Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22236
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dc.contributor.advisor李宗翰zh_TW
dc.contributor.advisorTsung-Han Leeen_US
dc.contributor.author王上知zh_TW
dc.contributor.authorWang, Shang-Chicen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T07:17:30Z-
dc.date.available2014-06-06T07:17:30Z-
dc.identifier.urihttp://hdl.handle.net/11455/22236-
dc.description.abstract吳郭魚(Oreochromis mossambicus)的天然棲地為淡水水域環境,但廣鹽性的特質使其能夠長期適應於淡水(fresh water; FW)或海水(seawater; SW; 35&;#8240;)中。然而過去的研究顯示,吳郭魚要從淡水適應到海水環境的過程,必須藉由間接的方式:先轉移至半淡鹹水(brackish water; BW; 15&;#8240;)環境中,適應過後才可再轉移進入海水以長期生存。如果直接從淡水轉移到海水,吳郭魚在&;#63953;個小時之內即會死亡。間接的轉移過程有助於魚體發展適當的滲透壓調節機制,以進一步適應海水。而目前的研究已知,在海水中適應達&;#63864;個星期(14 days)的吳郭魚,在轉移入淡水96小時後(SW-FW-96h)&;#63845;發展出適應淡水的滲透壓調節能&;#63882;。 在魚&;#63952;中,鰓是最主要的滲透壓調節器官;而鰓的富含&;#63993;線體細胞(mitochondrion-rich cells)為最主要進&;#64008;&;#63978;子吸收(淡水型)或&;#63978;子排除(海水型)的位置。由於在先前的實驗觀察中發現,將從海水移入淡水達96小時的吳郭魚(SW-FW-96h),再直接轉移回海水,吳郭魚的存活&;#63841;達百分之百。因此本實驗觀察將吳郭魚(SW-FW-96h)轉移回海水後反應時程的七個時間點(time-course)中,鰓上幾種與滲透壓相關的蛋白質表現以及MR cells型態的變化,試圖描繪出魚體是如何進&;#64008;滲透壓調節,使得這種適應過海水的吳郭魚在由淡水轉移回海水後,能突破&;#63953;個小時的限制,得以順&;#63965;存活。 實驗結果顯示,吳郭魚鰓上與&;#63978;子運輸相關的蛋白質表現&;#63870;(包括Na+/K+-ATPase、Na+/K+/2Cl- cotransporter及cystic fibrosis transmembrane conductance regulator)在轉移後24個小時內有顯著的提昇;相對的,鰓上的&;#63898;結蛋白(claudin 3-, 4-like proteins)則在轉移後1個小時內&;#63845;明顯地下&;#64009;,並且維持到至少轉移後24個小時。&;#63965;用掃瞄式電子顯微鏡觀察MR cells的結構,則顯示原本呈現部份凸起的細胞膜頂端,在轉移進入海水後&;#63845;開始凹陷,在3個小時內轉為凹&;#64005;型,12小時後則呈現深&;#64005;型(deep-hole)。以上結果&;#63855;明,在面&;#63990;海水環境時,與細胞&;#63898;結相關的蛋白質可能比&;#63978;子運輸相關的蛋白質作&;#63745;有效&;#63841;的調整,以重塑細胞結構促使魚體進&;#64008;&;#63978;子排除。除此之外,熱休克蛋白(heat shock protein; HSP)為伴護蛋白(molecular chaperones)的一種,能夠調節與穩定細胞內蛋白質的構型和功能;所以本實驗亦同時觀察其表現情形。結果證實,吳郭魚(SW-FW-96h)在轉移回海水的第6個小時開始,HSP70和HSP90蛋白質表現&;#63870;有顯著的提昇,且維持到轉移後第24個小時。 綜合上述結果可知,有效&;#63841;地活化伴護蛋白能夠幫助調節細胞內蛋白質表現&;#63870;,並且在面&;#63990;滲透壓逆境時,協助鰓表皮細胞調節其細胞間隙的滲透能&;#63882;。此外,本篇實驗是到目前為止,首次觀察到並探究,吳郭魚可以在如此劇&;#63903;環境鹽&;#64001;變化下持續生存的生&;#63972;現象和可能的作用機制。zh_TW
dc.description.abstractGill is the major osmoregulatory organs in teleost and mitochondrion-rich (MR) cells in the gill epithelia are the main sites to regulate ion movements. Tilapia (Oreochromis mossambicus) is a euryhaline teleost with fresh water (FW) preference. Furthermore, previous studies observed tilapia died within 6 h after direct transfer from FW to full-strength seawater (SW). The process of pre-acclimation in hypertonic brackish water is necessary for tilapia to develop the appropriate osmoregulatory mechanisms before SW acclimation. In the present study, we found that when SW-acclimated tilapia were transferred back to FW for 96h (SW-FW-96h), the osmoregulatory status were similar to FW-acclimated tilapia with lower Na+/K+-ATPase (NKA) responses, higher expression of claudin 3- and 4-like proteins, and apical Na+/Cl- cotransporter (NCC) expressed in the MR cells without basolateral Na+/K+/2Cl- cotransporter (NKCC) and apical cystic fibrosis transmembrane conductance regulator (CFTR). Inaddition, SW-FW-96h tilapia could be directly transferred to SW with no mortality. Therefore, the aim of our study is to investigate the integrated oamoregulatory mechanisms in the gills of tilapia to illustrate the mechanisms that could be regulated efficiently by SW-FW-96h tilapia for successful acclimation of direct transfer to full-strength SW. The results of this study revealed that the ion secretion mechanisms of transporter protein-dependent (i.e., NKA, NKCC and CFTR) were activated at 24h post-transfer. In contrast, abundance of branchial claudin 3- and 4-like proteins declined evidently at 1h post-transfer and sustained to 24 h post-transfer. On the other hand, the scanning electron microscopic observation also showed that when SW-FW-96h tilapia were transferred to SW, the convex structure of apical membrane of MR cells was indentation to form the concave