Osmoregulatory function of prolactin and prolactin-releasing peptide on gill cells of silver sea bream, Sparus sarba.

催乳素是一種負責廣鹽性硬骨魚低滲環境適應的重要激素。最近發現的催乳素釋放肽被報導在魚類生理活動中參與了刺激催乳素釋放和滲透調節過程。本研究以黃錫鯛為實驗動物，旨在詮釋催乳素和催乳素釋放肽在廣鹽性硬骨魚中的滲透調節作用和調控機制。實驗第一部份將黃錫鯛催乳素在大腸桿菌中過量表達並用鎳離子親和層析法將之純化。此重組黃錫鯛催乳素可被抗黃錫鯛催乳素抗體特異識別。將此重組催乳素注射入活體紅箭魚可導致其血漿鈉離子濃度和滲透壓顯著提高，從而證明了此催乳素的生物活性。在第二部份的研究中，黃錫鯛催乳素受體和催乳素釋放肽受體的全長編碼基因被克隆和分析。黃錫鯛催乳素受體和硬骨魚二型催乳素受體蛋白質相似性較高（可達47%至94%），但同魚類一型催乳素受體及其他高等脊椎動物催乳素受體同源性較低，相似性只有22%至37%。此催乳素受體廣泛表達在各種組織器官中，包括腦組織、腮、心臟、腸、腎、肌肉及性腺。黃錫鯛催乳素釋放肽受體屬於G-蛋白偶聯受體家族，它具備該家族成員的各種保守序列特徵。並且它含有同其他催乳素釋放肽受體一樣的PDZ域結合位點。組織表達分佈結果顯示此受體在中樞神經系統各部份及腮、心臟、肝臟和性腺中有表達。本研究最後一部份以黃錫鯛原代培養腮細胞為實驗對象，分別探究了腮細胞對於不同滲透壓力、催乳素和催乳素釋放肽處理的反應。在這些實驗中，我們檢測了腮細胞的死亡及凋亡狀況，并用實時定量PCR分析了一些滲透調節相關基因的表達狀況。另外，我們還採用了蛋白免疫印跡法檢測了腮細胞處於不同滲透壓條件下，其熱休克蛋白70的蛋白質豐度變化。我們的結果顯示：低滲壓力會引起腮細胞死亡包括誘導凋亡。催乳素和催乳素釋放肽處理均可以顯著降低低滲壓力所誘導的細胞死亡，並且催乳素還可以阻止一部份細胞進行凋亡。腮細胞中鈉鉀泵兩個亞基、熱休克同族蛋白70及催乳素受體的基因表達均被催乳素和催乳素釋放肽處理上調。但是水通道蛋白3的基因表達并未出現明顯的變化。當腮細胞暴露于高滲透壓力環境時，水通道蛋白3、囊性纤维化跨膜转运调节因子、鈉鉀泵兩個亞基和熱休克同族蛋白70的基因表達都明顯上調。與之相反，這些基因的表達在處於低滲環境的腮細胞中均被下調。本研究提供了許多關於催乳素和催乳素釋放肽在魚類腮滲透調節過程中的詳細作用，并且首次報導了催乳素釋放肽可以直接作用于魚類滲透調節組織發揮功能從而參與滲透調節過程，而不只是如以往被認為的通過旁分泌或者自分泌方式刺激催乳素的表達來發揮生理功能。本研究的探索和發現使我們更加深刻地理解催乳素和催乳素釋放肽在廣鹽性硬骨魚鹽度適應過程中的調控機制。Prolactin (PRL) is well known as a crucial hormone responsible for fresh (hypoosmotic) water acclimation in euryhaline teleosts. The recently discovered prolactin-releasing peptide (PrRP) has been reported to take part in stimulating PRL release and affecting osmoregulatory processes in fish. This study aims to investigate the osmoregulatory effects and regulatory mechanisms of PRL and PrRP in a euryhaline teleost, silver sea bream (Sparus sarba).First, silver sea bream prolactin (ssPRL) was over-expressed in E. coli by IPTG induction and purified by Ni-based immobilized metal ion affinity chromatography. This recombinant silver sea bream prolactin (rssPRL) was recognized by specific antibodies against ssPRL. Subsequently, its bioactivity was confirmed by in vivo injection to swordtails, which resulted in significant increase in plasma Na⁺ level and osmolality. In the second part, full-length cDNAs of silver sea bream PRL receptor (ssPRLR) and PrRP receptor (ssPrRPR) were cloned and characterized. ssPRLR shares high amino acid identities (47% to 94%) with teleost PRLR2s but low identities (22% to 37%) with piscine PRLR1s and higher vertebrate PRLRs. It is widely distributed in brain, gill, heart, gut, kidney, muscle and gonad. ssPrRPR belongs to G protein-coupled receptor (GPCR) family with all the conserved features of GPCRs, and possesses the special PDZ domain-binding motifs of other PrRPRs. Its expression was detected in the central nervous system, gill, heart, liver and gonad. Lastly, a primary gill cell culture of silver sea bream was developed and used as a tool for studying responses of gill cells following exposure to media of different osmotic stress, rssPRL and ssPrRP, respectively. Then, cytotoxicity and apoptosis assays were performed, and effects of these treatments on expression profiles of osmoregulatory genes were analyzed by real time PCR. Influence of osmotic stresses on protein abundance of heat shock protein 70s was examined by Western blot. The results showed that hypoosmotic challenge could induce cell death including apoptosis. Both PRL and PrRP treatment markedly decreased the induced cell death, and PRL treatment prevented some gill cells from apoptosis. Expression levels of Na⁺-K⁺-ATPase alpha (NKA-α) and beta (NKA-β) subunits, heat shock cognate 70 (HSC70) and PRLR were up-regulated in PRL- and PrRP- treated gill cells, however no significant effect on expression of aquaporin3 (AQP3) mRNA was apparent. After hyperosmotic exposure, expression levels of AQP3, cystic fibrosis transmembrane conductance regulator (CFTR), NKA-α, NKA-β, and HSC70 were significantly increased. In contrast, hypoosmotic exposure considerably down-regulated expression levels of these genes.The present study represented considerable addition of details about the precise actions of PRL and PrRP in branchial osmoregulation. Furthermore, it first reported that PrRP can exert direct effects on fish osmoregulatory epithelia, instead of its oft presumed autocrine or paracrine action. All these efforts provide new insights into the control mechanisms of PRL and PrRP in euryhaline teleost during salinity acclimation.