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標題: | 日本鰻卵巢發育過程中Kit-Ligand基因表現之研究 Expression of Kit Ligand in the Ovarian Development of Japanese eel (Anguilla japonica) |
作者: | Tien-Tsan Lin 林天讚 |
指導教授: | 王永松 |
關鍵字: | 日本鰻,人工誘導,卵巢發育,Kit ligand,細胞培養, Japanese eel,artificial induction,ovary development,kit ligand,cell culture, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 旁分泌因子Kit ligand (kitlg) 在哺乳類已被廣泛研究,於卵巢組織中主要表現在顆粒細胞 (Granulosa cell),能夠喚醒休眠的卵細胞並促進生長。本研究驗證人工催熟藥物對日本鰻 (Anguilla japonica) 卵巢kitlga表現的影響,並首次嘗試培養日本鰻濾泡細胞作為實驗基礎。我們選殖出日本鰻之kitlga基因序列,得到cDNA全長1931個鹼基對,預測能轉譯出275個胺基酸。將胺基酸序列與其他物種進行比較並分析親緣關係,結果最接近的是三刺魚 (Gasterosteus aculeatus) 與日本鰻有50%的相同度 (Identity),其次是大黃魚(Larimichthys crocea) 有48%的相同度。由於鰻魚在人工養殖環境下無法自然達到性成熟,需要施打鮭魚腦下垂體均質液 (Salmon pituitary homogenate, SPH) 來誘導生殖腺發育,因此我們認為人工催熟過程中施打SPH應能刺激kitlga基因表現,進而幫助卵巢繼續發育。為了驗證此假說,我們將日本鰻海水馴化後分為控制組與SPH處理組,進行7針的人工催熟。比較卵巢組織切片,發現人工催熟後僅部分個體進入卵黃蓄積早期,其餘則停留在前卵黃蓄積期。卵巢基因表現方面,kitlga在不同個體的表現差異甚大,各組間無法達到顯著差異。為此本研究嘗試分離並培養日本鰻濾泡細胞,以SPH進行處理觀察基因表現的變化。結果顯示濾泡細胞在高劑量 (1 mg/ml) 的SPH處理後可顯著增加cyp19a1 mRNA表現,卻有明顯抑制kitlga mRNA的效果。我們的結果得知注射SPH確實能刺激類固醇生合成 (Steroidogenesis),但也發現卵子生成作用 (Oogenesis) 可能因kitlga表現下降而受到抑制,間接影響卵濾泡的生長。至於SPH為何會影響kitlga的基因表現則有待進一步研究。 Japanese eel, Anguilla japonica, is one of economical animal in Asian aquaculture. The reproductive mechanism and strategies of Japanese eel are still unclear, and salmon pituitary homogenate (SPH) induction is necessary to promote ovarian follicle growth. Recently, kit ligand (kitlg) is mainly secreted by the granulosa cells of the ovarian tissue and identified as a cytokine to initiate primordial follicle activation and govern the development of dormant oocytes. However, the precise function and characteristics of kit ligand in the reproduction of Japanese eel remain unclear. This study assumed that the exogenous SPH can stimulate kitlga expression to promote the ovary development, therefore, we characterized the expression patterns of kitlga in the Japanese eel. To investigate the effect of kitlga on the reproductive physiology of Japanese eel, the in vivo and in vitro studies were performed in this research. Japanese female eel were intraperitoneal injected with 7 weekly SPH induction, and the isolated follicular cell from ovary was also established a cell culture platform to investigate the effect of SPH on kitlga gene expression. First, we cloned kitlga cDNA form ovary by reserve transcription polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). Next, in order to examine the effect of SPH induction on kitlga expression, we analyzed expression pattern of kitlga messenger RNA (mRNA) by real-time quantitative PCR (qPCR) in ovary tissue and follicular cells. We cloned the full length (1931bp) of kitlga cDNAs of Japanese eel ovary, and the amino acid sequence (275 a.a.) shows high identity with that of other reported teleost kitlga. The results of qPCR found that kitlga gene expression were not significant differences in the control- and SPH group after 7 weekly inductions, and the histological section indicated that the development stage of ovaries has an individual differences. Follicular cells treated with SPH of different concentrations have showed that SPH at a high concentration (1 mg/ml) enhanced the expression of key molecule of steroidogenesis such as cyp19a1 (P450 aromatase), however, the kitlga expression was down-regulated. These results reveal that SPH can promote ovarian steroidogenesis, but the decreased kitlga may have a different role in the ovary development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59799 |
DOI: | 10.6342/NTU201700193 |
全文授權: | 有償授權 |
顯示於系所單位: | 漁業科學研究所 |
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