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研究生:吳貫忠
研究生(外文):Guan-Chung Wu
論文名稱:黑鯛性別決定相關基因對生殖表現與性轉變機制之關係研究
論文名稱(外文):Expression of sex determining gene and sex development gene in protandrous black porgy (Acanthpagrus schlegeli) : the mechanism of sex change
指導教授:孫藍天張清風張清風引用關係
指導教授(外文):Lian-Tien SunChing-Fong Chang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:98
中文關鍵詞:性別分化性別決定
外文關鍵詞:sex differentiationsex determination
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本研究之目的在探討黑鯛不同生殖階段性別調控相關基因之變化,並以長時間投餵雌激素 (E2) 誘導性轉變方式及不同性類固醇注射,以此進ㄧ步探討黑鯛雄性性別分化與性轉變之分子機制。
黑鯛性別分化於孵化四個月後性別調控相關基因開始大量表現,Dmrt1表現量維持在高峰,Sox9無顯著差異,直到性轉變階段之精卵巢出現差異,Sf-1和Dax-1之表現在幼齡黑鯛明顯高於其它繁殖季,而Dax-1和Cyp11B1隨著生殖週期具季節性變化,性類固醇受器ARβ、ERβ與生殖細胞相關之Vasa基因亦在孵化四個月後開始大量表現,ERα則隨著生殖腺的成熟大量表現。性轉變過程中,精巢之性類固醇受器AR、ER下降,Cyp19a在卵巢顯著增加。長時間投餵E2誘導性轉變實驗中,精巢中大部分雄性性別調控相關基因皆受其負向調控而降低,而Sox9則無顯著差異,故與性轉變有密切之關係。不同性類固醇注射實驗中,精卵巢中Vasa皆受刺激增加表現,精巢Sf-1亦受不同性類固醇刺激增加表現,而只有高劑量T對卵巢之Sf-1顯著作用,低劑量T和11-KT可增加精巢Cyp11B1表現,E2則抑制表現,而E2可增加卵巢Cyp19a表現。綜合以上推論可發現,黑鯛之性別決定分化以精巢組織之調控為主,精巢組織對外在刺激之感受性較高,而卵巢組織處於停滯狀態直到性轉變階段經由E2刺激卵巢組織發育。在整個精巢組織的調控上,Dmrt1、Dax-1與Sf-1基因在精巢組織的誘發上扮演重要角色,Sox9和ER基因在性轉變期間中精卵巢之消長則與卵巢組織發育的調控相關。
The objectives of the present study were to investigate the molecular mechanism of sex determination and the role of sex development factors in sex differentiation, sex development and sex change of the protandrous black porgy (Acanthopagrus schlegeli).
At 120 dah (days after hatching), the expression of sex differentiation genes increased significantly. Dmrt1 levels were generally higher in testis while Sox9 levels were similar in both testes and ovaries. Both Dax-1 and Sf-1 gene expression levels were highest in 0-year old fish. The expression levels of both Dax-1 and Cyp11B1 increased significantly during the pre-spawning season while estrogen receptor alpha levels showed a significant increase during the spawning season. At sex change, both estrogen receptor and androgen receptor showed a significant decrease in testes while in the ovaries, Sox9 expression levels decreased dramatically while Cyp19a levels increased significantly. Long-term oral administration of E2 resulted in a significant decrease in the expression of sex differentiation genes in the testes except for Sox9 which was maintained at high levels. Injection of sex steroids caused a significant increase in the Vasa expression levels in both testes and ovaries and also an increase in Sf-1 expression levels in testes. Injection of a high dose of testosterone resulted in an increase in Sf-1 expression in the ovaries. E2 treatment resulted in an increase in Cyp19a expression levels in ovaries, while Cyp11B1 levels decreased in testis. Thus, the results suggest that the fundamental cause of sex differentiation is regulated in the testis. Dmrt1, Dax-1 and Sf-1 induce testis development, while Sox9 and estrogen receptor play an important role in sex change and maturation.
中文摘要……………………………………………………………………………… i
英文摘要……………………………………………………………………………. ii
圖表目錄……………………………………………………………...……………… iii

