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.

Fung Chun Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 117-139). / Abstracts in English and Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of figures --- p.vi / List of tables --- p.viii / List of abbreviations --- p.ix / Chapter Chapter I --- General introduction --- p.1 / Chapter 1.1 --- Different fish habitats with various salinities --- p.1 / Chapter 1.2 --- Osmotic challenges faced by teleosts --- p.2 / Chapter 1.3 --- High ionic strength results in DNA damage and excess water gain --- p.3 / Chapter 1.4 --- Osmoregulatory organs and mechanisms for osmotic balance --- p.4 / Chapter 1.5 --- Different tolerance to salinity changes --- p.8 / Chapter 1.6 --- Effective communication among osmoregulatory organs --- p.9 / Chapter 1.7 --- Introduction to GH and PRL --- p.9 / Chapter 1.8 --- Structure of the GHR and PRLR --- p.10 / Chapter 1.9 --- Hypoosmoregulatory action of GH/IGF-I axis in teleosts --- p.11 / Chapter 1.10 --- Hyperosmoregulatory action of PRL in teleosts --- p.11 / Chapter Chapter II --- Research rationale --- p.13 / Chapter 2.1 --- Physiological importance of osmoregulation in fish --- p.13 / Chapter 2.1.1 --- Energy metabolism --- p.13 / Chapter 2.1.2 --- Growth --- p.14 / Chapter 2.1.3 --- Immunity --- p.14 / Chapter 2.1.4 --- Reproduction --- p.15 / Chapter 2.2 --- Aquaculture importance --- p.15 / Chapter 2.3 --- Unknown molecular regulatory mechanism of hormones during salinity changes in fish --- p.16 / Chapter 2.4 --- Animal model --- p.17 / Chapter Chapter III --- In vivo studies of sbGHR and sbPRLR expression in osmoregulatory organs in response to salinity changes --- p.18 / Chapter 3.1 --- Introduction --- p.18 / Chapter 3.1.1 --- Dynamic change of GH level during salinity changes --- p.18 / Chapter 3.1.2 --- Dynamic change of PRL level during salinity changes --- p.19 / Chapter 3.1.3 --- In vitro studies of GH and PRL release from teleost pituitary in response to extracellular osmolality changes --- p.20 / Chapter 3.1.4 --- Biological actions of GH and PRL through the GHR and PRLR --- p.21 / Chapter 3.2 --- Materials and methods --- p.23 / Chapter 3.3 --- Results --- p.28 / Chapter 3.4 --- Discussion --- p.36 / Chapter 3.4.1 --- Plasma osmolality change during salinity changes --- p.36 / Chapter 3.4.2 --- Gene expression after HSW exposure --- p.38 / Chapter 3.4.3 --- Ionic mediators of the gene expression --- p.43 / Chapter 3.4.4 --- Gene expression after BW exposure --- p.44 / Chapter 3.4.5 --- Dynamic changes of the GHR and PRLR in response to salinity changes --- p.45 / Chapter 3.4.6 --- Regulation of the gene expression in response to salinity changes --- p.46 / Chapter Chapter IV --- Gene expression of sbGHR in liver during salinity changes --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.1.1 --- Responses of the somatotropic axis to salinity changes in fish --- p.49 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.3 --- Results --- p.56 / Chapter 4.4 --- Discussion --- p.60 / Chapter 4.4.1 --- Inhibition of GHR and IGF-I gene expression in liver during HSW exposure --- p.60 / Chapter 4.4.2 --- Downregulation of GHR gene expression by hyperosmotic stress --- p.62 / Chapter 4.4.3 --- Growth retardation of fish during hyperosmotic environment --- p.64 / Chapter Chapter V --- Gene expression studies of sbPRLR in gill organ culture --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.1.1 --- Functions of gill in fish osmoregulation --- p.68 / Chapter 5.1.2 --- Gill culture as a model for osmoregulation studies --- p.69 / Chapter 5.2 --- Materials and methods --- p.70 / Chapter 5.3 --- Results --- p.71 / Chapter 5.4 --- Discussion --- p.73 / Chapter Chapter VI --- Regulation of gene expression of sbGHR in liver during hyperosmotic stress: promoter studies --- p.75 / Chapter 6.1 --- Introduction --- p.75 / Chapter 6.1.1 --- What is a promoter? --- p.75 / Chapter 6.1.2 --- Promoter studies of GHR gene --- p.76 / Chapter 6.2 --- Materials and methods --- p.78 / Chapter 6.3 --- Results --- p.85 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter VII --- General discussion and future perspectives --- p.111 / References --- p.117

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.