Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa

BACKGROUND: Cryopreservation/thawing of bovine spermatozoa induces a reduction in cell viability and is possibly associated with a form of programmed cell death that we previously named 'apoptosis-like phenomenon'. METHODS: In this study, we specified, by flow cytometry, the moment of appearance of some characteristics of apoptosis during the cryopreservation process. We also studied the presence and/or activation in bovine sperm cells of specific proteins involved in somatic cell apoptosis by western blot and fluorimetry. RESULTS: A decrease of the mitochondrial membrane potential (Delta Psi(m)) was detectable 5 min after sperm dilution in the cryopreservation medium, caspase activation after 3 h of equilibration and an increase in plasma membrane permeability after the complete process of cryopreservation/thawing. The presence of the pro-apoptotic factor Bax, a protein that facilitates the formation of mitochondrial pores, was observed in bovine spermatozoa, but the anti-apoptotic factor Bcl-2 was not detectable. Moreover, it was observed that bovine spermatozoa contain cytochrome c and apoptosis-inducing factor (AIF), two proteins usually released from the mitochondria during the apoptotic process. Activated caspase-9, involved in the mitochondrial pathway, was detected in bovine spermatozoa but not caspase-3 and -8. CONCLUSIONS: The early features of apoptosis appear as ordered events during the cryopreservation/thawing process of bovine sperm cells. Bovine spermatozoa contain the machinery necessary to proceed to apoptosis involving especially the mitochondrial pathway.