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 chromom ycin A(3) (CMA(3)) and 4'6-diamidino-2-phenylindole (DAPI) fluorescenc e. This was accomplished by performing a competition assay between sal mon protamine and fluorochromes on decondensed spermatozoa that had th eir nuclear proteins extracted and were fixed on slides. Various conce ntrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon p rotamine were added to either the CMA, or DAPI staining solutions. Flu orescence emission measurements of stained sperm nuclei were then perf ormed using a microfluorometer. When the treated decondensed sperm hea ds were stained with either CMA(3) or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA(3) fluorescence, while the intensity of D API staining was decreased to similar to 50% at the highest concentrat ions of protamine. The addition of increasing amounts of salmon protam ine also induced the sperm nuclei to regain their initial condensed ap pearance. This study shows that protamine retains a strong affinity fo r sperm DNA in situ and that CMA(3) fluorescence is a strong indicator of the protamination state of spermatozoa.