ABSTRACT
Methods are given for the preparation and staining of human spermatozoa for flow cytometric DNA measurements. Using agents for the reductive cleavage of disulfide crosslinks and suitable proteolytic enzymes an effective decondensation of the sperm chromatin and a DNA-proportional uptake of fluorochromes is achieved. Thus reliable and precise measurements of the relative DNA content of human spermatozoa are possible and the two subpopulations of haploid spermatozoa can be distinguished according to the difference in their DNA content.
Subject(s)
DNA/analysis , Spermatozoa/analysis , Haploidy , Histocytochemistry , Humans , Male , Staining and LabelingABSTRACT
The Dna-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescene distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.