter 3.2.9 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- Cloning and sequence characterization of zebrafish Smad 4 (zfSmad 4) --- p.60 / Chapter 3.3.2 --- Smads regulate FSHβ transcription in LβT2 cells --- p.61 / Chapter 3.3.3 --- Smads regulate LHβ transcription in LPβT2 cells --- p.61 / Chapter 3.3.4 --- Functionality of the two forms of Smad 4 cloned --- p.62 / Chapter 3.3.5 --- Estrogen and ERs regulate zJFSHβ transcription in LβT2 cells --- p.63 / Chapter 3.3.6 --- Estrogen and ERs regulate zfLHβ transcription in LβT2 cells --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter CHAPTER 4 --- PROMOTER ANALYSIS FOR SMAD RESPONSIVE ELEMENT AND ESTROGEN RESPONSIVE ELEMENT IN ZEBRAFISH FSHB AND LHB PROMOTERS --- p.82 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials and Methods --- p.85 / Chapter 4.2.1 --- Chemicals and animals --- p.85 / Chapter 4.2.2 --- Construction of SEAP reporter plasmids containing different lengths of zfFSHβ promoter --- p.85 / Chapter 4.2.3 --- Construction of SEAP reporter plasmids containing different lengths of zfLHβ promoter --- p.85 / Chapter 4.2.4 --- Site-directed mutagenesis --- p.86 / Chapter 4.2.5 --- cell culture and transient transfection --- p.87 / Chapter 4.2.6 --- SEAP reporter gene assay --- p.87 / Chapter 4.2.7 --- P-galactosidase reporter gene assay --- p.87 / Chapter 4.2.8 --- Data analysis --- p.88 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Localization of Smad-responsive element (SRE) on zfFSHβ promoter --- p.88 / Chapter 4.3.2 --- Localization of estrogen-responsive element (ERE) on zfLHβ promoter --- p.89 / Chapter 4.3.3 --- Localization of estrogen-responsive element (ERE) on zfFSHβ promoter --- p.90 / Chapter 4.3.4 --- Confirmation of SRE by site-directed mutagenesis --- p.91 / Chapter 4.3.5 --- Confirmation of ERE by site-directed mutagenesis --- p.92 / Chapter 4.4 --- Discussion --- p.92 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.106 / Chapter 5.1 --- Overview --- p.106 / Chapter 5.2 --- Contribution of the present research --- p.107 / Chapter 5.3 --- Future research direction --- p.108 / REFERENCE: --- p.111