第一章 緒論
1. 性別的決定與分化動物的性別……………………………….…………………. 1
2. 生殖腺的分化與發育…………………………………………………………….. 2
2.1 體細胞層次的性別分化與決定因子(Sex - determined factor)………………. 3
2.2 生殖細胞層次的性別分化……………………………….…………………… 7
2.3 內分泌系統對性別分化發育的影響…………………………………………. 8
3. 魚類的性別決定與分化………………………………….………………………. 9
4. 研究源起……………………………………………….………………………… 10
5. 研究目的…………………………………………………………………………. 11

第二章 基因選殖與即時定量系統之建立
1. 前言……………………………………………………………………………… 12
2. 材料與方法……………………………………………………………………… 13
2.1 實驗魚種.…………………………………………………………………….. 13
2.2 基因選殖.…………………………………………………………………….. 13
2.3 即時定量系統之建立(Real - time quantification PCR)…………………….. 14
3. 結果……………………………………………………………………………… 16
3.1 黑鯛Sox9、 Dax-1、 Sf-1、Vasa和11β-hydroxylase之基因選殖.…….. 16
3.2 決對定量Real-time PCR系統之建立.……………………………………… 17
4. 討論……………………………………………………………………………… 18
4.1 黑鯛基因選殖………………………………………………………………… 18
圖..………………………………………………………………………………… 20

第三章 黑鯛性別決定與分化相關基因之季節性變化
1. 前言……………………………………………………………………………… 31
2. 材料與方法………………………………………………………………………. 32
2.1 實驗魚種.…………………………………………………………………….. 32
2.2 比較不同生殖階段生殖腺之季節性變化…………………………………… 34
2.3 利用即時定量系統分析黑鯛基因在生殖腺之季節性變化………………… 35
3. 結果.……………………………………………………………………………… 36
3.1 幼魚(0齡魚)至第一個繁殖季(1齡魚)中性別分化相關基因在
生殖腺之季節性變化……………………………………………………….. 36
3.2 第三個繁殖季前期(2齡魚)至第三個繁殖季(3齡魚)中性別分
化相關基因在生殖腺之季節性變化.………………………………………. 36
3.3 黑鯛幼鱼(0齡魚)至第三個繁殖季(3齡魚)中性別分化相關基
因在生殖腺之季節性變化………………………………………………….. 37
4. 討論.……………………………………………………………………………… 37
圖...………………………………………………………………………………… 41

第四章 以投餵雌二醇對誘導黑鯛性轉變過程之生殖腺影響
1. 前言……………………………………………………………………………… 49
2. 材料與方法………………………………………………………………………. 50
2.1 實驗魚種.…………………………………………………………………….. 50
2.2 實驗方法.…………………………………………………………………….. 50
2.3 利用即時定量系統分析黑鯛基因在生殖腺之變化.……………………….. 51
3. 結果.……………………………………………………………………………… 51
3.1 投餵E2對黑鯛幼魚(0齡魚)至第一個繁殖季對生殖腺中基因
表現量之影響.………………………………………………………………. 51
3.2 停餵E2對黑鯛幼魚(0齡魚)至第一個繁殖季對生殖腺中基因
表現量之影響……………………………………………………………….. 51
4. 討論.……………………………………………………………………………… 52
圖...………………………………………………………………………………… 54

第五章 以注射不同劑量、種類之類固醇激素探討黑鯛生殖內分泌的影響
1. 前言……………………………………………………………………………… 64
2. 材料與方法……………………………………………………………………… 65
2.1 實驗魚種.…………………………………………………………………….. 65
2.2 實驗方法.…………………………………………………………………….. 65
2.3 利用即時定量系統分析黑鯛基因在生殖腺之變化.……………………….. 66
2.4 細胞活性分析(In vivo labeling with BrdU)………………………………… 66
3. 結果.……………………………………………………………………………… 67
3.1 注射不同劑量、種類的類固醇激素對黑鯛生殖腺中基因表現
量之影響…………………………………………………………………….. 67
3.2 注射不同種類之類固醇激素對黑鯛生殖腺細胞活性之影………………… 67
4. 討論.……………………………………………………………………………… 68
圖...………………………………………………………………………………… 69

第六章 結論………………………………………………………………………. 77
圖...………………………………………………………………………………… 79
參考文獻……………………………………………………………………………. 80
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