Yam Kwan Mei. / Thesis submitted in: August 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-129). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xv / Symbols and Abbreviations --- p.xviii / Scientific names --- p.xx / 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.3 / Chapter 1.1.3 --- Regulation --- p.9 / Chapter 1.1.3.1 --- GnRH --- p.9 / Chapter 1.1.3.2 --- Steroids --- p.11 / Chapter 1.1.3.3 --- Activin --- p.12 / Chapter 1.2 --- Activin Family of Growth Factors --- p.14 / Chapter 1.2.1 --- Structure --- p.14 / Chapter 1.2.2 --- Function --- p.17 / Chapter 1.3 --- Objectives --- p.22 / Chapter Chapter 2 --- Cloning of Goldfish Activin βA cDNA and the Expression of Its mRNA in Gonadal and Non-gonadal Tissues --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Cloning of goldfish activin βA cDNA --- p.26 / Chapter 2.2.1.1 --- Cloning of the 5' and 3' cDNA ends --- p.26 / Chapter 2.2.1.2 --- Extension of the 5' and 3' fragments --- p.28 / Chapter 2.2.2 --- Sequencing of the cDNA --- p.28 / Chapter 2.2.2.1 --- Generation of pKS/GactβA constructs with insert in different orientations --- p.28 / Chapter 2.2.2.2 --- Generation of overlapping subclones --- p.29 / Chapter 2.2.2.3 --- Cycle sequencing --- p.30 / Chapter 2.2.2.4 --- Sequence analyses --- p.30 / Chapter 2.2.3 --- Isolation of total and messenger RNA --- p.30 / Chapter 2.2.3.1 --- Isolation of total RNA --- p.30 / Chapter 2.2.3.2 --- Isolation of messenger RNA --- p.31 / Chapter 2.2.4 --- Southern blot analysis --- p.32 / Chapter 2.2.5 --- Northern blot analysis --- p.33 / Chapter 2.2.6 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Cloning and sequence analysis of activin β A cDNA --- p.35 / Chapter 2.3.2 --- Distribution of activin βA mRNA in different tissues --- p.49 / Chapter 2.4 --- Discussion --- p.53 / Chapter Chapter 3 --- Establishment and Characterization of Stable Cell Lines for the Recombinant Production of Goldfish Activin A --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Construction of expression plasmid --- p.60 / Chapter 3.2.2 --- Cell culture --- p.62 / Chapter 3.2.3 --- Transfection of CHO cells --- p.62 / Chapter 3.2.4 --- G418 selection of transfected CHO cells --- p.62 / Chapter 3.2.5 --- Activin bioassay (EDF-assay) --- p.63 / Chapter 3.2.6 --- Cloning of pBK/GactβA-transfected CHO cells by limited dilution --- p.63 / Chapter 3.2.7 --- Isolation of total RNA --- p.65 / Chapter 3.2.8 --- Northern blot analysis --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Optimization of G418 concentration for selection --- p.66 / Chapter 3.3.2 --- Expression of activin activity by pBK/GactpβA- transfected CHO cells --- p.67 / Chapter 3.3.3 --- Establishment and characterization of CHO cell lines that stably produce recombinant goldfish activin A --- p.67 / Chapter 3.4 --- Discussion --- p.73 / Chapter Chapter 4 --- Differential Regulation of Goldfish Gonadotropin (GTH-Iβ and GTH-IIβ) Gene Expression by Recombinant Goldfish Activin --- p.79 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.82 / Chapter 4.2.1 --- Animals --- p.82 / Chapter 4.2.2 --- Drug treatment --- p.83 / Chapter 4.2.3 --- Primary culture of dispersed pituitary cells --- p.84 / Chapter 4.2.4 --- Southern blot analysis --- p.85 / Chapter 4.2.5 --- Isolation of total RNA --- p.86 / Chapter 4.2.6 --- Northern blot analysis --- p.86 / Chapter 4.2.7 --- Dot blot analysis --- p.87 / Chapter 4.2.8 --- Data analyses --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Probe specificity --- p.88 / Chapter 4.3.2 --- Effects of goldfish activin on pituitary GTH-Iβ and -IIβ mRNA expression --- p.88 / Chapter 4.3.3 --- Blockade of activin effects by follistatin --- p.92 / Chapter 4.4 --- Discussion --- p.96 / Chapter Chapter 5 --- General Discussion --- p.101 / Chapter 5.1 --- Overview --- p.101 / Chapter 5.2 --- Contribution of the Present Research --- p.103 / Chapter 5.2.1 --- Cloning of full-length goldfish activin βA cDNA --- p.103 / Chapter 5.2.2 --- Establishment of stable cell lines for the recombinant production of goldfish activin A --- p.104 / Chapter 5.2.3 --- Differential regulation of goldfish gonadotropin (GTH-Iβ and GTH-IIβ) gene expression by recombinant goldfish activin --- p.105 / Chapter 5.3 --- Future Research Direction --- p.107 / Chapter 5.3.1 --- Activin studies --- p.107 / Chapter 5.3.2 --- Gonadotropin studies --- p.107 / References --- p.108