INFLUENCE OF INCUBATION ON THE CHROMATIN CONDENSATION AND NUCLEAR-STABILITY OF HUMAN SPERMATOZOA BY FLOW-CYTOMETRY

Flow cytometry analysis was used for the acurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS + EDTA or SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS + DTT. in conclusion, the post-ejaculation and incubation time have to be taken into account when clinical interpretation of the effect of different treatments on sperm chromatin condensation is made.