韓玉山臺灣大學:漁業科學研究所薛莉瑋Hipolito, Sheryll GrospeSheryll GrospeHipolito2010-05-102018-07-062010-05-102018-07-062009U0001-2107200917482100http://ntur.lib.ntu.edu.tw//handle/246246/181721日本鰻 (Anguilla japonica) 為降海洄游魚類，棲息於日本、台灣、中國大陸及韓國。由於它一生橫跨淡水及海水水域且歷經多次變態，此特殊的生活史，使日本鰻非常適合做為硬骨魚類內分泌調控系統之研究。魚的腦下垂體包含了多樣的分泌細胞，分泌細胞藉由活化下視丘上的特殊目標細胞，引發成長荷爾蒙、生殖荷爾蒙、原嗎啡黑色素皮質素、促甲狀腺素與催乳激素等賀爾蒙的分泌，在生長、發育、代謝、生殖和神經系統等功能上具有重要性。先前研究對於這些荷爾蒙於滲透壓調節、生殖作用、成長分化與能量代謝等所扮演的角色已被深入的研究與了解，但其過程中彼此間可能的交互作用資訊仍缺乏，且須更進一步的研究。此研究主要的目標是評估野生雌黃鰻腦下垂體中成長賀爾蒙(GH)、促濾泡激素β次單元(FSHβ)、促黃體激素β次單元(LHβ)、荷爾蒙醣蛋白α次單元(GPα)與原嗎啡黑色素皮質素(POMC)，其表現量和可能的交互作用。驗樣本由2006年9月至2008年3月採集於台灣高屏溪下游之野生鰻，測量魚體全長、體重、並計算形態與生理指數：眼徑指數 (OI)、胸鰭指數 (FI)、肝指數 (HSI) 及生殖腺指數 (GSI)。自一個或數個腦下垂體萃取出total RNA並以Real-time-qPCR分析腦下垂體賀爾蒙之表現量。魚體全長和OI 呈顯著的正相關；FI呈負相關；但與HIS 及GSI 無關連性。另一方面，魚體全長和 GH、FSHβ、LHβ、GPα、POMC 表現量則無相關性。GSI和GH、FSHβ、LHβ呈顯著的正相關。在相關性分析中GH、FSHβ、LHβ及POMC的表現量兩兩呈現顯著正相關，但GPα表現量則和上述荷爾蒙無相關。研究結果顯示，野生雌黃鰻之腦下垂體荷爾蒙，生長激素、生殖激素及前腦啡黑色素腎上腺皮質素具有交互作用。腦下垂體荷爾蒙間交互作用有可能受到下游之信號因子傳遞、彼此鄰近的荷爾蒙分泌細胞藉由旁泌作用交流或是腦下垂體神經內分泌荷爾蒙具有雙重作用所造成。Anguilla japonica, known as the Japanese eel, is a catadromous fish that can be found in Japan, Taiwan, China and Korea. Owing to its unique life cycle, spending parts of its life in freshwater and seawater environment where they undergo multiple morphophysiological changes made it a suitable species for investigating endocrine regulatory system of teleost fish. el pituitary gland contains a variety of secretory cells which through the actions of hypothalamus target specific cell types that triggers hormonal secretions including growth hormone, reproductive hormones, proopiomelanocortin, thyrotrophin and prolactin that are important for growth, development, metabolism, reproductive and nervous system functions. Although considerable advances have been made in understanding the roles of hormones in the context of osmoregulation, reproduction, growth and differentiation and energy, some information concerning these processes and their possible interactions with each other are still lacking and needs further studies. The main objective of this study is to evaluate the potential interaction between growth hormone (GH), follicle stimulating hormone β-subunit (FSHβ), luteinizing hormone β-subunit (LHβ), glycoprotein hormone α-subunit (GPα) and proopiomelanocortin (POMC) at the pituitary level in wild caught female yellow stage Japanese eels.ild Japanese eels were obtained from the Kaoping River of Taiwan between September 2006 and March 2008. Total length and body weights were measured and morphometric indices including ocular index (OI), fin index (FI), hepatosomatic index (HSI) and gonadosomatic index (GSI) were calculated. Total RNA was extracted from individual or pooled pituitary glands and the expression levels of pituitary hormones were determined by Real-Time-qPCR. Total length showed a significant and positive correlation with OI and a negative correlation with FI while no correlations were observed with HSI and GSI. On the other hand, total length revealed no correlations with GH, FSHβ, LHβ, GPα and POMC. The results showed a significant and positive correlation of GH, FSHβ and LHβ to GSI. Correlation analysis between pituitary hormones revealed significant and positive correlations among GH, FSHβ, LHβ and POMC. However, no correlations were observed between these hormones and GPα. he results demonstrated that, at the pituitary level of yellow stage of wild female Japanese eels, hormone interaction occurs on growth hormone, reproductive hormones and proopiomelanocortin. Their interactions may be affected by downstream signaling factors, by anatomical distribution of the secretory cells resulting in paracrine communication or by dual actions of hypothalamic neuroendocrine hormones.Acknowledgement…………………………………………………………… ihinese Abstract ……………………………………………………………. iinglish Abstract …………………………………………………………….. iv. Introduction ………………………………………………………………. 1 1. Japanese Eel Life Cycle ……………………………………………….. 1 2. Eel Endocrine System …………………………………………………. 1 3. Eel Pituitary Gland and Its Hormonal Secretion ……………………… 2 3.1 Pituitary Gland ……………………………………………………. 2 3.2 Hormonal Secretions of the Pituitary Gland ……………………… 3 3.2.1 Growth Hormone ……………………………………………. 3 3.2.2 Gonadotropin Hormones ……………………………………. 4 3.2.3 Proopiomelanocortin ………………………………………… 4 4. Interplay Between Pituitary Hormones ………………………………... 5 5. Objectives ……………………………………………………………… 6I. Materials and Methods ……………………………………………………. 6 1. Experimental Animals …………………………………………………. 6 2. Total RNA Extraction and Reverse Transcription …………………… 8 2.1 RNA Extraction ………………………………………………….. 8 2.1.1 Homogenization …………………………………………….. 8 2.1.2 Phase Separation ……………………………………………. 8 2.1.3 RNA Isolation ………………………………………………. 