Morphological and biochemical analysis of cell death in human ejaculated spermatozoa.

We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies.