TY - JOUR
AU - Taubert,, Anja
AB - Abstract Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility. Summary Sentence Confrontation of bovine PMN with sperm cell preparation results in NOX-, PAD4-, and Ca++-dependent NET formation. Introduction Despite remarkable scientific development achieved within the field of artificial insemination and reproduction biology in humans and domestic animals in the last decades, infertility problems still have increased worldwide. Infertilities can occur by various reasons [1] and at least 10–20% of infertility/subfertility disorders lack any exact etiology [2]. Deposition of billions of spermatozoa during natural coitus or artificial insemination in the female reproduction tract results in early host innate immune reactions as visualized by rapid PMN infiltration into female reproduction tract. Following insemination, PMN seem capable to remove excess spermatozoa in vivo via phagocytosis in mammals (including humans) to maintain a microenvironment favorable to embryo development [3]. Additionally, various sterile or infectious inflammatory processes, including vestibulitis, vulvovaginitis, vaginitis, cervicitis, and endometritis, may also result in PMN infiltration into the female reproduction tract. Thus, recruited PMNs are directly exposed to sperm which frequently causes a decreased fertility due to PMN-derived sperm phagocytosis as previously reported [4]. Alongside phagocytosis, PMNs have evolved several other potent effector mechanisms to attack sperm, such as reactive oxygen species synthesis via NOX activation, antimicrobial peptide/protease degranulation/extrusion, and the release of neutrophil extracellular traps (NETs) [5]. NETs were firstly described in 2004 in a landmark study by Brinkmann et al. who demonstrated an extrusion of DNA-rich web-like structures after the stimulation of PMN with phorbol myristate acetate or Gram-positive bacteria [6]. NET formation is generally linked to PMN cell death, reported as NETosis, and is considered as an effector mechanism acting in the extracellular compartment. NETosis was described as an NOX- and PAD4-dependent cell death process, leading to the extrusion of a mixture of nuclear DNA and cytoplasmic granule contents resulting in the formation of DNA-rich web-like extracellular structures which are decorated with locally enriched global H3 and granular molecules, such as PTX, lactoferrin, NE, or MPO among others [7, 8]. Recently, NOX-independent NETosis was also reported [9–11]. Compared with NOX-dependent NET formation, NOX-independent NETosis is characterized by substantial lower levels of ERK 1/2- and Akt-activation, while activation of p38 seems similar in both signaling pathways. In addition, so-called “vital” NETosis circumventing PMN death was described. During this type of NETosis, PMN phagocytic/rolling capacities are maintained and exclusively DNA of mitochondrial origin is extruded [12]. While most NETosis-related investigations were performed on bacterial, viral, fungal, and parasitic infections, on metabolic diseases, cancer progression, autoimmunity, and coagulopathies, little is known on its role in reproduction. Since leukocytosis in the reproductive tract was implicated in human infertility [5, 13–17] and since artificial insemination is overall used in large domestic animals, interactions between PMN and sperm have largely been analyzed in several animal species, such as cattle and horses. As such, it was reported in horses that repeated deposition of sperm in the presence of PMN can lead to diminished fertility driven by NETosis [18, 19]. Furthermore, Strzemienski demonstrated that bovine seminal plasma (SP) reduced the capability of bovine PMN to eliminate spermatozoa via phagocytosis [20]. Moreover, other studies deciphering the mode of action of SP evidenced that equine SP also contained compounds which were able to diminish NET-related binding to spermatozoa in vitro [21], thereby presumably allowing a greater number of healthy mobile spermatozoa to reach the oviduct and fulfill oocyte fertilization [16]. Furthermore, Alghamdi and colleagues reported on