Andrologie

, Volume 15, Issue 2, pp 178–184 | Cite as

Cinétique et caractérisation du phénomène “apoptosis-like” induit au cours de la congélation des spermatozoïdes bovins

  • Guillaume Martin
  • Odile Sabido
  • Philippe Durand
  • Rachel Levy
Cryopréservation Congélation et Apoptose

Resume

La congélation des spermatozoïdes éjaculés bovins induit une mortalité cellulaire élevée. Au cours d’une étude préliminaire, nous avions décrit qu’elle s’accompagne d’un processus où apparaissent plusieurs caractéristiques précoces de l’apoptose: i) diminution du potentiel de membrane mitochondrial (Δψm, ii) activation des caspases, iii) augmentation de la perméabilité membranaire, sans apparition des caractéristiques tardives: iv) pas de fragmentation de l’ADN.

Pour préciser ce mécanisme, nous avons étudlé séparément l’effet des différentes étapes de la congélation des spermatozoïdes bovins: dilution dans le milieu de congélation, équilibration et congélation dans l’azote liquide/décongélation. L’apoptose et la réaction acrosomique ont été analysées en cytométrie en flux et la présence d’AIF (Apoptosis Inducing Factor) enwestern blot. Nous avons observé que la dilution dans le milieu de congélation induisait immédiatement une augmentation massive de la proportion de spermatozoïdes avec un Δψm faible.

Puls, dès l’équilibration, la proportion de spermatozoïdes contenant des caspases actives commence à augmenter. Après le processus complet de congélation/décongélation, cette population est maximale et la perméabilité membranaire apparaît. Ces résultats montrent que le processus “apoptosis-like” est déclenché dès les premières étapes de la congélation. Nous avons aussi mis en évidence la présence d’AIF, ce qui suggère l’implication d’une voie indépendante des caspases, annexe à la voie des caspases.

Mots clés

apoptose spermatozoïdes bovins congélation cytométrie en flux 

Kinetics and characterisation of the “apoptosis-like” phenomenon induced during the cryopreservation process of bovine spermatozoa

Abstract

Cryopreservation of ejaculated bovine spermatozoa induces severe cell death. In a preliminary study, we observed that cryopreservation and/or thawing was also associated with early apoptotic features in living spermatozoa: i) decrease of the mitochondrial membrane potentialαψm), ii) caspase activation, iii) increase of membrane permeability, without the appearance of late characteristics: iv) no DNA fragmentation. This process has been called “apoptosis-like”.

In this study, we evaluated the consequences of each step of cryopreservation of bovine spermatozoa: dilution in cryopreservation medium, equilibration and cryopreservation in liquid nitrogen/thawing. Apoptosis and acrosomal reaction were analysed by flow cytometry and the presence of AIF (Apoptosis Inducing Factor) was analysed by western blot. We observed that dilution in cryopreservation medium induced a marked and immediate increase of the proportion of living spermatozoa with a lowΔψm. After equilibration, the proportion of living spermatozoa with active caspases then began to increase. After the complete cryopreservation/thawing process, this population reached a maximum, and a significant increase of membrane permeability was observed.

These results, showing that some features of the “apoptosis-like” phenomenon are initiated in the early steps of cryopreservation, suggest that ice formation may not be the only factor affecting spermatozoa. The consequence of thisΔψm decrease could be the release and/or activation of various pro-apoptotic factors in the cytoplasm. Presence of the pro-apoptotic AIF factor in bovine spermatozoa suggests a possible role of this protein during the cryopreservation process.

We also confirmed that cryopreservation of bovine spermatozoa induced an acrosomal reaction. It would be of interest to investigate the relationship between this acrosomal reaction and membrane permeability. A better understanding of the cellular mechanisms involved in sperm cryopreservation would help to improve the preservation of bovine sperm.

Key-words

apoptosis bovine spermatozoa cryopreservation flow cytometry 

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Copyright information

© Société d’Andrologie de Langue Française 2005

Authors and Affiliations

  • Guillaume Martin
    • 3
    • 1
  • Odile Sabido
    • 2
  • Philippe Durand
    • 1
  • Rachel Levy
    • 3
  1. 1.INSERM U418-INRA UMR1245Hôpital DebrousseLyon
  2. 2.Centre Commun de Cytométrie en FluxUniversité Jean MonnetSaint-Etienne
  3. 3.Laboratoire de Biologie de la ReproductionHôpital NordSaint-Etienne

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