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Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility

      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.

      Key Words

      Although remarkable scientific developments have been made in the field of reproductive biology, the problem of infertility increases worldwide (
      • Boivin J.
      • Bunting L.
      • Collins J.A.
      • Nygren K.G.
      International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.
      ). This problem can occur at any time during the reproductive period of men or women. The worldwide prevalence of infertility is estimated to be between 3.5% and 16.7% in developed countries and between 6.9% and 9.3% in less developed nations (
      • Boivin J.
      • Bunting L.
      • Collins J.A.
      • Nygren K.G.
      International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.
      ). Despite recent progress made in the diagnosis and management of infertility, in 10%–20% of cases no exact etiology can yet be identified (
      • Boivin J.
      • Bunting L.
      • Collins J.A.
      • Nygren K.G.
      International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.
      ).
      Various inflammatory diseases may originate within the female reproductive tract, including infectious forms of vaginitis such as bacterial vaginosis (
      • Mitchell C.
      • Marrazzo J.
      Bacterial vaginosis and the cervicovaginal immune response.
      ), candidiasis (
      • de Repentigny L.
      • Goupil M.
      • Jolicoeur P.
      Oropharyngeal candidiasis in HIV infection: analysis of impaired mucosal immune response to Candida albicans in mice expressing the HIV-1 transgene.
      ), and sexually transmitted diseases (STDs). Leukocytosis occurs in all these STDs, with spermatozoa being directly exposed to polymorphonuclear neutrophils (PMN), which usually results in significantly decreased fertility (
      • Alghamdi A.S.
      • Foster D.N.
      Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps.
      ).
      Alongside phagocytosis, PMN have several other potent effector mechanisms to combat and eventually kill pathogens and spermatozoa, such as the oxidative burst activity resulting in the production of reactive oxygen species (ROS), the release of antimicrobial peptides/proteins, and the formation of neutrophil extracellular traps (NETs). NETs are released after PMN cell death process, reported as NETosis, and are primarily situated in the extracellular space. NETosis has been shown to be an NADPH oxidase (NOX)-dependent mechanism, which ultimately leads to the extrusion of a mixture of nuclear as well as cytoplasmic granule contents resulting in the formation of DNA-rich web-like structures that are adorned with histones (H1, H2A/H2B, H3, H4) and granular effector molecules, such as neutrophil elastase (NE), pentraxin, lactoferrin, MPO, and others (
      • Hermosilla C.
      • Caro T.M.
      • Silva L.M.
      • Ruiz A.
      • Taubert A.
      The intriguing host innate immune response: novel anti-parasitic defence by neutrophil extracellular traps.
      ,
      • Munoz-Caro T.
      • Mena Huertas S.J.
      • Conejeros I.
      • Alarcon P.
      • Hidalgo M.A.
      • Burgos R.A.
      • et al.
      Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent.
      ). Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of ERK activation and a rather moderate level of Akt activation, whereas the activation of p38 is similar in both pathways (
      • Douda D.N.
      • Khan M.A.
      • Grasemann H.
      • Palaniyar N.
      SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx.
      ). Interestingly, it has been recently demonstrated that PMN, which undergo NETosis without cell lysis, are still live and retain their capability of phagocytosis (
      Wold Health Organization
      WHO laboratory manual for the examination and processing of human semen.
      ). Consistent with these reports, PMN also seem to be capable of extruding NETs of mitochondrial origin, which are of smaller size than those originating from classical induced NETosis by different forms of NETs, such as aggregated (aggNETs), diffused (diffNETs), and spread (sprNETs) NETs, that have been recently reported (
      • Schauer C.
      • Janko C.
      • Munoz L.E.
      • Zhao Y.
      • Kienhofer D.
      • Frey B.
      • et al.
      Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines.
      ,
      • Munoz-Caro T.
      • Lendner M.
      • Daugschies A.
      • Hermosilla C.
      • Taubert A.
      NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.
      ).
      Much less is known about the molecular mechanisms behind spermatozoa-triggered NETosis. Thus, the aim of the present study was to not only investigate the direct interaction of live male spermatozoa with human PMN, but also to provide evidence on effector molecules and signaling pathways involved in this novel cell death process. In addition, we assessed the detrimental NETs-derived effects, such as the sperm motility, on entrapped spermatozoa.

      Materials and methods

      This study was approved by the appropriate University Review Boards and Ethics Committee of the Universidad de La Frontera (UFRO), Temuco, Chile, and the Justus Liebig University (JLU) Giessen, Germany.

       Reagents

      All reagents were obtained from Sigma Chemical unless otherwise stated.

       Samples of Blood and Spermatozoa

      In each experiment, blood samples were used from healthy female volunteers (n = 6) between 20 and 35 years of age. Thirty milliliters of blood were extracted from the cephalic vein of each donor. Isolation of the PMN started immediately after extraction at the JLU Giessen, Germany.
      The sperm samples were obtained from men (n = 4) with normal semen parameters, ages 20–26 years. Semen samples were obtained by masturbation after less than 3 days of sexual abstinence. The samples were collected in sterile plastic tubes (Deltalab) and transported immediately to the laboratory no later than 60 minutes after collection. The samples were evaluated according to recommendations by the World Health Organization (WHO), and only those with semen parameters within the normal ranges established by WHO (
      • Lu J.C.
      • Huang Y.F.
      • Lu N.Q.
      WHO laboratory manual for the examination and processing of human semen: its applicability to andrology laboratories in China.
      ) were used for NET-related experiments. Experiments were performed at JLU Giessen, Germany, and Universidad de La Frontera, Temuco, Chile.