NET-induced spermatozoa aggregates, which were inhibited by SP [18, 21]. Similar findings were more recently reported for humans in vitro and ex vivo. Thus, PMN and monocytes co-cultured with sperm resulted in spermatozoa aggregation thereby demonstrating the pivotal role of NET-derived spermatozoa entrapment [16, 22, 23]. Since SP is well-known for its fertility-promoting factors, such as DNase I or DNase I-like proteins, it was hypothesized that these enzymes may cleave sperm-triggered NETs in vivo and in vitro. First evidence for this assumption came from the equine system demonstrating that DNase I-like proteins from horse SP seemed able to digest plasmid DNA [18]. In agreement, entrapped human and bovine spermatozoa were efficiently released from NET structures by DNase I treatments indicating that this enzyme might be linked to NET evasion [16, 24]. Differences in functionality of DNases in different species could be observed by Alghamdi et al.: Bull DNase proved less active than that of horses but its affinity to spermatozoa was higher which is thought to be related to the fact that the natural site of insemination is the vagina [24]. With the current study, we aimed to investigate the direct interactions of bovine sperm with PMN on the level of NETosis and to provide novel data on NET-related molecules and signaling pathways to be involved in sperm-mediated NETosis in cattle. Materials and methods Ethic statements All animal procedures were performed according to the Justus Liebig University Animal Care Committee guidelines, approved by the Ethic Commission for Experimental Animal Studies of the State of Hessen (G16/2017) and in accordance to the current European Animal Welfare Legislation: ART13TFEU. Originality The material has not been submitted for publication elsewhere while under consideration of Biology of Reproduction. Guidelines of BOR We followed the guidelines of BOR described here: https://academic.oup.com/biolreprod/pages/Author_Guidelines Bovine PMN isolation Blood sampling for PMN isolation was performed on healthy adult dairy cows by jugular vein puncture. About 3–21 blood donors, 1–3 sperm donors, and 1–4 straws per bull from 1–2 ejaculates replicates were used. A number of replicates were 3–21. Precise n-numbers for each experiment are given in “experimental set-up.” Blood samples were not pooled. Heparinized blood was diluted in an equal volume of sterile phosphate buffered saline (PBS) containing 0.02% EDTA (Gibco, Dreieich, Germany), layered on Biocoll Separating Solution (Biochrome AG, Berlin, Germany) and centrifuged [45 min, 800 × g, room temperature]. Top layers containing plasma and peripheral blood mononuclear cells were removed, and remaining pellet composed of PMN and erythrocytes was isolated. The cell pellet was gently resuspended and shaken in 25-mL distilled water for 40 s to lyse erythrocytes. Osmolarity was immediately adjusted by adding the required volume of 10x Hanks Balanced Salt Solution (Biochrom AG). Afterwards, PMNs were washed twice (400 × g, 10 min), resuspended in RPMI 1640 cell culture medium, and counted in a Neubauer hemocytometer chamber. Preparation of bovine sperm samples All sperm samples originated from 1–3 healthy bulls with proven fertility. About 1–4 straws per bull were used for the experiments. Precise n-numbers for each experiment are given in “experimental set-up.” Sperm samples were used immediately after retrieval for “fresh/frozen-thawed” comparisons (experiment 1H) or we stored in liquid nitrogen (−196 °C) until further use. Frozen sperm samples were thawed for 20 s in a 35 °C water bath and were either immediately used for experimentation [=SCP, containing each substance of standard insemination portions] or centrifuged thrice (325 × g, 3 min, 22 °C) to separate different fractions. The supernatants (SNs) containing semen extender and traces of SP were kept at room temperature, and the sperm cells (SCs) were washed thrice in PBS or biladyl extender (Minitüb, Tiefenbach, Germany) (325 × g, 3 min, 22 °C) to obtain pure spermatozoa. To analyze the effect on sperm motility, motile and immotile sperm