BACKGROUND: Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. METHODS: A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. RESULTS: Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. CONCLUSIONS: This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.
BACKGROUND: Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. METHODS: A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. RESULTS: Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. CONCLUSIONS: This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.