用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法，取少量精标本经洗后制片，用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理，使精子头部染色质去凝集。然后，与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号；用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号，头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子），有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ，可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法，可以用于测定接触致突变剂和非整倍体诱导剂后，人精子染色体非整倍体率的变化。

Using the method of two color fluorescence in situ hybridization to detect aneuploidy of human sperms

Objective To develop the method of two color fluorescence in situ hybridization(FISH) assay and use it for detecting the aneuploidy frequency of human sperm chromosome. Methods Sperm sample was washed three times and the slides were prepared. The sperm heads were decondensed with dithiothreitol(DTT) and lithium diiodosalicylate(LIS). Then,the sperm nuclei were hybridized with biotin labeled alpha satellite X chromosome DNA probe (DXZ1) and digoxigenin labeled alpha satellite Y chromosome DNA probe (DYZ3). The hybridization signals were detected with CY3 Streptavidin, goat antistrepavidin for biotin labeled probe and with mouse antidigoxigenin, rabbit antimouse FITC for digoxigenin labeled probe.Results Under the Nikon fluorescence microscope, the hybridization signals in the sperm heads were clearly visible. The sperm with one red hybridization singal was X chromosome sperm (X sperm),and the sperm with one green hybridization signal in the sperm head was Y chromosome sperm (Y sperm). In the case of two hybridization signals in one sperm head, the sperm should be a numerical abnormal one. By using two color FISH with one euchromosome probe and one sex chromosome probe, the sperm with same color of two hybridization signals in one sperm head could be identified as aneuploidy sperm or diploid sperm. Conclusion The two color FISH assay may be used to detect the aneuploidy frequency of human sperms that were exposed to mutagents and environmental potential aneuoploidogenic agents.