Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa.
Fe(2)(+)/ascorbate, hydrogen peroxide (H(2)O(2)), 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 microM Fe(2)(+) (hydroxyl radical generator); 1 mM, 100, and 10 microM H(2)O(2); and 100, 10, and 1 mU/ml XOD (superoxide and H(2)O(2) generator), incubated at 37 degrees C for 180 min. Intracellular reactive oxygen species (ROS; H(2)DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Deltapsi(m)) were considerably decreased by H(2)O(2) (1 mM and 100 microM) and XOD (100 and 10 mU/ml). Only 1 mM H(2)O(2) reduced viability. The antioxidant Trolox (10 microM) reduced intracellular ROS, but could not prevent the H(2)O(2) or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H(2)O(2) increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Deltapsi(m) loss, confirming that H(2)O(2) 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 H(2)O(2) increased their proportion after 60 min. There were important differences between ROS generators, H(2)O(2) being the most cytotoxic. Although H(2)O(2) and XOD caused Deltapsi(m) dissipation, this was not reflected in increasing apoptotic markers.
Martínez-Pastor
Felipe
F
Biology of Reproduction Group, National Wildlife Research Institute (IREC), CSIC-UCLM-JCCM, and Institute for Regional Development (IDR), Ciencia y Tecnología Agroforestal, ETSIA, University of Castilla-La Mancha, Albacete, Spain. felipe.martinez@uclm.es
Aisen
Eduardo
E
Fernández-Santos
María Rocío
MR
Esteso
Milagros C
MC
Maroto-Morales
Alejandro
A
García-Alvarez
Olga
O
Garde
J Julián
JJ
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000032:analysis; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003670:Deer / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D017665:Hydroxyl Radical / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000032:analysis / Q000494:pharmacology*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology; D013997:Time Factors; D014969:Xanthine Oxidase / Q000494:pharmacology
D000975:Antioxidants; D015415:Biomarkers; D017382:Reactive Oxygen Species; D013481:Superoxides; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D007501:Iron; D002374:Catalase; D014969:Xanthine Oxidase; D020169:Caspases