PROBIEN   20416
INSTITUTO DE INVESTIGACION Y DESARROLLO EN INGENIERIA DE PROCESOS, BIOTECNOLOGIA Y ENERGIAS ALTERNATIVAS
Unidad Ejecutora - UE
artículos
Título:
ROS GENERATORS AFFECT DIFFERENTLY QUALITY PARAMETERS AND APOPTOSIS MARKERS IN RED DEER SPERMATOZOA
Autor/es:
F. MARTÍNEZ-PASTOR; E. AISEN; M. R. FERNÁNDEZ-SANTOS; M. C. ESTESO; A. MAROTO-MORALES; O. GARCÍÝA-ALVAREZ; J. J. GARDE
Revista:
REPRODUCTION
Editorial:
BIOSCIENTIFICA LTD
Referencias:
Año: 2009 p. 225 - 235
ISSN:
1470-1626
Resumen:
Fe2+/ascorbate, hydrogen peroxide (H2O2), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 µM Fe2+ (hydroxyl radical generator); 1 mM, 100, and 10 µM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential ( m) were considerably decreased by H2O2 (1 mM and 100 µM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 µM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented m loss, confirming that H2O2 was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused m dissipation, this was not reflected in increasing apoptotic markers.