Thesis (Ph. D.)--Murdoch University, 2003. / Title from PDF title page (viewed Mar. 6, 2005). Includes bibliographical references (p. 195-212).

Leung Ling Yan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 218-259). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Man, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 101-126). / Abstracts in English and Chinese. / Chapter I. --- Title page --- p.i / Chapter II. --- Thesis committee --- p.ii / Chapter III. --- Acknowledgements --- p.iii / Chapter IV. --- Abstract (English) --- p.iv / Chapter V. --- Abstract (Chinese) --- p.vi / Chapter VI. --- Table of contents --- p.vii / Chapter VII. --- List of abbreviations --- p.xiii / Chapter VIII. --- List of figures --- p.xv / General introduction --- p.1 / Chapter Chapter 1: --- Literature review --- p.4 / Chapter 1.1. --- Cadmium --- p.5 / Chapter 1.1.1. --- Cadmium - Ways of uptake in human and aquatic life --- p.5 / Chapter 1.1.2. --- Cadmium - Toxic effects in fish --- p.6 / Chapter 1.2. --- Cortisol --- p.11 / Chapter 1.2.1. --- Cortisol - General information and its regulations --- p.11 / Chapter 1.2.2. --- Cortisol - Functions --- p.12 / Chapter 1.3. --- Thyroid hormones --- p.14 / Chapter 1.3.1. --- THs - General information and its regulations --- p.14 / Chapter 1.3.2. --- THs - Functions --- p.15 / Chapter 1.4. --- Growth hormone --- p.18 / Chapter 1.4.1. --- GH - General information and its regulations --- p.18 / Chapter 1.4.2. --- GH - Functions --- p.20 / Chapter 1.5. --- Insulin-like growth factor --- p.22 / Chapter 1.5.1. --- IGF-I - General information and its regulations --- p.22 / Chapter 1.5.2. --- IGF-I - Functions --- p.24 / Chapter 1.6 --- Metallothioneins --- p.26 / Chapter 1.6.1. --- MTs - Definition and Classification --- p.26 / Chapter 1.6.2. --- MTs - Functions --- p.27 / Chapter 1.7. --- Glucose-6-phosphate dehydrogenase --- p.31 / Chapter 1.7.1. --- G6PDH - General information and its regulations --- p.31 / Chapter 1.7.2. --- G6PDH ´ؤ Functions --- p.32 / Chapter Chapter 2: --- "Effects of cadmium exposure on the endocrine status of silver sea bream, Sparus sarba" --- p.34 / Chapter 2.1. --- Introduction --- p.35 / Chapter 2.2. --- Materials and methods --- p.37 / Chapter 2.2.1. --- Overall experimental design --- p.37 / Chapter 2.2.2. --- In vivo exposure to waterborne cadmium --- p.37 / Chapter 2.2.2.1. --- Experimental animals --- p.37 / Chapter 2.2.2.2. --- Adaptation --- p.37 / Chapter 2.2.2.3. --- Tissue sampling --- p.38 / Chapter 2.2.2.4. --- Enzyme-linked immunosorbent assay (ELISA) --- p.38 / Chapter 2.2.2.4.1. --- Serum cortisol analysis --- p.39 / Chapter 2.2.2.4.2. --- Serum triiodothyronine analysis --- p.39 / Chapter 2.2.2.4.3. --- Serum thyroxine analysis --- p.39 / Chapter 2.2.2.5. --- Protein extraction and Protein quantification --- p.40 / Chapter 2.2.2.6. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.40 / Chapter 2.2.2.7. --- RNA extraction --- p.41 / Chapter 2.2.2.8. --- Reverse transcription for first-strand cDNAs from total RNAs samples from liver --- p.42 / Chapter 2.2.2.9. --- Real-time quantitative PCR assays of IGF-I mRNA expression --- p.43 / Chapter 2.2.3. --- In vivo experiments involving cadmium injection --- p.44 / Chapter 2.2.3.1. --- Experimental animals --- p.44 / Chapter 2.2.3.2. --- Adaptation --- p.44 / Chapter 2.2.3.3. --- Tissue sampling --- p.45 / Chapter 2.2.3.4. --- Enzyme-linked immunosorbent assay (ELISA) --- p.45 / Chapter 2.2.3.4.1. --- Serum cortisol analysis --- p.45 / Chapter 2.2.3.4.2. --- Serum triiodothyronine analysis --- p.45 / Chapter 2.2.3.4.3. --- Serum thyroxine analysis --- p.46 / Chapter 2.2.3.5. --- Protein extraction and Protein quantification --- p.46 / Chapter 2.2.3.6. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.46 / Chapter 2.2.3.7. --- RNA extraction --- p.46 / Chapter 2.2.3.8. --- Reverse transcription for the first-strand cDNAs from total RNAs samples from liver --- p.46 / Chapter 2.2.3.9. --- Real-time quantitative PCR of IGF-I mRNA expression --- p.46 / Chapter 2.2.4. --- In vitro part of the project (Primary cell culture: hepatocytes exposed to cadmium medium) --- p.47 / Chapter 2.2.4.1. --- Experimental animals --- p.47 / Chapter 2.2.4.2. --- Primary hepatocytes culture preparation --- p.47 / Chapter 2.2.4.3. --- Cadmium treatment and cell harvest --- p.48 / Chapter 2.2.4.4. --- "RNA extraction, reverse transcription for the first-strand cDNAs from total RNAs samples from lysed cells and real-time quantitative PCR of IGF-I mRNA expression" --- p.48 / Chapter 2.2.5. --- Statistical analysis --- p.48 / Chapter 2.3. --- Results --- p.49 / Chapter 2.3.1. --- Serum cortisol level --- p.49 / Chapter 2.3.2. --- Serum triiodothyronine level --- p.49 / Chapter 2.3.3. --- Serum thyroxine level --- p.49 / Chapter 2.3.4. --- Pituitary growth hormone level --- p.50 / Chapter 2.3.5. --- Hepatic insulin-like growth factor mRNA expression --- p.50 / Chapter 2.4. --- Discussion --- p.58 / Chapter 2.4.1. --- Serum cortisol level --- p.58 / Chapter 2.4.2. --- Thyroid hormones --- p.61 / Chapter 2.4.3. --- Growth hormone --- p.64 / Chapter 2.4.4. --- Insulin-like growth factor-I --- p.67 / Chapter 2.5. --- Conclusion --- p.70 / Chapter Chapter 3: --- "Effects of cadmium exposure on MT and G6PDH mRNA expressions of silver sea bream, Sparus sarba" --- p.71 / Chapter 3.1. --- Introduction --- p.72 / Chapter 3.2. --- Materials and methods --- p.74 / Chapter 3.2.1. --- Overall experimental design --- p.74 / Chapter 3.2.2. --- In vivo experiments involving exposure to waterborne cadmium --- p.74 / Chapter 3.2.2.1. --- Experimental animals --- p.74 / Chapter 3.2.2.2. --- Adaptation --- p.74 / Chapter 3.2.2.3. --- Tissue sampling --- p.74 / Chapter 3.2.2.4. --- RNA extraction --- p.74 / Chapter 3.2.2.5. --- Reverse transcription for first-strand cDNAs from total RNAs samples from gill and liver --- p.75 / Chapter 3.2.2.6. --- Amplification of partial fragments of metallothionein (MT) --- p.75 / Chapter 3.2.2.7. --- Rapid amplification of 5´ة and 3´ة cDNA ends of metallothionein (MT) --- p.76 / Chapter 3.2.2.7.1. --- Amplification of 5´ة cDNA end --- p.76 / Chapter 3.2.2.7.2. --- Amplification of 3´ة cDNA end --- p.78 / Chapter 3.2.2.8. --- Real-time quantitative PCR of MT and G6PDH mRNA expressions --- p.79 / Chapter 3.2.3. --- In vivo injection of cadmium --- p.80 / Chapter 3.2.3.1. --- "Experimental animals, adaptation and tissue sampling" --- p.80 / Chapter 3.2.3.2. --- RNA extraction --- p.80 / Chapter 3.2.3.3. --- Reverse transcription for first-strand cDNAs from total RNAs samples from gill and liver --- p.81 / Chapter 3.2.3.4. --- Real-time quantitative PCR of MT and G6PDH mRNA expression --- p.81 / Chapter 3.2.4. --- In vitro exposure of primary hepatocyte culture to cadmium --- p.81 / Chapter 3.2.4.1. --- Experimental animals --- p.81 / Chapter 3.2.4.2. --- Preparation of the hepatocytes for cell culture --- p.81 / Chapter 3.2.4.3. --- Cadmium treatment and cell harvest --- p.81 / Chapter 3.2.4.4. --- "RNA extraction, reverse transcription for first-strand cDNAs from total RNAs samples from lysed cells and real-time quantitative PCR of MT and G6PDH mRNA expression" --- p.82 / Chapter 3.2.5. --- Statistical analysis --- p.82 / Chapter 3.3. --- Results --- p.83 / Chapter 3.3.1. --- MT cloning and characterization --- p.83 / Chapter 3.3.2. --- Metallothionein mRNA expression --- p.83 / Chapter 3.3.3. --- Hepatic glucose-6-phosphate dehydrogenase mRNA expression --- p.84 / Chapter 3.4. --- Discussion --- p.91 / Chapter 3.4.1. --- MT cloning and characterization --- p.91 / Chapter 3.4.2. --- Metallothioneins mRNA expression --- p.92 / Chapter 3.4.3. --- Hepatic glucose-6-phosphate dehydrogenase mRNA expression --- p.95 / Chapter 3.5. --- Conclusion --- p.98 / General conclusion --- p.99 / References --- p.101

Zhang, Chaoxiong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 115-132). / Abstracts in English and Chinese. / Chapter I --- Title page --- p.i / Chapter II --- Thesis committee --- p.ii / Chapter III --- Abstract --- p.iii / Chapter IV --- Abstract (Chinese version) --- p.v / Chapter V --- Acknowledgement --- p.vii / Chapter VI --- Table of content --- p.viii / Chapter VII --- List of figure --- p.xiii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.7 / Chapter 2.1 --- An introduction to myostatin --- p.8 / Chapter 2.1.1 --- A general introduction --- p.8 / Chapter 2.1.2 --- Myostatin identification --- p.9 / Chapter 2.1.3 --- Structural studies of myostatin --- p.10 / Chapter 2.1.4 --- Phenotype of myostatin-null animals or transgenic animal --- p.10 / Chapter 2.2 --- Regulation of myostatin --- p.12 / Chapter 2.2.1 --- Biosynthesis of myostatin --- p.12 / Chapter 2.2.2 --- Regulation of myostatin expression --- p.13 / Chapter 2.2.3 --- Regulation of myostatin protein --- p.16 / Chapter 2.3 --- Myostatin effect --- p.20 / Chapter 2.3.1 --- Myostatin Signaling Pathway --- p.20 / Chapter 2.3.2 --- Cellular Responses to Myostatin Signaling --- p.23 / Chapter 2.4 --- Possible functions in tissues other than muscle --- p.26 / Chapter 2.5 --- Myostatin in fishes --- p.27 / Chapter 2.5.1 --- Introduction of silver sea bream --- p.27 / Chapter 2.5.2 --- Studies carried out in fishes --- p.27 / Chapter 2.5.3 --- Possible novel functions of myostatin in fishes --- p.30 / Chapter Chapter 3 --- Characterization of myostatin gene in the silver seabream (Sparus sarba) --- p.31 / Chapter 3.1 --- Abstract --- p.32 / Chapter 3.2 --- Introduction --- p.33 / Chapter 3.3 --- Materials and methods --- p.35 / Chapter 3.3.1 --- Experimental fish --- p.35 / Chapter 3.3.2 --- Total RNA extraction and cDNA cloning of myostatin-1 and myostatin-2 in silver sea bream --- p.35 / Chapter 3.3.3 --- Multiple sequence alignment --- p.38 / Chapter 3.3.4 --- Real-time PCR for quantification of myostatin-1 and myostatin-2 mRNA expression --- p.38 / Chapter 3.3.5 --- 1 --- p.39 / Chapter 3.3.6 --- Data processing and statistical analysis --- p.40 / Chapter 3.4 --- Results --- p.40 / Chapter 3.4.1 --- Cloning of myostatin-l and myostatin-2 cDNA --- p.40 / Chapter 3.4.2 --- Myostatin tissue distribution and seasonal pattern --- p.42 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4 --- "Effects of growth hormone, 11-ketotestosterone and cortisol on myostatin mRNA expression in silver sea bream (Sparus sarba)" --- p.61 / Chapter 4.1 --- Abstract --- p.62 / Chapter 4.2 --- Introduction --- p.63 / Chapter 4.3 --- Materials and methods --- p.65 / Chapter 4.3.1 --- Experimental fish --- p.65 / Chapter 4.3.2 --- Growth hormone injection --- p.65 / Chapter 4.3.3 --- 11-ketotestosterone and cortisol injection --- p.66 / Chapter 4.3.4 --- Muscle explants culture and hormone exposure --- p.67 / Chapter 4.3.5 --- Primary pituitary cell culture and cortisol exposure --- p.68 / Chapter 4.3.6 --- Measurement of growth hormone secretion by ELISA --- p.69 / Chapter 4.3.7 --- Data processing and statistical analysis --- p.70 / Chapter 4.4 --- Results --- p.71 / Chapter 4.4.1 --- Growth hormone injection --- p.71 / Chapter 4.4.2 --- 11-ketotestosterone injection --- p.71 / Chapter 4.4.3 --- Cortisol injection --- p.71 / Chapter 4.4.4 --- "In vitro hormone treatment-growth hormone, 11-ketotestosterone and cortisol" --- p.72 / Chapter 4.4.5 --- Pituitary cell growth hormone secretion under cortisol treatment --- p.72 / Chapter 4.5 --- Discussion --- p.81 / Chapter Chapter 5 --- Expression of myostatin mRNA in silver sea bream in different salinity --- p.87 / Chapter 5.1 --- Abstract --- p.88 / Chapter 5.2 --- Introduction --- p.89 / Chapter 5.3 --- Materials and Methods --- p.91 / Chapter 5.3.1 --- Experimental fish --- p.92 / Chapter 5.3.2 --- Long term salinity adaptation --- p.92 / Chapter 5.3.3 --- Abrupt transfer form seawater to freshwater --- p.92 / Chapter 5.3.4 --- Data processing and statistical analysis --- p.93 / Chapter 5.4 --- Results --- p.93 / Chapter 5.4.1 --- Long term adaptation to different salinities --- p.93 / Chapter 5.4.2 --- Abrupt transfer from 33ppt to 6ppt - 24 h --- p.93 / Chapter 5.4.3 --- Abrupt transfer from 33ppt to 6ppt - 72 h --- p.94 / Chapter 5.5 --- Discussion --- p.104 / Chapter Chapter 6 --- General discussion and conclusion --- p.108 / References --- p.115

