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。为了完全分析DNA阵列分析的数据，我们试图通过Photoshop的睾丸图像“后代化”来量化ISH信号，这是一种适合定量的器官，因为生殖细胞经历了同步发育，并且经常显示阶段特异性的基因表达。在此模型实验中，将rRNA选为石蜡切片中的杂交RNA。可与探针杂交的rRNA量可以表示为数字，在初级原代精子细胞早期的5级，原代精子细胞中的4级，外载精子细胞中的3级3级和精子中的2级或1级。在DI（2-乙基己基）邻苯二甲酸酯（DEHP）诱导的萎缩中，通过超微结构，组织化学和生化方法研究了大鼠睾丸。 DEHP给药6-9小时后，在生精小管中出现了退化的原代精子细胞和多核精子，并且在给药12小时后增加了TUNEL阳性精子细胞。 DEHP增加了活性氧的产生，睾丸中谷胱甘肽和抗坏血酸的浓度随之降低。 DEHP代谢产物MEHP诱导了从睾丸的分离线粒体中释放细胞色素c。 DNA阵列分析表明，DEHP处理诱导的几种基因的降低，但是，原位杂交组织化学并未显示与基因谱相吻合的模式。这些结果表明，通过DEHP诱导的精子细胞诱导的凋亡引起的氧化应激，从而导致睾丸萎缩。