       PMN Isolation from Human Blood

      The blood samples were kept in a vacutainer with heparin (10 U/mL). The isolation method employed was according to Oh et al. (
      • Oh H.
      • Siano B.
      • Diamond S.
      Neutrophil isolation protocol.
      ). Briefly, 6 mL of Histopaque 1119 (Gibco) were placed in a 15-mL Falcon tube and 7 mL of heparinized blood were carefully added. This mixture was centrifuged at 800 × g for 20 minutes without braking. After aspiration and discard of the clear yellowish top layer, the lower reddish phase containing granulocytes was transferred into fresh Falcon tubes. The cells were then washed by filling the Falcon tubes with sterile phosphate-buffered saline (PBS) and centrifuged for 10 minutes at 300 × g. A 100% Percoll solution (Amersham) was prepared by mixing 18 mL Percoll with 2 mL 10× PBS. Four-milliliter solutions of 85%, 80%, 75%, 70%, and 65% Percoll gradients were thereafter diluted with 1× PBS. Then two Falcon tubes (for each blood sample) containing descending Percoll gradients (85%, 80%, 75%, 70%, and 65%, respectively) were layered with the blood. After centrifugation (10 minutes at 300 × g), the supernatant fraction was discarded. The pellets were then resuspended in 4 mL of PBS. Two milliliters of the resuspension from each of the gradients was collected and centrifuged again at 800 × g for 20 minutes without braking. After centrifugation, the top layer and most of the 65% layer containing mononuclear peripheral blood cells (PBMCs) were discarded, while the remaining white interphases belonging to the 85% layer were collected in a new Falcon tube. The cells were then washed by filling up the Falcon tubes with PBS and centrifuging for 10 minutes at 300 × g. Finally, the supernatant was removed and the cell pellets were resuspended in 2 mL of PBS. PMN were counted with a Neubauer hemocytometer (
      • Munoz-Caro T.
      • Mena Huertas S.J.
      • Conejeros I.
      • Alarcon P.
      • Hidalgo M.A.
      • Burgos R.A.
      • et al.
      Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent.
      ).

       Immunofluorescence of NETs and Detection of Histone H3, NE, and MPO in Human Spermatozoa-Induced NET Structures

      After incubation of PMN with human spermatozoa at a ratio of 1:6 (25 × 104:15 × 105) for 2 hours, the samples were fixed in 4% paraformaldehyde on coverslips previously coated with poly-L-lysine for 15 minutes at room temperature (RT). Cells were then incubated with 500 μL of 2% bovine serum albumin for 15 minutes at 37°C. Then two washes were performed with 500 mL PBS for each sample, and primary monoclonal antibodies were added: anti-NE antibodies (anti-rabbit ab 68672 Abcam), anti-MPO antibodies (anti-rabbit orb 11073 Biorbyt), and anti-histone H3 antibodies (anti-mouse clone H11-4, Merck Millipore). All samples were incubated for 1 hour at RT. Samples were washed twice with 500 μL of PBS, and secondary conjugated antibodies were then added (AlexaFluor 488 goat anti-rabbit IgG, reference no. A11008, Life Technologies, for detection of NE and MPO; AlexaFluor 488 goat anti-mouse IgG, reference no. A11001, molecular probes for the detection of H3). All samples were incubated for 1 hour at RT in the dark. Finally, the samples were washed twice in 500 μL PBS and mounted face down onto a glass coverslip to which one drop of antifading mounting medium (Molecular Probes, reference no. PB6941) had previously been added containing DAPI for staining the cell nuclei. Samples were immediately observed thereafter by using an Olympus IX81 inverted fluorescence microscope equipped with a digital camera.

       NETs Quantification

      To quantify the NETs induced by spermatozoa, PMN and spermatozoa were cocultured at different ratios (1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]) and for different time points (30, 60, 90, 120, and 180 minutes, respectively). To assess dose-dependent effects on NETs, cells were incubated at different ratios (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105]). Zymosan 1 μg/mL (Invitrogen) was used to induce the formation of NETs in human PMN (positive control) as described elsewhere (
      • Munoz-Caro T.
      • Lendner M.
      • Daugschies A.
      • Hermosilla C.
      • Taubert A.
      NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.
      ). The effect of different inhibitors of PMN/sperm samples ratio 1:6 (25 × 104:15 × 105) was also assessed. Before exposure to human spermatozoa samples, PMN were incubated for 30 minutes with diphenylene iodonium (DPI) 10 μM (NADPH oxidase inhibitor), Suc-Ala-Ala-Pro-Val chloromethyl ketone (CMK) 1 mM (NE inhibitor), and 4-aminobenzoic acid hydrazide (ABAH) 100 μM (MPO inhibitor). For DNase I (90 U) control, this enzyme was added 15 minutes before the end of incubation. Additionally, for negative control of NETs quantification, different settings were used: PMN alone, PMN/sperm without inhibitors, and sperm alone. After different incubation times, the samples were treated with 0.1 U/μL of micrococcal nuclease (New England Biolabs) for 15 minutes at 37°C. Then 100 μL of supernatant from each sample were transferred in duplicates to a 96-well cell culture plate (Greiner). Finally, 50 μL of PicoGreen (1:200 in 10 mM Tris base buffered with 1 mM EDTA) were added to samples, protected from light, and incubated for 4 minutes in complete darkness. Quantification of NETs formation was determined by spectrofluorometric analysis at an excitation wavelength of 484 nm and an emission wavelength of 520 nm using an automated plate monochrome reader (Varioskan Flash; Thermo Scientific), as described elsewhere (
      • Munoz-Caro T.
      • Mena Huertas S.J.
      • Conejeros I.
      • Alarcon P.
      • Hidalgo M.A.
      • Burgos R.A.
      • et al.
      Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent.
      ,
      • Munoz-Caro T.
      • Lendner M.
      • Daugschies A.
      • Hermosilla C.
      • Taubert A.
      NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.
      ,
      • Silva L.M.
      • Caro T.M.
      • Gerstberger R.
      • Vila-Vicosa M.J.
      • Cortes H.C.
      • Hermosilla C.
      • et al.
      The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps.
      ).