Yuen, Wing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-155). / Abstract also in Chinese. / Chapter I --- Title page --- p.i / Chapter II --- Acknowledgements --- p.ii / Chapter III --- Abstract --- p.iii / Chapter IV --- Abstract (Chinese version) --- p.vi / Chapter V --- Table of contents --- p.viii / Chapter VI --- List of abbreviations --- p.xv / Chapter VII --- List of figures --- p.xvi / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.5 / Chapter 2.1 --- Cystic fibrosis transmembrane conductance regulator in human --- p.5 / Chapter 2.1.1. --- Pathology of cystic fibrosis --- p.5 / Chapter 2.1.2. --- CFTR gene and the encoded protein --- p.6 / Chapter 2.1.3. --- Hypothetical model for CFTR function --- p.7 / Chapter 2.1.4. --- Functions of CFTR --- p.7 / Chapter 2.1.5. --- Regulation of CFTR gene expression --- p.8 / Chapter 2.1.6 --- Regulation of CFTR protein --- p.9 / Chapter 2.1.7. --- Discovery of CFTR homologues in other vertebrates --- p.10 / Chapter 2.2 --- Cystic fibrosis transmembrane conductance regulator in teleosts --- p.10 / Chapter 2.2.1. --- Evidence for the presence of CFTR homologue in teleosts --- p.10 / Chapter 2.2.2. --- Molecular cloning of teleost CFTR genes --- p.11 / Chapter 2.2.3. --- Role of teleost CFTR in osmoregulation --- p.13 / Chapter 2.2.3.1. --- Tissue distribution of CFTR --- p.13 / Chapter 2.2.3.2. --- Changes in CFTR expression in response to ambient salinities --- p.14 / Chapter 2.2.3.3. --- Immunocytochemical studies of CFTR --- p.15 / Chapter 2.2.3.4. --- Regulation of CFTR --- p.17 / Chapter 2.3 --- Osmoregulation in teleosts --- p.19 / Chapter 2.3.1. --- Importance of osmoregulation --- p.19 / Chapter 2.3.2. --- Major components of chloride cells in marine teleosts --- p.20 / Chapter 2.3.2.1. --- Overview --- p.20 / Chapter 2.3.2.2. --- Sodium-potassium adenosine triphosphatase (Na+，K+-ATPase) --- p.21 / Chapter 2.3.2.3. --- Cystic fibrosis transmembrane conductance regulator (CFTR) --- p.22 / Chapter 2.3.2.4. --- Na+/K+/2Cr cotransporter (NKCC) --- p.23 / Chapter 2.3.2.5. --- Potassium (K+) channel --- p.25 / Chapter 2.4 --- Endocrine control of osmoregulation --- p.26 / Chapter 2.4.1. --- Overview --- p.26 / Chapter 2.4.2. --- Growth hormone (GH) and insulin-like growth factor I (IGF-I) --- p.27 / Chapter 2.4.2.1. --- Role of GH in osmoregulation --- p.27 / Chapter 2.4.2.2. --- Mediation through IGF-I --- p.29 / Chapter 2.4.2.3. --- Synergic effect with cortisol --- p.30 / Chapter 2.4.3. --- Prolactin (PRL) --- p.30 / Chapter 2.4.3.1. --- Role of PRL in osmoregulation --- p.30 / Chapter 2.4.3.2. --- Synergic effect with cortisol --- p.33 / Chapter 2.4.4. --- Cortisol --- p.33 / Chapter 2.4.4.1. --- Role of cortisol in osmoregulation --- p.33 / Chapter 2.4.4.2. --- Dual functions of cortisol --- p.34 / Chapter Chapter 3 --- Cloning and tissue distribution of silver sea bream CFTR gene --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and methods --- p.38 / Chapter 3.2.1. --- Part A: Cloning of silver sea bream CFTR gene --- p.38 / Chapter 3.2.1.1. --- Fish and culture conditions --- p.38 / Chapter 3.2.1.2. --- Sampling of fish --- p.38 / Chapter 3.2.1.3. --- Preparation of first strand cDNA --- p.38 / Chapter 3.2.1.4. --- Design of primers --- p.39 / Chapter 3.2.1.5. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.40 / Chapter 3.2.1.6 --- Cloning --- p.41 / Chapter 3.2.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.41 / Chapter 3.2.2.1. --- Fish and culture conditions --- p.41 / Chapter 3.2.2.2. --- Tissue sampling --- p.42 / Chapter 3.2.2.3. --- Preparation of first strand cDNA --- p.42 / Chapter 3.2.2.4 --- Design of primers --- p.42 / Chapter 3.2.2.5. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.43 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1. --- Part A: Cloning of silver sea bream CFTR gene --- p.44 / Chapter 3.3.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.60 / Chapter 3.4 --- Discussion --- p.62 / Chapter 3.4.1. --- Part A: Cloning of silver sea bream CFTR --- p.62 / Chapter 3.4.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.64 / Chapter Chapter 4 --- Effect of salinity on CFTR mRNA expression in gill and posterior intestine of silver sea bream --- p.68 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and methods --- p.70 / Chapter 4.2.1. --- Part A: Effect of long-term exposure to different salinities on CFTR expression --- p.70 / Chapter 4.2.1.1. --- Experimental fish and salinity adaptation --- p.70 / Chapter 4.2.1.2. --- Tissue sampling --- p.70 / Chapter 4.2.1.3. --- Serum ion levels --- p.71 / Chapter 4.2.1.4. --- Preparation of first strand cDNA --- p.71 / Chapter 4.2.1.5. --- Design of primers --- p.71 / Chapter 4.2.1.6. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.71 / Chapter 4.2.1.7. --- Statistical analysis --- p.72 / Chapter 4.2.2. --- Part B: Effect of abrupt transfer on CFTR expression --- p.73 / Chapter 4.2.2.1. --- Experimental fish --- p.73 / Chapter 4.2.2.2. --- Experimental design --- p.73 / Chapter 4.2.2.2.1 --- Experiment 1: Abrupt transfer from seawater (SW) to 6 ppt --- p.73 / Chapter 4.2.2.2.2. --- Experiment 2: Abrupt transfer from 6 ppt to SW --- p.73 / Chapter 4.2.2.3. --- Tissue sampling --- p.74 / Chapter 4.2.2.4. --- Serum ion levels --- p.74 / Chapter 4.2.2.5. --- Preparation of first strand cDNA --- p.74 / Chapter 4.2.2.6. --- Design of primers --- p.75 / Chapter 4.2.2.7. