Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters.
BACKGROUND
Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF.


METHODS
To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy.


RESULTS
FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF.


CONCLUSIONS
Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.
Marchetti
Carole
C
INSERM U459, Faculté de Médecine, 1 Place Verdun, F-59045 Lille Cedex, France.
Gallego
Miguel-Angel
MA
Defossez
André
A
Formstecher
Pierre
P
Marchetti
Philippe
P
D000590:Amino Acid Chloromethyl Ketones; D017209:Apoptosis; D001723:Birth Rate; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D020287:In Situ Nick-End Labeling; D007248:Infertility, Male / Q000473:pathology*; D008297:Male; D012661:Semen; D013094:Spermatozoa / Q000201:enzymology / Q000473:pathology*; D013194:Staining and Labeling
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate