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

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DNA阵列分析的数据，我们试图通过Photoshop对睾丸中的图像进行量化来量化ISH信号，因为生殖细胞经历了同步发育，并且经常表现出阶段特异性的基因表达，因此睾丸是一个适合进行量化的器官。在本模型实验中，选择rRNA作为石蜡切片中的杂交RNA。与该探针杂交的rRNA的数量可以用数字表示，早期初级精母细胞为5级，初级精母细胞为4级，双线期精母细胞为3级，精子细胞为2级或1级。为探讨氧化应激在邻苯二甲酸二(2-乙基己基)酯(DEHP)诱导的精子萎缩中的可能作用，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增加了睾丸中活性氧的生成，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可诱导睾丸线粒体细胞色素c的释放。DNA阵列分析显示DEHP诱导的几个基因显著减少，然而，原位杂交组织化学显示出与基因图谱不一致的模式。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。

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.