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Research of gene expression during rat spermatogenesis and the role of reactive oxygen species in rat testis under the physiological and pathological conditions

生理病理条件下大鼠精子发生过程中基因表达及活性氧在大鼠睾丸中作用的研究

基本信息

批准号：
14570013
负责人：
NOMURA Takako
金额：
$ 2.24万
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：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

DNA array analysis can reveal underlying genes. and proteins involved in physiological and pathological conditions. To analyze the data from DNA array analysis in full, we tried to quantify ISH signals via Photoshop "posterization" of the images in testis, a suitable organ for the 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 in paraffin sections. The amount of rRNA hybridizable with the probe can be expressed as numerals, grade 5 in early primary spermatocytes, grade 4 in primary spermatocytes, grade 3 in diplotene spermatocytes, and grade 2 or grade 1 in spermatids.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. MEHP, metabolite of DEHP, induced the release of cytochrome c from isolated mitochondria of the testis. DNA array analysis revealed that DEHP treatment induced marked a decrease of several genes, however, in situ hybridization histochemistry did not show patterns coincident with the gene profile. These results indicate that oxidative stress elicited by the administration of DEHP induced apoptosis in spermatocytes, thereby causing atrophy of the testis.

DNA阵列分析可以揭示潜在的基因。以及参与生理和病理条件的蛋白质。为了全面分析来自DNA阵列分析的数据，我们试图通过睾丸中图像的Photoshop“后处理”来量化ISH信号，睾丸是一个适合量化的器官，因为生殖细胞经历同步发育并且经常显示阶段特异性基因表达。在该模型实验中，选择rRNA作为石蜡切片中的可杂交RNA。为探讨氧化应激在邻苯二甲酸二（2-乙基己基）酯（DEHP）诱导的大鼠睾丸萎缩中的作用，采用超微结构、组织化学和生化方法对DEHP诱导的大鼠睾丸萎缩进行了研究。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增加了活性氧的产生，同时降低了睾丸中谷胱甘肽和抗坏血酸的浓度。DEHP的代谢产物MEHP可诱导睾丸线粒体释放细胞色素c。DNA阵列分析显示，DEHP处理诱导了几个基因的显着减少，然而，原位杂交组织化学没有显示出与基因谱一致的模式。这些结果表明，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。