Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm

Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential ((delta)(psi)m),caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% (plus-minus) 17% (vs. 11.3% (plus-minus) 10.6% before cryopreservation) of sperm cells showed low (delta)(psi)m , 12% (plus-minus) 6.3% (vs. 2.2% (plus-minus) 1.0% before) contained active caspases, and 10.8% (plus-minus) 5.8% (vs. 1.4% (plus-minus) 1.1% before) exhibited high membrane permeability. However, cryopreservation had no effect on DNA fragmentation (9.1% (plus-minus) 7.7% before vs. 11.1% (plus-minus) 5.7% after cryopreservation) or on nucleus condensation (46% (plus-minus) 12.7% before vs. 43.8% (plus-minus) 13.1% after). Cryopreservation acts as an apoptotic mechanism inducer in bovine sperm cells, where the earliest but not the latest features of cells undergoing apoptosis occur. We have named this abortive process an apoptosis-like phenomenon.