Fabiola Zambrano1, Tessa Carrau2, Ulrich Gärtner3, Anika Seipp3, Anja Taubert2, Ricardo Felmer4, Raúl Sanchez5, Carlos Hermosilla2. 1. Laboratory of Reproduction, Centre of Reproductive Biotechnology, Universidad de La Frontera, Temuco, Chile. 2. Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany. 3. Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany. 4. 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. 5. 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.
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. DESIGN: Experimental study. SETTING: University-based laboratory. PATIENT(S): Semen samples from four men and blood samples from six healthy female donors. INTERVENTION(S): 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]). MAIN OUTCOME MEASURE(S): 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]). RESULT(S): 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. CONCLUSION(S): 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.
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. DESIGN: Experimental study. SETTING: University-based laboratory. PATIENT(S): Semen samples from four men and blood samples from six healthy female donors. INTERVENTION(S): 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]). MAIN OUTCOME MEASURE(S): 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]). RESULT(S): 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. CONCLUSION(S): 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: S Hahn; P Hasler; L Vokalova; S V van Breda; O Lapaire; N G Than; I Hoesli; S W Rossi Journal: Clin Exp Immunol Date: 2019-03-13 Impact factor: 4.330
Authors: Stuart Wigby; Susan S Suarez; Brian P Lazzaro; Tommaso Pizzari; Mariana F Wolfner Journal: Curr Top Dev Biol Date: 2019-05-15 Impact factor: 4.897
Authors: Carolina Galan; Ryan W Serra; Fengyun Sun; Vera D Rinaldi; Colin C Conine; Oliver J Rando Journal: PLoS Genet Date: 2021-03-04 Impact factor: 5.917