Department of Obstetrics & Gynecology, University Hospital Freiburg, Germany.
Toxicol Appl Pharmacol. 2012 May 1;260(3):232-40. doi: 10.1016/j.taap.2012.02.016. Epub 2012 Mar 6.
Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17β-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.
胚胎着床是人类生殖的关键步骤，依赖于子宫内膜的适时接受能力。人类子宫内膜在成年组织中独一无二，因为它在每个月经周期中都会发生动态变化。它是着床过程的宿主，由孕激素调控，而 17β-雌二醇则调控子宫内膜的前期增殖。这两种类固醇的受体都是药物和内分泌干扰化学物质的作用靶点。具有非期望抗孕作用的化学物质对胚胎着床有潜在危害，因为许多药物抗孕激素会对子宫内膜的接受能力产生不利影响。这种风险可以通过人体组织特异性的体外检测来解决。作为工作基础，我们汇总了与 PR 相互作用的化学物质的数据。在我们的实验工作中，我们开发了一种基于人子宫内膜 Ishikawa 细胞的灵活体外模型。通过 RT-qPCR 获得的 S 形浓度-反应曲线，对抗孕激素化合物对预选靶基因的影响进行了特征描述。通过微阵列分析，鉴定出雌激素硫酸转移酶 (SULT1E1) 是最敏感的靶基因。RU486（米非司酮）和 ZK137316 浓度依赖性拮抗孕激素对 SULT1E1 mRNA 的激动作用，而 4-壬基酚、双酚 A 和芹菜素的作用较弱。阴性对照甲基乙酰乙酸酯则没有效果。通过 Western blot 从蛋白质水平验证了孕激素和 RU486 的作用。我们证明了我们的 Ishikawa 模型适用于研究抗孕激素样化学物质对子宫内膜靶基因的影响，与药物参考化合物相比具有定量优势。该测试可用于危害识别，并有助于减少动物研究。
