- 學位論文
外源性雌激素對青鱂魚胚胎發育與促性腺釋放激素神經細胞的影響
Effects of xenoestrogens on embryonic development and gonadotropin-releasing hormone neurons in medaka (Oryzias latipes)
摘要
外源性雌激素(Xenoestrogens)是一種存在環境中的化學物質，具有像雌激素一樣的活性，可干擾人類和野生物的生殖功能。本研究探討的是，這些外源性雌激素是否會對青鱂魚(Oryzias latipes)的胚胎發育以及促性腺釋放激素(Gonadotropoin-releasing hormone，GnRH)基因的表現造成影響。青鱂魚的GnRH基因有三種，GnRH1經由腦下腺的促性腺激素調控生殖作用，而GnRH2和GnRH3的功能則與生殖行為有關。本研究使用基因轉殖螢光青鱂魚去追蹤GnRH3神經細胞的發展，其螢光來自綠色螢光蛋白(Green fluorescent protein，GFP)，由GnRH3基因的啟動子所控制(gnrh3-GFP)。因青鱂魚胚胎是透明的，所以在胚胎發育過程中，可以利用GFP的表現來監控GnRH神經細胞。我們將gnrh3-GFP青鱂魚胚胎以不同濃度的雙酚A (bisphenol-A，BPA)、壬基苯酚(nonylphenol，NP)和雌激素(17β-estradiol，E2)處理之後，觀察、記錄其螢光強度、心跳速率、眼睛和頭部的發育，以及胚胎孵化的時間等。之後再將孵化的幼魚磨碎抽取RNA，以即時定量聚合酶連鎖反應(quantitative real-time reverse transcription-PCR，qRT-PCR)分析GnRH (1、2和3)、GnRH受體(I、II和III) ，以及雌激素受體(α、β1和β2) mRNA的表現。我們的研究結果顯示，BPA、NP和E2都顯著地降低GFP螢光的表現，使胚胎心跳速率變慢，影響眼睛和頭部的發育，並且延長胚胎孵化的時間。此外，BPA、NP和E2大幅降低GnRH1、GnRH2和GnRH3基因的表現(P < 0.001)；NP和E2也顯著增加雌激素受體α的mRNA產量(P < 0.01)，但BPA則無影響。因此，本研究的結論是，BPA、NP和E2可能藉由雌激素受體α與細胞其他傳訊路徑，改變GnRH基因的表現，干擾胚胎的發育。
並列摘要
Xenoestrogens are chemicals in the environment that can exert estrogenic activity and disrupt the reproductive function of humans and wildlife. In this study, we investigated whether xenoestrogens had any effect on gonadotropin-releasing hormone (GnRH) gene expression during embryonic development in medaka (Oryzias latipes). The medaka fish express three forms of GnRH: GnRH1 regulates reproduction via gonadotropin release, while GnRH2 and GnRH3 are involved in sexual behavior. Here we used a transgenic medaka to track the development of the GnRH3 neurons, in which the green fluorescent protein (GFP) was placed under the control of the gnrh3 promoter. As the medaka embryos are transparent, the GnRH neurons expressing GFP can be monitored in vivo during embryonic development. We treated the gnrh3-GFP medaka embryos with various concentrations of bisphenol-A (BPA), nonylphenol (NP), and 17β-estrogen (E2), and recorded the fluorescence intensity, heart rate, eye and head development, and the time for the embryos to hatch. We also investigated the mRNA levels of GnRH1, GnRH2, GnRH3, GnRH receptors (RI, RII, RIII) and estrogen receptors (ERα, ERβ1, ERβ2) in above hatchlings with quantitative real-time reverse transcription-PCR (qRT-PCR). Our results showed that all three xenoestrogens significantly reduced the GnRH/GFP fluorescence intensity, decreased the heart rate, affected the eye and the head development, and lengthened the time to hatch (P<0.05). In addition, the levels of GnRH1, GnRH2, and GnRH3 mRNA were all significantly decreased (P<0.001), while the ERα mRNA levels were upregulated only by NP and E2 (P<0.01). In conclusion, BPA, NP, and E2 may disrupt the embryonic development and alter the expression of the GnRH genes through ERα and other cellular signaling pathways.