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 (DeltaPsim) 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.
Martin
G
G
Laboratoire de Biologie de la Reproduction-GIMAP, Hôpital Nord, Saint-Etienne, France.
Cagnon
N
N
Sabido
O
O
Sion
B
B
Grizard
G
G
Durand
P
P
Levy
R
R
D020101:Acrosome Reaction; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival; D015925:Cryopreservation; D045304:Cytochromes c / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008297:Male; D010743:Phospholipids / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D012662:Semen Preservation / Q000009:adverse effects*; D013094:Spermatozoa / Q000378:metabolism; D020298:U937 Cells; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
D051033:Apoptosis Inducing Factor; D010743:Phospholipids; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9