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Hum. Reprod. Advance Access originally published online on August 19, 2005
Human Reproduction 2005 20(12):3459-3468; doi:10.1093/humrep/dei245
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© The Author 2005. 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@oupjournals.org

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

Guillaume Martin1,2, Odile Sabido3, Philippe Durand2 and Rachel Levy1,4

1 Laboratoire de Biologie de la Reproduction, GIMAP, Hôpital Nord, 42055 Saint-Etienne, 2 INSERM U418 – INRA UMR1245, Hôpital Debrousse, 29 rue Sur Bouvier, 69322 Lyon, 3 Centre Commun de Cytométrie en Flux, Université Jean Monnet, 15 rue Ambroise Paré, 42023 Saint-Etienne Cedex 2, France

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

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 [GenBank] led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187 [GenBank] . Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp–fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187 [GenBank] . However, A23187 [GenBank] significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187 [GenBank] , is mainly related to the acrosome reaction rather than to apoptosis.

Key words: acrosome reaction/apoptosis/calcium ionophore A23187/capacitation/phosphatidylserine exposure


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