ANALYSIS OF MICRONUCLEI INDUCED IN MOUSE EARLY SPERMATIDS BY MITOMYCIN-C, VINBLASTINE SULFATE OR ETOPOSIDE USING FLUORESCENCE IN-SITU HYBRIDIZATION

Non-radioactive in situ hybridization with mouse centromere specific ( major) gamma satellite DNA probe was used to analyze the mechanism of induction of spermatid micronuclei (MN) caused by the alkylating agent mitomycin C (MMC), the spindle poison vinblastine sulfate (VBL) or th e DNA topoisomerase II inhibitor etoposide (VP-16). Male mice were tre ated with a single i.p. injection of 25 mg/kg VP-16, 5 mg/kg MMC or 2 mg/kg VBL, respectively. After 24 h (VP-16, VBL) or 13 days (MMC) stag e I spermatid slides were prepared and in situ hybridization was perfo rmed using a polymerase chain reaction amplified mouse (major) gamma s atellite DNA probe. The observed MN frequencies for VP-16 and MMC, 6.2 /1000 and 7.5/1000 round spermatids, respectively, show a strong mutag enic effect on mouse germ cells compared with controls (1.4/1000 sperm atids). VBL, on the contrary, induced a much lower total frequency of MN (2.8/1000 spermatids) compared with previous results on mouse somat ic cells. Of MN in controls, 24% carried a FISH signal. After correcti ng for background, MMC induced 38.6% signal-positive MN, consistent wi th a predominantly clastogenic mode of action, while VBL induced 67.9% signal-positive MN, consistent with a mainly aneugenic mechanism. VP- 16 induced 65.5% signal-positive MN, indicating that its MN-inducing c apacity is mainly due to whole chromosome lagging.