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研究生中文姓名:曾鵬瑋
研究生英文姓名:Tseng, Peng-Wei
中文論文名稱:點帶石斑魚環化酵素啟動子的甲基化程度與性轉變之關係
英文論文名稱:Increased DNA methylation levels of cyp19a1a 5’-flanking region during sex change in orange-spotted grouper, Epinephelus coioides
指導教授姓名:張清風
吳貫忠
口試委員中文姓名:教授︰張清風
副教授︰吳貫忠
教授︰黃鵬鵬
副教授︰游智凱
助理教授︰曾庸哲
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學號:10633032
請選擇論文為:學術型
畢業年度:108
畢業學年度:107
學期:
語文別:中文
論文頁數:73
中文關鍵詞:表觀遺傳性逆轉轉路調控
英文關鍵字:Aromatase5'-flanking regionepigeneticsfoxl3sex reversal
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點帶石斑魚是一種雌雄同體硬骨魚,性別分化後會先進入雌性的生殖階段,當成長到足夠的體型大小時,會性轉變成雄性。甲基睪固酮 (17α - Methyltestosterone, MT) 和環化酵素抑制劑 (Aromatase inhibitor)可以誘導雌性石斑提早性轉變為雄性,但停止處理後會性逆轉為雌性。環化酵素可以將雄性素 (Testosterone)轉化成雌性素 (Estradiol),在魚類的雌性生殖腺分化與發育扮演重要角色。DNA甲基化是一種表觀遺傳修飾,發生在基因啟動子上的甲基化通常可以抑制基因的轉錄。本實驗通過研究環化酵素5’-flanking regions在性轉變及性逆轉的甲基化程度,來瞭解表觀遺傳修飾在點帶石斑魚性別變化中的作用。半定量分析的結果顯示,環化酵素的轉錄調控會受到轉錄因子之外的因素參與。我們在環化酵素的5’-flanking regions上找到一段有8個CpG sites的CpG rich regions。透過亞硫酸氫鈉處理並定序,在卵巢中有著顯著低於下視丘、肝臟與MT誘導精巢的甲基化程度。我們透過不同組織的甲基化程度,定義出低甲基clone (甲基化程度0-40%)、半甲基clone (甲基化程度41-80%)及高甲基clone (甲基化程度81-100%)。在雌往雄的性轉變過程中,甲基化程度會隨著卵細胞的發育而下降並在卵黃堆積時期有最多的低甲基clone,而當性轉變為精巢後會呈現高甲基化的狀態。我們透過Real-time PCR得知卵巢的細胞懸浮實驗後貼盤細胞有著高比例的體細胞而懸浮細胞有著高比例的生殖細胞,而這兩種細胞的甲基化模式沒有明顯的差異。原雄性(不會經歷成熟雌性階段)與MT誘導雄性皆比一般自然性轉變雄魚有著較小的年齡與體型,然而MT誘導雄性會發生性逆轉而原雄性不會,比較兩者在環化酵素5’-flanking regions的甲基化模式,沒有發現可以用來區分兩者的差異性。我們確認了環化酵素5’-flanking regions的甲基化程度與基因表現量呈現負相關,並且性轉變過程中甲基化模式的變化可能與細胞種類的相對數量無關。在歐洲海鱸中,會受到foxl2調控轉錄的基因也會受到foxl3的調控,因此我們想瞭解在點帶石斑魚中foxl3可能的功能及是否參與環化酵素的調控。組織分佈的結果顯示foxl3會在生殖腺中表達,且精巢中有著顯著高於卵巢的基因表達量。組織學顯示在生殖腺中只有卵原細胞及精原細胞有著foxl3基因的表達,顯示了精巢顯著高於卵巢的基因表達量可能是精巢中精原細胞的相對數量大於卵巢中卵原細胞的相對數量所造成的。根據實驗結果我們認為foxl3在點帶石斑中可能參與了生殖腺的早期發育。
Grouper is the protogynous teleost. 17α-methyltestosterone (MT) or aromatase inhibitor (AI) were induced female-to-male sex change in grouper. However, sex reversal was observed after withdrawal chemical administration. The Cyp19a1a catalyzes the conversion of androgens to estrogens. The transcription factor foxl2 and sf-1 has been characterized as an upstream regulator of a cyp19a1a that would activate cyp19a1a expression. In addition, the paralog of foxl2, foxl3, may involve in cyp19a1a regulation. To understand the potential role of epigenetics on sex change, the DNA methylation profile of cyp19a1a 5'-flanking region is used. DNA methylation typically acts to repress gene transcription. Our data showed the 5'-flanking regions of cyp19a1a had lower methylation levels and higher gene expression in ovary compared with the hypothalamus, liver and testis. Furthermore, methylation levels of cyp19a1a were significantly decreased in MT-induced female-to-male sex change. According to our bisulfite sequencing results, no differences were found in proto-male and MT-induced male and suspension cells and adhesive cells in ovary. Moreover, in situ hybridization shows that foxl3 is expressed in oogonia and spermatogonia but not somatic cells and other germ cells in both sexes. Taken together, our results demonstrate that hypermethylated 5'-flanking region of cyp19a1a is associated to male characteristic but not related to the sexual phase.
謝詞 I
摘要 II
Abstract III
目錄 IV
圖目錄 V
表目錄 VI
第一章、前言 1
一、文獻整理 1
1.性別決定 1
2.性別分化 1
3.雌雄同體 1
4.雌激素與環化酵素(Cyp19a1a)對性轉變的影響 2
5.表觀遺傳機制對cyp19a1a的轉錄調控 3
二、研究源起 7
1.研究背景 7
2.研究目的 7
第二章、實驗材料與方法 8
一、實驗設計 8
二、材料方法 13
第三章、結果 27
第四章、討論 32
第五章、參考文獻 59
附錄 68

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