structure within 3h post-transfer and the deep-hole structure at 12h post-transfer. It might appear that tight junction proteins rather than transporter protein-dependent mechanisms were regulated efficiently to play a crucial role in reshaping the gill phenotype to leakier epithelium for promoting ion secretion in SW-FW-96h tilapia acutely exposed to SW. Furthermore, the mechanisms of molecular chaperones regulated protein quality control were investigated. Protein expression of gill heat shock protein 70 (HSP70) and HSP90 significantly elevated at 6h and the levels were sustained to 24 h after SW transfer. Meanwhile, the levels of aggregated protein were constant among different time points. Taken together, efficient activation of molecular chaperones regulated protein quality control might protect branchial cells from osmtic stress and lead to gill cells have the ability to regulate the mechanisms of paracellular permeability in SW-FW-96h tilapia transferred to SW. This study is the first to observe and investigate tilapia can survive on the experience of drastic changes in environmental salinity. Key words: tilapia, mitochondrion-rich cells, Na+/K+/2Cl− cotransporter, cystic fibrosis transmembrane conductance regulator, claudin and heat shock proteinen_US
dc.description.tableofcontentsContents Contents...............................................i Figures contents..............................................ii Chapter 1. The survival strategies of tilapia when transferred from fresh water to full-strength seawater 中文摘要...............................................1 Abstract...............................................3 Introduction...........................................5 Materials and methods................................................8 Results...............................................16 Discussion............................................20 References............................................26 Figures...............................................34 Chapter 2. Evidences for deep-hole subtype of mitochondrion-rich cells with different functions in gills of tilapia, Oreochromis mossambicus 中文摘要....................................................50 Abstract..............................................52 Introduction..........................................54 Materials and methods...............................................57 Results...............................................66 Discussion............................................70 References............................................76 Figures...............................................81 Publications..........................................95 Figures contents Chapter 1. Fig. 1. Survival rate of the SW-FW-96h fish directly transferred to SW.....................................34 Fig. 2. NKA protein expression in gills of tilapia acclimated to FW, SW and SW-FW-96h...................................................35 Fig. 3. NKA activity in gills of tilapia acclimated to FW, SW and SW-FW-96h......................................36 Fig. 4. Expression of claudin 3-like protein in gills of tilapia acclimated to FW, SW and SW-FW-96h...................................................37 Fig. 5. Expression of claudin 4-like protein in gills of tilapia acclimated to FW, SW and SW-FW-96h...................................................38 Fig. 6. Changes of NKA protein expression in gills of tilapia transfer from SW-FW-96h to SW....................................................39 Fig. 7. Changes of NKA activity in gills of tilapia transfer from SW-FW-96h to SW.........................40 Fig. 8. Confocal micrographs of immunofluorescence staining for NCC/NKCC during SW-FW-96h acclimated tilapia transfer to SW....................................................41 Fig. 9. Confocal micrographs of immunofluorescence staining for CFTR during SW-FW-96h acclimated tilapia transfer to