臺灣博碩士論文加值系統

動物之生長、發育及生殖皆與內分泌系統相關，生殖系統是由下視丘-腦下腺-性腺軸（gonadotropin releasing hormone (GnRH) -gonadotropins-gonads axis）所控制，下視丘調控腦下垂體促濾泡素（follicle-stimulating hormone, FSH）及促黃體素（luteinizing hormone, LH）之分泌，其分別與促濾泡素受體（FSH receptor, FSHR）及促黃體素受體（LH receptor, LHR）結合，進而促使相關固醇類荷爾蒙之生成（steroidogenesis），以利進行精子之製造、卵巢及濾泡之發育與成熟。目前此方面於哺乳類及魚類均有頗多之研究，但對本省之經濟魚種-黃錫鯛（Silver sea bream, Sparus sarba）於此並無相關之研究，因此本研究除將針對黃錫鯛之 FSHR 及 LHR 進行基因選殖，並經由 RT-PCR、RNA 原位雜合反應法（RNA in situ hybridization）及免疫組織化學分析法（immunohistochemistry）進行表現分析。本研究以 5´RACE 及 3´RACE 之方式選殖出 FSHR 序列全長 2,402 bp，胺基酸長度為 704 a.a.，LHR 序列全長 2,514 bp，胺基酸長度為 691 a.a.，於 GenBank 之登錄編號分別為 DQ522160 及 DQ522161。將 FSHR 譯碼區（coding region）之胺基酸序列與其他物種（哺乳、禽類、魚類、兩棲及爬蟲類）進行比對後，分別具有 51.2%、50.5%、69.5%、50.6% 及 48.5% 以上之相同度（identity），而 LHR 譯碼區（coding region）之胺基酸序列與其他物種（哺乳、禽類及魚類）做比對後，亦分別具有 50.7%、52.1% 及 59.5% 以上之相同度。RT-PCR 分析之結果顯示，非繁殖季（nonspawning, 每年四月至十一月）及繁殖季（spawning, 每年十二月至隔年三月）之黃錫鯛 FSHR 2.1 kb 片段主要表現於腦部及性腺組織，且繁殖季黃錫鯛雄魚睪丸之表現量高出非繁殖季者1.1倍，於雌魚則無明顯差異； LHR 2.0 kb 片段於非繁殖季雄魚表現於腦、肝臟及睪丸，雌魚表現於腦、腸、腎及卵巢組織，而繁殖季雄魚主要表現於腦、心、肝及睪丸，雌魚表現於腦、心、及卵巢等組織中，且繁殖季雄魚睪丸表現量高於非繁殖季者 0.7 倍，雌魚卵巢表現量高於非繁殖季者 1.8 倍，此結果顯示黃錫鯛之 FSHR 及 LHR 表現量均受繁殖季之影響。另以 RNA 原位雜合分析之結果顯示 FSHR 及 LHR 於性腺組織均有表現；以免疫組織化學分析其蛋白質表現與分佈之結果顯示，FSHR 及 LHR 除表現於性腺外，亦發現於其他組織，如腦、小腸、肝臟、心臟、腎臟、脾臟及膽等皆有蛋白之表現，此部分之結果可與 RT-PCR 之分析結果相對驗證之。本研究除選殖黃錫鯛促性腺激素受體基因外，另並就其繁殖季節、基因序列、結構、表現組織及表現量進行分析，盼其可為促性腺素受體、生殖及生物醫學相關研究之基礎，並藉以深入之瞭解魚類生殖系統之基因調控，另期對生物防治之相關發展奠定基礎。

