Evaluation of sister-chromatid exchanges in mouse spermatogonia: a comparison between the classical fluorescence plus Giemsa staining and an immunocytochemical approach

Abstract

Sister-chromatid exchanges (SCE) were analyzed in mouse spermatogonia using two different protocols for bromodeoxyuridine (BrdU) exposure and detection. With the classical approach, based on subcutaneous implantation of agar-coated BrdU tablets and fluorescence plus Giemsa (FPG) staining a satisfactory differentiation of spermatogonial metaphases was obtained with 50 or 25 mg BrdU per mouse (two or one tablets respectively). Alternatively, the immunodetection of BrdU was carried out after exposure to a very low BrdU concentration (three injections i.p., 3 mg/kg b.w. each, at 5-h intervals), and after exposure to one BrdU tablet; SCE frequencies evaluated in this way were lower than those found after classical FPG staining, even when the mice were exposed to the same BrdU concentration (one tablet, 25 mg BrdU). We concluded that the two methodologies may have different sensitivities with respect to SCE detection. In addition, when the effect of a treatment with mitomycin C was tested (1 mg/kg b.w, at time intervals ranging from 24 h to 5 days), no sister-chromatid differentiation was obtained with the multiple injection protocol, or with one BrdU tablet. By contrast, with two BrdU tablets and FPG, well differentiated metaphases were found at any time interval tested after MMC treatment, and the peak frequency of SCE (3.4 times the baseline) was observed at 55 h after treatment, as expected on the basis of cell cycle duration in spermatogonia. In summary, even though the same of medium-low concentrations of BrdU was successful in untreated animals, these protocols appeared inadequate to detect SCE induction by MMC. It is possible that, in the presence of cell cycle delay induced by the treatment, interferences with the rate of BrdU uptake produce an unsatisfactory differentiation of sister chromatids.