日本鰻濾泡細胞內雌二醇引起的DNA甲基化調控對細胞存活率之影響

Effects of estrogen-induced DNA methylation and cell viability in Japanese eel (Anguilla japonica) follicle cells

藍伯彊(Po-Chiang Lan)

指導教授 ： 王永松

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801571

摘要

日本鰻(Anguilla japonica)人工催熟常使用注射鮭魚腦下垂體萃取物(salmon pituitary extracts, SPE)作為人工催熟的方式促進卵巢卵濾泡發育，但是SPE處理會造成濾泡細胞死亡，進而造成卵母細胞數量明顯減少。本研究結果顯示，從卵巢組織切片中觀察SPE人工誘導確實可以使卵濾泡發育，我們自卵巢組織抽取total RNA，使用qPCR結果表示參與卵濾泡發育的ER與FSHR的mRNA表現量均有提升；反觀在細胞凋亡相關基因檢測中，促凋亡基因Bax表現量有增加之現象。在濾泡細胞培養實驗中，使用雌二醇(estradiol, E2)處理比使用甲基睪固酮(methyltestosterone, MT)處理更容易造成細胞存活數減少。為了解DNA甲基化(DNA methylation)是否影響濾泡細胞生長，我們使用卵巢組織檢測DNA甲基轉移酶(DNA methyltransferase, DNMT)，結果顯示SPE人工誘導會使DNMT1 mRNA表現量提升。混用E2與DNMT1抑制劑5’-Azacytidine(5’-AZA)處理濾泡細胞，含有5’-Azacytidine的處理組均可使細胞存活數回升，而E2也使濾泡細胞的Bcl2基因甲基化程度提升。本研究結果表示，E2確實透過使Bcl2甲基化程度提升對於濾泡細胞發育有負面的影響。

關鍵字

日本鰻 ； 人工催熟 ； 濾泡細胞 ； 雌二醇 ； 甲基化 ； Bcl2

並列摘要

Japanese eel (Anguilla japonica) usually use salmon pituitary extracts (SPE) promotes ovarian follicular development for artificial maturation. However, SPE treatment can cause the death of follicular cells, which in turn leads to a significant reduction in the number of oocytes. In this study, experimental results reveal that SPE can artificially induced ovary growth in Japanese eel at the early vitellogenic stage, in comparison to with a control group of eel in the previtellogenic stage. The results of qPCR also showed that the levels of mRNA expression of ER and FSHR increased in the ovarian tissue after SPE induction. However, we found an increased expression of the pro-apoptotic gene Bax in SPE treatment. Estradiol (E2) treatment is more likely to cause a decrease in cell survival than in the use of methyltestosterone (MT) in follicular cell culture experiments. To understand how DNA methylation affects the growth of follicular cells, this study detected the mRNA expression of DNA methyltransferase (DNMT) in ovarian tissue. The results showed that artificial induction of SPE increased the mRNA expression of DNMT1. In cell culture, the DNA methylation level of Bcl2 gene was found to be significantly higher in E2-treated follicular cells than in the control group. Furthermore, E2-induced hypermethylation on Bcl2 gene and repressed follicular cell counts are suppressed by adding DNMT inhibitor (5’-AZA). The results of this study indicate that E2 has a negative impact on the reproductive system of female eels by regulating epigenetic mechanism during artificial maturation.

並列關鍵字

Japanese eel(Anguilla japonica) ； artificial maturation ； follicular cells ； estradiol ； methylation ； Bcl2

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