Gonadotropins, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are the key regulators of vertebrate reproduction, and they are synthesized and secreted from the gonadotrophs in the anterior pituitary. FSH and LH exert their effects on ovarian or testicular functions through the membrane receptors (FSH receptor, FSHR; LH receptor, LHR) on the gonadal cells. Most studies on the reproduction of mammals and fish, but scant about of commercial fish – silver sea bream (Sparus sarba) in Taiwan. Here we report on the first cloning of full length FSHR and LHR sequences of silver sea bream, and on the characterization and expression of these two genes. Besides, the effect of spawning was also examined. Silver sea bream FSHR and LHR have been cloned by 5´RACE and 3´RACE, and their expression was analysis by semi-quantitative RT-PCR, RNA in situ hybridization and immunohistochemistry. The cloned FSHR cDNA sequence has 2,402 bp, which composed of a coding region of 704 a.a. (GenBank accession no. DQ522160), and LHR cDNA sequence has 2,514 bp, whose coding region has 691 a.a. (GenBank accession no. DQ522161). Amino acid sequences of the coding region of silver sea bream FSHR has a 51.2% identities with that of mammals, 50.5% with bird, 69.5% with fish, 50.6% with amphibian and 48.5% with reptiles; and amino acid sequence of the coding region of LHR has a 50.7% identities with that of mammals, 52.1% with bird and 59.5% with fish. The expression of FSHR 2.1 kb fragment in nonspawning (April to November) and spawning (December to next March) seasons was detected by RT-PCR, which was mainly expressed in the brain and gonads of both genders; the expression level in testes in spawning season was 1.1 fold higher than that in nonspawning season, but no difference existed between those of ovaries in the seasons. LHR 2.0 kb fragment in nonspawning season of male silver sea bream was detected in brain, liver and testis, while in brain, intestines, kidney and ovary in female, in spawning season male silver sea bream detected in brain, heart, liver and testis, while in brain, heart and ovary in female. The LHR expression level in testes and ovaries in spawning season was 0.7 and 1.8 fold, respectively, higher than that in nonspawning season. These data indicated that the expression of gonadotropin receptors could be affected by seasons. Expression of the FSHR and LHR RNA and proteins were also detected by in situ hybridization and immunohistochemistry in the tissues mentioned before. These results are consistent with those analyzed by RT-PCR. These basic data could be applied in the studies of reproductive regulation and dysfunction, and of biomedicine, and further, it is also an alternative in the development of biocontrol of transgenic animals.

