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Re-Evaluation of Sperm Chromatin Structure Assay (SCSA)

Satoru Kaneko, Kiyoshi Takamatsu

The Sperm Chromatin Structure Assay (SCSA) based on the polychromatic fluorescence of Acridine Orange (AO) is widely employed to observe DNA damages in human sperm nuclei. To re-evaluate the principle of the SCSA, human motile sperm with a negative DNA fragmentation rate of 87% were prepared as the Negative Control (NC) and the Positive Control (PC) was prepared through heat denaturation of the NC. The AO stainability for the controls was compared on the basis of microscopic, electrophoretic, and flow cytometric observations. Human motile sperm were separated by means of Percoll density gradient centrifugation and subsequent swim up. Then, DNA fragmentation was observed by the aid of Single-cell pulse-field gel electrophoresis.

We found that the AO molecule emitted red and green fluorescence depending on the excitation wavelength. Both controls were changed colour from green to red as the AO concentration increased and lacked red fluorescence after in-gel digestion with trypsin. The change of colour of the NC from red to green and DNA fragmentation proceeded concurrently during photo-bleaching. The cytograms of NC and PC were similar each other. These facts conflict with the SCSA principle. Green and red fluorescence were emitted due to the intercalation of AO into DNA and adsorption of AO to proteins, respectively, and their merge led to the colour variation. Discoloration from red to green during photo-bleaching was possibly observed because green fluorescence from the intercalated AO was more tolerant to reactive oxygen species than red fluorescence from adsorption of AO to some proteins. Overall, SCSA cannot detect DNA damage in the human sperm nucleus.