       Scanning Electron Microscopy (SEM)

      Human PMN were incubated with vital human spermatozoa (ratio 1:1) for 120 minutes on poly-L-lysine (0.01%; Sigma-Aldrich) precoated coverslips (10 mm diameter). Samples were then fixed in 2.5% glutaraldehyde (Merck) and washed in distilled water. Samples were then dehydrated, critical-point dried by CO2 treatment, and sputter-coated with gold. The samples were examined using a Philips XL30 scanning electron microscope at the Institute of Anatomy and Cell Biology, JLU Giessen, Giessen, Germany.

       Transmission Electron Microscopy (TEM)

      Pellets of PMN and human spermatozoa were fixed in Karnosvsky solution (
      • Karnovsky M.J.
      The ultrastructural basis of capillary permeability studied with peroxidase as a tracer.
      ), postfixed in 1% OsO4 at 4°C and embedded in Epon-Araldite. Ultrathin sections were stained with uranyl acetate and leas citrate. The samples were examined using a Hitachi EM-700 electron microscope at the Institute of Pathology and Histology, Austral University, Valdivia, Chile, and Hitachi STEM SU-3500 at the Scientific and Technological Bioresource Nucleus at UFRO, Temuco, Chile.

       Assessment of Sperm Motility

      The Computer Aided Sperm Analysis system was used with Integrated Sperm Analysis System software (Proiser) equipped with phase-contrast microscopy. The software default configuration for human semen was used for the evaluation. Unselected sperm samples were used diluted in modified human tubal fluid medium and kept at 37°C (
      • Quinn P.
      • Kerin J.F.
      • Warnes G.M.
      Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid.
      ). Progressive motility was evaluated in four groups with different PMN/sperm ratios: control group, sperm without exposure to PMN; PMN/sperm (1:18 [25 × 104:45 × 105]); PMN/sperm (6:18 [15 × 105:45 × 105]); and PMN/sperm (9:18 [252 × 104:45 × 105]). Cells were incubated for 3 hours, and progressive motility was evaluated at 0, 1, 2, and 3 hours for each group. For the study, 10 μL of each group was taken and placed onto a slide, and then the sample was covered with a coverslip of 22 × 22 mm. The samples were quickly assessed with a microscope equipped with a thermal plate at 37°C. At least 200 spermatozoa were considered for each evaluation group and taken from at least five different power vision fields. Spermatozoa that formed aggregates or that attached to PMN were excluded from analysis. Only spermatozoa free in the medium were considered in the current sperm motility assessment experiments.

       Statistical Analysis

      The results were analyzed by descriptive statistics of the mean ± SD calculated for each of the variables using GraphPad Prism version 5.0a Software. Gaussian distribution was analyzed with D'Agostino's test. One-way analysis of variance (ANOVA) was used to test for statistically significant differences (P<.05) between groups. In cases where statistically significant differences were observed, Tukey's test was used to determine where those differences existed. Arcsine transformation was carried out to evaluate spermatozoa motility, and a two-way ANOVA was performed to assess this assay.

