pyrimidinones and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

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.. 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.. Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Topics: Acrosome; Acrosome Reaction; Amino Acid Chloromethyl Ketones; Apoptosis; Biomarkers; Calcimycin; Calcium; Caspase Inhibitors; Cell Membrane; DNA Fragmentation; Enzyme Inhibitors; Flow Cytometry; Fluorescein-5-isothiocyanate; Humans; In Situ Nick-End Labeling; Ionophores; Male; Membrane Cofactor Protein; Microscopy, Fluorescence; Phosphatidylserines; Propidium; Pyrimidinones; Sperm Capacitation; Spermatozoa