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.75 / Chapter 4.2.2.8. --- Statistical analysis --- p.75 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1. --- Part A: Effect of long-term exposure to different salinities on CFTR expression --- p.76 / Chapter 4.3.1.1. --- Serum ion levels --- p.76 / Chapter 4.3.1.2. --- CFTR expression in gill --- p.76 / Chapter 4.3.1.3. --- CFTR expression in posterior intestine --- p.76 / Chapter 4.3.2. --- Part B: Effect of abrupt salinity transfer on CFTR expression --- p.83 / Chapter 4.3.2.1. --- Experiment 1: Abrupt transfer from SW to 6 ppt --- p.83 / Chapter 4.3.2.1.1. --- Serum ion levels --- p.83 / Chapter 4.3.2.1.2. --- CFTR in gill --- p.83 / Chapter 4.3.2.1.3. --- CFTR in posterior intestine --- p.83 / Chapter 4.3.2.2. --- Experiment 2: Abrupt transfer from 6 ppt to SW --- p.89 / Chapter 4.3.2.2.1. --- Serum ion levels --- p.89 / Chapter 4.3.2.2.2. --- CFTR in gill --- p.89 / Chapter 4.3.2.2.3. --- CFTR in posterior intestine --- p.89 / Chapter 4.4 --- Discussion --- p.95 / Chapter 4.4.1. --- Long-term exposure to various salinities --- p.95 / Chapter 4.4.2. --- Abrupt salinity transfer --- p.98 / Chapter 4.4.2.1. --- Abrupt hypo-osmotic transfer (33 ppt to 6 ppt) --- p.98 / Chapter 4.4.2.2. --- Abrupt seawater transfer (6 ppt to 33 ppt) --- p.99 / Chapter 4.4.3. --- CFTR mRNA expression in posterior intestine --- p.101 / Chapter 4.4.4. --- Conclusion --- p.101 / Chapter Chapter 5 --- Effect of hormones on CFTR expression in gill and posterior intestine of silver sea bream --- p.102 / Chapter 5.1 --- Introduction --- p.102 / Chapter 5.2 --- Materials and methods --- p.104 / Chapter 5.2.1. --- Part A: In vivo effect of hormones on CFTR expression --- p.104 / Chapter 5.2.1.1. --- Experimental fish and salinity adaptation --- p.104 / Chapter 5.2.1.2. --- Hormone treatment --- p.104 / Chapter 5.2.1.3. --- Tissue sampling --- p.105 / Chapter 5.2.1.4. --- "Serum ion levels, preparation of first strand cDNA, design of primers and semi-quantitative reverse transcriptase (RT)-PCR" --- p.105 / Chapter 5.2.1.5. --- Statistical analysis --- p.105 / Chapter 5.2.2. --- Part B: In vitro effect of hormones on CFTR expression --- p.106 / Chapter 5.2.2.1. --- Fish and culture conditions --- p.106 / Chapter 5.2.2.2. --- Gill and posterior intestine preparations --- p.106 / Chapter 5.2.2.3. --- Hormone treatment --- p.106 / Chapter 5.2.2.4. --- "Preparation of first strand cDNA, design of primers and semi-quantitative reverse transcriptase (RT)-PCR" --- p.107 / Chapter 5.2.2.5. --- Statistical analysis --- p.107 / Chapter 5.3 --- Results --- p.108 / Chapter 5.3.1. --- Part A: In vivo effect of hormones on CFTR expression --- p.108 / Chapter 5.3.1.1. --- Serum ion levels --- p.108 / Chapter 5.3.1.1.1. --- Serum [Na+] level --- p.108 / Chapter 5.3.1.1.2. --- Serum [K+] level --- p.108 / Chapter 5.3.1.1.3. --- Serum [Cl' ] level --- p.108 / Chapter 5.3.1.2. --- CFTR expression in gill --- p.109 / Chapter 5.3.1.3. --- CFTR expression in posterior intestine --- p.109 / Chapter 5.3.2. --- Part B: In vitro effect of hormones on CFTR expression --- p.115 / Chapter 5.3.2.1. --- CFTR expression in gill --- p.115 / Chapter 5.3.2.2. --- CFTR expression in posterior intestine --- p.115 / Chapter 5.4 --- Discussion --- p.122 / Chapter 5.4.1. --- Effects of cortisol on CFTR expression --- p.122 / Chapter 5.4.2. --- Effects of growth hormone on CFTR expression --- p.124 / Chapter 5.4.3. --- Effects of prolactin on CFTR expression --- p.127 / Chapter 5.4.4. --- "Overall effect of cortisol, growth hormone and prolactin on CFTR expression" --- p.128 / Chapter 5.4.5 --- Conclusion --- p.130 / Chapter Chapter 6 --- General discussion and conclusion --- p.132 / References --- p.136

Chau, Kai Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-159). / Abstract also in Chinese. / Chapter I --- Abstract --- p.i / Chapter II --- Acknowledgements --- p.vi / Chapter III --- Table of Contents --- p.vii / Chapter IV --- List of Figures --- p.xv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.7 / Chapter 2.1 --- Na+-K+ ATPase --- p.7 / Chapter 2.1.1 --- Introduction / Chapter 2.1.2 --- Structure of Na+-K+ ATPase --- p.9 / Chapter 2.1.1.2 --- Na+-K+ ATPase a subunit --- p.9 / Chapter 2.1.1.3 --- Na+-K+ ATPase β subunit --- p.11 / Chapter 2.1.1.4 --- Composition of the a subunit and β subunit --- p.12 / Chapter 2.1.1.5 --- Isomers of Na+-K+ ATPase --- p.13 / Chapter 2.1.1.6 --- Mechanism of ion exchange --- p.15 / Chapter 2.2 --- Aquaporins --- p.17 / Chapter 2.2.1 --- Introduction --- p.17 / Chapter 2.2.2 --- Structure of AQP-1 --- p.18 / Chapter 2.2.3 --- Distribution and function of AQP-1 --- p.19 / Chapter 2.3 --- Hormone --- p.22 / Chapter 2.3.1 --- Prolactin --- p.22 / Chapter 2.3.1.1 --- Structure of prolactin --- p.22 / Chapter 2.3.1.2. --- Functions of prolactin --- p.24 / Chapter 2.3.2 --- Growth hormone --- p.27 / Chapter 2.3.2.1 --- Structure --- p.27 / Chapter 2.3.2.2 --- Function of growth hormone --- p.28 / Chapter 2.3.3 --- Cortisol --- p.30 / Chapter 2.3.3.1 --- Structure --- p.30 / Chapter 2.3.3.2 --- Functions of cortisol --- p.31 / Chapter 2.4 --- Sparus sarba --- p.34 / Chapter 2.5 --- Urinary bladder of fish --- p.36 / Chapter Chapter 3: --- Effect of salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Chronic effect of salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.40 / Chapter 3.2.1 --- Materials and Methods --- p.40 / Chapter 3.2.1.1 --- Fish --- p.40 / Chapter 3.2.1.2 --- Tissue sampling --- p.41 / Chapter 3.2.1.3 --- Protein extraction and quantification --- p.41 / Chapter 3.2.1.4 --- Na+-K+ ATPase ATPase activity --- p.42 / Chapter 3.2.1.5 --- RNA extraction and first strand cDNA synthesis --- p.43 / Chapter 3.2.1.6 --- Validation of semi-quantitative RT-PCR --- p.45 / Chapter 3.2.1.7 --- Semi-quantification of expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.47 / Chapter 3.2.1.8 --- Statistical analysis --- p.47 / Chapter 3.2.2 --- Results --- p.48 / Chapter 3.2.2.1 --- Na+-K+ ATPase activity --- p.48 / Chapter 3.2.2.2 --- Relative expression of Na+-K+ ATPase and aquaporin-1 in urinary bladder --- p.48 / Chapter 3.2.3 --- Discussion --- p.54 / Chapter 3.2.3.1 --- Chronic effect of salinity on Na+-K+ ATPase in urinary bladder --- p.54 / Chapter 3.2.3.2 --- Chronic effect of salinity on AQP-1 expression in urinary bladder --- p.59 / Chapter 3.3 --- Effect of abrupt transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.61 / Chapter 3.3.1. --- Materials and Methods --- p.61 / Chapter 3.3.1.1 --- Fish --- p.61 / Chapter 3.3.1.2 --- Tissue sampling --- p.62 / Chapter 3.3.1.3 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.62 / Chapter 3.3.1.4 --- Statistical analysis --- p.63 / Chapter 3.3.2 --- Results --- p.64 / Chapter 3.3.2.1 --- Effect of abrupt hypo-osmotic transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.64 / Chapter 3.3.2.2 --- Effect of abrupt hyper-osmotic transfer on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.65 / Chapter 3.3.3 --- Discussion --- p.73 / Chapter 3.4 --- Effect of in vitro salinity on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.78 / Chapter 3.4.1 --- Materials and Methods --- p.78 / Chapter 3.4.1.1 --- Fish --- p.78 / Chapter 3.4.1.2 --- Tissue sampling --- p.78 / Chapter 3.4.1.3 --- Preparation of culture medium --- p.79 / Chapter 3.4.1.4 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.79 / Chapter 3.4.1.5 --- Statistical analysis --- p.80 / Chapter 3.4.2 --- Results --- p.81 / Chapter 3.4.3 --- Discussion --- p.85 / Chapter 3.5 --- Conclusion --- p.86 / Chapter Chapter 4: --- Effect of hormones on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.88 / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- In vivo effect of hormones on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.91 / Chapter 4.2.1 --- Material and method --- p.91 / Chapter 4.2.1.1 --- Fish --- p.91 / Chapter 4.2.1.2 --- Tissue sampling --- p.92 / Chapter 4.2.1.3 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.92 / Chapter 4.2.1.4 --- Statistical analysis --- p.92 / Chapter 4.2.2 --- Results / Chapter 4.2.2.1 --- Hormonal effect on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder of sea water adapted fish --- p.93 / Chapter 4.2.2.2 --- Hormonal effect on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder of brackish water adapted fish --- p.97 / Chapter 4.2.3 --- Discussion --- p.101 / Chapter 4.2.3.1 --- Effect of prolactin on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.101 / Chapter 4.2.3.2 --- Effect of growth hormone on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.104 / Chapter 4.2.3.3 --- Effect of cortisol on mRNA expression of Na+-K+ ATPase and AQP-1 in urinary bladder --- p.106 / Chapter 4.3 --- In vitro effect of hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder of silver sea bream Sparus sarba --- p.109 / Chapter 4.3.1 --- Materials and methods --- p.109 / Chapter 4.3.1.1 --- Fish --- p.109 / Chapter 4.3.1.2 --- Tissue sampling --- p.110 / Chapter 4.3.1.3 --- Preparation of culture medium --- p.110 / Chapter 4.3.1.4 --- "RNA extraction, first strand cDNA synthesis and RT-PCR" --- p.111 / Chapter 4.3.1.5 --- Statistical analysis --- p.111 / Chapter 4.3.2 --- Results --- p.112 / Chapter 4.3.2.1 --- Effect of prolactin on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.112 / Chapter 4.3.2.2 --- Effect of growth hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.113 / Chapter 4.3.2.3 --- Effect of cortisol on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.123 / Chapter 4.3.3 --- Discussion --- p.124 / Chapter 4.3.3.1 --- Effect of prolactin on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.124 / Chapter 4.3.3.2 --- Effect of growth hormone on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.125 / Chapter 4.3.3.3 --- Effect of cortisol on the expression of Na+-K+ ATPase and aquaporin-1 in the urinary bladder --- p.127 / Chapter 4.4 --- Conclusion --- p.129 / Chapter Chapter 5 --- General Conclusions --- p.131 / References --- p.136

The present study provided information for the role of the RAS in seabream osmoregulatory responses. The structure of angiotensinogen suggested that flounder type Ang II was the prevalent form in seabream. However, HPLC analysis suggested that different forms of angiotensins were present in seabream adapted to different salinities. The status of RAS was revealed in seabream adapted to different salinities and a higher status was found in hypersaline environment. Local renal RAS was identified and it may be activated in hyposmotic media and associated with an increase in glomerular and tubular function to excrete excess water. In general, the RAS in seabream displays differential status, both at systemic and local levels, which modulates osmoregulatory functions under acute and chronic salinity perturbation. / The renin angiotensin system (RAS) is involved in the control of body fluid homeostasis in silver seabream. Seabream angiotensinogen was cloned and sequenced in the present study. The sequence alignment showed that the angiotensinogen of seabream is most similar to that of pufferfish. Differential status of RAS was found among different salinities, with relatively higher RAS activity among hyperosmotic adapted seabream. Circulating angiotensin II (Ang II) was higher in hyperosmotic adapted seabream, with the highest value observed in seabream adapted to double-strength seawater. Although the level of immunoreactive angiotensins in freshwater adapted seabream was higher than that of brackish-water, Ang III, but not Ang II, was the prevalent circulating form in freshwater adapted seabream according to HPLC analysis. Hepatic angiotensinogen expression, however, did not show any statistical difference among different salinities. A positive feedback control for angiotensinogen by Ang II is present in the hepatic tissue of seabream as Ang II increased the expression of angiotensinogen in isolated hepatocyte but captopril