SOCE-inhibitor reduced human sperm-induced formation of neutrophil extracellular traps

in Reproduction
Authors:
Fabiola ZambranoCenter of Excellence in Translational Medicine-Scientific and Technological Bioresource Nucleus (CEMT – BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile

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Liliana SilvaInstitute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany

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Pamela UribeCenter of Excellence in Translational Medicine-Scientific and Technological Bioresource Nucleus (CEMT – BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Department of Internal Medicine, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile

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Ulrich GärtnerInstitute of Anatomy and Cell Biology, Justus Liebig University Giessen, Giessen, Germany

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Anja TaubertInstitute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany

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Mabel SchulzCenter of Excellence in Translational Medicine-Scientific and Technological Bioresource Nucleus (CEMT – BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile

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Raúl SánchezCenter of Excellence in Translational Medicine-Scientific and Technological Bioresource Nucleus (CEMT – BIOREN), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile
Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile

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Carlos HermosillaInstitute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany

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Correspondence should be addressed to R Sanchez; Email: raul.sanchez@ufrontera.cl
Free access

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

Abstract

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

Introduction

Polymorphonuclear neutrophils (PMN) play an important role as the first line of innate immune defence in mammals. The formation of neutrophil extracellular traps (NETs) is a novel effector mechanism of PMN (Brinkmann et al. 2004). NETs are sticky extracellular fibrous structures that are released from activated PMN in response to pathogenic or non-pathogenic stimuli in both mammals and invertebrates (Brinkmann et al. 2004).

The main components of NETs include DNA, histones (H1, H2A/H2B, H3, H4), myeloperoxidase (MPO), neutrophil elastase (NE), pentraxin 3 (PTX3), cathepsin G, matrix metalloproteinase-9 (MMP9), peptidoglycan recognition proteins, and peptidyl arginine deiminase type IV (PAD4) (Brinkmann et al. 2004). The signalling cascade triggering NETosis comprises the production of reactive oxygen species (ROS) by neutrophil NADPH oxidase (NOX), translocation of NE and MPO to the nucleus, association of the citrullinating activity of PAD4 on histones, and induction of chromatin decondensation (Leshner et al. 2012). Besides DNA-related analysis, co-localisation experiments have demonstrated the concomitant presence of H3, PTX3, NE, and MPO in bovine sperm-induced NETosis, confirming the classical molecular properties of NETs (Brinkmann et al. 2004). Interestingly, PTX3 was previously shown to be expressed in the male genital tract and to be bound to human spermatozoa (Doni et al. 2009). In agreement with this report, a recent study found PTX3 in a co-culture of bovine sperm and PMN (Fichtner et al. 2020).

Human spermatozoa are known to act as antigens in the female reproductive tract (FRT), and sperm immunisation can even result in infertility (Hahn et al. 2012). Human PMN are actively recruited to the FRT following insemination in a process similar to sterile inflammation, and leukocytes act as efficient phagocytes, since they are involved in the removal of excess spermatozoa in vivo (Strzemienski 1989, Alghamdi et al. 2009). As human spermatozoa within the FRT are directly exposed to PMN and are disposable targets of PMN-derived NETs (Alghamdi et al. 2009), an interaction might occur with unknown consequences for sperm physiology. Given that NET-related cytotoxicity via direct damage to membrane integrity has been previously described for several invasive pathogens such as bacteria (Marin-Esteban et al. 2012), fungi (Urban et al. 2006), and protozoan/metazoan parasites (Silva et al. 2014), it is plausible that human NETs may also harm spermatozoa via antimicrobial effector molecules (Saffarzadeh et al. 2012). Moreover, in vitro, human spermatozoa induce NETosis in PMN, resulting in the formation of different types of NETs (i.e. aggregated NETs (aggNETs), diffuse NETs (diffNETs) and spread NETs (sprNETs)) (Zambrano et al. 2016). Recently, similar types of NETs have been demonstrated in the seminal fluid (ex vivo) of infertile patients (Schulz et al. 2019b) and in seminal samples from patients with epididymitis (Zambrano et al. 2020).

Analysis of sperm/PMN co-cultures have confirmed the essential role of NOX, MPO, and NE in the formation of NETs, which adversely affects sperm function and motility (Zambrano et al. 2016); however, very little information is available concerning the role of ROS production, which is known to be dependent on store-operated Ca2+ entry (SOCE) and which is a key factor for the adequate release of NETs by PMN (Gupta et al. 2014). The aim of this study was to establish the role of SOCE in human sperm-triggered NETosis and its impact on sperm integrity and oocyte binding capacity.

Materials and methods

Ethics statement

This study was approved by the Scientific Ethics Committee of the Universidad de La Frontera, Temuco, Chile, and the Justus Liebig University (JLU) Giessen, Germany. The healthy sperm and PMN donors included in this study were volunteers from these two universities. All donors provided written informed consent according to the ethical protocols of both institutions.

Reagents

All reagents were obtained from Sigma Chemicals Ltd., unless otherwise stated.

Blood and semen samples

In each experiment, blood samples were obtained from healthy women volunteers (n = 3), aged 20–35 years. Thirty millilitres of blood were extracted from the cephalic vein of each donor at the blood bank unit of the JLU, and PMN were immediately isolated. We used female PMN to simulate the interaction that would occur during the natural reproductive process, wherein the sperm interacts with PMN in the FRT. The sperm samples were obtained from healthy male volunteers (n = 3) with normal semen parameters (WHO 2010), aged 20–26 years. Samples were collected in sterile plastic tubes (Deltalab®) via masturbation after at least 3 days of sexual abstinence.

In total, semen samples from 12 male donors and peripheral blood samples from 12 healthy female donors were used in this study.

