Chapter 10 Sperm Chromatin Structure Assay: DNA Denaturability

Studies over the past decade have proven the usefulness of measurements of the integrity of sperm chromatin structure. The flow cytometry (FCM) measurement of chromatin structure is based on the principle that abnormal sperm chromatin has a greater susceptibility to physical induction of partial DNA denaturation in situ. The extent of DNA denaturation following heat or acid treatment is determined by measuring the metachromatic shift from green fluorescence [Acridine orange (AO) intercalated into double-stranded nucleic acid] to red fluorescence. Apparently acid conditions that cause partial denaturation of protamine-complexed DNA in sperm with abnormal chromatin structure do not cause denaturation of histone-complexed somatic cell DNA. The FCM measurement of sperm chromatin structure has been termed the sperm chromatin structure assay (SCSA) to distinguish it from other AO staining protocols. This protocol was formerly divided into SCSAacid and SCSAheat to distinguish the physical means of inducing DNA denaturation. The two methods give essentially the same results but the SCSAacid method is much easier to use and is the method of choice.