title: Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system.
abstract: 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.
authors.affiliation: Department of Biological Chemistry,  University of Padova, Viale G. Colombo 3, 35131 Padova,  Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Donà
authors.forename: Cristina
authors.initials: C
authors.lastname: Fiore
authors.forename: Alessandra
authors.initials: A
authors.lastname: Andrisani
authors.forename: Guido
authors.initials: G
authors.lastname: Ambrosini
authors.forename: AnnaMaria
authors.initials: A
authors.lastname: Brunati
authors.forename: Eugenio
authors.initials: E
authors.lastname: Ragazzi
authors.forename: Decio
authors.initials: D
authors.lastname: Armanini
authors.forename: Luciana
authors.initials: L
authors.lastname: Bordin
authors.forename: Giulio
authors.initials: G
authors.lastname: Clari
mesh_terms: D020101:Acrosome Reaction; D006801:Humans; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D055101:Semen Analysis; D013075:Sperm Capacitation; D014443:Tyrosine / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D014443:Tyrosine; D019255:NADPH Oxidases