Isolation of PMN

Blood samples were placed in a Vacutainer® containing heparin (10 U/mL). The isolation method employed was as described in a previous report (Oh et al. 2008). Briefly, 6 mL of Histopaque® 1119 (Gibco) was placed in a 15 mL Falcon® plastic tube and 7 mL of heparinised blood was carefully layered on top. This mixture was centrifuged at 800 g for 20 min. Next, the clear yellowish top layer was aspirated and discarded, and the lower reddish phase containing PMN was transferred into fresh Falcon® plastic tubes. The cells were then washed by filling the Falcon® tubes with sterile PBS and centrifuged for 10 min at 300 g. Percoll solution (100%; Amersham) was prepared by mixing 18 mL of Percoll with 2 mL of 10x PBS. Four millilitres each of 85%, 80%, 75%, 70%, and 65% Percoll gradients were then diluted with 1x PBS. Next, two Falcon® tubes (for each blood sample) containing descending Percoll gradients (85%, 80%, 75%, 70%, and 65%, respectively) were layered with blood. After centrifugation (10 min at 300 g), the supernatant was discarded, and the pellets were resuspended in 4 mL of PBS. Two millilitres of resuspended pellet from each of the gradients was collected and centrifuged at 800 g for 20 min. After centrifugation, the top layer and most of the 65% layer containing peripheral blood mononuclear cells (PBMCs) were discarded, while the remaining white interphase, comprising the 85% layer, was collected in a new Falcon® tube. Cells were then washed by filling up the Falcon® tube with PBS and centrifuging for 10 min at 300 g. Finally, the supernatant was removed, and cell pellets were resuspended in 2 mL of RPMI 1640 medium. PMN were counted using a Neubauer haemocytometer.

Spermatozoa and PMN co-culturing assay

Motile spermatozoa were isolated using the swim-up technique. For this, 500 μL of modified human tubal fluid (HTF) medium with gentamicin-HEPES (HTF; Irvine Scientific) was placed in a 1.7 mL polypropylene tube. Then, 150 μL of semen was placed at the bottom of the tube. The tube was tilted to 45º and incubated at 37ºC for 60 min. After incubation, 350 μL of the sample was recovered from the upper part of the tube without aspirating the contents at the bottom of the tube. Only samples with ≥75% spermatozoa showing progressive motility and suitable sperm counts were included in the experiments. Human PMN and spermatozoa were co-cultured at a ratio of 1:6 (2.5 × 105 PMN: 1.5 × 106 spermatozoa) in serum-free RPMI 1640 cell culture medium (Gibco) without phenol red, as described previously (Munoz-Caro et al. 2015b). The co-culture was incubated for 180 min at 37ºC with 5% CO2 and saturated humidity.

Immunofluorescence analysis of sperm-triggered NETosis

After co-incubation of PMN with human spermatozoa on coverslips (22 mm × 60 mm; Nunc, Thermo Fisher Scientific) previously coated with poly-L-lysine (0.01%), 15 min at room temperature (RT), the samples were fixed in 4% paraformaldehyde (15 min, RT). Then, the samples were blocked with 2% BSA (15 min, 37ºC), washed twice in PBS, and incubated with the following primary monoclonal antibodies: anti-global histone (H1, H2A/H2B, H3, H4) antibody (anti-mouse, clone H11–4, diluted 1:1000 in PBS) and anti-PTX3 antibody (SAB2104614-50UG, diluted 1:1000 in PBS). The samples were washed twice in PBS and then incubated with the following secondary antibodies: Alexa Fluor® 488 goat anti-mouse IgG (ref. A11001, Molecular Probes) for the detection of histones and Alexa Fluor 488 goat anti-rabbit IgG (ref. A11008, Life Technologies) for the detection of PTX3. All samples were incubated for 1 h at RT in the dark. Finally, the samples were washed twice in PBS and stained for 20 min with Sytox Orange® (Invitrogen), as described previously (Martinelli et al. 2004, Lippolis et al. 2006). After two washes with PBS, each sample was mounted face-down on a glass coverslip and immediately analysed using an inverted fluorescence microscope equipped with a digital camera (Olympus IX81®).

Quantification of NETs

To quantify NETs induced by spermatozoa, PMN and spermatozoa were co-cultured at a ratio of 1:6 for 180 min. The experimental groups were as follows: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (previously treated with 2 aminoethoxydiphenyl borate (2-APB) for SOCE inhibition (100 μM, Sigma-Aldrich)) + sperm and (5) (PMN + PMA 500 nM (Adipogen®)) + sperm. Stimulation with PMA was used as positive control, as described in a previous report (Munoz-Caro et al. 2015a). All samples were incubated for 180 min at 37ºC. After incubation, samples were treated with 0.1 U/μL of micrococcal nuclease (New England Biolabs) for 15 min at 37ºC and centrifuged (300 g, 5 min). Then, 100 μL of supernatant of each sample was transferred in duplicate to a 96-well cell culture plate (Greiner). Finally, 50 μL of PicoGreen® (1:200 in 10 mM of Tris base buffered with 1 mM EDTA) was added to the samples, and the samples were incubated for 4 min in total darkness. NET formation was quantified by spectrofluorometric analysis (484 nm excitation wavelength and 520 nm emission wavelength) using an automated plate monochrome reader (Varioskan Flash®; Thermo Scientific) as previously described (Silva et al. 2014, Munoz-Caro et al. 2015a,b).

Immunofluorescence staining to confirm the formation of NETs

Immunofluorescence staining was performed according to a previously described protocol (Fichtner et al. 2020). Monoclonal anti-NE antibody (AB68672, 1:200, Abcam) was used as the primary antibody to detect human NE during 1 h; subsequently, the samples were incubated 1 h with a secondary antibody conjugated with Alexa Fluor 488 (anti-rabbit ref. A31556, Invitrogen). To view the leucocyte nuclei and the DNA released into the extracellular space, antifade mounting medium with DAPI was used (ProLong®). The samples were analysed using an Olympus IX81® inverted fluorescence microscope equipped with an Olympus XM10® digital camera.

Sperm plasma membrane and acrosome integrity

Sperm plasma membrane integrity was evaluated using the LIVE/DEAD Sperm Viability Kit® (Molecular Probes). The different groups (sperm, PMN/sperm, (PMN + 2APB) + sperm and (PMN + DNase (90 U of DNase I (Roche Diagnostics)) + sperm), were resuspended in 400 μL of HTF medium and incubated with SYBR-14 at 1 nM concentration for 10 min, Acrosome evaluation assay was performed using PNA/FITC (300 μg/mL) and propidium iodide (PI) (18 mM) for 15 min at 37°C in darkness. PI was added and incubated for 15 min. Finally, the spermatozoa were washed by centrifugation at 500 g for 5 min and resuspended in 300 μL of PBS.

