An attempt has been made to follow some of the metabolic changes occuring during acclimation of goldfish (Carassius auratus) to temperature. The goldfish were acclimated to certain temperatures and then reacclimated to different temperatures. The change in the unsaturation of their body fats was found using Wij's iodine value determinations. The fats of goldfish fully acclimated to different temperatures show different iodine values. In general the fats are more saturated at higher temperatures. Changes in the amount of unsaturation during the acclimation process have been followed. It has been found upon increase in temperature there is a decrease in the amount of unsaturation. / Science, Faculty of / Zoology, Department of / Graduate

An attempt has been made to alter the degree of unsaturation of the body fats of goldfish (Carassius auratus) and correlate these changes with any modifications of heat tolerance subsequently exhibited by the fish. The goldfish were fed three different diets each containing a fat of different degree of unsaturation. The fats used were pilchard oil (iodine value of 181.7), herring oil (iodine value of 128.4.) and lard (iodine value of 66.2). Heat resistance was tested by holding the fish at a constant high temperature and observing the time to death. Variations in the ability of the groups to withstand high temperature were then compared to differences in the degree of unsaturation of their extracted fats. It was found that while diet could effect changes in the degree of unsaturation of the goldfish fats to approximately 54% of the theoretical level, and that these changes in turn modified the heat resistance of the goldfish, no quantitative relationship was established. / Science, Faculty of / Zoology, Department of / Graduate

Goldfish were hypophysectomlzed and subsequently treated, with luteinizing, thyrotropic, adrenocorticotropic and lactotropic (prolactin) hormones; likewise some fish were treated with crude salmon pituitary extract. It was found that, hypophysectomized goldfish had a lower heat, resistance than normal fish, and that replacement therapy using these hormones produced no statistically significant effect on the heat resistance of hypophysectomized fish. Experiments were undertaken in which fish were subjected to either a long or short photoperiod. The results for the photoperiod manipulations were not always statistically significant at the 0.05 level, but there was always the suggestion that fish with intact pituitaries under the 16 hour photoperiod were more heat resistant than fish held under an 8 hour photoperiod. Experiments were carried out in which the pituitary was autotransplanted into the musculature of the caudal peduncle. Fish so treated had a heat resistance like that of pituitary intact 16 hour photoperiod fish while hypophysectomized fish had a heat resistance like that of pituitary intact 8 hour photoperiod fish. From a study of the endocrine (target) organs of these animals it is concluded that the autotransplanted pituitary secretes thyrotropic hormone in supernormal amounts, adrenocorticotropic hormone in subnormal amounts gonadotropic hormone not at all and prolactin in at least normal amounts. Based on information in the literature it seems that growth hormone is secreted in very small amounts by the transplanted pituitary. From all these experiments it is concluded that the pituitary is involved in heat resistance. It is suggested that it must be located in its normal position, for a photoperiod effect. By combining the histological information with the heat resistance results of the pituitary autotransplant experiments it is suggested that growth, gonadotropic, thyrotropic and adrenocorticotropic hormones play no major role in heat resistance. It is suggested that prolactin is the hormone most directly concerned with the heat resistance of goldfish; this hormone may also be involved in the photoperiod induced change in heat resistance. / Science, Faculty of / Zoology, Department of / Graduate

