Effects of the DNA topoisomerase II inhibitor merbarone in male mouse meiotic divisions in vivo: cell cycle arrest and induction of aneuploidy.

Abstract

In order to clarify possible risks of aneuploidy induction in germ cells by cancer chemotherapy we studied effects of a non complex-stabilizing DNA topoisomerase II (topo II) inhibitor merbarone in male mouse meiotic divisions in vivo. Two cytogenetic approaches were used: (1) C-banding on meiotic chromosome preparations and (2) analysis of spermatid micronuclei (MN) combined with immunocytochemical staining of kinetochore proteins using CREST serum. For comparison, another topo II inhibitor, VP-16, known to form cleavable complexes, was studied. The microdissection technique of mouse seminiferous tubules enabled us to carefully examine effects at specific phases of meiosis. Merbarone injections increased percentages of polyploid and hypoploid metaphase II spermatocytes at time intervals corresponding to the treatment of the first meiotic division and diplotene-diakinesis. The highest level of MN induction (5.8 MN/1000 spermatids, P < 0.001) was observed in animals injected 48 hours before the harvest, corresponding to the treatment of diplotene-diakinesis spermatocytes. Most of the induced MN (80%) contained kinetochore signals, indicating that they resulted from detachment of a whole bivalent or chromosome from the meiotic spindle. The high frequency of MN with two kinetochore signals at opposite sides (33%) most likely denotes lagging of whole bivalents during MI. Inhibition of cell proliferation was determined by scoring cells arrested at different phases of MI and MII. All groups of treated animals showed a clear increase in the frequency of arrested divisions compared to controls (P < 0.001). Thus, merbarone was shown to severely damage normal meiotic processes.