Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

中文翻译：

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使用新型转基因 cyp19a1a-eGFP 斑马鱼评估内分泌干扰化学品对芳香酶基因表达和繁殖的影响的经合组织测试指南的改进。

转基因鱼是强大的模型，可以提供有关测试化学品内分泌活动的机械信息。在这项研究中，我们的目标是在经合组织鱼类短期繁殖试验 (TG 229) 中使用新开发的表达 eGFP 的转基因斑马鱼系，该系在 cyp19a1a 启动子的控制下，以提供有关测试物质的额外机械信息。为此，我们将成年转基因斑马鱼暴露于 TG 229 的参考物质，即 prochloraz（PCZ；1.7、17.2 和 172.6 μg/L）。除了 TG 229 中使用的“经典”端点（生殖输出，卵黄蛋白）外，还使用体内成像在 4 个不同的暴露时间监测卵巢的荧光强度。我们的数据显示，172。6 μg/L PCZ 显着降低了每只雌性每天产卵的数量和雌性卵黄原蛋白的浓度，反映了由于抑制卵巢芳香酶活性而导致 E2 合成减少。在第 7 天和第 14 天，卵巢中的 GFP 强度与治疗组相似，但在 21 天后显着增加，分别为 17.2 和 172.6 μg/L。对于通过 qPCR 测量的内源性 cyp19a1a 表达，观察到类似的概况，从而证实了 GFP 测量用于评估芳香酶基因表达的可靠性。cyp19a1a 基因的过表达可能反映了对 PCZ 对芳香酶活性的抑制作用的补偿反应。总体，该研究说明了使用 cyp19a1a-eGFP 转基因系评估 PCZ 在 OECD 测试指南中的效果的可行性，同时提供有关该化合物的时间和浓度依赖性影响的补充信息，而不会干扰鱼类的繁殖。在不增加个体数量的情况下，实现了通过卵巢的体内荧光成像获取关键靶基因的额外机制信息。