DNA fragmentation

DNA fragmentation was evaluated by the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, using the in situ Cell Death Detection Kit® (Roche Biochemical) according to the manufacturer’s instructions. Briefly, spermatozoa were either untreated (controls) or were treated with PMN (180 min), washed with 1% PBS/BSA (w/v) (pH 7.4) (400 g, 5 min), resuspended in 1 mL of PBS/BSA (1%) and fixed in 0.2% paraformaldehyde for 45 min. The samples were centrifuged at 400 g for 5 min and washed with 500 μL of 1% PBS/BSA. Subsequently, the spermatozoa were incubated for 1 h at RT in permeabilisation solution (PBS supplemented with 1% BSA, 0.3% Triton and 0.1% sodium citrate), and then the samples were washed in HTF medium (500 g for 5 min) and resuspended in 500 µL of 1% PBS/BSA. All samples were then incubated with 5 µL of enzyme solution and 45 µL of label solution for 60 min in a humidity chamber at 38.5°C in the darkness. Finally, the samples were centrifuged at 500 g for 5 min and resuspended in 300 µL of PBS for flow cytometry evaluation. To verify successful cell permeabilisation, samples were counterstained with PI (18 mM) for the last 5 min of incubation.

Hemizona binding assay for assessment of spermatozoa function

The hemizona binding assay was carried out according to a previous report (Franken et al. 1989), with slight modifications. Only discarded oocytes obtained by in vitro fertilisation program, stored in salt storage solution were used (Yanagimachi et al. 1979). The oocytes were placed in 100 mm petri dishes in 50 µL drops of modified HTF medium supplemented with 0.5% albumin and bisected into two hemizonae by a scalpel cut, with the blade at right angle (perpendicular to the cutting surface). The spermatozoa of each group were incubated for 180 min with PMN and NETosis inhibitor as described previously. After the completion of this incubation period, NET-entrapped spermatozoa were released from extracellular structures by treatment with DNase I (addition of 90 U/well of DNase I for 15 min) in order to dissolve the NETs. In each assessment group, 60,000 spermatozoa were added to each hemizona for 4 h and incubated in a humidified chamber at 37°C and 5% CO2. The hemizonae were then washed three times in 1x PBS and gently placed on a coverslip containing 15 µL of mounting medium with DAPI (sperm nucleus marker). Hemizona imaging analysis was performed using an epifluorescence microscope (NIKON eclipse TS100F®).

Flow cytometry

All fluorescence analyses were performed using a BD FACS Canto II Flow Cytometer (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). One straw was used for all treatments in each experiment, and each experiment was repeated three times on different days. A minimum of 10,000 spermatozoa were included in each analysis. When evaluating plasma membrane, acrosome, and DNA integrity in the co-cultures, the sperm populations were separated from the PMN based on cell size (Forward Scatter, FSC) and cell complexity (Side Scatter, SSC) to ensure that only fluorescence emitted by the sperm was considered while excluding that emitted by the PMN.

Statistical analysis

The current data were analysed by descriptive statistics and represented as the mean ± s.d. calculated for each of the variables using the GraphPad Prism software, version 5.0a. Gaussian distribution was analysed by D’Agostino’s test. One-way ANOVA was performed to determine statistically significant differences (P < 0.05) between groups. In cases where statistically significant differences were observed, Tukey’s test was performed for more detailed analysis.

Results

Scanning electron microscopy (SEM) and localisation of histones and PTX3 in human sperm-induced NETs

SEM and immunofluorescence analyses revealed the release of both fine and thick NETs by human PMN and the presence of entrapped spermatozoa after 180 min (Fig. 1). Following a 3 h incubation period, PMN were detected with initial cell membrane damage occurring prior to NET extrusion (Fig. 1A). Further, we observed localisation of histones (Fig. 1C and D) and PTX3 (green fluorescence) (Fig. 1E and F) with DNA-containing structures (red fluorescence (Fig. 1C and E)). In addition, different types of NETs, such as sperm-induced spread NETs (sprNETs), were observed in sperm/PMN co-cultures (Fig. 1C and D).

Figure 1
Figure 1

Visualisation and analyses of human sperm-triggered NETs by SEM (A and B) and epifluorescence microscopy (C, D, E and F). Images (A and B) show human PMN entrapping spermatozoa; image (A) shows a human PMN carrying out NETosis, and thereby entrapping a spermatozoon within thin extracellular fibres (B). NET-derived structures showing DNA labelling with Sytox Orange® dye (red fluorescence, (C) and global histones (H1, H2A/H2B, H3, H4) labelled with Alexa Fluor 488 (green fluorescence, (D). Image (F) shows PTX3 immunofluorescence labelling. Green fluorescence represents PTX3 labelling; in (E) orange fluorescence represents DNA staining (cell nuclei and NETs). Red arrows indicate PMN; white arrows indicate NETs; yellow arrows indicate spermatozoa. Epifluorescence images, magnification 40x (C and D) and 60x (E and F).

Citation: Reproduction 161, 1; 10.1530/REP-20-0185

Effect of SOCE inhibitor on human sperm-induced NETosis

The quantification of NET formation confirmed human spermatozoa as potent inducers of NETs (Fig. 2). Thus, a significantly enhanced quantity of extracellular DNA was observed in sperm/PMN co-cultures compared to PMN or sperm alone (*P < 0.05). Stimulation of PMN with PMA showed significantly enhanced NET formation compared to all other groups. The relevance of SOCE in sperm-triggered NETosis was examined via functional inhibitor treatment. Pretreatment of PMN with 2-ABP and subsequent co-culturing with spermatozoa led to significantly reduced NET formation compared with that in the control PMN/sperm group (*P < 0.05), thus proving the key role of SOCE in sperm-mediated NETosis.

Figure 2
Figure 2

(A) Graph representing quantification of NETs showing a significantly enhanced quantity of extracellular DNA in sperm/PMN co-cultures compared to PMN or sperm alone (*P < 0.05) at 180 min. Stimulation of PMN with PMA shows significantly enhanced NET formation compared with all the other groups. Treatment of PMN/sperm with 2-ABP (for SOCE inhibition (100 μM)) significantly reduced NET formation compared with that in control PMN/sperm (*P < 0.05). (B) Epifluorescence microscopy images showing NET structures of different groups: DNA (blue staining, DAPI); NE (green staining, Alexa Fluor 488). Epifluorescence images, magnification 60x.

Citation: Reproduction 161, 1; 10.1530/REP-20-0185

Effect of NETs on sperm plasma membrane, acrosome integrity and DNA integrity

Sperm plasma membrane integrity was significantly reduced after 180 min of co-culture with PMN (Figs 3A and 4A) in comparison to spermatozoa which were not exposed to PMN (P < 0.05). This effect could be partially prevented by pretreatment of PMN with the SOCE inhibitor (2-APB) (P < 0.05).