lowered the angiotensinogen expression in intact fish. Branchial Na-K-ATPase activities were elevated by Ang II and the activities among different salinities showed a pattern similar to that of circulating angiotensins. However, upon abrupt hyposmotic transfer, branchial Na-K-ATPase elevated along with a decrease in circulating Ang II, an observation implying that the relationship between Na-K-ATPase and Ang II may only be causal. Captopril blockade not only lowered not only circulating Ang II levels but also that of cortisol, indicating RAS activity may limit cortisol secretion. An elevation in the circulating cortisol may be related to the increase in branchial Na-K-ATPase activities after abrupt hyposmotic transfer. The stimulatory effect on branchial Na-K-ATPase activity and the vasopressor effect of Ang II were more potent in hyposmotic than hyperosmotic adapted seabream, which indicates hyposmotic adapted seabream is more sensitive to RAS activation. The renal RAS in silver seabream functions independently from the systemic RAS as the pattern of renal angiotensins was dissimilar to that of systemic angiotensins. The renal RAS was activated in brackish water conditions and abrupt hyposmotic transfer significantly increased renal RAS activities. Kidney morphometrics also indicated that hyposmotic adaptation increase the filtering capacity of seabream nephrons. The number and diameter of glomeruli increase significantly in freshwater adapted seabream, which may vastly increase the filtering surface of the nephrons. Collecting tubules were more prevalent in the kidney of hyposmotic adapted seabream, with higher number, diameter and thickness, suggesting a lower water permeability of collecting tubules is essential for the formation of copious and diluted urine in hyposmotic environment. / Wong Kwok Shing. / "December 2005." / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6144. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 130-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Various aspects of the biology of the tarwhine Rhabdosargus sarba and western yellowfin bream Acanthopagrus latus were studied. The studies on R. sarba have focused on populations in temperate coastal marine waters at ca 32oS and the lower reaches of an estuary (Swan River Estuary) located at the same latitude and in a subtropical embayment (Shark Bay) at ca 26oS, while those on A. latus were conducted on the population in the latter embayment. A combination of a macroscopic and histological examination of the gonads demonstrated that R. sarba is typically a rudimentary hermaphrodite in Western Australian waters, i.e. the juveniles develop into either a male or female in which the ovarian and testicular zones of the gonads, respectively, are macroscopically undetectable. This contrasts with the situation in the waters off Hong Kong and South Africa, in which R. sarba is reported to be a protandrous hermaphrodite. However, it is possible that a few of the fish that are above the size at first maturity and possess, during the spawning period, ovotestes with relatively substantial amounts of both mature testicular and immature ovarian tissue, could function as males early in adult life and then change to females. Although R. sarba spawns at some time between late winter and late spring in Western Australia, spawning peaks later in the Swan River Estuary than in coastal, marine waters at the same latitude and Shark Bay, in which salinities are always close to or above that of full strength sea water, i.e. 35 ñ . While the males and females attain sexual maturity at very similar lengths in the Swan River Estuary and Shark Bay, i.e. L50s all between 170 and 177 mm, they typically reach maturity at an earlier age in the former environment, i.e. 2 vs 3 years old. Thus, length and consequently growth rate influence the timing of maturity rather than age. During the spawning period, only 9 % of the fish caught between 180 and 260 mm in nearshore, shallow marine waters had become mature, whereas 91 % of those in this length range over reefs were mature, indicating that R. sarba tends to move offshore only when it has become gphysiologically ready to mature. The L50s at first maturity indicate that the current minimum legal length in Western Australia (230 mm) is appropriate for managing this species. Oocyte diameter frequency distributions, stages in oocyte development, duration of oocyte hydration and time of formation of post-ovulatory follicles in mature ovaries of Rhabdosargus sarba in the lower Swan River Estuary (32o 03fS, 115o 44fE) were used, in conjunction with data on tidal cycles, to elucidate specific aspects of the reproductive biology of this sparid in an estuarine environment. The results demonstrated the following. (i) Rhabdosargus sarba has indeterminate fecundity sensu Hunter et al. (1985). (ii) Oocyte hydration commences at about dusk (18:30 h) and is completed by ca 01:30-04:30 h, at which time ovulation, as revealed by the presence of hydrated oocytes in the ovarian duct and appearance of newlyformed post-ovulatory follicles, commences. (iii) The prevalence of spawning was positively correlated with tidal strength and was greatest on days when the tide changed from flood to ebb at ca 06:00 h, i.e. approximately when spawning ceases. Spawning just prior to strong ebb tides would lead to the transport of eggs out of the estuary and thus into salinities that remain at ca 35 ñ . The likelihood of eggs being transported downstream is further enhanced by R. sarba spawning in deeper waters in the estuary, where the flow is greatest. (iv) Although mature ovaries were found in R. sarba in the estuary between early July and December, the prevalence of atretic oocytes was high until September, when salinities started rising markedly from their winter minima. Batch fecundities ranged from 2,416 for a 188 mm fish to 53,707 for a 266 mm fish. The average daily