title: Peroxiredoxins prevent oxidative stress during human sperm capacitation.
abstract: Do peroxiredoxins (PRDXs) control reactive oxygen species (ROS) levels during human sperm capacitation?
  
  
  
  PRDXs are necessary to control the levels of ROS generated during capacitation allowing spermatozoa to achieve fertilizing ability.
  
  
  
  Sperm capacitation is an oxidative event that requires low and controlled amounts of ROS to trigger phosphorylation events. PRDXs are antioxidant enzymes that not only act as scavengers but also control ROS action in somatic cells. Spermatozoa from infertile men have lower levels of PRDXs (particularly of PRDX6), which are thiol-oxidized and therefore inactive.
  
  
  
  Semen samples were obtained from a cohort of 20 healthy nonsmoker volunteers aged 22-30 years old over a period of 1 year.
  
  
  
  Sperm from healthy donors was capacitated with fetal cord serum ultrafiltrate (FCSu) in the absence or presence of thiostrepton (TSP), inhibitor of 2-Cys PRDXs or 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium (MJ33), inhibitor of calcium independent-phospholipase A2 (Ca2+-iPLA2) activity of PRDX6, added at different times of incubation. Capacitation was also induced by the dibutyryl cAMP+3-isobuty1-1-methylxanthine system. Sperm viability and motility were determined by the hypo-osmotic swelling test and computer-assisted semen analysis system, respectively. Capacitation was determined by the ability of spermatozoa to undergo the acrosome reaction triggered by lysophosphatidylcholine. Percentages of acrosome reaction were obtained using the FITC-conjugated Pisum sativum agglutinin assay. Phosphorylation of tyrosine residues and of protein kinase A (PKA) substrates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblotting with specific antibodies. Actin polymerization was determined by phalloidin labeling.
  
  
  
  TSP and MJ33 prevented sperm capacitation and its associated actin polymerization in spermatozoa incubated with 10% FCSu (capacitation inducer) compared to non-capacitated controls (P < 0.05) without altering sperm viability. PKA substrates and tyrosine phosphorylations were prevented in FCSu-treated spermatozoa in a differential fashion depending on the type and the time of addition of the inhibitor used compared to non-capacitated controls (P < 0.05). TSP and MJ33 promoted an increase of lipid peroxidation in spermatozoa (P < 0.01) and these levels were higher in those spermatozoa incubated with the inhibitors and FCSu compared to those capacitated spermatozoa incubated without the inhibitors (P < 0.0001). Inhibition of 2-Cys PRDXs by TSP generated an oxidative stress in spermatozoa, affecting their viability compared to controls (P < 0.05). This oxidative stress was prevented by nuclephile D-penicillamine (PEN). MJ33 also promoted an increase of lipid peroxidation and impaired sperm viability compared to non-treated controls (P < 0.05) but its effect was not circumvented by PEN, suggesting that not only peroxidase but also Ca2+-iPLA2 activity of PRDX6 are necessary to guarantee viability in human spermatozoa.
  
  
  
  Not applicable.
  
  
  
  We focused on the global effect of PRDXs inhibitors on human sperm capacitation and in two of its associated phosphorylation events. Thus, other phosphorylation events and mechanisms necessary for capacitation may also be affected.
  
  
  
  PRDXs are the major antioxidant system in ejaculated spermatozoa and are necessary to allow spermatozoon to achieve fertilizing ability (capacitation and acrosome reaction).
  
  
  
  This research was supported by Canadian Institutes of Health Research (MOP 133661) and the Fonds de Recherché en Santé Quebec (FRSQS #22151) to C.O. The authors have nothing to disclose.
authors.affiliation: The Research Institute of the McGill University Health Centre, Montréal, Canada.|Department of Surgery (Urology Division), McGill University, Montréal, Québec, Canada.
authors.forename: Donghyun
authors.initials: D
authors.lastname: Lee
authors.affiliation: The Research Institute of the McGill University Health Centre, Montréal, Canada.|Department of Surgery (Urology Division), McGill University, Montréal, Québec, Canada.|Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
authors.forename: Adel R
authors.initials: AR
authors.lastname: Moawad
authors.affiliation: The Research Institute of the McGill University Health Centre, Montréal, Canada.|Department of Surgery (Urology Division), McGill University, Montréal, Québec, Canada.
authors.forename: Tania
authors.initials: T
authors.lastname: Morielli
authors.affiliation: The Research Institute of the McGill University Health Centre, Montréal, Canada.|Department of Surgery (Urology Division), McGill University, Montréal, Québec, Canada.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Fernandez
authors.affiliation: The Research Institute of the McGill University Health Centre, Montréal, Canada.|Department of Surgery (Urology Division), McGill University, Montréal, Québec, Canada.|Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.
authors.forename: Cristian
authors.initials: C
authors.lastname: O'Flaherty
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D020101:Acrosome Reaction / Q000187:drug effects; D000328:Adult; D002470:Cell Survival / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005312:Fetal Blood / Q000737:chemistry; D005786:Gene Expression Regulation; D005994:Glycerophosphates / Q000494:pharmacology; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008297:Male; D018384:Oxidative Stress; D010396:Penicillamine / Q000494:pharmacology; D054464:Peroxiredoxins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013075:Sperm Capacitation / Q000187:drug effects / Q000235:genetics*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D013883:Thiostrepton / Q000494:pharmacology
chemical_list: D005994:Glycerophosphates; D008244:Lysophosphatidylcholines; D017382:Reactive Oxygen Species; C078587:1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D054464:Peroxiredoxins; D017868:Cyclic AMP-Dependent Protein Kinases; D010396:Penicillamine; D013883:Thiostrepton; D015056:1-Methyl-3-isobutylxanthine
keywords: 2-Cys peroxiredoxins; calcium independent phospholipase A2 activity; oxidative stress; redox signaling; spermatozoa