A risk assessment of endocrine disrupters using analytical methods for gene expression in the poultry embryo

使用家禽胚胎基因表达分析方法对内分泌干扰物进行风险评估

• 批准号: 14580577
• 负责人: TAKAHASHI Shinji
• 金额: $ 1.86万
• 依托单位: National Institute for Environmental Studies
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580577/
• 关键词: Endocrine disrupting chemicals; Endocrine disrupters; Avian; Estrogen; Gonad; Sexual differentiation; Gene expression; Sex-determining gene; 内分泌攪乱化学物質

The present study has been performed to develop a risk assessment of endocrine disrupters for avian species. To elucidate the relationship between gene activation during embryogenesis and reproductive abnormalities in wild birds, we have examined expressions of the genes for proposed sex-determining factors, steroidogenic enzymes and the estrogen receptor in the urogenital system during chicken embryogenesis (days 4-16 of incubation), using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Although we found several physiological differences that expressions of the genes for more downstream enzymes in a steroidogenic pathway were clearly different between the sexes, in ovo treatment of a xenoestrogen, diethyistilbestrol, had no effect on the expressions of all the genes examined here. Therefore, it has been considered that new candidates are necessary for target genes and the mechanisms of reproductive disorder in wildlife.In order to find unknown target gene … More s, we used a fluorescence differential display method which could detect the difference in the level of mRNA transcript. Fertilized chicken eggs treated with ethynylestradiol (10 ng-100 μg/egg) were incubated and RNA sample was extracted from the gonads in each embryo on day 12. Twelve and one mRNAs which were increased and decreased by xenoestrogen treatment, respectively, were detected in male embryos. The cDNA samples were collected and followed by DNA sequencing analysis. Eight of the cDNAs were derived from one mRNA which has a significant homology with the chaperonin protein gene in human. In addition, one of the cDNAs increased by xenoestrogen has a significant homology with the kynurenine aminotransferase II gene in the mouse, however, the others show no sequence homology with any known genes. It has been reported that chaperonin mRNA transcript in human breast cancer cell lines (MCF-7) increases in dependence on 178-estradiol (Charpentier et al., 2000). These genes reported here are expected to be candidates of new targets for estrogenic actions. Less

本研究的目的是建立一个鸟类内分泌干扰物的风险评估。为了阐明基因激活过程中的胚胎发育和繁殖异常的野生鸟类之间的关系，我们已经研究了基因的表达建议性别决定因子，类固醇生成酶和雌激素受体在泌尿生殖系统鸡胚胎发育（孵化4-16天），使用半定量逆转录-聚合酶链反应（RT-PCR）。虽然我们发现了几个生理差异，在类固醇生成途径中的下游酶的基因的表达明显不同的性别之间，在卵治疗的异种雌激素，diethyistilbestrol，没有影响的所有基因的表达在这里检查。因此，研究野生动物生殖障碍的靶基因和机制，需要寻找新的候选基因 ...更多信息 采用荧光差异显示技术检测mRNA转录水平的差异。将用乙炔基炔雌醇（10 ng-100 μg/蛋）处理的受精鸡蛋孵育，并在第12天从每个胚胎的性腺中提取RNA样品。在雄性胚胎中分别检测到12种和1种mRNA，它们在外源性雌激素处理后分别增加和减少。收集cDNA样品并随后进行DNA测序分析。其中8个cDNA来源于与人伴侣蛋白基因有显著同源性的mRNA。此外，其中一个由外源性雌激素增加的cDNA与小鼠中的犬尿氨酸氨基转移酶II基因具有显著的同源性，然而，其他cDNA与任何已知基因没有序列同源性。据报道，人乳腺癌细胞系（MCF-7）中的伴侣蛋白mRNA转录物依赖于178-雌二醇而增加（Charpentier等人，2000）。本文报道的这些基因有望成为雌激素作用的新靶点候选基因。少