In-situ competition between protamine and fluorochromes for sperm DNA

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.