Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23084
標題: 吳郭魚(Oreochromis mossambicus)適應不同環境鹽度時鰓上的滲透壓調節機制及其離子調節細胞功能之探討
Osmoregulatory mechanisms and the ion-transporting functions of mitochondrion-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to environments of different salinities.
作者: 王上知
Wang, Shang-Chic
關鍵字: 吳郭魚
tilapia
熱休克蛋白
滲透壓
氯細胞
廣鹽性
mitochondrion-rich cells
Na+/K+/2Cl−cotransporter
cystic fibrosis transmembrane conductance regulator
claudin
heat shock protein
出版社: 生命科學系所
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摘要: 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‰)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‰)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,吳郭魚在六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,在海水中適應達兩個星期(14 days)的吳郭魚,在轉移入淡水96小時後(SW-FW-96h)便發展出適應淡水的滲透壓調節能力。 在魚類中,鰓是最主要的滲透壓調節器官;而鰓的富含粒線體細胞(mitochondrion-rich cells)為最主要進行離子吸收(淡水型)或離子排除(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,吳郭魚的存活率達百分之百。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行滲透壓調節,使得這種適應過海水的吳郭魚在由淡水轉移回海水後,能突破六個小時的限制,得以順利存活。 實驗結果顯示,吳郭魚鰓上與離子運輸相關的蛋白質表現量(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,鰓上的連結蛋白(claudin 3-, 4-like proteins)則在轉移後1個小時內便明顯地下降,並且維持到至少轉移後24個小時。利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,在轉移進入海水後便開始凹陷,在3個小時內轉為凹洞型,12小時後則呈現深洞型(deep-hole)。以上結果說明,在面臨海水環境時,與細胞連結相關的蛋白質可能比離子運輸相關的蛋白質作更有效率的調整,以重塑細胞結構促使魚體進行離子排除。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蛋白質表現量有顯著的提昇,且維持到轉移後第24個小時。 綜合上述結果可知,有效率地活化伴護蛋白能夠幫助調節細胞內蛋白質表現量,並且在面臨滲透壓逆境時,協助鰓表皮細胞調節其細胞間隙的滲透能力。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烈環境鹽度變化下持續生存的生理現象和可能的作用機制。
Gill 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 protein
URI: http://hdl.handle.net/11455/23084
其他識別: U0005-2008201017420700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2008201017420700
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