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Analysis of gene expression in the rat testis during exposure to endocrine-disrupting chemicals

暴露于内分泌干扰化学物质期间大鼠睾丸的基因表达分析

基本信息

批准号：
15591748
负责人：
SASAKI Junzo
金额：
$ 2.18万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591748/
关键词：
endocrine-disrupting chemicals DNA array rat testis ISH DEHP ribosomal RNA quantification 内分泌攪乱物質フタル酸エステル in situハイブリダイゼーション標的物質複合暴露 in situ ハイブリダイゼーション

项目摘要

As DNA array analysis can demonstrate the qualitative change in gene expression among several samples, quantitative analysis such as quantitative RT-PCR is used. However, it is not easy to quantify transcripts or proteins on tissue sections. To analyze the data from DNA array analysis in full, we tried to quantify in situ hybridization (ISH) signals via Photoshop "posterization" of the images in rat testis, a suitable organ for such quantification because germ cells undergo synchronized development and often show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA on paraffin sections. The amount of ribosomal RNA (rRNA) can be expressed numerically, grade 5 in early primary spermatocytes, grade 4 in primary spermatocytes, grade 3 in diplotene spermatocytes, and grade 2 or grade 1 in spermatids. In the present study, more accurate quantification of rRNA signals was performed using fluorescent probes and this method led to a patent applicat … More ion from Okayama University (Application number;2004-360631, application date : Dec.13,2004). The process includes the determination of ISH signals on tissue sections by comparison with signals from spots containing the standard concentrations of oligonucleotides on a slide glass.To test the possible involvement of oxidative stress in di(2-ethylhexyl) phthalate (DEHP)-induced atrophy, the rat testis was investigated by ultrastructural, histochemical and biochemical methods. Degenerated primary spermatocytes and multinucleated spermatids appeared in the seminiferous tubules after 6-9 hrs of DEHP administration, and TUNEL-positive spermatocytes increased after 12 hr of administration. DEHP increased the generation of Reactive Oxygen Species with a concomitant decrease in the concentration of glutathione and ascorbic acid in the testis.Mono(2-ethylhexyl) phthalate (MEHP), metabolite of DEHP, induced the release of cytochrome c from isolated mitochondria of the testis. DNA array analysis demonstrated that DEHP treatment induced marked changes in several genes, however, in situ hybridization histochemistry did not show patterns coincident with the gene profile. These findings indicate that oxidative stress elicited by the administration of DEHP induced apoptosis in spermatocytes, thereby causing atrophy of the testis. Less

由于DNA阵列分析可以显示几个样品之间基因表达的定性变化，因此使用定量分析，如定量RT-PCR。然而，在组织切片上定量转录物或蛋白质并不容易。为了全面分析DNA阵列分析的数据，我们试图通过Photoshop中的“色调分离”的图像在大鼠睾丸，一个合适的器官，这样的量化，因为生殖细胞进行同步发展，并经常显示特定阶段的基因表达的原位杂交（ISH）信号进行量化。在该模型实验中，选择rRNA作为石蜡切片上的可杂交RNA。核糖体RNA（rRNA）的量可以用数字表示，早期初级精母细胞中为5级，初级精母细胞中为4级，双线期精母细胞中为3级，精子细胞中为2级或1级。在目前的研究中，使用荧光探针进行了更准确的rRNA信号定量，该方法导致了一项专利申请。 ...更多信息申请号：2004-360631，申请日：2004年12月13日。该过程包括通过与载玻片上含有标准浓度寡核苷酸的斑点的信号进行比较来确定组织切片上的ISH信号，以测试氧化应激在邻苯二甲酸二（2-乙基己基）酯（DEHP）诱导的萎缩中的可能参与，通过超微结构、组织化学和生物化学方法研究了大鼠睾丸。DEHP染毒6-9 h后，生精小管内出现退化的初级精母细胞和多核精子细胞，染毒12 h后TUNEL阳性精母细胞增多。DEHP可增加睾丸中活性氧的产生，降低睾丸中谷胱甘肽和抗坏血酸的浓度，其代谢产物邻苯二甲酸单（2-乙基己基）酯（MEHP）可诱导睾丸线粒体释放细胞色素c。DNA阵列分析表明，DEHP处理诱导了几个基因的显着变化，然而，原位杂交组织化学没有显示出与基因谱一致的模式。这些发现表明，DEHP给药引起的氧化应激诱导精母细胞凋亡，从而导致睾丸萎缩。少