      Results

       Visualization of Human Spermatozoa-Triggered NETs

      SEM as well as fluorescence microscopy analyses revealed that the exposure of human PMN to vital human spermatozoa trigger the formation of NETs (Fig. 1, white arrows) that are firmly attached to the spermatozoa, seemingly trapping them (Fig. 1, yellow arrows). Kinetic analyses revealed morphologically different types of spermatozoa-derived NET formation: diffused, spread, and aggregated NETs (Fig. 2 and Supplemental Fig. 1). The spermatozoa were frequently captured either by their tails or heads. Furthermore, it was shown that by exposing PMN to spermatozoa (ratio 1:1, for 2 hours), different levels of PMN activation were observed. Some PMN still exhibited the morphology of intact cells, whereas others became activated as illustrated by their rough membrane surface. Later on, spermatozoa are entrapped in a network of long drawn-out fibers originating from disrupted PMN (Fig. 1) and conglomerates of spermatozoa and rather chunky mesh works of PMN-derived filaments (Fig. 2). Close contact of gametes with the PMN surface was also detected, whereas others were clearly phagocytizing spermatozoa (Fig. 1).
      Figure thumbnail gr1
      Figure 1Visualization of human spermatozoa-triggered NETs by SEM and TEM. NETs induced by vital human spermatozoa after 2 hours of exposure to human PMN. SEM analysis shows (A-D) the activation of PMN in the presence of human spermatozoa. Spermatozoa-triggered NETs formation and the capture of sperm by NETs structures. White arrows indicate NETs structures, red arrows indicate PMN, and yellow arrows indicate sperm being trapped by NETs. TEM (E and F) shows the activation of PMN by human sperm as the surface becomes rough and forms foldings and crinkles: (E) sperm phagocytosis and (F) completely covered sperm by NET-like extrusions. White arrows indicate NET-like extrusions, red arrows indicate intracellular sperm within PMN after phagocytosis, and yellow arrows indicate a sperm being entrapped by thicker NET-like extrusion (×98.500).
      Figure thumbnail gr2
      Figure 2Representative immunofluorescence images of PMN exposed to sperm. Coincubation PMN/sperm ratio 1:6 for 2 hours. Phase contrast (A, E, and I), blue fluorescence stains the nuclei of cells (DAPI, B, F, and J), green fluorescence (Alexafluor 488) shows different components of NETs, (C) HIS (histone H3), (G) MPO, and (K) NE. Colocalization areas are shown in panels D, H, and L by mixing both channels (merge).
      The ultrastructural analyses via TEM (Fig. 1) revealed additional interactions of activated PMN and sperm. As such, sperm-induced phagocytosis by exposed PMN was also observed (Fig. 1E and 1F), as well as the early formation of NET-like structures derived from the PMN surface.

       Quantification of NETs Induced by Spermatozoa

      Quantification analyses on total NETs formation (Table 1) showed that human spermatozoa significantly induced NETosis in human PMN (P<.05). As expected, this reaction was significantly and almost entirely resolved by DNase I treatment in sperm-exposed PMN (sperm vs. DNase I; P<.05). Furthermore, quantitative assessment of sprNETs and diffNETs revealed that exposure of PMN to vital sperm significantly induced NETosis when compared with the negative control (P<.05). Moreover, results of NETs quantification (fluorescence intensity of PicoGreen [AU]) exposed to spermatozoa at different ratios for 2 hours of incubation showed that NETs formation increased significantly (P<.05) at a ratio of 1:6 compared with negative controls (unexposed PMN). This significant increase of sperm-mediated NETosis was comparable to the ones induced by the positive control Zymosan.
      Table 1Kinetic experiments of spermatozoa-triggered NETosis.
      KineticsFluorescence intensity (AU), mean ± SDRatioFluorescence intensity (AU), mean ± SDInhibitorsFluorescence intensity (AU), mean ± SD
      PMN/sperm1/61/18PMN5.7 ± 1.8aPMN6.2 ± 1.5a,e
      PMN5.7 ± 1.7a3.9 ± 0.4a1:110.2 ± 5.0aPMN/sperm23.2 ± 23.2b,c
      60 min10.6 ± 4.1a83.8 ± 5.1b1:311.5 ± 5.4aSperm19.2 ± 4.0a,b,d,e
      90 min10.7 ± 3.0a80.0 ± 2.7b1:621.5 ± 9.0bPMN/sperm + DPI13.3 ± 1.8a,c,d,f
      120 min22.3 ± 4.1b86.0 ± 6.0bZymosan33.0 ± 11.5bPMN/sperm + CMK7.9 ± 1.7a,e
      180 min22.2 ± 2.1b88.6 ± 2.8cPMN/sperm + ABAH8.8 ± 6.7e
      Zymosan37.7 ± 20.7b99.5 ± 13.9cPMN/sperm+ DNAse I0.6 ± 0.1e
      Zymosan74.6 ± 15.6g
      Note: PMN were incubated with spermatozoa at different ratios (ratio 1:6; ratio 1:18), Zymosan (1 mg/mL, positive control), or PMN (negative control) for different time periods. PMN and spermatozoa were incubated at different ratios (PMN: spermatozoa (PMN/sperm) = 1:1, 1:3, 1:6). NET inhibition assays were performed by preincubating PMN with inhibitors of NOX (DPI, 10 μM), NE (CMK, 1 mM), and MPO (ABAH, 100 mM) before spermatozoa exposure and NET measurement. DNase I (90 U) control was also included. For positive controls, Zymosan treatment of PMN was used. PMN in plain medium as well as spermatozoa alone served as negative controls. After incubation, samples were analyzed for extracellular DNA by quantifying PicoGreen-derived fluorescence intensities (AU). Data are the mean ± SD (three biological replicates).
      a-gData followed by different letters in the same column are significantly different (P<.05).
      Quantification of fluorescence intensities mirroring NETs formation revealed that exposure of PMN to spermatozoa significantly increased the amount of extracellular DNA when compared with spermatozoa-free controls (P<.05; Table 1). Furthermore, spermatozoa-induced NETosis was dose-dependent, as increasing the amount of spermatozoa led to enhanced fluorescence intensities (Table 1). Quantification of NETs in PMN exposed to sperm at a ratio 1:6 showed significant differences in time (P<.05) from 120 minutes onwards (PMN/sperm 120 minutes, 22.35 AU). Zymosan showed increases (37.76 AU) in NETs formation similar to that seen at 120 and 180 minutes (PMN/sperm 180 minutes, 22.26 AU). The PMN/sperm ratio 1:18 showed consistent significant increases of NETs (P<.05) when exposed for 60, 90, 120, and 180 minutes (83.89, 80.00, 86.02, and 88.62 AU, respectively) compared with the control (PMN, 3.9 AU). The NETs increase observed at 180 minutes was similar to the one observed for the positive control (Zymosan, 99.58 AU; Table 1).

