Propidium iodide and the thiol-specific reagent DACM as a dye pair for fluorescence resonance energy transfer analysis: an application to mouse sperm chromatin.

The dyes N-(-7-dimethyl-amino-4-methyl-coumarinyl) maleimide and propidium iodide, specific for the thiol group and DNA, respectively, were considered as a donor-acceptor couple suitable for investigating "in situ" the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable spectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double-stained samples, can be applied to evaluate the relative efficiency of the energy transfer process and its topological distribution. The results obtained indicate that during the maturation process: 1) the basic arrangement of protamine-DNA complex does not undergo structure changes, and 2) the oxidation of sulfhydryl to disulfide groups, resulting in chromatin stabilization, first involves the protamine thiols spatially closer to DNA. Fluorescence energy transfer imaging suggests that chromatin stabilization starts in the midportion of the sperm head, then spreads towards the periphery.