The objective of this study was to determine the involvement of prostaglandins in the sexual development of both male and female goldfish. Carassius auratus. A chromatographic method was developed to separate the different prostaglandin groups. To standardize the procedure, extraction and separation recoveries were measured using tritiated-prostaglandins. Radioimmunoassay was used to measure the PGB1, PGE1 and PGF2* in the plasma and gonad. Initially, a seasonal study was undertaken to assess the importance of prostaglandins during sexual maturation. Samples of plasma and gonad were assayed monthly for prostaglandins (from December to March) from two groups of fish, one held under natural photoperiod and the other under long photoperiod (16L:8D). Although monthly variations occurred in all three prostaglandins examined, these changes did not correlate with changes in gonadal maturationi Prostaglandins were then measured in serial plasma samples of non-gravid and ovulating female goldfish. Ovulation was induced in gravid fish by increasing the water temperature from 14° C to 20° C and by injecting human chorionic gonadotropin (HCG) . It was found that: 1) PGF2 increased over 14 fold, 12 hours after the onset of ovulation (from pre-injection levels of 300 pg/ml to more than 4,000 pg/ml); however, this increase appeared to commence immediately after ovulation. There was no change in plasma P1GF20< levels in non-gravid control fish. The concentration of PGF2^in the ovarian fluid was over 9,000 pg/ml. 2) PGE1 decreased almost three-fold between the time of HCG injection (an average of 10 hours before ovulation) and 24 hours later. The plasma PGE 1 levels in the non-gravid females were up to 20 times less than the gravid ovulating females. The concentration of PGE1 in the ovarian fluid was 630 pg/ml. 3) PGB1 levels decreased in the plasma of non-gravid and ovulating goldfish, following HCG injection. The ovarian fluid contained 300 pg/ml of PGB1. Parallel experiments were performed on male goldfish that were spermiating. There were no significant changes in plasma PGF20C within 24 hours of HCG injection, whereas PGB1 decreased slightly (as for the females), and PGE1 increased significantly 10 hours after HCG injection. The findings of this study suggest that PGF2 and PGE1 in the ovarian fluid are the agents controlling ovulation in the female goldfish and that corresponding levels in the blood contribute to other events associated with ovulation. The experiments on males indicate a possible role for PGE1 during spermiation; however, it is difficult to ascertain its precise involvement at present. / Science, Faculty of / Zoology, Department of / Unknown