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Qu, Zhe.Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 108-142).Abstracts also Chinese.Title page --- p.iThesis committee --- p.iiAcknowledgement --- p.iiiAbstract --- p.vAbstract-Chinese version --- p.viiiTable of contents --- p.xList of figures --- p.xivList of abbreviations --- p.xviChapter Chapter 1 --- General introduction --- p.1Chapter Chapter 2 --- Literature review --- p.6Chapter 2.1 --- The role of gill in teleost osmoregulation --- p.7Chapter 2.1.1 --- An overview of fish osmoregulation --- p.7Chapter 2.1.2 --- The gill structure and epithelial ionocytes --- p.8Chapter 2.1.3 --- Osmoregulatory molecules in gill cells --- p.10Chapter 2.2 --- Piscine prolactin --- p.16Chapter 2.2.1 --- General features and distribution --- p.16Chapter 2.2.2 --- Prolactin receptor --- p.17Chapter 2.2.3 --- Prolactin and teleost osmoregulation --- p.18Chapter 2.3 --- Prolactin-releasing peptide in teleost --- p.21Chapter 2.3.1 --- Discovery and characteristics --- p.21Chapter 2.3.2 --- Distribution and its receptor --- p.22Chapter 2.3.3 --- Functions of PrRP --- p.23Chapter A. --- PrRP and prolactin release --- p.23Chapter B. --- PrRP and neuroendocrine stress response --- p.24Chapter C. --- Effects on food intake and energy metabolism --- p.26Chapter D. --- PrRP and cardiovascular system --- p.27Chapter Chapter 3 --- Production and characterization of recombinant silver sea bream (Sparus sarba) prolactin --- p.29Chapter Abstract --- p.30Chapter 3.1 --- Introduction --- p.31Chapter 3.2 --- Materials and methods --- p.33Chapter 3.2.1 --- Experimental animals --- p.33Chapter 3.2.2 --- Construction of recombinant silver sea bream prolactin expression vector --- p.33Chapter 3.2.3 --- Large-scale production and purification of rssPRL --- p.34Chapter 3.2.4 --- SDS polyacrylamide gel electrophoresis and Western blotting analysis of rssPRL --- p.35Chapter 3.2.5 --- In vivo bioassay of rssPRL --- p.36Chapter 3.2.6 --- Statistical analysis --- p.37Chapter 3.3 --- Results --- p.38Chapter 3.3.1 --- Overexpression and purification of rssPRL --- p.38Chapter 3.3.2 --- Biological activity assay of rssPRL in vivo --- p.38Chapter 3.4 --- Discussion --- p.43Chapter Chapter 4 --- Molecular cloning and characterization of prolactin receptor (PRLR) and prolactin-releasing peptide receptor (PrRPR) in silver sea bream (Sparus sarba) --- p.46Chapter Abstract --- p.47Chapter 4.1 --- Introduction --- p.48Chapter 4.2 --- Materials and methods --- p.51Chapter 4.2.1 --- Fish and tissue samples --- p.51Chapter 4.2.2 --- Molecular cloning of full-length ssPRLR and ssPrRPR cDNA --- p.51Chapter 4.2.3 --- Multiple sequence alignments and phylogenetic analysis --- p.53Chapter 4.2.4 --- Tissue distribution of ssPRLR and ssPrRPR --- p.53Chapter 4.3 --- Results --- p.54Chapter 4.3.1 --- Cloning and characterization of silver sea bream PRLR and PrRPR cDNA --- p.54Chapter 4.3.2 --- Tissue distribution of ssPRLR and ssPrRPR mRNA --- p.56Chapter 4.4 --- Discussion --- p.65Chapter Chapter 5 --- Influences of osmotic stress and hormones on gill cell of silver sea bream (Sparus sarba) --- p.69Chapter Abstract --- p.70Chapter 5.1 --- Introduction --- p.72Chapter 5.2 --- Methods and materials --- p.77Chapter 5.2.1 --- Fish and primary gill cell culture --- p.77Chapter 5.2.2 --- Experiments using gill cell culture --- p.78Chapter 5.2.3 --- Cell survival assay and apoptosis relevant detections --- p.79Chapter 5.2.4 --- Real time PCR analysis --- p.80Chapter 5.2.5 --- Western blot analysis of HSP70 --- p.82Chapter 5.2.6 --- Data processing and statistical analysis --- p.82Chapter 5.3 --- Results --- p.83Chapter 5.3.1 --- Hypoosmotic stress induced cell death --- p.83Chapter 5.3.2 --- Influences of PRL and PrRP on hypoosmotic challenge induced cell death --- p.84Chapter 5.3.3 --- Effects of hormones and osmotic stresses on expression levels of branchial genes --- p.84Chapter 5.3.4 --- Osmotic stresses induced changes in branchial protein amount of HSP70s --- p.85Chapter 5.4 --- Discussion --- p.94Chapter Chapter 6 --- General discussion and conclusions --- p.102References --- p.10