Figure 3
Figure 3

Effects of NETs on membrane integrity, acrosome integrity, and DNA fragmentation in human spermatozoa in base medium (spermatozoa without PMN), in co-cultures of PMN/sperm (1:6) for 180 min, using NETosis inhibitor (SOCE inhibitor (2-APB, 100 μM)) and DNase control group. The data are represented as the mean percentage ± s.d.; statistical significance was considered at *P < 0.05.

Citation: Reproduction 161, 1; 10.1530/REP-20-0185

Figure 4
Figure 4

Flow cytometry images, (A) percentage of sperm population with intact plasma membrane (viable, SYBR14+/PI−). (B) The viable sperm population with intact acrosome (PI-/PNA/FITC−). (C) The percentage of sperm population with fragmented DNA (TUNEL+).

Citation: Reproduction 161, 1; 10.1530/REP-20-0185

The integrity of the acrosomal membrane was found to be significantly (P < 0.05) reduced after 180 min of PMN/spermatozoa co-culture, indicating the adverse effects of NETs (Figs 3B and 4B). However, in this experimental setting treatment of PMN with 2-APB prior to co-culture with spermatozoa failed to significantly prevent this reaction. Interestingly, DNA integrity was not affected in spermatozoa exposed to PMN (Figs 3C and 4C).

Capacity of sperm to bind to the hemizona after exposure to NETs

Spermatozoa previously exposed to PMN had a significantly lower capacity to bind to zona pellucida than control spermatozoa (*P < 0.05; Fig. 5A and B). Thus, 48% less spermatozoa (relative to the control) bound in the hemizona assay. This effect could be partially prevented by pretreatment of PMN with the SOCE inhibitor before incubation with spermatozoa. This result indicated the relevance of SOCE in sperm-mediated NETosis. Thus, the mean number of spermatozoa binding to the zona pellucida increased significantly after 2-APB treatment when compared to the untreated PMN/sperm group (*P < 0.05). However, the mean number of the PMN + SOCE inhibitor-treated group (prior to exposure with spermatozoa) did not reach that of the control spermatozoa and still showed significantly reduced levels of hemizona binding when compared to unexposed spermatozoa (*P < 0.05). PMN group with sperm and DNase maintained sperm function similar to sperm group without PMN.

Figure 5
Figure 5

Hemizona binding ability of sperm. (A) Spermatozoa exposed to PMN had a significantly lower capacity to bind to zona pellucida than control spermatozoa did (*P < 0.05). The mean number of spermatozoa binding to the zona pellucida increased significantly after 2-APB treatment compared with that in the untreated PMN/sperm group (all: *P < 0.05). However, inhibitor-treated PMN/sperm showed significantly reduced levels of hemizona binding compared with unexposed spermatozoa (*P < 0.5). (B) Shows images of sperm binding to the hemizona (sperm cell nuclei stained blue with DAPI); epifluorescence microscopy, magnification 40x. Three biological replicates were included in each assay. The data are represented as mean percentage ± s.d.; statistical significance was considered at *P < 0.05.

Citation: Reproduction 161, 1; 10.1530/REP-20-0185

Discussion

Human spermatozoa induce NETosis in PMN, resulting in the formation of different types of NETs (aggNETs, sprNETs and diffNETs), which adversely affect sperm motility (Zambrano et al. 2016). NETosis-derived sperm entrapment might be linked to PTX3 activity, since the latter could form molecular complexes with other NET components cooperating as a binding matrix to enhance entrapment (Daigo & Hamakubo 2012). In this study, we documented for the first time, the presence of PTX3 in human sperm-triggered NETosis. PTX3 is a known antimicrobial factor of the mammalian innate immune system, wherein it plays a role in microbe recognition and facilitates the entrapment of invasive pathogens (Bottazzi et al. 2009). This finding is in accordance with the current microscopy evidence, showing effective sperm-entrapping capacities of NETs.

Various studies have described that spermatozoa can interact with their environment and that these interactions have an impact on sperm viability (Taylor et al. 2008). Herein, we use a concentration of neutrophils which establishes the point at which NETs have a negative effect on male gamete viability, which is consistent with the previously determined decreased progressive motility (Zambrano et al. 2016). These results confirm an unfavourable influence of inflammatory cells on the quality of spermatozoa; this may reduce their fertilising potential, given the trapping of sperm on the one hand, and on the other, the damage caused by NETs to spermatozoa that come in contact with them (Zambrano et al. 2016). The dynamic nature of the sperm membrane plays a fundamental role in sperm maturation, capacitation and fertilisation. However, exposure to NETs causes loss of membrane integrity, as has been reported in macrophages and dendritic cells. It has been reported that specific damage is mediated principally by the high contents of NETs and the presence of cathepsin G in the extracellular network (Donis-Maturano et al. 2015). Furthermore, excessive exposure to ROS may cause oxidative stress with irreparable damage to the sperm membrane (Muller et al. 1999). This is an interesting aspect since we think that the production of ROS from NETs could be one of the most critical points affecting sperm physiology. The greatest damage might be caused by membrane lipid peroxidation, which principally impacts long-chain polyunsaturated fatty acids (PUFAs), such as docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) (Waterhouse et al. 2006). This leads to loss of viability and motility and diminished capability of the spermatozoon to fuse with the oocyte (Maldjian et al. 2005) and may partly explain the loss of sperm function in this model, where in the presence of NETs caused direct damage to the sperm membrane. Furthermore, the trapping of spermatozoa by NETs may be accentuated by the DNA present in the NETs: the NET surface has a negative charge and contains molecules that may mediate binding, for example, with microorganisms, possibly through electrostatic interactions between the cationic components of the NETs and their anionic surfaces (Brinkmann & Zychlinsky 2007). These interactions also explain the diminished viability of the spermatozoa that causes their agglomeration and/or immobilisation, which are thought to be closely related to unexplained infertility (Schulz et al. 2019a,b). These responses may be similar to infection processes in the male genital tract in which sperm parameters are altered, generally due to inflammation, and increased formation of different NET structures, as described previously (Zambrano et al. 2020).