prevalence of spawning amongst mature females during the spawning period of R. sarba caught in the lower estuary, i.e. July to end of October, was 36.5 %. Thus, individual female R. sarba spawned, on average, at intervals of ca 2.7 days in each spawning season. Female R. sarba with total lengths of 200, 250 and 300 mm were estimated to have a batch fecundity of 7,400, 20,100 and 54,800 eggs, respectively and annual fecundities of 332,000, 903,000 and 2,461,000 eggs, respectively. Rhabdosargus sarba is shown to undergo size-related movements in each of the three very different environments in which it was studied. In temperate coastal waters, R. sarba settles in unvegetated nearshore areas and then moves progressively firstly to nearby seagrass beds and then to exposed unvegetated nearshore areas and finally to areas around reefs where spawning occurs. Although R. sarba spawns in the lower Swan River Estuary, relatively few of its early 0+ recruits remain in the estuary and substantial numbers of this species do not start reappearing in the estuary until they are ca 140 mm. In Shark Bay, R. sarba uses nearshore mangroves as a nursery area and later moves into areas around reefs. The maximum ages recorded for R. sarba in coastal marine waters (11 years) and Shark Bay (13 years) were far greater than in the lower Swan River Estuary (6 years). However, the maximum lengths recorded in these three environments were all ca 350 mm. Due to the production by size-related movements of differences amongst the lengths of R. sarba at given ages in different habitats in coastal marine waters, the composite suite of lengths at age was not fully representative of the population of this species as a whole in this environment. A von Bertalanffy growth curve, which was adjusted to take into account size related changes in habitat type, significantly improved the fit to the lengths at age of individuals in the composite samples for the population beyond that provided by the unadjusted von Bertalanffy growth curve. This resulted in the maximum difference between the estimates of length at age from the two growth curves, relative to the L‡ derived from the unadjusted von Bertalanffy curve, reaching a value equivalent to 8 %. However, the maximum differences for the corresponding curves for populations in the lower Swan River Estuary and Shark Bay were far less, i.e. 1.7 and 3.2 %, respectively, and thus not considered biologically significant. Rhabdosargus sarba grew slightly faster in the lower Swan River Estuary than in either coastal marine waters or Shark Bay, possibly reflecting the greater productivity of estuarine environments. Acanthopagrus latus is a protandrous hermaphrodite. Detailed macroscopic and histological examination of the gonads of a wide size range of fish, together with a quantification of how the prevalences of the different categories of gonad change with size and age and during the year, were used to elucidate the sequence of changes that occur in the ovotestes of A. latus during life. The scheme proposed in the present study for the protandrous changes in A. latus differed from those proposed for this species elsewhere, but was similar to that of Pollock (1985) for the congeneric Acanthopagrus australis. The ovotestes of functional males develop from gonads which, as in older juveniles, contain substantial amounts of testicular and ovarian tissue. Such ovotestes, and particularly their testicular component, regress markedly after spawning and then, during the next spawning season, either again become ovotestes in which the testicular zone predominates and contains spermatids and spermatozoa (functional males), or become ovotestes in which the ovarian zone predominates and contains vitellogenic oocytes (functional females). Once a fish has become a functional female, it remains a female throughout the rest of its life. The trends exhibited during the year by reproductive variables demonstrate that A. latus in Shark Bay typically spawns on a very limited number of occasions during a short period in August and September and has determinate fecundity. The potential annual fecundities of 24 A. latus ranged from 764,000 in a 600 g fish to 7,910,000 in a 2,050 g fish and produced a mean }1SE of 1,935,000 } 281,000. The total length at which 50 % of A. latus become identifiable as males (245 mm) is very similar to the current minimum legal length (MLL) of 250 mm, which corresponds to an age of 2.5 years less than the age at which 50 % of males become females. Current spawning potential ratios calculated over a range of alternative values for natural mortality (M) for A. latus in Shark Bay suggests that the present fishing pressure is sustainable, but that the current MLL should be reviewed if recreational fishing pressure continues to increase. The age composition and von Bertalanffy growth parameters for Acanthopagrus latus have been determined. The relevant parameters were inserted into the empirical equations of Pauly (1980) and Ralston (1987) for estimating natural mortality (M). Total mortality (Z) was calculated using Hoenigfs (1983) equations, relative abundance analysis and a simulation based on maximum age and sample size.The two point estimates for M for A. latus, which were both 0.70 year-1, greatly exceeded all estimates for Z (range 0.18 to 0.30 year-1), which is clearly an erroneous result. To resolve this problem of inconsistent estimates, a Bayesian approach was developed, which, through combining the likelihood distributions of the various mortality estimates, produced integrated estimates for M and Z that are more consistent and precise than those produced for these two variables using the above methods individually. This approach now yielded lower values for M than Z and a measure of fishing mortality that appears to be consistent with the current status of the fishery. This approach is equally applicable to other fish species.

