NOMURA Takako Okayama Univ., Graduate School of Medicine and Dentistry. Dept.of Cytology and Histology, Assistant, 大学院・医歯学総合研究科, 助手 (20116437)
YAMADA Teruo Okayama Univ., Graduate School of Medicine and Dentistry. Dept.of Cytology and Histology, Assistant, 大学院・医歯学総合研究科, 助手 (00033225)
KOSAKA Jun Okayama Univ., Graduate School of Medicine and Dentistry. Dept.of Cytology and Histology, Associate Pro., 大学院・医歯学総合研究科, 助教授 (40243216)
|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2004 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2003 : ¥2,100,000 (Direct Cost : ¥2,100,000)
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
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