Suppression of Radiation-Induced Testicular Germ Cell Apoptosis by 2,5-Hexanedione Pretreatment. III. Candidate Gene Analysis Identifies a Role for Fas in the Attenuation of X-ray–Induced Apoptosis

• Published in: Toxicological sciences Vol. 117; no. 2; pp. 466 - 474
• Main Authors: Campion, Sarah N., Sandrof, Moses A., Yamasaki, Hideki, Boekelheide, Kim
• Format: Journal Article
• Language: English
• Published: United States Oxford University Press 01-10-2010
• Subjects: 5-hexanedione; Animals; Apoptosis - drug effects; Apoptosis - genetics; Apoptosis - radiation effects; Fas; Fas Ligand Protein - genetics; Fas Ligand Protein - metabolism; fas Receptor - genetics; fas Receptor - metabolism; Gene Expression Regulation - drug effects; Gene Expression Regulation - radiation effects; Hexanones - pharmacology; laser capture microdissection; Male; Microdissection; Oligonucleotide Array Sequence Analysis - methods; Radiation-Protective Agents - pharmacology; Rats; Rats, Inbred F344; Reproductive and Developmental Toxicology; RNA, Messenger - metabolism; Seminiferous Tubules - drug effects; Seminiferous Tubules - pathology; Seminiferous Tubules - radiation effects; Spermatozoa - drug effects; Spermatozoa - pathology; Spermatozoa - radiation effects; testis; Testis - drug effects; Testis - metabolism; Testis - radiation effects; x-radiation
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfq205

Germ cell apoptosis directly induced by x-radiation (x-ray) exposure is stage specific, with a higher incidence in stage II/III seminiferous tubules. A priming exposure to the Sertoli cell toxicant 2,5-hexanedione (HD) results in a marked reduction in x-ray–induced germ cell apoptosis in these affected stages. Because of the stage specificity of these responses, examination of associated gene expression in whole testis tissue has clear limitations. Laser capture microdissection (LCM) of specific cell populations in the testis is a valuable technique for investigating the responses of different cell types following toxicant exposure. LCM coupled with quantitative real-time PCR was performed to examine the expression of apoptosis-related genes at both early (3 h) and later (12 h) time points after x-ray exposure, with or without the priming exposure to HD. The mRNAs examined include Fas, FasL, caspase 3, bcl-2, p53, PUMA, and AEN, which were identified either by literature searches or microarray analysis. Group 1 seminiferous tubules (stages I–VI) exhibited the greatest changes in gene expression. Further analysis of this stage group (SG) revealed that Fas induction by x-ray is significantly attenuated by HD co-exposure. Selecting only for germ cells from seminiferous tubules of the most sensitive SG has provided further insight into the mechanisms involved in the co-exposure response. It is hypothesized that following co-exposure, germ cells adapt to the lack of Sertoli cell support by reducing the Fas response to normal FasL signals. These findings provide a better understanding and appreciation of the tissue complexity and technical difficulties associated with examining gene expression in the testis.