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

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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.