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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77399完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅秀婉 | zh_TW |
| dc.contributor.author | 李尚樫 | zh_TW |
| dc.contributor.author | Shang-Chien Lee | en |
| dc.date.accessioned | 2021-07-10T21:59:59Z | - |
| dc.date.available | 2024-02-19 | - |
| dc.date.copyright | 2019-02-19 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77399 | - |
| dc.description.abstract | 摘要
目前日本鰻人工繁殖研究，尚無法達到商業化量產的需求，鰻苗仍須取自天然環境，以鮭魚腦下垂體激素 (salmon pituitary homogenate, SPH) 人工誘導未成熟鰻已成為主軸方法迫使卵巢發育及成熟。本研究先以高通量次世代定序(Next generation DNA sequencing, NGS) 分析成鰻卵巢初發育的相關重要基因，於未注射SPH及SPH注射後的基因表現量差異。以SPH注射後迫使卵巢發育，進行體內實驗，並以甲基睪固酮 (17α-methyltestosterone, MT) 結合SPH注射，研究雄性激素如何影響卵巢早期發育的基因表現及卵巢組織觀察。體外實驗則於注射SPH注射後，進行24小時的體外組織培養，分別於控制組、follicle-stimulating hormone (FSH) 、MT及 FSH+MT處理後，以RT-qPCR測定arα、arβ及 fshr 的表現量。雄性素受體（ARs）的序列結構也以胺基酸的比對分析其結構差異。 NGS的卵巢轉錄子分析可發現，未注射SPH及SPH注射後的雌鰻有顯著的基因表現差異，此差異基因與卵巢器官形成、卵濾胞初級發育相關。其中，細胞外間質（extracellular matrix）Decorin, Integrin, Nidogen 基因於SPH注射後表現上升，成長因子相關基因EGFR, VEGFR於未注射SPH的成鰻呈現較高的表現量。PI3K-Akt 訊息傳導及AR coregulator 基因等，皆在未注射SPH時，處於較高的表現。日本鰻的AR 較AR 於N-terminal 多出51個胺基酸，其中AR 具有FxxLF-like motif ，兩者之轉錄活性可能因結構差異而有所不同。 組織學比較顯示，原始轉初級卵濾胞皆可於未注射激素時，初始狀態下的卵巢中觀察到。雖然SPH及SPH+MT的注射單皆可使卵巢發育，相對於單獨的SPH注射，SPH+MT注射組的雌鰻呈現較同步的卵濾胞發育。arα的表現於SPH注射前與微幅增加的 GSI 呈現顯著正相關，而arβ 於SPH注射後與上升的GSI呈現顯著正相關。 fshr 的表現量於SPH+MT 注射後顯著上升，並且也於MT單獨注射後顯著上升。然而，fshr 的表現量於FLUT注射後下降。本研究顯示，雄性激素透過其受體作用與卵巢內發育因子，參與細胞外間質發育，影響卵巢的器官功能成熟及卵濾胞的初發育。此外，亦調節FSHR的表現，影響卵細胞初級發育至卵黃蓄積期。 | zh_TW |
| dc.description.abstract | Abstract
In Japanese eels, the seed for aquaculture still totally depends on the nature supply, and the artificial seed production is insufficient for commercial requirement. Artificial hormone induction using salmon pituitary homogenate (SPH) is a pivotal method for forcing the ovarian induction. In cultivated adult eels, a detailed understanding of initial ovarian development is still unclear before artificial SPH inductions. In this study, ovarian transcriptome was analyzed via high-throughput next generation sequencing (NGS). In vivo weekly injection of SPH was treated as the main method to force ovarian development. Androgen effect was investigated by adding 17α-methyltestosterone (MT) with SPH. Gene expression was detected after the hormone treatment, and the early ovarian development was observed with histological analysis. In vitro experiments were undertaken within 24 hours. Gene expression patterns were detected in the control, follicle-stimulating hormone (FSH)-, MT-, and FSH+MT-treated tissues respectively. In addition, arα, arβ, and fshr were analyzed using RT-qPCR. Differences of ARs’ peptide sequences were analyzed by amino acid sequence alignment and phylogenic tree construction. In the result of NGS analysis, gene expression relevant to organogenesis and the primary follicle development were detected. Differentially expressed genes were seen in the function of extracellular matrix, Decorin, Integrin, Nidogen increases after SPH injections. EGFR and VEGFR presents higher expression levels prior to SPH injections. PI3K-Akt signaling pathway and AR co-regulator presents higher expression levels before SPH injections. The peptide sequence of AR contain a FxxLF-like motif in its N-terminal side, therefore different