On the integrity of sperm DNA

Abstract

AbstractEjaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X‐100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na2SO4 were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na2SO3 or sodium dodecylsulphate and dithiothreitol instead of with Triton X‐100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer‐washed spermatozoa, but much less degradation was observed if EDTA, Na2SO4, thioglycollic acid, Na2SO3 or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer‐washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ−32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose‐washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.