Another important event in the formation of NETs is NOX activation and the subsequent production of ROS, both processes dependent on Ca2+ influx mediated by SOCE (Brechard & Tschirhart 2008). In our study, sperm-induced NETosis leads to a partial loss of sperm plasma membrane and acrosome integrity in addition to reduced capacity of sperm adhesion and binding to the oocyte. The implications of these effects will most likely include reduced fertilisation success. Interestingly, NET-induced damage to the spermatozoa was significantly prevented by the SOCE inhibitor 2-APB, underscoring our previous data on the relevance of SOCE-derived Ca2+ in sperm-induced NETosis (Conejeros et al. 2011, Munoz-Caro et al. 2015a). These data demonstrate that reduced contact of sperm with toxic or adverse NET-derived molecules is beneficial to sperm function. As a result, the use of SOCE inhibitor in sperm-exposed human PMN helped these cells to maintain the dynamics and integrity of the sperm membrane, and, therefore, subsequent capacitation and fertilisation (Zhou et al. 2010). Despite the positive effects of 2-APB on sperm function, this treatment failed to significantly improve acrosome integrity. This may be due to the very stable and rigid nature of this structure compared to the sperm plasma membrane (Zambrano et al. 2016). Using the current experimental conditions, we did not find any impact of NETs on sperm DNA fragmentation. This was reasonable given that high ROS levels are associated with plasma membrane damage, and DNA fragmentation indicates the final stage of cell death (Galluzzi et al. 2012).

Conclusion

We demonstrated the key role of SOCE and, for the first time, the presence of PTX3 in human sperm-triggered NETs. NETosis induced damage in the sperm membrane and acrosome, thus negatively impacting sperm function. However, the use of SOCE inhibitor maintains sperm function with a high oocyte binding capacity. This new data will improve the current understanding of the role of PMN in female and male reproductive tracts and may contribute to the development of alternative strategies to block the formation of NETs in situations of reduced fertilisation success.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

F Z, A T, R S and C H involved in conception and design of the study. F Z, L S, P U, U G and M S contributed to acquisition of data; F Z, A T, R S, C H involved in analysis and interpretation of data. F Z and P U contributed drafting the manuscript. F Z, R S and C H revised the manuscript critically for important intellectual content. R S finally approval of the version to be submitted.

Acknowledgements

We would like to express our gratitude to Joachim Misterek (Blood Bank of the JLU Giessen, Germany) for helping us collect human blood samples and Anika Seipp (Institute of Anatomy and Cell Biology, JLU Giessen, Germany) for helping with the scanning electron microscopy analysis. Fabiola Zambrano was supported by a postdoctoral scholarship from the Universidad de La Frontera, Chile.

References

  • Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC & Foster DN 2009 Species-specific interaction of seminal plasma on sperm-neutrophil binding. Animal Reproduction Science 114 331344. (https://doi.org/10.1016/j.anireprosci.2008.10.015)

    • Search Google Scholar
    • Export Citation
  • Bottazzi B, Garlanda C, Cotena A, Moalli F, Jaillon S, Deban L & Mantovani A 2009 The long pentraxin PTX3 as a prototypic humoral pattern recognition receptor: interplay with cellular innate immunity. Immunological Reviews 227 918. (https://doi.org/10.1111/j.1600-065X.2008.00719.x)

    • Search Google Scholar
    • Export Citation
  • Brechard S & Tschirhart EJ 2008 Regulation of superoxide production in neutrophils: role of calcium influx. Journal of Leukocyte Biology 84 12231237. (https://doi.org/10.1189/jlb.0807553)

    • Search Google Scholar
    • Export Citation
  • Brinkmann V & Zychlinsky A 2007 Beneficial suicide: why neutrophils die to make NETs. Nature Reviews: Microbiology 5 577582. (https://doi.org/10.1038/nrmicro1710)

    • Search Google Scholar
    • Export Citation
  • Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y & Zychlinsky A 2004 Neutrophil extracellular traps kill bacteria. Science 303 15321535. (https://doi.org/10.1126/science.1092385)

    • Search Google Scholar
    • Export Citation
  • Conejeros I, Patterson R, Burgos RA, Hermosilla C & Werling D 2011 Induction of reactive oxygen species in bovine neutrophils is CD11b, but not dectin-1-dependent. Veterinary Immunology and Immunopathology 139 308312. (https://doi.org/10.1016/j.vetimm.2010.10.021)

    • Search Google Scholar
    • Export Citation
  • Daigo K & Hamakubo T 2012 Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps. Frontiers in Immunology 3 378. (https://doi.org/10.3389/fimmu.2012.00378)

    • Search Google Scholar
    • Export Citation
  • Doni A, Paffoni A, Nebuloni M, Ragni G, Pasqualini F, Valentino S, Bonetti S, Mantovani A, Somigliana E & Garlanda C 2009 The long PENTRAXIN 3 is a soluble and cell-associated component of the human semen. International Journal of Andrology 32 255264. (https://doi.org/10.1111/j.1365-2605.2007.00845.x)

    • Search Google Scholar
    • Export Citation
  • Donis-Maturano L, Sanchez-Torres LE, Cerbulo-Vazquez A, Chacon-Salinas R, Garcia-Romo GS, Orozco-Uribe MC, Yam-Puc JC, Gonzalez-Jimenez MA, Paredes-Vivas YL & Calderon-Amador J et al.2015 Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells. Springerplus 4 161. (https://doi.org/10.1186/s40064-015-0932-8)

    • Search Google Scholar
    • Export Citation
  • Fichtner T, Kotarski F, Gartner U, Conejeros I, Hermosilla C, Wrenzycki C & Taubert A 2020 Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4- and Ca++-dependent process. Biology of Reproduction 102 902914. (https://doi.org/10.1093/biolre/ioaa003)

    • Search Google Scholar
    • Export Citation
  • Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck LL, Kruger TF, Rosenwaks Z & Hodgen GD 1989 Hemizona assay using salt-stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa. Gamete Research 22 1526. (https://doi.org/10.1002/mrd.1120220103)

    • Search Google Scholar
    • Export Citation
  • Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS & Fulda S et al.2012 Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death and Differentiation 19 107120. (https://doi.org/10.1038/cdd.2011.96)

    • Search Google Scholar
    • Export Citation
  • Gupta AK, Giaglis S, Hasler P & Hahn S 2014 Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. PLoS ONE 9 e97088. (https://doi.org/10.1371/journal.pone.0097088)

    • Search Google Scholar
    • Export Citation
  • Hahn S, Giaglis S, Hoesli I & Hasler P 2012 Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss. Frontiers in Immunology 3 362. (https://doi.org/10.3389/fimmu.2012.00362)

    • Search Google Scholar
    • Export Citation
  • Leshner M, Wang S, Lewis C, Zheng H, Chen XA, Santy L & Wang Y 2012 PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Frontiers in Immunology 3 307. (https://doi.org/10.3389/fimmu.2012.00307)