致謝……………………………………….……..............i
中文摘要………………………………….…….............ii
英文摘要………………………………………….…..……...iv
圖目錄……………………………………….…..………...ix
表目錄…………………………………………...…………...xi
第一章 序論…………………………………………...…..…1
第二章 文獻回顧……………………………….....…... .2
2.1 黃錫鯛之簡介.................................2
2.1.2 黃錫鯛之相關研究…………………………..2
2.2 生殖內分泌系統之調控……………………………...3
2.2.1 下視丘-腦下線-性腺軸之調控方式……………3
2.2.2 促濾泡素（follicle-stimulating hormone receptor, FSH）及促黃體素（luteinizing hormone receptor, LH）之功能………...………...............................…….....3
2.2.2.1 促濾泡素（FSH）及促黃體素（LH）於魚類之相關研究......................................................4
2.2.2.2 促濾泡素（FSH）及促黃體素（LH）於小鼠之相關研究......................................................4
2.2.3 促濾泡素受體（FSH receptor, FSHR）及促黃體素受體
（LH receptor, LHR）之功能及研究……………………………….........................…...5
2.2.3.1 G 蛋白耦合性受體（G protein-coupled receptors）…....….................................................5
2.2.3.2 促濾泡素受體（FSHR）及促黃體素受體（LHR）之表
現位置…………………………………………………..........…5
2.2.3.3 FSHR及LHR之表現時間………………….………...….……6
2.2.3.4 FSHR及LHR之生理功能……………..……………......7
2.3 研究方向與策略……………………..…………..………8
第三章 材料與方法……………………………………….....…9
3.1 試驗材料………...…………..................…9
3.1.1生物材料（Animal)……...……….….............….9
3.1.2菌株 (Competent cells).........................9
3.1.3質體(Plasmids)………..……….............9
3.1.4 酵素（Enzymes）…………………….……...9
3.1.5化學藥劑（Chemicals）…………….………..9
3.1.6生物反應試劑組（Kits）………………..….10
3.1.7引子（Primer...........................10
3.1.8培養基 ..…....…………………....10
3.1.9抗生素（Antibiotics）…………………...10
3.1.10反應溶液（Reaction solusion）及緩衝溶液（Buffer）..10
3.2 試驗方法…………………………………………13
3.2.1細菌之培養 ...........................13
3.2.2小量質體DNA之抽取 .................13
3.2.3大量質體DNA之抽取 ..................... 14
3.2.4限制酶切割分析 ...................... 14
3.2.5 DNA之回收 ...............................14.
3.2.6 DNA之黏接反應（Ligation） .................. 15
3.2.7大腸桿菌勝任細胞之製備 ...........................15
3.2.8 轉形作用（Transformation）.......................15
3.2.9洋菜膠體電泳分析（Agarose gel electrophoresis）...16
3.2.10 Rapid amplification of cDNA ends（RACE）........16
3.2.11 組織RNA之萃取...................................16
3.2.12 反轉錄聚合酶連鎖反應（Reverse transcription polymerase chain reaction, RT-PCR）...................17
3.2.13親緣演化樹之建立.................................17
3.2.14 RNA原位雜合反應（RNA in situ hybridization).... 17
3.2.15免疫組織化學法（(Immunohistochemistry）..........20
3.2.16半定量（Semi-quantitation）及統計分析............21
第四章 結果............................................22
4.1黃錫鯛促性腺素受體FSHR及LHR 全長序列之選殖 .........22
4.1.1 黃錫鯛FSHR之cDNA 選殖..............................22
4.1.2 黃錫鯛LHR之cDNA 選殖............................. 22
4.2促性腺素受體FSHR及LHR 之序列分析....................23
4.2.1黃錫鯛 FSHR 之 cDNA 序列分析......................23
4.2.2 黃錫鯛LHR之序列分析..............................25
4.3促性腺素受體FSHR及LHR 之演化樹分析..................26
4.4 促性腺素受體 FSHR及LHR 於繁殖季（spawning）及非繁殖季
（nonspawning）之組織分佈（tissue distribution）分析…………….….......................................27
4.4.1繁殖季之黃錫鯛短片段 FSHR 1.5 kb 及 LHR 1.8 kb 於組織
之分佈……………………………………………...….….... 27
4.4.1.1 繁殖季黃錫鯛 FSHR 1.5 kb之組織分佈分析…….. 27
4.4.1.2 繁殖季黃錫鯛 LHR 1.8 kb之組織分佈分析 ....... 28
4.4.2 繁殖季之黃錫鯛長片段FSHR 2.1 kb 及 LHR 2.0 kb
於組織之分佈情形………………………..……………………. 28
4.4.2.1 繁殖季黃錫鯛 FSHR 2.1 kb 之組織分佈分析........28
4.4.2.2 繁殖季黃錫鯛 LHR 2.0 kb 之組織分佈分析....... 29
4.4.3 非繁殖季之黃錫鯛 FSHR 1.5 kb 及 LHR 1.8 kb 組織分布情形.....................................................29
4.4.3.1 非繁殖季黃錫鯛 FSHR 1.5 kb 之組織分佈分析......29
4.4.3.2 非繁殖季黃錫鯛 LHR 1.8 kb 之組織分佈分析.......30
4.4.4 非繁殖季之黃錫鯛 FSHR 2.1 kb及 LHR 2.0 kb組織分布情形.................................................... 30
4.4.4.1 非繁殖季黃錫鯛 FSHR 2.1 kb之組織分佈分析.......30
4.4.4.2 非繁殖季黃錫鯛 LHR 2.0 kb之組織分佈分析........30
4.5 促性腺素受體 FSHR 及 LHR 之 RNA 原位雜合反應法 (RNA in situ hybridization)分析................................30
4.6促性腺素受體FSHR及LHR 之免疫組織化學分析............31
4.6.1繁殖季黃錫鯛 FSHR及 LHR之免疫組織化學分析.........31
4.6.2非繁殖季黃錫鯛 FSHR及 LHR之免疫組織化學分析.......31
第五章 討論………………………………………………….…….32
第六章 結論…………………………………………...….…….37
參考文獻…………………………………………………….…. 39
圖表…………………………………………………………….….50

