Finally, the isolation and cloning of black sea bream SL receptor using PCR cloning and protein pull down assay were also attempted. Based on the PCR cloning results, the phylogenetic analysis of nonsalmonids fish GHR1 and SLR protein sequence, the GHR1 data of tissue distribution and effects of environmental salinity and fasting in tilapia, along with the results of far western blot, black sea bream GHR1 is probably a receptor for SL, however there is also a SL specific receptor in black sea bream. / In hormone treated primary cell culture of nonspawning black sea bream pituitary, 10-8 M E2 significantly increases SL mRNA level but 10-10 M, 10-9 M, 10-8 M of E2 inhibit GH mRNA level in female black seabream; 10-8 M E2 also inhibits SL and GH mRNA expression in bisexual black sea bream; 10-8 M MT inhibits SL mRNA expression in male black sea bream but any concentration of MT detected shows no significant effect on GH mRNA level. / Key words. somatolactin (SL), monthly changes, SL promoter, pit-1 and SL receptor / Somatolactin, SL, is a novel member of GH family of pituitary hormone only found in fish. It is considered to be a member of the GH gene family after gene duplication. Two types of SL, SL alpha and SL beta were identified, and SL 13 seems only in fresh water fish, such as goldfish, catfish, rainbow trout, eel and zebrafish. Black sea bream is a marine fish, and there is only SL alpha found from sequencing of over 100 SL cDNA clones. / The cDNAs encoding for transcription factor pit-1 variants were cloned and the transactivation of these Pit-1 isoforms on SL gene promoter were studied. Three variants of Pit-1 are first identified in fish. Pit-1b and Pit-1c can enhance SL promoter activity in Hepa-T1 cells respectively to about 2 fold and 12 fold, but pit-1a failed to activate the SL gene it in the same cells. All the three pit-1s of black sea bream couldn't reverse the inhibition of SL promoter in GH3 cells. The data suggest that N terminal 60 amino acid residues are critical in transactiation on SL promoter and SL promoter activity is possibly limited to fish SL secreting cells. / The SL gene promoter was obtained for gene regulation studies aiming to search for possible regulatory elements controlling the transcription of SL gene in black seabream. SL gene promoter is active in HepaT1 cells, but is inhibited in GH3 cells. Seven putative pit-1 response elements were confirmed with EMSA and super shift assay. / To study the physiological function of SL in black seabream, we initiated a study of monthly expressions of SL mRNA and gonadal somatic index (GSI) to determine whether SL is related to reproduction in black seabream, with GH mRNA levels were also detected for comparison. The results imply that function of SL is possibly related to early development of testis, while GH probably plays some roles in testis and ovary maturation. / by Tian, Jing. / "October 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4574. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-170). / 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.

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor

These studies were conducted to examine the influence of several variables on the growth hormone binding protein (GHBP) in serum of pigs. Continuous long term porcine somatotropin (pST) injections (daily for 6-7 wk) increased GHBP activity (p < .05). However, periodic short term pST injections (daily, every second d or every fourth d for 2 wk) did not cause significant change in GHBP levels (p > .40). No difference was observed between fed animals and animals fasted for 5 days (p > .3). Between 0 and 6 mo of age boar and gilt serum GHBP activity were not significantly different from each other, but increased significantly with age in both sexes(p < .0001). There was no significant correlation between serum GHBP and body weight in this study (p > .30). In pregnant sows, GHBP concentrations were highest at the beginning (day 72) of the third trimester (p< .05). These values were compared with information in the literature on serum growth hormone (GH) concentrations and GH receptor activity under similar conditions. Growth hormone receptor activity reported by other researchers and GHBP activity appear to vary concurrently except during fasting which may indicate alternate regulation of either the GHBP or the GH receptor. / Graduation date: 1992

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Not available / text

published_or_final_version / Zoology / Master / Master of Philosophy

In dairy cattle, treatment with exogenous growth hormone (GH) affects growth and function of mammary gland. The actions of GH are mediated via interaction with GH receptors (GHR). The first step in signal transduction is homodimerization of two GHR molecules by GH. This step is critical since mutation in either GH or GHR can block dimerization and thus target cell activation. However, association between milk related traits and combination of GH and GHR variations are not known. Accordingly, DNA genotypes in the GH and GHR genes were investigated for association with milk, fat and protein lactation yields in Holsteins. The marker data were obtained on 873 progeny tested bulls by using PCR-RFLP and PCR-SSCP analysis. There were five markers in GH and three in GHR. Estimated breeding values (EBVs) were obtained from Canadian Dairy Network for milk, fat, and protein lactation yields for the 873 genotyped bulls. / There was significant difference among GH6.1 alleles (C-to-G transversion at position 2141) for the milk yield (P < 0.05) and protein yield (p < 0.05). There were significant differences in GHR AluI (A-to-T transversion at -1182) for milk (p < 0.05) and fat (p < 0.05), and GHR StuI (C-to-T transversion at -232) for fat (p < 0.0001) and protein (p < 0.05). Allele frequencies for GH6.1 (C), GHR AluI (A) and GHR StuI (C) alleles in bulls genotyped were 0.95, 0.63 and 0.95, respectively. Bulls with GH6.1 (C/G) genotype had higher milk EBV (p < 0.05) compared to C/C bulls. Bulls with GHR AluI (A/A) genotype had higher milk EBV (p < 0.01) and fat EBVs (p < 0.05). Bulls with StuI (C/C) genotype had higher fat EBV (p < 0.0001) and protein EBV (p < 0.05) compared to StuI (C/T). This study indicates that the combination of GH and GHR markers could serve as a tool to aid in selection for improving milk, fat, and protein production.

The effect of zinc deficiency on the growth promoting effect of circulating IGF-I and the direct growth effect of GH on long bone growth were investigated. Food intake was decreased by lack of zinc in the diet. Tissue zinc content and plasma alkaline phosphatase activity were reduced by zinc deficiency. Systemic administration of human IGF-I increased the body weight, tail length and tibia epiphyseal cartilage width of control animals. This somatogenic action was impaired by zinc deficiency, as evidenced by continued weight loss, no increase in tail length and decreased tibial epiphyseal cartilage width of zinc deficient animals. Unilateral arterial infusion of GH increased the tibial epiphyseal width of the treated limb but not of the non-treated limb in control rats. However, no difference was found between the infused and the non-infused limb of zinc deficient animals, suggesting the occurrence of GH resistance on long bone growth in zinc deficiency. We conclude that zinc deficiency inhibits the growth promoting action of circulating IGF-I and the direct growth effect of GH on long bone growth.

Graduation date: 1992