    • Search Google Scholar
    • Export Citation
  • Lippolis JD, Reinhardt TA, Goff JP & Horst RL 2006 Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Veterinary Immunology and Immunopathology 113 248255. (https://doi.org/10.1016/j.vetimm.2006.05.004)

    • Search Google Scholar
    • Export Citation
  • Maldjian A, Pizzi F, Gliozzi T, Cerolini S, Penny P & Noble R 2005 Changes in sperm quality and lipid composition during cryopreservation of boar semen. Theriogenology 63 411421. (https://doi.org/10.1016/j.theriogenology.2004.09.021)

    • Search Google Scholar
    • Export Citation
  • Marin-Esteban V, Turbica I, Dufour G, Semiramoth N, Gleizes A, Gorges R, Beau I, Servin AL, Lievin-Le Moal V & Sandre C et al.2012 Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells. Infection and Immunity 80 18911899. (https://doi.org/10.1128/IAI.00050-12)

    • Search Google Scholar
    • Export Citation
  • Martinelli S, Urosevic M, Daryadel A, Oberholzer PA, Baumann C, Fey MF, Dummer R, Simon HU & Yousefi S 2004 Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. Journal of Biological Chemistry 279 4412344132. (https://doi.org/10.1074/jbc.M405883200)

    • Search Google Scholar
    • Export Citation
  • Muller K, Pomorski T, Muller P & Herrmann A 1999 Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment. Journal of Cell Science 112 1120.

    • Search Google Scholar
    • Export Citation
  • Munoz-Caro T, Lendner M, Daugschies A, Hermosilla C & Taubert A 2015a NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation. Developmental and Comparative Immunology 52 245254. (https://doi.org/10.1016/j.dci.2015.05.007)

    • Search Google Scholar
    • Export Citation
  • Munoz-Caro T, Mena Huertas SJ, Conejeros I, Alarcon P, Hidalgo MA, Burgos RA, Hermosilla C & Taubert A 2015b Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent. Veterinary Research 46 23. (https://doi.org/10.1186/s13567-015-0155-6)

    • Search Google Scholar
    • Export Citation
  • Oh H, Siano B & Diamond S 2008 Neutrophil isolation protocol. Journal of Visualized Experiments 23 745. (https://doi.org/10.3791/745)

  • Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, Lohmeyer J & Preissner KT 2012 Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS ONE 7 e32366. (https://doi.org/10.1371/journal.pone.0032366)

    • Search Google Scholar
    • Export Citation
  • Schulz M, Zambrano F, Schuppe HC, Wagenlehner F, Taubert A, Gaertner U, Sanchez R & Hermosilla C 2019a Monocyte-derived extracellular trap (MET) formation induces aggregation and affects motility of human spermatozoa in vitro. Systems Biology in Reproductive Medicine 65 357366. (https://doi.org/10.1080/19396368.2019.1624873)

    • Search Google Scholar
    • Export Citation
  • Schulz M, Zambrano F, Schuppe HC, Wagenlehner F, Taubert A, Ulrich G, Sanchez R & Hermosilla C 2019b Determination of leucocyte extracellular traps (ETs) in seminal fluid (ex vivo) in infertile patients – a pilot study. Andrologia 51 e13356. (https://doi.org/10.1111/and.13356)

    • Search Google Scholar
    • Export Citation
  • Silva LM, Caro TM, Gerstberger R, Vila-Vicosa MJ, Cortes HC, Hermosilla C & Taubert A 2014 The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitology Research 113 27972807. (https://doi.org/10.1007/s00436-014-3939-0)

    • Search Google Scholar
    • Export Citation
  • Strzemienski PJ 1989 Effect of bovine seminal plasma on neutrophil phagocytosis of bull spermatozoa. Journal of Reproduction and Fertility 87 519528. (https://doi.org/10.1530/jrf.0.0870519)

    • Search Google Scholar
    • Export Citation
  • Taylor U, Rath D, Zerbe H & Schuberth HJ 2008 Interaction of intact porcine spermatozoa with epithelial cells and neutrophilic granulocytes during uterine passage. Reproduction in Domestic Animals 43 166175. (https://doi.org/10.1111/j.1439-0531.2007.00872.x)

    • Search Google Scholar
    • Export Citation
  • Urban CF, Reichard U, Brinkmann V & Zychlinsky A 2006 Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cellular Microbiology 8 668676. (https://doi.org/10.1111/j.1462-5822.2005.00659.x)

    • Search Google Scholar
    • Export Citation
  • Waterhouse KE, Hofmo PO, Tverdal A & Miller RR 2006 Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction 131 887894. (https://doi.org/10.1530/rep.1.01049)

    • Search Google Scholar
    • Export Citation
  • World Health Organization 2010 WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva, Switzerland: WHO Press.

  • Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA & Nicolson GL 1979 Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertility and Sterility 31 562574. (https://doi.org/10.1016/s0015-0282(1644004-5)

    • Search Google Scholar
    • Export Citation
  • Zambrano F, Carrau T, Gartner U, Seipp A, Taubert A, Felmer R, Sanchez R & Hermosilla C 2016 Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility. Fertility and Sterility 106 1053 .e11060.e1. (https://doi.org/10.1016/j.fertnstert.2016.06.005)

    • Search Google Scholar
    • Export Citation
  • Zambrano F, Schulz M, Pilatz A, Wagenlehner F, Schuppe HC, Conejeros I, Uribe P, Taubert A, Sanchez R & Hermosilla C 2020 Increase of leucocyte-derived extracellular traps (ETs) in semen samples from human acute epididymitis patients – a pilot study. Journal of Assisted Reproduction and Genetics 37 22232231. (https://doi.org/10.1007/s10815-020-01883-7)

    • Search Google Scholar
    • Export Citation
  • Zhou X, Xia XY & Huang YF 2010 Updated detection of the function of sperm plasma membrane. Zhonghua Nan Ke Xue (National Journal of Andrology) 16 745748.

    • Search Google Scholar
    • Export Citation

 

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    Figure 1

    Visualisation and analyses of human sperm-triggered NETs by SEM (A and B) and epifluorescence microscopy (C, D, E and F). Images (A and B) show human PMN entrapping spermatozoa; image (A) shows a human PMN carrying out NETosis, and thereby entrapping a spermatozoon within thin extracellular fibres (B). NET-derived structures showing DNA labelling with Sytox Orange® dye (red fluorescence, (C) and global histones (H1, H2A/H2B, H3, H4) labelled with Alexa Fluor 488 (green fluorescence, (D). Image (F) shows PTX3 immunofluorescence labelling. Green fluorescence represents PTX3 labelling; in (E) orange fluorescence represents DNA staining (cell nuclei and NETs). Red arrows indicate PMN; white arrows indicate NETs; yellow arrows indicate spermatozoa. Epifluorescence images, magnification 40x (C and D) and 60x (E and F).

