Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

Abstract

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.

Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

“…It has been reported that exposure of germ cells to ET results in a preferential localization of the aberrations near the centromere (20,23,24). We have observed a similar phenomenon in both MI and MII spermatocytes.…”

“…ET induced both aneuploidy and chromosomal structural aberrations in female germ cells (20,21) and, unlike any other chemical shown to induce aneuploidy in female germ cells (22), ET did so without causing a delay in meiotic progression (20). In male germ cells, ET induced fragmentation of centromeric DNA and micronuclei after exposure of primary spermatocytes (23,24). Diplotene-diakinesis I cells were the most sensitive to the action of ET (25).…”

Chromosome analysis of mouse one-cell androgenones derived from a sperm nucleus exposed to topoisomerase II inhibitors at pre- and post-fertilization stages

“…One is that these inhibitors 14 are not accessible to the tightly packed sperm nucleus. Another is that mature spermatozoa lack etoposide and merbarone targets.…”

Abnormal chromosome migration and chromosome aberrations in mouse oocytes during meiosis II in the presence of topoisomerase II inhibitor ICRF-193

“…Etoposide, a specific topoisomerase II inhibitor, caused both structural chromosome aberrations and aneuploidy in primary oocytes of the mouse [3,4] and the Chinese hamster [5]. Similar chromosomal effects of the inhibitor were found in mouse primary spermatocytes [6,7]. Another topoisomerase II inhibitor, merbarone, caused aneuploidy in mouse primary spermatocytes without formation of structural chromosome aberrations [8].…”