title: Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa.
abstract: Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.
authors.affiliation: MRC Reproductive Biology Unit, Edinburgh EH3 9ET, Scotland, UK.
authors.forename: P
authors.initials: P
authors.lastname: Vernet
authors.forename: N
authors.initials: N
authors.lastname: Fulton
authors.forename: C
authors.initials: C
authors.lastname: Wallace
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004822:Epididymis / Q000166:cytology*; D019344:Lactic Acid / Q000494:pharmacology; D007962:Leukocytes / Q000201:enzymology; D008293:Malates / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019802:Succinic Acid / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: D008293:Malates; D009840:Oligomycins; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D012402:Rotenone; D009243:NAD; D013481:Superoxides; D019344:Lactic Acid; D009249:NADP; C007517:diphenyleneiodonium; C030298:malic acid; D019802:Succinic Acid; D019255:NADPH Oxidases; D015032:Zinc