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

本研究旨在对禽类内分泌干扰物进行风险评估。为了阐明胚胎发育过程中的基因激活与野生鸟类生殖异常的关系，我们利用半定量逆转录聚合酶链式反应(RT-PCR)技术检测了鸡胚胎发生过程中(孵化4-16天)泌尿生殖系统中性别决定因子基因、类固醇合成酶基因和雌激素受体基因的表达。尽管我们发现了一些生理差异，即类固醇合成途径中更多下游酶的基因表达在性别之间明显不同，但在异种雌激素二乙基己烯雌酚的体外治疗中，对这里所研究的所有基因的表达都没有影响。因此，人们认为有必要对野生动物繁殖障碍的靶基因和机制寻找新的候选基因。为了寻找未知的靶基因…此外，S，我们还使用了荧光差异显示的方法，可以检测到基因转录水平的差异。用乙炔雌二醇(10 ng-100μg/枚)处理受精卵，于第12天从每个胚胎的性腺中提取RNA样本，在雄性胚胎中分别检测到12个和1个被外源雌激素处理后增加和减少的基因。收集样本，进行DNA测序分析。其中8个cDNA来自于一个与人类伴侣蛋白基因有显著同源性的mRNA。此外，外源雌激素增加的cDNA中有一个与小鼠的犬尿氨酸转氨酶II基因有显著的同源性，而其他的则没有与任何已知基因的序列同源性。据报道，人乳腺癌细胞系(MCF-7)中伴侣蛋白的mRNA转录增加依赖于178-雌二醇(Charpentier et al.，2000)。这些基因有望成为雌激素作用的新靶点。较少

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