  • View in gallery
    Figure 2

    (A) Graph representing quantification of NETs showing a significantly enhanced quantity of extracellular DNA in sperm/PMN co-cultures compared to PMN or sperm alone (*P < 0.05) at 180 min. Stimulation of PMN with PMA shows significantly enhanced NET formation compared with all the other groups. Treatment of PMN/sperm with 2-ABP (for SOCE inhibition (100 μM)) significantly reduced NET formation compared with that in control PMN/sperm (*P < 0.05). (B) Epifluorescence microscopy images showing NET structures of different groups: DNA (blue staining, DAPI); NE (green staining, Alexa Fluor 488). Epifluorescence images, magnification 60x.

  • View in gallery
    Figure 3

    Effects of NETs on membrane integrity, acrosome integrity, and DNA fragmentation in human spermatozoa in base medium (spermatozoa without PMN), in co-cultures of PMN/sperm (1:6) for 180 min, using NETosis inhibitor (SOCE inhibitor (2-APB, 100 μM)) and DNase control group. The data are represented as the mean percentage ± s.d.; statistical significance was considered at *P < 0.05.

  • View in gallery
    Figure 4

    Flow cytometry images, (A) percentage of sperm population with intact plasma membrane (viable, SYBR14+/PI−). (B) The viable sperm population with intact acrosome (PI-/PNA/FITC−). (C) The percentage of sperm population with fragmented DNA (TUNEL+).

  • View in gallery
    Figure 5

    Hemizona binding ability of sperm. (A) Spermatozoa exposed to PMN had a significantly lower capacity to bind to zona pellucida than control spermatozoa did (*P < 0.05). The mean number of spermatozoa binding to the zona pellucida increased significantly after 2-APB treatment compared with that in the untreated PMN/sperm group (all: *P < 0.05). However, inhibitor-treated PMN/sperm showed significantly reduced levels of hemizona binding compared with unexposed spermatozoa (*P < 0.5). (B) Shows images of sperm binding to the hemizona (sperm cell nuclei stained blue with DAPI); epifluorescence microscopy, magnification 40x. Three biological replicates were included in each assay. The data are represented as mean percentage ± s.d.; statistical significance was considered at *P < 0.05.

  • Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC & Foster DN 2009 Species-specific interaction of seminal plasma on sperm-neutrophil binding. Animal Reproduction Science 114 331344. (https://doi.org/10.1016/j.anireprosci.2008.10.015)

    • Search Google Scholar
    • Export Citation
  • Bottazzi B, Garlanda C, Cotena A, Moalli F, Jaillon S, Deban L & Mantovani A 2009 The long pentraxin PTX3 as a prototypic humoral pattern recognition receptor: interplay with cellular innate immunity. Immunological Reviews 227 918. (https://doi.org/10.1111/j.1600-065X.2008.00719.x)

    • Search Google Scholar
    • Export Citation
  • Brechard S & Tschirhart EJ 2008 Regulation of superoxide production in neutrophils: role of calcium influx. Journal of Leukocyte Biology 84 12231237. (https://doi.org/10.1189/jlb.0807553)

    • Search Google Scholar
    • Export Citation
  • Brinkmann V & Zychlinsky A 2007 Beneficial suicide: why neutrophils die to make NETs. Nature Reviews: Microbiology 5 577582. (https://doi.org/10.1038/nrmicro1710)

    • Search Google Scholar
    • Export Citation
  • Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y & Zychlinsky A 2004 Neutrophil extracellular traps kill bacteria. Science 303 15321535. (https://doi.org/10.1126/science.1092385)

    • Search Google Scholar
    • Export Citation
  • Conejeros I, Patterson R, Burgos RA, Hermosilla C & Werling D 2011 Induction of reactive oxygen species in bovine neutrophils is CD11b, but not dectin-1-dependent. Veterinary Immunology and Immunopathology 139 308312. (https://doi.org/10.1016/j.vetimm.2010.10.021)

    • Search Google Scholar
    • Export Citation
  • Daigo K & Hamakubo T 2012 Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps. Frontiers in Immunology 3 378. (https://doi.org/10.3389/fimmu.2012.00378)

    • Search Google Scholar
    • Export Citation
  • Doni A, Paffoni A, Nebuloni M, Ragni G, Pasqualini F, Valentino S, Bonetti S, Mantovani A, Somigliana E & Garlanda C 2009 The long PENTRAXIN 3 is a soluble and cell-associated component of the human semen. International Journal of Andrology 32 255264. (https://doi.org/10.1111/j.1365-2605.2007.00845.x)

    • Search Google Scholar
    • Export Citation
  • Donis-Maturano L, Sanchez-Torres LE, Cerbulo-Vazquez A, Chacon-Salinas R, Garcia-Romo GS, Orozco-Uribe MC, Yam-Puc JC, Gonzalez-Jimenez MA, Paredes-Vivas YL & Calderon-Amador J et al.2015 Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells. Springerplus 4 161. (https://doi.org/10.1186/s40064-015-0932-8)

    • Search Google Scholar
    • Export Citation
  • Fichtner T, Kotarski F, Gartner U, Conejeros I, Hermosilla C, Wrenzycki C & Taubert A 2020 Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4- and Ca++-dependent process. Biology of Reproduction 102 902914. (https://doi.org/10.1093/biolre/ioaa003)

    • Search Google Scholar
    • Export Citation
  • Franken DR, Burkman LJ, Oehninger SC, Coddington CC, Veeck LL, Kruger TF, Rosenwaks Z & Hodgen GD 1989 Hemizona assay using salt-stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa. Gamete Research 22 1526. (https://doi.org/10.1002/mrd.1120220103)

    • Search Google Scholar
    • Export Citation
  • Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS & Fulda S et al.2012 Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death and Differentiation 19 107120. (https://doi.org/10.1038/cdd.2011.96)

    • Search Google Scholar
    • Export Citation
  • Gupta AK, Giaglis S, Hasler P & Hahn S 2014 Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. PLoS ONE 9 e97088. (https://doi.org/10.1371/journal.pone.0097088)

    • Search Google Scholar
    • Export Citation
  • Hahn S, Giaglis S, Hoesli I & Hasler P 2012 Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss. Frontiers in Immunology 3 362. (https://doi.org/10.3389/fimmu.2012.00362)