SW....................................................42 Fig.10. Effect of salinity transfer on MWCs..................................................43 Fig.11. Changes of claudin 3-like protein expression in gills of tilapia transfer from SW-FW-96h to SW....................................................44 Fig.12. Changes of claudin 4-like protein expression in gills of tilapia transfer from SW-FW-96h to SW....................................................45 Fig.13. SEM micrographs of gill filaments in tilapia during SW-FW-96h acclimated tilapia transfer to SW....................................................46 Fig.14. Effect of salinity transfer on protein expression of branchial HSP70....................................47 Fig.15. Effect of salinity transfer on protein expression of branchial HSP90....................................48 Fig.16. Effect of salinity transfer on protein aggregation in gills..............................................49 Chapter 2. Fig. 1. SEM micrographs of gill filaments in tilapia acclimated to various salinities......................81 Fig. 2. Immunofluorescence staining of NKA, NCC/NKCC and CFTR in gill filaments of tilapia acclimated to hypotonic environment (5‰)......................................82 Fig. 3. Immunofluorescence staining of NKA, NCC/NKCC and CFTR in gill filaments of tilapia acclimated to isotonic environment (9‰)......................................83 Fig. 4. Double immunofluorescence staining of NCC and CFTR in gill filaments of tilapia acclimated to isotonic environment(9‰).......................................84 Fig. 5. Double immunofluorescence staining of NCC and CFTR at separate Z optical sections in gill filaments of tilapia acclimated to isotonic environment (9‰).......85 Fig. 6. Immunofluorescence staining of NKA, NCC/NKCC and CFTR in gill filaments of tilapia acclimated to hypertonic environment (15‰).....................................86 Fig. 7. Immunofluorescence staining of NKA, NCC/NKCC and CFTR in gill filaments of tilapia acclimated to hypertonic environment (35‰).....................................87 Fig. 8. Relative mRNA abundance of NCC and NKCC1a in gills of tilapia acclimated to various salinities............................................88 Fig. 9. Fluorescent microscope image of Chloride test in gill epithelium of 5‰- and 35‰-acclimated tilapia...............................................89 Fig.10. Scanning electron microscope image of Chloride test in gill epithelium of 5‰- and 35‰-acclimated tilapia...............................................90 Fig.11. Confocal scanning micrographs of immunofluorescence staining of NKA and the average sizes of gill MR cells of tilapia acclimated to various salinities............................................91 Fig.12. NKA responses in gills of tilapia acclimated to various salinities....................................92 Fig.13. Plasma and environmental osmolalities, and MWCs in tilapia acclimated to various salinities............................................93 Fig.14. Schematic diagrams of the different mitochondrion-rich cell types.......................................94zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.subject吳郭魚zh_TW
dc.subjecttilapiaen_US
dc.subject熱休克蛋白zh_TW
dc.subject滲透壓zh_TW
dc.subject氯細胞zh_TW
dc.subject廣鹽性zh_TW
dc.subjectmitochondrion-rich cellsen_US
dc.subjectNa+/K+/2Cl&ampen_US
dc.subjectampen_US
dc.subjectcotransporteren_US
dc.subjectcystic fibrosis transmembrane conductance regulatoren_US
dc.subjectclaudinen_US
dc.subjectheat shock proteinen_US
dc.title吳郭魚(Oreochromis mossambicus)適應&;#63847;同環境鹽&;#64001;時鰓上的滲透壓調節機制及其&;#63978;子調節細胞功能之探討zh_TW
dc.titleOsmoregulatory mechanisms and the ion-transporting functions of mitochondrion-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to environments of different salinities.en_US
dc.typeThesis and Dissertationzh_TW
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