In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry

The process of sperm chromatin decondensation occurs when a spermatozoonenters an ovum. Protamine disulphide bonds are reduced to SH and thepolycationic protamines combine with the polyanionic egg protein,nucleoplasmin, thus being stripped from DNA which then combines withhistones. Defective chromatin decondensation will thus prevent furtherdevelopment of the male pronucleus. In this study human sperm samples wereincubated in vitro at 28 degrees C (using a medium in which the polyanion,heparin, substitutes for nucleoplasmin and beta- mercaptoethanol for eggglutathione) for 10, 20 and 30 min before stopping the reaction withformalin (to 3.6%). The DNA of the fixed cells was stained with AcridineOrange by a one-step method and subjected to flow cytometry and dataanalysis, in which a zone characteristic of condensed chromatin is outlinedon red-green fluorescence contour plots. After 20 min of incubation 97% ofthe control spermatozoa that were in the mature window (WIN M) haddecondensed and moved out of this region. Defects in sperm decondensationwere seen in four semen samples of the 20 that were tested. In cases wherespermatozoa fail to produce a fertilized egg the cause may lie withdefective chromatin quality, including failure of the sperm chromatin todecondense. The method described here is a simple procedure for detectingsperm samples containing such defective cells.