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Hum. Reprod. Advance Access published online on November 8, 2006
Human Reproduction, doi:10.1093/humrep/del399
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received April 11, 2006
Revised June 7, 2006
Accepted June 30, 2006

Article

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

G. Martin 1, N. Cagnon 2, O. Sabido 3, B. Sion 4, G. Grizard 4, P. Durand 5, and R. Levy 2 *

1 Laboratoire de Biologie de la Reproduction--GIMAP, Hôpital Nord, Saint-Etienne, France; INSERM U418--INRA UMR1245, Hôpital Debrousse, Lyon, France
2 Laboratoire de Biologie de la Reproduction--GIMAP, Hôpital Nord, Saint-Etienne, France
3 Centre Commun de Cytométrie en Flux, Université Jean Monnet, Saint-Etienne Cedex 2, France
4 Laboratoire de Biologie de la Reproduction--EA 975, Faculté de Médecine, Clermont-Ferrand, France
5 INSERM U418--INRA UMR1245, Hôpital Debrousse, Lyon, France

* To whom correspondence should be addressed.
R. Levy, E-mail: rachel.levy{at}chu-st-etienne.fr


  Abstract

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 mitochon-drial pathway.

Keywords: bovine spermatozoa/cryopreservation/apoptosis/mitochondria/caspases.
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