       Spermatozoa-Induced NETosis is a NOX-, NE-, and MPO-Dependent Process

      The actual role of the NET-associated enzymes NOX, NE, and MPO was analyzed via functional inhibitor experiments using DPI, CMK, and ABAH, respectively. CMK and ABAH inhibitors treatments resulted in a significant decrease of spermatozoa-triggered NETs formation in the human system (P<.05; Table 1), thus demonstrating the key role of these enzymes in spermatozoa-induced NETs formation. Moreover, spermatozoa-induced NETs in human PMN were efficiently abolished by DNase I treatment (P<.05; Table 1), proving the typical characteristics of NETs.

       Immunofluorescence of NETs Structures

      Immunofluorescence analysis of PMN exposed to spermatozoa (ratio 1:6) for 2 hours showed the formation of different-sized aggregated NETs (aggNETs) entrapping vast amounts of spermatozoa, activated PMN, and NETs structures (Fig. 2). Areas of colocalization are also observed in these clusters with intercalated blue fluorescence (DAPI, DNA brand, Fig. 2B, 2F, and 2J) and green fluorescence (Alexa Fluor 488, HIS, MPO, and NE brands, Fig. 2C, 2G, and 2K) forming a turquoise color (Fig. 2D, 2H, and 2L), which confirms the formation of NETs. Also shown are representative images of how the NETs (Supplemental Fig. 1, white arrow) efficiently capture the spermatozoa by ensnaring diverse parts (head, middle piece, and flagellum; Supplemental Fig. 1A). Areas of colocalization (Supplemental Fig. 1C, red arrow indicates colocalization of DNA and NE) of cell aggregates in nebulae form also show the formation of these structures (Supplemental Fig. 1).

       Motility Assay

      Sperm motility (Fig. 3) was measured at 0, 1, 2, and 3 hours of exposure of the spermatozoa to increasing proportions of PMN (PMN/sperm, 1:18, 6:18, 9:18). This parameter was unchanged in the different groups when measured at time 0. In the first hour of incubation the two groups with the highest proportion of PMN significantly decreased in sperm motility (PMN/sperm 6:18 [21.67%] and 9:18 [18.67%]) compared with the control group (40.53%). At 2 and 3 hours, a significant reduction of progressive motility was evident in all sperm groups exposed to PMN (PMN/sperm 1:18, 6:18, and 9:18, with motility 26.87%, 18.60%, and 14.23%, respectively); meanwhile, the control group at this period maintained 35.23% of progressively motile sperm.
      Figure thumbnail gr3
      Figure 3Effect in time on the progressive motility of human sperm of incubation of PMN/sperm with different ratios (1:18, 6:18, 9:18). Data are shown as percentages of progressively motile sperm (progressive motility [%]). Graph with three biological replicas. The results correspond to the mean ± SD. Differences were regarded as significant (*, ***) at a level of P<.001.