8 2.1.4 RNA Wash and Resuspension ………………………………. 9 2.2 Reverse Transcription …………………………………………….. 9 2.2.1 Preparation of RNA Target and Primer ……………………… 9 2.2.2 Preparation of Reverse Transcription Reaction Mix ………… 10 2.2.3 Reverse Transcription ……………………………………….. 10 3. Real Time - Quantitative Polymerase Reaction (qPCR) for GH, FSHβ, LHβ, GPα and POMC ……………………………………………………. 10 4. Statistical Analysis …………………………………………………….. 12II. Results ………………………………………………...…………………. 13 1. Total Length and Morphometric Indices ………………………………. 13 2. Correlation Between Total Length and Morphometric Indices ……….. 13 3. Correlation Between Total Length and Pituitary Hormones ………….. 13 4. Correlation Between Pituitary Hormones and Morphometric Indices … 14 5. Correlation Between Pituitary Hormones ……………………………... 14V. Discussion ……………………………………………………………….. 14 1. Total Length and Morphometric Indices ………………………………. 14 2. Total Length and Pituitary Hormones …………………………………. 15 3. Pituitary Hormones and Morphometric Indices ………………………. 16 4. Interaction Between Pituitary Hormones ……………………………… 16 4.1 Downstream Signaling ……………………………………………. 16 4.2 Anatomical Organization of Secretory Cells ……………………... 18 4.3 Neuroendocrine Regulatory Factors - Dual Actions ……………… 19. Conclusion ………………………………………………………………... 20I. References ……………………………………………………………….. 21ppendix A. Tables ………………………………………………………….. 29ppendix B. Figures …………………………………………………………. 37ppendix Aist of Tablesable Gene-specific primers for quantitative assay of Japanese eel pituitaryhormones by Real-Time-qPCR ……………………………………… 30 Individual morphometric and physiological data and calculated morphometric indices. TL (total length), BW (body weight), HED (horizontal eye diameter), VED (vertical eye diameter), FL (fin length), LW (liver weight), GW (gonad weight), OI (ocular index), FI (fin index), HSI (hepatosomatic index) and GSI (gonadosomatic index) ………………………………………………………………… 31 Calculated relative values of pituitary hormones. GH (growth hormone), FSHβ (follicle stimulating hormone β-subunit), LHβ (luteinizing hormone β-subunit), GPα (glycoprotein α-subunit) and POMC (proopiomelanocortin) ……………………………………….. 34 ppendix Bist of Figuresigure Diagram representing common external morphometric characters of eel. PA: pre-anal length, LD: distance from the tip of the lower jaw to the origin of the dorsal fin, AD: distance between the verticals through the anus and origin of the dorsal fin, HL: head length, TR: trunk length, HED: horizontal eye diameter, VED: vertical eye diameter, ML: mouth length, FL: fin length and TL: total length ………………. 38 Geographical description of collection site, the Kaoping River (120o 50’ E and 22o 40’ N) ………………………………………………….. 39 Schematic diagram representing interaction between pituitary hormones ……………………………………………………………… 40 Linear regression of TL on OI of Japanese eel at yellow stage ……….. 41 Linear regression of TL on FI of Japanese eel at yellow stage ……….. 42 Linear regression of TL on HSI of Japanese eel at yellow stage ……… 43 Linear regression of TL on GSI of Japanese eel at yellow stage ……… 44 Linear regression of TL on GH of Japanese eel at yellow stage ………. 45 Linear regression of TL on FSHβ of Japanese eel at yellow stage ……. 460 Linear regression of TL on LHβ of Japanese eel at yellow stage ……... 471 Linear regression of TL on GPα of Japanese eel at yellow stage ……... 482 Linear regression of TL on POMC of Japanese eel at yellow stage …... 493 Linear regression of GH on GSI of Japanese eel at yellow stage ……... 504 Linear regression of FSHβ on GSI of Japanese eel at yellow stage …… 515 Linear regression of LHβ on GSI of Japanese eel at yellow stage …….. 526 Linear regression of GPα on OI of Japanese eel at yellow stage ……… 537 Linear regression of GH on FSHβ of Japanese eel at yellow stage …… 548 Linear regression of GH on LHβ of Japanese eel at yellow stage …….. 559 Linear regression of GH on GPα of Japanese eel at yellow stage …….. 560 Linear regression of GH on POMC of Japanese eel at yellow stage ….. 571 Linear regression of FSHβ on LHβ of Japanese eel at yellow stage ….. 582 Linear regression of FSHβ on GPα of Japanese eel at yellow stage ….. 593 Linear regression of FSHβ on POMC of Japanese eel at yellow stage ... 604 Linear regression of LHβ on GPα of Japanese eel at yellow stage ……. 615 Linear regression of LHβ on POMC of Japanese eel at yellow stage …. 626 Linear regression of GPα on POMC of Japanese eel at yellow stage …. 63application/pdf1481086 bytesapplication/pdfen-US日本鰻生長激素促濾泡刺激素黃體成熟激素醣蛋白原嗎啡黑色素皮質素Japanese eelgrowth hormonefollicle stimulating hormoneluteinizing hormoneglycoproteinproopiomelanocortinhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181721/1/ntu-98-R96b45032-1.pdf