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


RESULTS
Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 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. 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. However, A23187 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, is mainly related to the acrosome reaction rather than to apoptosis.
Martin
Guillaume
G
Laboratoire de Biologie de la Reproduction, GIMAP, Hôpital Nord, 42055 Saint-Etienne, France.
Sabido
Odile
O
Durand
Philippe
P
Levy
Rachel
R
D000177:Acrosome / Q000378:metabolism; D020101:Acrosome Reaction / Q000187:drug effects*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D015415:Biomarkers; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors; D002462:Cell Membrane / Q000378:metabolism*; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007476:Ionophores / Q000494:pharmacology*; D008297:Male; D051916:Membrane Cofactor Protein / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000494:pharmacology*; D011419:Propidium / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D000590:Amino Acid Chloromethyl Ketones; D015415:Biomarkers; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007476:Ionophores; D051916:Membrane Cofactor Protein; D010718:Phosphatidylserines; D011744:Pyrimidinones; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; D000001:Calcimycin; C003954:merocyanine dye; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium