A SIMPLE METHOD TO DETECT SPERM CHROMATIN ABNORMALITIES - CYTOCHEMICAL MECHANISM AND POSSIBLE VALUE IN PREDICTING SEMEN QUALITY IN ASSISTEDREPRODUCTIVE PROCEDURES

Protamine crosslinking by disulphide (-SS-) bonds is the main factor r esponsible for the stability of chromatin structure in mammalian sperm atozoa. Sperm chromatin containing arginine/cysteine-rich protamines s hows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatin. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smears from human fertile donors exhibited a pale blue (orthochromatic ) colour, while a few sperm heads exhibited violet-blue or violet (met achromatic) staining. Smears from infertile patients showed an increas ed amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, su ggesting that DNA phosphates then become available for TB stacking. Mi cro- and macro-spectrophotometric studies confirmed the microscopic co lour reaction of sperm nuclei. The ortho-/metachromatic ratio seems a useful parameter for evaluation of altered chromatin structure in sper matozoa cells. Taking into account the current interest in complementa ry staining tests for evaluation of semen quality, the metachromatic T B reaction represents a simple cytochemical approach for detecting spe rm chromatin abnormalities based on differences in -SS- crosslinking.