圖目錄

圖一、FSHR 退化性引子之設計 ..................50
圖二、以 FSHR 退化性引子進行 PCR 之結果.......51
圖三、LHR 退化性引子設計之位置...............52
圖四、以 LHR 退化性引子進行 PCR 之結果....... 53
圖五、LHR RACE PCR 之結果.....................54
圖六、本研究所選殖之核苷酸及胺基酸 FSHR 全長序列...55
圖七、FSHR 胺基酸序列之分析結果....................59
圖八、以TMHMM Server v. 2.0預測 FSHR 七個螺旋膜區域（TMD）
之結果................................................64
圖九、本研究所選殖之核苷酸及胺基酸 LHR 全長序列...... 65
圖十、LHR 胺基酸序列分析之結果........................69
圖十一、以TMHMM Server v. 2.0預測 LHR 七個螺旋膜區域
（TMD）之結果.........................................74
圖十二、FSHR、LHR 及 TSHR 之演化分析..................75
圖十三、鄰聚法親緣關係樹（Neighbor-joining tree）.... 76
圖十四、黃錫鯛性腺於繁殖季（A）及非繁殖季（B）之相關資料.....................................................77
圖十五、繁殖季雄性黃錫鯛 FSHR 1.5 kb於各組織之表現分佈.78
圖十六、繁殖季雌性黃錫鯛 FSHR 1.5 kb於各組織之表現分佈 .79
圖十七、繁殖季雄性黃錫鯛 LHR 1.8 kb於各組織之表現分佈...80
圖十八、繁殖季雌性黃錫鯛 LHR 1.8 kb於各組織之表現分佈...81
圖十九、繁殖季雄性黃錫鯛 FSHR 2.1 kb於各組織之表現分佈..82
圖二十、繁殖季雌性黃錫鯛 FSHR 2.1 kb於各組織之表現分佈..83
圖二十一、繁殖季雄性黃錫鯛 LHR 2.0 kb於各組織之表現分佈.84
圖二十二、繁殖季雌性黃錫鯛 LHR 2.0 kb於各組織之表現分佈.85
圖二十三、非繁殖季雄性黃錫鯛 FSHR 1.5 kb於各組織之表現分佈......................................................86
圖二十四、非繁殖季雌性黃錫鯛 FSHR 1.5 kb於各組織之表現分佈......................................................87
圖二十五、非繁殖季雄性黃錫鯛 LHR 1.8 kb於各組織之表現分佈.......................................................88
圖二十六、非繁殖季雌性黃錫鯛 LHR 1.8 kb於各組織之表現分佈.......................................................89
圖二十七、非繁殖季雄性黃錫鯛 FSHR 2.1 kb於各組織之表現分佈...................................................... 90
圖二十八、非繁殖季雌性黃錫鯛 FSHR 2.1 kb於各組織之表現分佈.......................................................91
圖二十九、非繁殖季雄性黃錫鯛 LHR 2.0 kb於各組織之表現分佈...................................................... 92
圖三十、非繁殖季雌性黃錫鯛 LHR 2.0 kb於各組織之表現分佈..................................................... 93
圖三十一、雄性黃錫鯛 FSHR 及 LHR 睪丸組織之RNA原位雜合反應
（RNA in situ hybridization, RNA ISH）分析之結果........94
圖三十二、雌性黃錫鯛 FSHR 及 LHR 卵巢組織之RNA原位雜合反應
（RNA ISH）分析之結果...................................95
圖三十三、黃錫鯛腦部組織之 FSHR 及 LHR免疫組織化學分析之結果......................................................96
圖三十四、黃錫鯛睪丸組織之 FSHR 及 LHR免疫組織化學分析之結果......................................................97
圖三十五、黃錫鯛卵巢組織之 FSHR 及 LHR免疫組織化學分析之結果......................................................98
圖三十六、黃錫鯛心臟及肝臟之LHR免疫組織化學分析之結果...99
圖三十七、黃錫鯛脾臟之 LHR免疫組織化學分析之結果.......100
圖三十八、非繁殖季黃錫鯛腦部組織之 FSHR 及 LHR免疫組織化學分析之結果..............................................101
圖三十九、非繁殖季黃錫鯛睪丸組織之 FSHR 及 LHR免疫組織化學分析之結果..............................................102
圖四十、非繁殖季黃錫鯛卵巢組織之 FSHR 及 LHR免疫組織化學分析之結果................................................103
圖四十一、非繁殖季黃錫鯛腸組織之 FSHR 及 LHR免疫組織化學分析之結果................................................104
圖四十二、非繁殖季黃錫鯛心臟及脾臟之 FSHR免疫組織化學分析之結果....................................................105
圖四十三、非繁殖季黃錫鯛膽組織之FSHR免疫組織化學分析之結果......................................................106
圖四十四、非繁殖季黃錫鯛肝臟及腎臟之 LHR免疫組織化學分析之結果....................................................107

表目錄

表一、NCBI 公佈各物種之 FSHR 序列.....................108
表二、NCBI 公佈各物種之 LHR 序列......................108
表三、選殖黃錫鯛 FSHR 及 LHR 所用之退化性及專一性引子序列..................................................109
表四、黃錫鯛 FSHR 胺基酸序列與其他物種之比對結果......110
表五、黃錫鯛 LHR 胺基酸序列與其他物種之比對結果.......111
表六、FSHR 及LHR組織分佈分析所用之引子................112

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