      Discussion

      In the present work we confirmed that, as previously described, the efficient phagocytic PMN consume spermatozoa directly (Fig. 1) or trap them by enmeshing them through NETs (
      • Alghamdi A.S.
      • Foster D.N.
      Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps.
      ,
      • Marey M.A.
      • Liu J.
      • Kowsar R.
      • Haneda S.
      • Matsui M.
      • Sasaki M.
      • et al.
      Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.
      ). This direct cell contact between the PMN and the spermatozoon, which can result in the entrapment via the head, middle piece, or flagellum, represents the first stage of sperm phagocytosis (spermiophagy) (
      • Marey M.A.
      • Liu J.
      • Kowsar R.
      • Haneda S.
      • Matsui M.
      • Sasaki M.
      • et al.
      Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.
      ). TEM as well as SEM analyses confirmed that both effector mechanisms are displayed by PMN exposed to vital spermatozoa, namely, phagocytosis and the NETosis process. We found both fine and coarse networks; some NETs merged when they were released by nearby PMN, and this may also establish a neutrocyte cooperation system for more effective sperm elimination as postulated elsewhere (
      • Piasecka M.
      • Fraczek M.
      • Gaczarzewicz D.
      • Gill K.
      • Szumala-Kakol A.
      • Kazienko A.
      • et al.
      Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.
      ). The immunofluorescence images showed that the neutrophils establish multiple phagocyte centers of differing sizes to form cluster-like structures; they may occur on the head or the tail and prevent the spermatozoa from moving. These clusters consist of neutrophils, spermatozoa, and NETs, as we confirmed by marking extracellular DNA, NE, histones, and MPO (Fig. 2), which are essential components of NETs (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      ). The cell clusters are similar to those formed when PMN are in the presence of bacteria or parasites. They may turn into phagocyte centers which can capture, trap, or swallow male gametes, suggesting that they may be a cooperation mechanism by which PMN form a different unit to work more efficiently to capture damaged spermatozoa (
      • Piasecka M.
      • Fraczek M.
      • Gaczarzewicz D.
      • Gill K.
      • Szumala-Kakol A.
      • Kazienko A.
      • et al.
      Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.
      ).
      Our results for NETs quantification showed that human spermatozoa exposed to PMN are capable of activating them, triggering NETosis (
      • Fuchs T.A.
      • Abed U.
      • Goosmann C.
      • Hurwitz R.
      • Schulze I.
      • Wahn V.
      • et al.
      Novel cell death program leads to neutrophil extracellular traps.
      ). This biological phenomenon further implies the death of PMN as the cell loses its compact circular shape (Fig. 1), the euchromatin and the heterochromatin become homogenized, the nuclear membrane ruptures, and the cytoplasmic granules dissolve. This allows the cellular and nuclear components to mingle to produce the final rupture of the cell membrane, releasing them in the extracellular medium where they form NETs (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      ). We also found that the stimulus for NETs formation provided by spermatozoa is dose dependent and increases over time (Table 1). This agrees with other reports in which NETs formation over time was assessed in bovine spermatozoa (
      • Alghamdi A.S.
      • Foster D.N.
      Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps.
      ) and is also confirmed by findings reported in bacteria (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      ) and parasites (
      • Hermosilla C.
      • Caro T.M.
      • Silva L.M.
      • Ruiz A.
      • Taubert A.
      The intriguing host innate immune response: novel anti-parasitic defence by neutrophil extracellular traps.
      ). A recent report has further found the elimination of human spermatozoa with NETs formation in leukocytospermia and leukocytospermia with bacteriospermia (
      • Piasecka M.
      • Fraczek M.
      • Gaczarzewicz D.
      • Gill K.
      • Szumala-Kakol A.
      • Kazienko A.
      • et al.
      Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.
      ). Although the generation of NETs depends on the stimulus, it has been observed that the formation time varies from 10 minutes to 4 hours (
      • von Kockritz-Blickwede M.
      • Nizet V.
      Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps.
      ). In this work, we determined that using a PMN/sperm ratio of 1:6, the NETs increased after 120 minutes, while with a ratio of 1:18 they increased after 60 minutes (Table 1). The response to the release of NETs is similar to that documented for bacteria or their toxins such as the hemotoxins of Escherichia coli described as NETs inducers (
      • Goldmann O.
      • Medina E.
      The expanding world of extracellular traps: not only neutrophils but much more.
      ,
      • Zawrotniak M.
      • Rapala-Kozik M.
      Neutrophil extracellular traps (NETs)—formation and implications.
      ,
      • Cheng O.Z.
      • Palaniyar N.
      NET balancing: a problem in inflammatory lung diseases.
      ), as well as fungi, protozoa, and some mediators such as interleukin 8, lipopolysaccharide, or hydrogen peroxide (
      • Arazna M.