    • Search Google Scholar
    • Export Citation
  • Leshner M, Wang S, Lewis C, Zheng H, Chen XA, Santy L & Wang Y 2012 PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Frontiers in Immunology 3 307. (https://doi.org/10.3389/fimmu.2012.00307)

    • Search Google Scholar
    • Export Citation
  • Lippolis JD, Reinhardt TA, Goff JP & Horst RL 2006 Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Veterinary Immunology and Immunopathology 113 248255. (https://doi.org/10.1016/j.vetimm.2006.05.004)

    • Search Google Scholar
    • Export Citation
  • Maldjian A, Pizzi F, Gliozzi T, Cerolini S, Penny P & Noble R 2005 Changes in sperm quality and lipid composition during cryopreservation of boar semen. Theriogenology 63 411421. (https://doi.org/10.1016/j.theriogenology.2004.09.021)

    • Search Google Scholar
    • Export Citation
  • Marin-Esteban V, Turbica I, Dufour G, Semiramoth N, Gleizes A, Gorges R, Beau I, Servin AL, Lievin-Le Moal V & Sandre C et al.2012 Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells. Infection and Immunity 80 18911899. (https://doi.org/10.1128/IAI.00050-12)

    • Search Google Scholar
    • Export Citation
  • Martinelli S, Urosevic M, Daryadel A, Oberholzer PA, Baumann C, Fey MF, Dummer R, Simon HU & Yousefi S 2004 Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. Journal of Biological Chemistry 279 4412344132. (https://doi.org/10.1074/jbc.M405883200)

    • Search Google Scholar
    • Export Citation
  • Muller K, Pomorski T, Muller P & Herrmann A 1999 Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment. Journal of Cell Science 112 1120.

    • Search Google Scholar
    • Export Citation
  • Munoz-Caro T, Lendner M, Daugschies A, Hermosilla C & Taubert A 2015a NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation. Developmental and Comparative Immunology 52 245254. (https://doi.org/10.1016/j.dci.2015.05.007)

    • Search Google Scholar
    • Export Citation
  • Munoz-Caro T, Mena Huertas SJ, Conejeros I, Alarcon P, Hidalgo MA, Burgos RA, Hermosilla C & Taubert A 2015b Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent. Veterinary Research 46 23. (https://doi.org/10.1186/s13567-015-0155-6)

    • Search Google Scholar
    • Export Citation
  • Oh H, Siano B & Diamond S 2008 Neutrophil isolation protocol. Journal of Visualized Experiments 23 745. (https://doi.org/10.3791/745)

  • Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, Lohmeyer J & Preissner KT 2012 Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS ONE 7 e32366. (https://doi.org/10.1371/journal.pone.0032366)

    • Search Google Scholar
    • Export Citation
  • Schulz M, Zambrano F, Schuppe HC, Wagenlehner F, Taubert A, Gaertner U, Sanchez R & Hermosilla C 2019a Monocyte-derived extracellular trap (MET) formation induces aggregation and affects motility of human spermatozoa in vitro. Systems Biology in Reproductive Medicine 65 357366. (https://doi.org/10.1080/19396368.2019.1624873)

    • Search Google Scholar
    • Export Citation
  • Schulz M, Zambrano F, Schuppe HC, Wagenlehner F, Taubert A, Ulrich G, Sanchez R & Hermosilla C 2019b Determination of leucocyte extracellular traps (ETs) in seminal fluid (ex vivo) in infertile patients – a pilot study. Andrologia 51 e13356. (https://doi.org/10.1111/and.13356)

    • Search Google Scholar
    • Export Citation
  • Silva LM, Caro TM, Gerstberger R, Vila-Vicosa MJ, Cortes HC, Hermosilla C & Taubert A 2014 The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitology Research 113 27972807. (https://doi.org/10.1007/s00436-014-3939-0)

    • Search Google Scholar
    • Export Citation
  • Strzemienski PJ 1989 Effect of bovine seminal plasma on neutrophil phagocytosis of bull spermatozoa. Journal of Reproduction and Fertility 87 519528. (https://doi.org/10.1530/jrf.0.0870519)

    • Search Google Scholar
    • Export Citation
  • Taylor U, Rath D, Zerbe H & Schuberth HJ 2008 Interaction of intact porcine spermatozoa with epithelial cells and neutrophilic granulocytes during uterine passage. Reproduction in Domestic Animals 43 166175. (https://doi.org/10.1111/j.1439-0531.2007.00872.x)

    • Search Google Scholar
    • Export Citation
  • Urban CF, Reichard U, Brinkmann V & Zychlinsky A 2006 Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cellular Microbiology 8 668676. (https://doi.org/10.1111/j.1462-5822.2005.00659.x)

    • Search Google Scholar
    • Export Citation
  • Waterhouse KE, Hofmo PO, Tverdal A & Miller RR 2006 Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction 131 887894. (https://doi.org/10.1530/rep.1.01049)

    • Search Google Scholar
    • Export Citation
  • World Health Organization 2010 WHO Laboratory Manual for the Examination and Processing of Human Semen. Geneva, Switzerland: WHO Press.

  • Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi CA & Nicolson GL 1979 Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: the use of salt-stored eggs for assessing the fertilizing capacity of spermatozoa. Fertility and Sterility 31 562574. (https://doi.org/10.1016/s0015-0282(1644004-5)

    • Search Google Scholar
    • Export Citation
  • Zambrano F, Carrau T, Gartner U, Seipp A, Taubert A, Felmer R, Sanchez R & Hermosilla C 2016 Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility. Fertility and Sterility 106 1053 .e11060.e1. (https://doi.org/10.1016/j.fertnstert.2016.06.005)

    • Search Google Scholar
    • Export Citation
  • Zambrano F, Schulz M, Pilatz A, Wagenlehner F, Schuppe HC, Conejeros I, Uribe P, Taubert A, Sanchez R & Hermosilla C 2020 Increase of leucocyte-derived extracellular traps (ETs) in semen samples from human acute epididymitis patients – a pilot study. Journal of Assisted Reproduction and Genetics 37 22232231. (https://doi.org/10.1007/s10815-020-01883-7)

    • Search Google Scholar
    • Export Citation
  • Zhou X, Xia XY & Huang YF 2010 Updated detection of the function of sperm plasma membrane. Zhonghua Nan Ke Xue (National Journal of Andrology) 16 745748.

    • Search Google Scholar
    • Export Citation