Also, the branchial hsp70 levels in fish following chronic salinity acclimation and abrupt hypo-osmotic exposure to 6 ppt were assessed by Western blotting. Upon chronic salinity acclimation, the lowest branchial hsp70 level was found in fish cultured in an iso-osmotic salinity of 12 ppt and the highest was in 50 ppt and 6 ppt environments. Freshwater acclimation resulted in return to lower hsp70 level. The results indicated that iso-osmotic salinity would bring about the least stress level while 50 ppt and 6 ppt were the most stressful salinities to Sparus sarba as indicated by using hsp70 expression as a biomarker of stress. Compared to 50 ppt and 6 ppt, the stress level of fish in fresh water was lower. On the other hand, Sparus sarba exhibited a significant increase in branchial hsp70 level immediately after abrupt hypo-osmotic exposure to 6 ppt when compared with seawater fish sampled at the same time point and increased hsp70 level was sustained throughout the sampling period, indicating the exposure was stressful to the fish. / In the present study, pituitary and serum levels of prolactin in a marine teleost, Sparus sarba, chronically acclimated to various salinities: fresh water (0 ppt), hypo-osmotic (6 ppt), iso-osmotic (12 ppt), normal seawater (33 ppt) and hypersaline (50 ppt) or abruptly exposed to a hypo-osmotic environment of 6 ppt were quantified by the developed peptide-based indirect ELISAs. Progressive increases in pituitary and serum prolactin were found as chronic salinity acclimation progressed from seawater to fresh water. Also, prolactin secretion was immediately induced by abrupt hypo-osmotic exposure to 6 ppt and remained significantly elevated up to 5 days post-exposure to 6 ppt. The results underline the importance of prolactin in marine teleosts kept in fresh water or waters of low salinity. However, there was no significant difference in pituitary prolactin during the course of the abrupt hypo-osmotic exposure experiment. The results may indicate that prolactin might be secreted rapidly from pituitary in large quantities to cope with abrupt exposure to a low-salinity environment. / In the present study, the effects of pharmacological drugs on prolactin levels in pituitary and serum of Sparus sarba were investigated. An increase in prolactin synthesis and release but a decrease in branchial hsp70 expression were found after treatment with sulpiride, a DA-D2 receptor antagonist. In contrast, a reduction in prolactin levels in pituitary and serum but an elevation in hsp70 level in gill were observed following administration of bromocriptine, a DA-D2 receptor agonist. Since hsp70 expression indicates the stress levels, the results of these studies supported the notion that increased prolactin synthesis and release might be related to a reduced stress state and prolactin might have a protective effect on stress tolerance in fish. / Lastly, the role of prolactin in regulating apoptosis in Sparus sarba branchial cells was examined. Successful induction of apoptosis was indicated by an increase in the apoptotic parameter caspase-3 activity in primary cultures of Sparus sarba branchial cells treated with camptothecin, a specific inducer of apoptosis. In this study, prolactin was shown to be anti-apoptotic in Sparus sarba branchial cells as co-treatment with ovine prolactin (oPRL) and camptothecin has been observed to attenuate the elevated caspase-3 activity in gill cell primary cultures. Also, prolactin was found to protect the branchial cells from apoptosis by maintaining the hsp70 level in the cells treated with camptothecin. / The objectives of the present study were to investigate the roles of prolactin in salinity adaptation, hsp70 expression and apoptosis in silver sea bream (Spaurs sarba). Firstly, specific peptide-based indirect ELISAs were developed for pituitary and serum prolactin of Sparus sarba. These assays had been validated by parallelism between the dilution response curves using serially diluted pituitary homogenate and serum sample with the standard curves of the synthetic peptide derived from the amino acid sequence of black sea bream (Acanthopagrus schlegelii ) prolactin. / Ng, Ho Yuen Andus. / "September 2007." / Adviser: N. Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4567. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 143-189). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

All these findings can help us to elucidate the mechanisms for the fish to detect changing osmotic conditions and transform signals to osmoregulatory responses. / In the first part of the study, PRL and PRL-releasing peptide (PrRP) cDNAs have been isolated from euryhaline silver sea bream. The PRL cDNA consists of 1360 bp encoding 212 amino acids whereas the PrRP cDNA contains 631 bp encoding prepro-PrRP with 122 amino acids. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of tissues. Expression levels of both PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50 ppt). In pituitary, both PRL and PrRP mRNA were synchronized in their expression, being significantly higher in fish adapted to low salinities (0 and 6 ppt), but the expression profile of hypothalamic PrRP in different salinities was different. These data suggested that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulating pituitary PRL expression in silver sea bream. / In the second part of the study, silver sea bream abruptly transferred from 33 to 6 ppt exhibited a remarkable pituitary PRL secretion following closely with the temporal changes in serum osmolality and ion levels. In order to investigate the direct effect of extracellular osmolality to pituitary PRL secretion, sea bream pituitary cells were dispersed and exposed to a medium with reduced ion levels and osmolality. PRL released from pituitary cells was found to be significantly elevated. In hyposmotic exposed anterior pituitary cells, cell volume exhibited a 20% increase when exposed to a medium with a 20% decrease in osmolality. These enlarged pituitary cells did not shrink until the surrounding hyposmotic medium was replaced, a phenomenon suggesting an osmosensing ability of silver sea bream PRL cells for PRL secretion in response to a change in extracellular osmotic pressure. / In the third part, olfactory rosette in the nasal cavity was surgically removed from silver sea bream adapted to 6 ppt and 33 ppt and mRNA expression of PRL and PrRP in silver sea bream were measured. The elevated pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish were abolished by this olfactory lamellectomy. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change but those in 33 ppt-adapted fish increase significantly after olfactory lamellectomy. These data suggest a possible osmosensing role of the olfactory system for regulation of PRL expression during hypo-osmotic acclimation of the fish. Besides, calcium-sensing receptor (CaSR) was cloned and its mRNA expression in olfactory system, as shown in other fish species previously, was investigated. However, no CaSR expression could be detected in olfactory rosette and nerve but its expression was demonstrated in osmoregulatory tissues and brain. There was no significant difference in CaSR mRNA expression in pituitary, kidney and anterior intestine of fish adapted to different salinities. These studies could not provide conclusive evidence to correlate CaSR with osmosensing in silver sea bream. / The present study used silver sea bream (Sparus sarba ) as a euryhaline fish model to investigate the regulation of prolactin (PRL) expression and secretion in fish adapted to different salinities. / Kwong, Ka Yee. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3248. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 154-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.