Kallio M, Lähdetie J
Department of Medical Genetics, University of Turku, Finland.
Mutagenesis. 1993 Nov;8(6):561-7. doi: 10.1093/mutage/8.6.561.
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 the DNA topoisomerase II inhibitor etoposide (VP-16). Male mice were treated 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) stage I spermatid slides were prepared and in situ hybridization was performed using a polymerase chain reaction amplified mouse (major) gamma satellite DNA probe. The observed MN frequencies for VP-16 and MMC, 6.2/1000 and 7.5/1000 round spermatids, respectively, show a strong mutagenic effect on mouse germ cells compared with controls (1.4/1000 spermatids). VBL, on the contrary, induced a much lower total frequency of MN (2.8/1000 spermatids) compared with previous results on mouse somatic cells. Of MN in controls, 24% carried a FISH signal. After correcting for background, MMC induced 38.6% signal-positive MN, consistent with 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 capacity is mainly due to whole chromosome lagging.
采用与小鼠着丝粒特异性（主要）γ卫星DNA探针的非放射性原位杂交技术，分析烷化剂丝裂霉素C（MMC）、纺锤体毒物硫酸长春碱（VBL）或DNA拓扑异构酶II抑制剂依托泊苷（VP - 16）诱导精子细胞微核（MN）的机制。分别给雄性小鼠腹腔注射25mg/kg VP - 16、5mg/kg MMC或2mg/kg VBL。24小时后（VP - 16、VBL）或13天后（MMC），制备I期精子细胞玻片，并使用聚合酶链反应扩增的小鼠（主要）γ卫星DNA探针进行原位杂交。与对照组（1.4/1000个精子细胞）相比，VP - 16和MMC观察到的微核频率分别为6.2/1000和7.5/1000个圆形精子细胞，显示出对小鼠生殖细胞有很强的诱变作用。相反，与先前小鼠体细胞的结果相比，VBL诱导的微核总频率要低得多（2.8/1000个精子细胞）。对照组的微核中，24%带有荧光原位杂交（FISH）信号。校正背景后，MMC诱导38.6%的信号阳性微核，这与主要的断裂作用模式一致，而VBL诱导67.9%的信号阳性微核，这与主要的非整倍体机制一致。VP - 16诱导65.5%的信号阳性微核，表明其诱导微核的能力主要是由于整条染色体滞后。