      • Pruchniak M.P.
      • Zycinska K.
      • Demkow U.
      Neutrophil extracellular trap in human diseases.
      ); however, it has been speculated that the regulatory factors of the neutrophil-spermatozoon reaction are different from those described for pathogenic microorganisms (
      • Marey M.A.
      • Liu J.
      • Kowsar R.
      • Haneda S.
      • Matsui M.
      • Sasaki M.
      • et al.
      Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.
      ). This has not yet been clarified due to a lack of information about this interaction.
      Our results (Table 1) showed that NE and MPO inhibitors inhibit NETs formation in spermatozoa exposed to PMNs and therefore could help to diminish the probabilities of spermatozoa becoming trapped or undergoing phagocytosis. We also confirmed that DNase I is a NETs inhibitor. The largest component of NETs is DNA, which is disintegrated by DNase (
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      ). In this work we observed that DNase inhibits NETs when the neutrophils are exposed to spermatozoa; this inhibition mechanism has been found in DNAses located in the bacterial membranes, which are able to degrade NETs in vitro (
      • Buchanan J.T.
      • Simpson A.J.
      • Aziz R.K.
      • Liu G.Y.
      • Kristian S.A.
      • Kotb M.
      • et al.
      DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.
      ).
      We determined a threshold neutrophil concentration, which establishes the time and ratio when the NETs start to have a negative effect on the progressive motility of the male gamete (Fig. 3). These results confirm an unfavorable influence of inflammatory cells on sperm quality, which may possibly reduce their fertilizing potential. New evidence confirms that the creation of NETs is associated with ROS (
      • Arazna M.
      • Pruchniak M.P.
      • Zycinska K.
      • Demkow U.
      Neutrophil extracellular trap in human diseases.
      ) and has also shown that these networks release ROS together with all their other components. In this context, it has been found that leukocytes are the principal source of ROS in seminal plasma and that the main leukocyte population in semen are neutrophils, since it has been shown that their presence at the moment of semen deposition reduces the fertility in different species, including cattle, horses, and pigs (
      • Alghamdi A.S.
      • Foster D.N.
      • Troedsson M.H.
      Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri.
      ,
      • Matilsky M.
      • Ben-Ami M.
      • Geslevich Y.
      • Eyali V.
      • Shalev E.
      Cervical leukocytosis and abnormal post-coital test: a diagnostic and therapeutic approach.
      ,
      • Ou M.C.
      • Su C.S.
      Implications of asymptomatic endocervical leukocytosis in infertility.
      ,
      • Rozeboom K.J.
      • Troedsson M.H.
      • Hodson H.H.
      • Shurson G.C.
      • Crabo B.G.
      The importance of seminal plasma on the fertility of subsequent artificial inseminations in swine.
      ,
      • Kasimanickam R.
      • Duffield T.F.
      • Foster R.A.
      • Gartley C.J.
      • Leslie K.E.
      • Walton J.S.
      • et al.
      A comparison of the cytobrush and uterine lavage techniques to evaluate endometrial cytology in clinically normal postpartum dairy cows.
      ). Different studies agree with this conclusion, stating that these extracellular traps associated with proteins trap the spermatozoa and hamper their motility (
      • Alghamdi A.S.
      • Foster D.N.
      Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps.
      ,
      • Marey M.A.
      • Liu J.
      • Kowsar R.
      • Haneda S.
      • Matsui M.
      • Sasaki M.
      • et al.
      Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.
      ,
      • Alghamdi A.S.
      • Lovaas B.J.
      • Bird S.L.
      • Lamb G.C.
      • Rendahl A.K.
      • Taube P.C.
      • et al.
      Species-specific interaction of seminal plasma on sperm-neutrophil binding.
      ). Because the structure of NETs includes DNA, their surface has a negative charge and contains molecules that may mediate bonds with microorganisms, possibly through electrostatic interactions between the cationic components of the NETs and their anionic surfaces (
      • Brinkmann V.
      • Zychlinsky A.
      Beneficial suicide: why neutrophils die to make NETs.
      ). These interactions also explain the diminished progressive motility of sperm at a time when they are exposed to increasing doses of PMNs. Likewise, the infection of the male genital tract decreases the number of sperm, generally because of the inflammation and obstruction of the output way of spermatozoa (
      • Weidner W.
      • Pilatz A.
      • Diemer T.
      • Schuppe H.C.
      • Rusz A.
      • Wagenlehner F.
      Male urogenital infections: impact of infection and inflammation on ejaculate parameters.
      ), but according to our results, this decrease could be enhanced by mass trapping of spermatozoa by the NETs, since phagocytosis is a slower process and limited to a very low number of spermatozoa by neutrophils.
      In conclusion, we show that the human spermatozoon is a sufficient stimulus to trigger the release of NETs; this response is dose dependent and increases with exposure time. The spermatozoa undergo changes in motility, suggesting that this interaction may be detrimental to the probability of successful fertilization.

