B

Fig. 4.2 Protein Tyr nitration associated with sperm capacitation is prevented by SOD, catalase, and l-NMMA. Percoll-washed spermatozoa are incubated in BWW medium supplemented (+, capacitating) or not (-, control) with FCSu (10% v/v) and in the absence (none) or presence of SOD (0.1 mg/mL), catalase (0.03 mg/mL), or l-NMMA (1 mM) for 3 h at 37°C. Then, sperm proteins (equivalent to 0.2 x 106 cells/well) are immunoblotted with an anti-nitro-Tyr antibody [46, 69]

exogenous NO' (NONOates) and supports a role for H2O2 in ONOO" formation [45]. A plausible explanation involves a subsequent oxidation of NO' to the nitroso-nium cation (NO+) which can then react with H2O2 (from O2'- dismutation) to give ONOO- [70, 71]. The nature of the Tyr-nitrated proteins and whether this modification is essential for, or only an effect of, capacitation is still under debate.

Therefore, as we attempted to stress above, it is possible to measure ROS produced during sperm capacitation even if levels are extremely low. These tests should not impair cell function and need real care and caution, not only for the choice of the probe and the technique (specificity, interferences, etc.) and tools (activators, inhibitors, chelators, etc.) but also to ascertain that the ROS measured effectively play a role in sperm capacitation (hyperactivation, acrosome reaction). The possibility of ROS reactions with cell components and between themselves should always be kept in mind as well as the need to preserve cell physiology.

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