transcription activity may be performed between AR and AR. Besides, from histological analysis, primordial to primary follicle transition was observed before hormone injection, and SPH+MT-injected eels showed greater synchronous follicle development than SPH- injected group. A positive correlation have been shown between arα expression and increasing gonadal somatic index (GSI) without SPH injection. On the other hand, another positive correlation was seen between arβ expression and GSI after three weekly SPH injections. fshr expression was high in the SPH+MT-injected group, and the effect of AR action on fshr expression was identified by flutamide treatment. The expression of arα and arβ revealed obviously different patterns after SPH induction. In this study, androgen modulation was found with regard to ARs’ actions in initial ovarian development, and ovarian organogenesis. The androgen/AR action is also affects fshr expression from the primary oocyte growth to vitellogenesis stage. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:59:59Z (GMT). No. of bitstreams: 1 ntu-108-F97b45012-1.pdf: 22285810 bytes, checksum: 13982f8c8930a98a746a83c9bd4936a3 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目 錄
口試委員會審定書……………………………………………………… # 誌謝……………………………………………………………………… # 中文摘要…………………………………………………………………..i 英文摘要………………………………………………………………….iii 第一章、研究背景………………………………………………………..1 1.1. 日本鰻人工繁殖……………………………………………………...1 1.2誘導過程中日本鰻的卵巢發育………………………………………2 1.3. 研究動機……………………………………………………………...5 第二章 、雌鰻前卵黃期早期發育轉錄體分析研究……………………13 2.1. 前言…………………………………………………………………..13 2.2. 材料與方法…………………………………………………………..14 2.2.1. 實驗動物…………………………………………………………...14 2.2.2. Total RNA萃取及cDNA 庫之建立………………………………15 2.2.3. Illumina 定序 (sequencing)、序列重新組裝 (de novo assembly) 及命名 (annotateon) ……………………………………………………...16 2.2.4. 基因表現量差異分析……………………………………………...17 2.3. 結果…………………………………………………………………..18 2.3.1. Illumina 定序 (sequencing)、序列重新組裝 (de novo assembly) 及命名 annotateon) ………………………………………………………18 2.3.2. 基因表現量差異分析……………………………………………...19 2.4. 討論…………………………………………………………………..19 第三章 、雄性激素對雌鰻卵巢前卵黃期早期發育之影響……………25 3.1. 前言…………………………………………………………………..25 3.2. 材料與方法…………………………………………………………..27 3.2.1實驗動物……………………………………………………………27 3.2.2. 激素注射誘導卵巢發育…………………………………………...27 3.2.3. 卵巢前卵黃期早期組織採樣與收集…………………………….28 3.2.4. 卵巢組織切片觀察與分期……………………………………….28 3.2.5. 顯微鏡觀察與檢視……………………………………………….29 3.2.6. 體內實驗 (in vivo experiments) ………………………………….30 3.2.7. 體內雄性素受體拮抗實驗 (in vivo experiments of androgen receptor antagonist) ………………...30 3.2.8. 體外卵巢組織培養及激素處理………………………………….31 3.2.9. Total RNA 萃取及 cDNA 合成…………………………………..32 3.2.10. 定量反轉錄聚合酶連鎖反應 ( Quantitative revese trnscriptasePCR ) ………………………………...33 3.2.11. 統計法…………………………………………………………...34 3.3. 結果…………………………………………………………………34 3.3.1. 前卵黃期早期卵濾胞分期……………………………………….34 3.3.2. 外源性激素對前卵黃期早期發育的觀察與比較……………….35 3.3.3. 以分期計數了解激素誘導前後卵濾胞於卵巢中的組成……….36 3.3.4. 以外源性激素誘導卵巢初步發育並調查 arα, arβ 及 fshr 之表現………………………………………………….37 3.3.5. 激素誘導卵巢初步發育後以雄性素受體拮抗劑注射偵測 arα, arβ 及 fshr之表現………………………………………………….38 3.3.6. 以短時間的體外組織培養瞭解arα 及arβ 之表現是否 受外源激素直接或間接影響…………………………………………...39 3.4. 討論…………………………………………………………………39 第四章 、雄性激素受體 (ARs) 的胺基酸序列的比對分析…………..48 4.1. 前言…………………………………………………………………48 4.2. 材料與方法…………………………………………………………49 4.2.1. AR序列的系統發生與比對分析………………………………...49 4.3. 結果…………………………………………………………………49 4.4. 