title: Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility.
abstract: OBJECTIVE
  To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  University-based laboratory.
  
  
  METHODS
  Semen samples from four men and blood samples from six healthy female donors.
  
  
  METHODS
  Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]).
  
  
  METHODS
  During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 104:45 × 105], 6:18 [15 × 105:45 × 105], 9:18 [252 × 104:45 × 105]).
  
  
  RESULTS
  The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils.
  
  
  CONCLUSIONS
  We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology, Universidad de La Frontera, Temuco, Chile.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Zambrano
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Tessa
authors.initials: T
authors.lastname: Carrau
authors.affiliation: Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Gärtner
authors.affiliation: Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Seipp
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Taubert
authors.affiliation: Laboratory of Reproduction, Centre of Reproductive Biotechnology, Universidad de La Frontera, Temuco, Chile; Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany; Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany; Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felmer
authors.affiliation: Laboratory in Reproductive Medicine and Molecular Endocrinology, Centre of Genetic and Immunology, Universidad de La Frontera, Temuco, Chile; Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile. Electronic address: Raul.Sanchez@ufrontera.cl.
authors.forename: Raúl
authors.initials: R
authors.lastname: Sanchez
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Carlos
authors.initials: C
authors.lastname: Hermosilla
mesh_terms: D000328:Adult; D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D065206:Extracellular Traps / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D013081:Sperm Motility; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013997:Time Factors; D055815:Young Adult
chemical_list: D004791:Enzyme Inhibitors; D006657:Histones; D009195:Peroxidase; D019255:NADPH Oxidases; D019272:Leukocyte Elastase
keywords: Human spermatozoa; neutrophil extracellular traps (NETs); reproduction; sperm motility