Metachromatic staining of human sperm nuclei after reduction of disulphide bonds

Intra- and interprotamine cross-linking by disulphide bonds are the main factors responsible for the highly compact and stable structure of chromatin in mammalian spermatozoa. Unfixed or methanol fixed smears of human sperm and sperm suspensions from fertile donors and oligospermic patients were subjected to a reductive cleavage of disulphide bonds by using 2-mercaptoethanol (ME) or dithiothreitol (DTT). Untreated (control) and ME or DTT treated samples were stained with toluidine blue (TB) and examined in light microscopy; spectral characteristics of TB stained sperm nuclei were also analyzed. Untreated smears from fertile donors showed an orthochromatic (pale blue) staining of most sperm heads, while a variable proportion of metachromatic nuclei was found in spermatozoa from patients with oligospermia. After treatment with DTT followed by TB staining, fixed and unfixed smears showed metachromatic sperm heads. ME treatment only induced a scarce colour shift, whereas a striking metachromatic reaction and variable nuclear swelling were observed in DTT treated sperm suspensions. These results indicate that after cleavage of disulphide bonds, phosphate groups from chromatin DNA are unmasked and available for TB binding and metachromatic staining.