      Acknowledgments

      The authors thank Rodolfo Villagra and Theresa Fichtner for contributing to the protocols of this study.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Coincubation PMN/sperm ratio 1:6, for 2 hours. (A) Blue fluorescence stains the DNA of nuclei cells and NETs (white arrows) of DAPI (NETs trapping sperm), (B and C), green fluorescence (Alexa fluor 488) shows different components of NETs: (B) green arrows, MPO; (C) red arrow, colocalization of DNA and NE.

      References

        • Boivin J.
        • Bunting L.
        • Collins J.A.
        • Nygren K.G.
        International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.
        Hum Reprod. 2007; 22: 1506-1512
        • Mitchell C.
        • Marrazzo J.
        Bacterial vaginosis and the cervicovaginal immune response.
        Am J Reprod Immunol. 2014; 71: 555-563
        • de Repentigny L.
        • Goupil M.
        • Jolicoeur P.
        Oropharyngeal candidiasis in HIV infection: analysis of impaired mucosal immune response to Candida albicans in mice expressing the HIV-1 transgene.
        Pathogens. 2015; 4: 406-421
        • Alghamdi A.S.
        • Foster D.N.
        Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps.
        Biol Reprod. 2005; 73: 1174-1181
        • Hermosilla C.
        • Caro T.M.
        • Silva L.M.
        • Ruiz A.
        • Taubert A.
        The intriguing host innate immune response: novel anti-parasitic defence by neutrophil extracellular traps.
        Parasitology. 2014; 141: 1489-1498
        • Munoz-Caro T.
        • Mena Huertas S.J.
        • Conejeros I.
        • Alarcon P.
        • Hidalgo M.A.
        • Burgos R.A.
        • et al.
        Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent.
        Vet Res. 2015; 46: 23
        • Douda D.N.
        • Khan M.A.
        • Grasemann H.
        • Palaniyar N.
        SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx.
        Proc Natl Acad Sci U S A. 2015; 112: 2817-2822
        • Wold Health Organization
        WHO laboratory manual for the examination and processing of human semen.
        5th ed. WHO Press, Geneva2010
        • Schauer C.
        • Janko C.
        • Munoz L.E.
        • Zhao Y.
        • Kienhofer D.
        • Frey B.
        • et al.
        Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines.
        Nat Med. 2014; 20: 511-517
        • Munoz-Caro T.
        • Lendner M.
        • Daugschies A.
        • Hermosilla C.
        • Taubert A.
        NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.
        Dev Comp Immunol. 2015; 52: 245-254
        • Lu J.C.
        • Huang Y.F.
        • Lu N.Q.
        WHO laboratory manual for the examination and processing of human semen: its applicability to andrology laboratories in China.
        Nat J Androl. 2010; 16: 867-871
        • Oh H.
        • Siano B.
        • Diamond S.
        Neutrophil isolation protocol.
        J Vis Exp. 2008; : 745
        • Silva L.M.
        • Caro T.M.
        • Gerstberger R.
        • Vila-Vicosa M.J.
        • Cortes H.C.
        • Hermosilla C.
        • et al.
        The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps.
        Parasitol Res. 2014; 113: 2797-2807
        • Karnovsky M.J.
        The ultrastructural basis of capillary permeability studied with peroxidase as a tracer.
        J Cell Biol. 1967; 35: 213-236
        • Quinn P.
        • Kerin J.F.
        • Warnes G.M.
        Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid.
        Fertil Steril. 1985; 44: 493-498
        • Marey M.A.
        • Liu J.
        • Kowsar R.
        • Haneda S.
        • Matsui M.
        • Sasaki M.
        • et al.
        Bovine oviduct epithelial cells downregulate phagocytosis of sperm by neutrophils: prostaglandin E2 as a major physiological regulator.
        Reproduction. 2014; 147: 211-219
        • Piasecka M.
        • Fraczek M.
        • Gaczarzewicz D.
        • Gill K.
        • Szumala-Kakol A.
        • Kazienko A.
        • et al.
        Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.
        Am J Reprod Immunol. 2014; 72: 348-358
        • Brinkmann V.
        • Reichard U.
        • Goosmann C.
        • Fauler B.
        • Uhlemann Y.
        • Weiss D.S.
        • et al.
        Neutrophil extracellular traps kill bacteria.
        Science. 2004; 303: 1532-1535
        • Fuchs T.A.
        • Abed U.
        • Goosmann C.
        • Hurwitz R.
        • Schulze I.
        • Wahn V.
        • et al.
        Novel cell death program leads to neutrophil extracellular traps.
        J Cell Biol. 2007; 176: 231-241
        • von Kockritz-Blickwede M.
        • Nizet V.
        Innate immunity turned inside-out: antimicrobial defense by phagocyte extracellular traps.
        J Mol Med. 2009; 87: 775-783
        • Goldmann O.
        • Medina E.
        The expanding world of extracellular traps: not only neutrophils but much more.
        Front Immunol. 2012; 3: 420
        • Zawrotniak M.
        • Rapala-Kozik M.
        Neutrophil extracellular traps (NETs)—formation and implications.
        Acta Biochim Pol. 2013; 60: 277-284
        • Cheng O.Z.
        • Palaniyar N.
        NET balancing: a problem in inflammatory lung diseases.
        Front Immunol. 2013; 4: 1
        • Arazna M.
        • Pruchniak M.P.
        • Zycinska K.
        • Demkow U.
        Neutrophil extracellular trap in human diseases.
        Adv Exp Med Biol. 2013; 756: 1-8
        • Buchanan J.T.
        • Simpson A.J.
        • Aziz R.K.
        • Liu G.Y.
        • Kristian S.A.
        • Kotb M.
        • et al.
        DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.
        Curr Biol. 2006; 16: 396-400
        • Alghamdi A.S.
        • Foster D.N.
        • Troedsson M.H.
        Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri.
        Reproduction. 2004; 127: 593-600
        • Matilsky M.
        • Ben-Ami M.
        • Geslevich Y.
        • Eyali V.
        • Shalev E.
        Cervical leukocytosis and abnormal post-coital test: a diagnostic and therapeutic approach.
        Hum Reprod. 1993; 8: 244-246
        • Ou M.C.
        • Su C.S.
        Implications of asymptomatic endocervical leukocytosis in infertility.
        Gynecol Obstet Invest. 2000; 49: 124-126
        • Rozeboom K.J.
        • Troedsson M.H.
        • Hodson H.H.
        • Shurson G.C.
        • Crabo B.G.
        The importance of seminal plasma on the fertility of subsequent artificial inseminations in swine.
        J Anim Sci. 2000; 78: 443-448
        • Kasimanickam R.
        • Duffield T.F.
        • Foster R.A.
        • Gartley C.J.
        • Leslie K.E.
        • Walton J.S.
        • et al.
        A comparison of the cytobrush and uterine lavage techniques to evaluate endometrial cytology in clinically normal postpartum dairy cows.
        Can Vet J. 2005; 46: 255-259
        • Alghamdi A.S.
        • Lovaas B.J.
        • Bird S.L.
        • Lamb G.C.
        • Rendahl A.K.
        • Taube P.C.
        • et al.
        Species-specific interaction of seminal plasma on sperm-neutrophil binding.
        Anim Reprod Sci. 2009; 114: 331-344
        • Brinkmann V.
        • Zychlinsky A.
        Beneficial suicide: why neutrophils die to make NETs.
        Nat Rev Microbiol. 2007; 5: 577-582
        • Weidner W.
        • Pilatz A.
        • Diemer T.
        • Schuppe H.C.
        • Rusz A.
        • Wagenlehner F.
        Male urogenital infections: impact of infection and inflammation on ejaculate parameters.
        World J Urol. 2013; 31: 717-723