title: Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.
abstract: OBJECTIVE
  Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa?
  
  
  RESULTS
  Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity.
  
  
  BACKGROUND
  MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed.
  
  
  METHODS
  The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry.
  
  
  RESULTS
  MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions.
  
  
  CONCLUSIONS
  The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors.
  
  
  CONCLUSIONS
  The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study in male infertility.
  
  
  METHODS
  None.
  
  
  BACKGROUND
  The study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT (F.T.). The authors declare no conflict of interest.
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: F
authors.initials: F
authors.lastname: Treulen
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: P
authors.initials: P
authors.lastname: Uribe
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Boguen
authors.affiliation: Center of Reproductive Biotechnology - Scientific and Technological Bioresource Nucleus (CEBIOR - BIOREN), Universidad de La Frontera, Temuco 4811230, Chile Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco 4781218, Chile juana.villegas@ufrontera.cl.
authors.forename: J V
authors.initials: JV
authors.lastname: Villegas
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D001713:Biphenyl Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D005452:Fluoresceins / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009596:Nitrophenols / Q000494:pharmacology*; D010539:Permeability; D010879:Piperazines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000819:agonists* / Q000378:metabolism; D055101:Semen Analysis; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C501332:ABT-737; D001713:Biphenyl Compounds; D005452:Fluoresceins; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D013449:Sulfonamides; C085925:calcein AM; D045304:Cytochromes c; D002118:Calcium
keywords: ABT-737; Bad mimetic; mitochondrial membrane potential; mitochondrial outer membrane permeabilization; reactive oxygen species