討論…………………………………………………………………..50 第五章 、結論……………………………………………………………53 參考文獻………………………………………………………………….55 專有名詞………………………………………………………………….96 表目錄 Table 1. Statistical summary of Anguilla japonica previtellogenic ovary transcriptome assembly and annotation.…………………………………73 Table 2. The differentially expressed genes of extracellular matrix.……………...74 Table 3. The differentially expressed genes of initial ovarian development. ……..75 Table 4. The differentially expressed genes of AR-related coregulators. …………77 Table 5. Categorization of initial ovarian development in previtellogenic ovary….78 Table 6. Schedule of in vivo experiments ………………………………………….79 Table 7. Schedule of in vitro experiments …………………………………………80 Table 8. Primers for the quantitative reverse transcription PCR…………81 圖目錄 Figure 1. Nr database BLAST hits in the Japanese eel ovary. …………………….82 Figure 2. Differential expressed genes (DEGs) after three weekly SPH injections. 83 Figure 3. Differential expressed genes (DEGs) on GO and KEGG after three weekly SPH injections ……………………………………………..84 Figure 4. Histological comparison of ovarian development among the control, three weekly SPH- and SPH+MT- injected groups.……………………………...85 Figure 5. Calculation of follicle stage among female eels undergoing hormonal induction of ovary development.………………………………………….86 Figure 6. The ovarian composition of female eel undergoing hormonal induction...87 Figure 7. In vivo mRNA expression of arα, arβ, and fshr after weekly hormone injection for three weeks. ………………………………………………...88 Figure 8. Correlations between gene (arα, arβ, and fshr) expression and GSI …….89 Figure 9. In vivo mRNA expression of arα and arβ following AR’s agonist and antagonist treatment. ……………………………………………………..90 Figure 10. In vivo mRNA expression of fshr following AR’s agonist and antagonist treatment. …………………………………………………………………91 Figure 11. In vitro detection of arα and arβ expression during ovarian tissue culture with or without hormone treatment. ……………………………………...92 Figure 12. Alignment of AR NTD sequences among teleost and nonteleost fish……93 Figure 13. Alignment of AR LBD sequences among teleost and nonteleost fish……94 Figure 14. The phylogenetic tree of fish ARs.……………………………………….95 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 雄性激素 | zh_TW |
| dc.subject | 卵巢 | zh_TW |
| dc.subject | 原始至初級卵濾胞發育 | zh_TW |
| dc.subject | 雄性素受體 | zh_TW |
| dc.subject | 卵巢初發育 | zh_TW |
| dc.subject | Androgen | en |
| dc.subject | androgen receptors | en |
| dc.subject | primordial to primary transition | en |
| dc.subject | ovary | en |
| dc.subject | primary growth | en |
| dc.title | 雄性激素對日本鰻卵巢前卵黃期早期發育影響之研究 | zh_TW |
| dc.title | The effects of androgen on the early previtellogenic development in Japanese eel, (Anguilla japonica) ovary | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-1 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 嚴宏洋;黃永森;蕭崇德;韓玉山 | zh_TW |
| dc.contributor.oralexamcommittee | ;;; | en |
| dc.subject.keyword | 雄性激素,雄性素受體,原始至初級卵濾胞發育,卵巢,卵巢初發育, | zh_TW |
| dc.subject.keyword | Androgen,androgen receptors,primordial to primary transition,ovary,primary growth, | en |
| dc.relation.page | 97 | - |
| dc.identifier.doi | 10.6342/NTU201900133 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-02-18 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 漁業科學研究所 | - |
| 顯示於系所單位： | 漁業科學研究所 | |
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