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Updated: Jul 11, 2026
Fluorescence in situ hybridization (FISH) Protocol in Human Sperm
Published on: September 1, 2009
[Using the method of two-color fluorescence in situ hybridization to detect aneuploidy of human sperms]
1Laboratory of Genetic Toxicology, Sun Yat-Sen University of Medical Sciences, Guangzhou, 510089, P. R. China. zhenglk@gzsums.edu.cn
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 signal 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.