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by Cheung Yeuk Siu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 摘要 --- p.iv / List of abbreviations --- p.v / Table of contents --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prolactin (PRL) --- p.1 / Chapter 1.1.1 --- General introduction --- p.1 / Chapter 1.1.2 --- Genomic organization of teleost PRL gene --- p.2 / Chapter 1.1.3 --- Conserved domains of fish PRL --- p.3 / Chapter 1.1.4 --- Structure of teleost PRL --- p.6 / Chapter 1.1.5 --- Tissue sources of PRL --- p.8 / Chapter 1.2 --- Prolactin receptor (PRLR) --- p.9 / Chapter 1.2.1 --- Tissue distribution in teleosts --- p.9 / Chapter 1.2.2 --- Receptor structure and multiple forms of PRLR --- p.11 / Chapter 1.2.3 --- Possible action mechanisms of PRL --- p.14 / Chapter 1.3 --- Control of PRL release --- p.17 / Chapter 1.4 --- Biological functions of PRL in vertebrates --- p.19 / Chapter 1.4.1 --- Biological effects on teleosts --- p.19 / Chapter 1.4.1.1 --- Osmoregulatory roles --- p.20 / Chapter 1.4.1.2 --- Non-osmoregulatory roles --- p.29 / Chapter 1.5 --- Aim of the present study --- p.32 / Chapter Chapter 2 --- "Recombinant goldfish (Carassius auratus) prolactin: subcloning, expression, purification and refolding of the recombinant protein" / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.3 --- Methods --- p.48 / Chapter 2.3.1 --- Subcloning of the gfPRL cDNA --- p.48 / Chapter 2.3.1.1 --- PCR cloning of gfPRL cDNA --- p.48 / Chapter 2.3.1.2 --- DNA sequencing of the subcloned fragment --- p.51 / Chapter 2.3.1.3 --- Subcloning of the gfPRL cDNA fragment into the expression vector --- p.52 / Chapter 2.3.2 --- Expression and purification of rgfPRL --- p.53 / Chapter 2.3.2.1 --- Transformation of pRSETA/gfPRL into BL21(DE3)pLysS cells --- p.53 / Chapter 2.3.2.2 --- Prokaryotic expression of rgfPRL --- p.53 / Chapter 2.3.2.3 --- Affinity purification of rgfPRL --- p.55 / Chapter 2.3.2.4 --- Western blot analysis of the purified rgfPRL --- p.57 / Chapter 2.3.2.5 --- Protein concentration determination of the rgfPRL --- p.59 / Chapter 2.3.3 --- Protein refolding --- p.60 / Chapter 2.4 --- Results --- p.61 / Chapter 2.4.1 --- Subcloning and DNA sequencing of the gfPRL --- p.61 / Chapter 2.4.2 --- Expression and purification of rgfPRL --- p.63 / Chapter 2.4.2.1 --- Prokaryotic expression of rgfPRL --- p.63 / Chapter 2.4.2.2 --- Affinity purification of rgfPRL --- p.66 / Chapter 2.4.2.3 --- Western blot analysis of the purified rgfPRL --- p.68 / Chapter 2.4.2.4 --- Protein concentration determination of the rgfPRL --- p.68 / Chapter 2.4.3 --- Protein refolding --- p.70 / Chapter 2.5 --- Discussion --- p.72 / Chapter Chapter 3 --- Production of polyclonal antibodies against rgfRPL / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials --- p.82 / Chapter 3.3 --- Methods --- p.84 / Chapter 3.3.1 --- Immunization of rabbits --- p.84 / Chapter 3.3.2 --- Collection of the polyclonal antisera --- p.85 / Chapter 3.3.3 --- Purification of IgG from the polyclonal antisera --- p.86 / Chapter 3.3.4 --- Enzyme linked immunosorbent assay (ELISA) --- p.87 / Chapter 3.3.5 --- Western blot analysis for cross-reactivity --- p.88 / Chapter 3.4 --- Results --- p.90 / Chapter 3.4.1 --- Isolation and purification of IgG from the polyclonal antisera --- p.90 / Chapter 3.4.2 --- ELISA --- p.93 / Chapter 3.4.3 --- Western blot analysis for cross-reactivity --- p.96 / Chapter 3.5 --- Discussion --- p.98 / Chapter Chapter 4 --- Isolation of native PRL from goldfish pituitaries / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials --- p.101 / Chapter 4.3 --- Methods --- p.103 / Chapter 4.3.1 --- Alkaline extraction --- p.103 / Chapter 4.3.2 --- Size exclusion chromatography --- p.104 / Chapter 4.3.3 --- Anion exchange chromatography --- p.104 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Size exclusion chromatography --- p.106 / Chapter 4.4.2 --- Anion exchange chromatography --- p.109 / Chapter 4.4.3 --- SDS-PAGE analysis and immuno-detection of the purified protein --- p.112 / Chapter 4.5 --- Discussion --- p.114 / Chapter Chapter 5 --- Receptor binding assays / Chapter 5.1 --- Introduction --- p.115 / Chapter 5.2 --- Materials --- p.117 / Chapter 5.3 --- Methods --- p.119 / Chapter 5.3.1 --- Gill membrane preparation --- p.119 / Chapter 5.3.2 --- Radioactive labelling of the primary ligand --- p.120 / Chapter 5.3.3 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.121 / Chapter 5.3.4 --- Membrane protein dependence assay --- p.122 / Chapter 5.3.5 --- Receptor binding study using rgfPRL --- p.124 / Chapter 5.4 --- Results --- p.125 / Chapter 5.4.1 --- Radioactive labelling of the primary ligand --- p.125 / Chapter 5.4.2 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.127 / Chapter 5.4.3 --- Membrane protein dependence assay --- p.129 / Chapter 5.4.4 --- Receptor binding study using rgfPRL --- p.131 / Chapter 5.5 --- Discussion --- p.133 / Chapter Chapter 6 --- General discussion and conclusion --- p.136 / References --- p.141

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

Chan, Yuk-Hang. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy