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

由于DNA阵列分析可以显示几个样本之间基因表达的质变，因此使用了定量RT-PCR等定量分析。然而，要量化组织切片上的转录本或蛋白质并非易事。为了全面分析DNA阵列分析的数据，我们试图通过Photoshop对大鼠睾丸中的图像进行“后处理”来量化原位杂交(ISH)信号，这是一个适合进行这种量化的器官，因为生殖细胞经历同步发育，并且经常表现出阶段特异性的基因表达。在本模型实验中，选择rRNA作为石蜡切片上的可杂交RNA。核糖体RNA(RRNA)在早期初级精母细胞中呈5级表达，在初级精母细胞中呈4级表达，在双线期精母细胞中呈3级表达，在精子细胞中呈2级或1级表达。在本研究中，使用荧光探针对rRNA信号进行了更准确的定量，该方法导致了专利申请…更多来自冈山大学的申请(申请号：2004-360631，申请日期：2004年12月13日)。该过程包括测定组织切片上的ISH信号，并与玻片上含有标准浓度寡核苷酸的斑点的信号进行比较。为了测试氧化应激在邻苯二甲酸二(2-乙基己基)酯(DEHP)诱导的睾丸萎缩中的可能参与，采用超微结构、组织化学和生化方法对大鼠睾丸进行了研究。染毒6~9h后，曲细精管内出现初级精母细胞变性和多核精子细胞，染毒12h后TUNEL阳性精母细胞增多。DEHP的代谢产物邻苯二甲酸单(2-乙基己基)酯(MEHP)可诱导睾丸线粒体细胞色素c的释放。DNA阵列分析表明，DEHP处理引起了几个基因的显著变化，然而，原位杂交组织化学显示的模式与基因图谱不一致。这些结果表明，DEHP引起的氧化应激诱导精母细胞凋亡，从而导致睾丸萎缩。较少

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