Russell L B, Hunsicker P R, Hack A M, Ashley T
Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Mutat Res. 2000 Jan 24;464(2):201-12. doi: 10.1016/s1383-5718(99)00185-0.
Unlike other chemicals that have been tested in mammalian germ cells, the type-II topoisomerase inhibitor etoposide exhibits significant mutagenicity in primary spermatocytes. Because this is the cell stage during which meiotic recombination normally occurs, and because topoisomerases play a role in recombination, we studied the effect of etoposide on crossing-over in male mice. Exposure to those meiotic prophase stages (probably early to mid-pachytene) during which specific-locus deletion mutations can be induced resulted in decreased crossing-over in the p-Tyr(c) interval of mouse chromosome 7. Accompanying cytological studies with fluorescent antibodies indicated that while there was no detectable effect on the number of recombination nodules (MLH1 foci), there were marked changes in the stage of appearance and localization of RAD51 and RPA proteins. These temporal and spatial protein patterns suggest the formation of multiple lesions in the DNA after MLH1 has already disappeared from spermatocytes. Since etoposide blocks religation of the cut made by type II topoisomerases, repair of DNA damage may result in rejoining of the original DNA strands, undoing the reciprocal exchange that had already occurred and resulting in reduced crossing-over despite a normal frequency of MLH1 foci. Crossing-over could conceivably be affected differentially in different chromosomal regions. If, however, the predominant action of etoposide is to decrease homologous meiotic recombination, the chemical could be expected to increase nondisjunction, an event associated with human genetic risk. Three periods in spermatogenesis respond to etoposide in different ways. Exposure of (a) late differentiating spermatogonia (and, possibly, preleptotene spermatocytes) results in cell death; (b) early- to mid-pachytene induces specific-locus deletions and crossover reduction; and, (c) late pachytene-through-diakinesis leads to genetically unbalanced conceptuses as a result of clastogenic damage.
与其他已在哺乳动物生殖细胞中进行测试的化学物质不同，II型拓扑异构酶抑制剂依托泊苷在初级精母细胞中表现出显著的致突变性。由于这是减数分裂重组正常发生的细胞阶段，且拓扑异构酶在重组中发挥作用，我们研究了依托泊苷对雄性小鼠交叉互换的影响。暴露于可诱导特定基因座缺失突变的减数分裂前期阶段（可能是粗线期早期至中期），导致小鼠7号染色体p-Tyr(c)区间的交叉互换减少。用荧光抗体进行的伴随细胞学研究表明，虽然对重组结节（MLH1灶）的数量没有可检测到的影响，但RAD51和RPA蛋白的出现阶段和定位有明显变化。这些时间和空间上的蛋白质模式表明，在MLH1已经从精母细胞中消失后，DNA中形成了多个损伤。由于依托泊苷会阻止II型拓扑异构酶切割后的重新连接，DNA损伤的修复可能导致原始DNA链重新连接，消除已经发生的相互交换，尽管MLH1灶的频率正常，但仍会导致交叉互换减少。可以想象，交叉互换在不同染色体区域可能会受到不同的影响。然而，如果依托泊苷的主要作用是减少同源减数分裂重组，那么这种化学物质可能会增加不分离现象，这是一种与人类遗传风险相关的事件。精子发生的三个时期对依托泊苷有不同的反应。暴露于（a）晚期分化的精原细胞（可能还有细线前期精母细胞）会导致细胞死亡；（b）粗线期早期至中期会诱导特定基因座缺失和交叉互换减少；以及（c）粗线期晚期至终变期会由于致断裂损伤导致基因不平衡的胚胎。
