title: The immobilization of human spermatozoa by STAT3 inhibitory compound V results from an excessive intracellular amount of reactive oxygen species.
abstract: We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca(2+) concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA, acrosomal integrity by FITC conjugated Pisum sativum agglutinin (PSA-FITC) labeling, intracellular pH, and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N-acetyl-L-cysteine (NAC), prevented Stattic V-induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.
authors.affiliation: Département d'Obstétrique, gynécologie et reproduction, Centre de recherche en biologie de la reproduction, Université Laval, Quebec, QC, Canada.|Axe reproduction, santé de la mère et de l'enfant, Centre de recherche du CHU de Québec, Québec, QC, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Lachance
authors.affiliation: Département d'Obstétrique, gynécologie et reproduction, Centre de recherche en biologie de la reproduction, Université Laval, Quebec, QC, Canada.|Axe reproduction, santé de la mère et de l'enfant, Centre de recherche du CHU de Québec, Québec, QC, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Goupil
authors.affiliation: Axe reproduction, santé de la mère et de l'enfant, Centre de recherche du CHU de Québec, Québec, QC, Canada.
authors.forename: R R
authors.initials: RR
authors.lastname: Tremblay
authors.affiliation: Département d'Obstétrique, gynécologie et reproduction, Centre de recherche en biologie de la reproduction, Université Laval, Quebec, QC, Canada.|Axe reproduction, santé de la mère et de l'enfant, Centre de recherche du CHU de Québec, Québec, QC, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Leclerc
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D020101:Acrosome Reaction / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors / Q000494:pharmacology; D003499:Cyclic S-Oxides / Q000494:pharmacology*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D061988:Proteasome Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors*; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003499:Cyclic S-Oxides; D007976:Leupeptins; D061988:Proteasome Inhibitors; D017382:Reactive Oxygen Species; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C517409:stattic; D000255:Adenosine Triphosphate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium; D000111:Acetylcysteine
keywords: intracellular pH; mitochondrial membrane depolarization; motility; oxidative stress; thiol oxidation