samples were needed. To produce immotile sperms, samples were thrice dipped into liquid nitrogen to completely reduce their motility. In all experimental settings, AndroVision (Minitüb, Tiefenbach, Germany)-based analysis of sperm motility was used. SCs were categorized as vital/motile when at least 50% showed motility, whereas they were considered as immotile when less than 3% were motile. Quantification of NETosis If not stated different, bovine PMNs were resuspended in serum-free RPMI 1640 medium without phenol red and then co-cultured for 60 min with bovine semen-derived samples (SCP/SC/SN) (37 °C, 5% CO2 atmosphere). In preliminary tests, the optimal ratios between PMN and SCs were determined (data not shown). For NET quantification experiments, 0.25 million SCs were used, if not stated different in the “experimental set-up” part. To evaluate dose-dependent effects of sperm, different PMN: sperm ratios were applied [1 + 1 (0.5 million: 0.5 million), 1 + 3 (0.5 million: 1.5 million), 1 + 6 (0.5 million: 3 million)]. For kinetic analyses, PMN and sperm were co-cultured either for a short time span (0, 15, 30, 45, and 60 min) or for prolonged time (60, 120, and 180 min). For NET quantification, samples were treated with micrococcal nuclease (0.1 U/μL, New England Biolabs, Frankfurt, Germany, 15 min, 37 °C) and thereafter centrifuged (300 × g, 5 min) [25, 26]. SNs were transferred into a 96-well plastic flat-bottom plate (100 μL per well in duplicates). Then, Pico Green (50 μL/sample, Thermo Scientific, Dreieich, Germany, diluted 1:200 in 10 nM Tris/1 mM EDTA buffer, in the dark) was added. NETs were determined by spectrofluorometric analysis at an extinction wavelength of 484 nm and an emission wavelength of 520 nm using an automated multiplate reader (Varioskan Flash, Thermo Scientific, Dreieich, Germany). For negative controls, PMNs in plain medium were used. For positive controls, bovine PMNs were stimulated with zymosan (1 mg/ml; Invitrogen, Dreieich, Germany) as demonstrated elsewhere [11, 25, 27, 28]. To dissolve NETosis and thereby prove the DNA nature of these structures, 90 U of DNase I (Roche Diagnostics, Unterhaching, Germany) were supplemented 15 min before the end of incubation period. For NETosis-related inhibition experiments, bovine PMNs were treated with inhibitors in serum-free medium RPMI 1640 cell culture medium without phenol red (30 min, 37 °C, 5% CO2 atmosphere) prior to sperm exposure, if not stated different. The following inhibitors were used: the NOX-inhibitor diphenyleniodonium chloride [DPI; Sigma-Aldrich, Taufkirchen, Germany, 25 and 50 μM]; the store-operated Ca2+ entry (SOCE)-inhibitor 2-aminoethoxydiphenyl borate [2-APB; Merck, Darmstadt, Germany, 100 μM, according to Conejeros [29, 30] and 125 μM, Locks medium]; the PAD4 inhibitor Cl-amidine (250 μM; Sigma-Aldrich, Locke’s solution); BAPTA, the chelator of intracellular Ca2+ [50 μM, Sigma-Aldrich]; EGTA, the chelator of extracellular Ca2+ [500 μM, Sigma-Aldrich]; the MPO-inhibitor 4-aminobenzoic acid hydrazide [ABAH; 100 μM, Merck, according to Parker [31] and Muñoz-Caro [32]]; the PI3K-mediated autophagy inhibitor 2-(4-morpholinyl)-8-phenyl-1(4)-benzopyran-4-one hydrochloride [LY294; 25 μM, Sigma-Aldrich]; and the ERK1/2 signaling pathway inhibitor U0126 [62.5 μM; Sigma-Aldrich]. Detection of H3, NE, MPO, and PTX via immunofluorescence Bovine PMNs were co-cultured with SC preparations on poly-L-lysine pre-coated coverslips allocated in six-well tissue plates (Greiner, Frickenhausen, Germany). Then, samples were fixed (4% paraformaldehyde, Merck), washed thrice in sterile PBS, blocked with BSA (2%, Sigma-Aldrich, 15 min, room temperature), and incubated in primary antibody solutions [1 h, room temperature; anti-histone (1: 500 in PBS, H11–4, Millipore, Darmstadt, Germany), anti-NE (1: 200 in PBS, ab68672, Abcam, Berlin, Germany), anti-MPO (1: 500 in PBS, Orb11073, Biorbyt, Eching, Germany)]. After incubation, the samples were washed again with PBS and incubated in secondary antibody solution (1: 500 in PBS, goat anti rabbit, Merck) for 30 [anti-H3] or 60 min (anti-NE, anti-MPO, and anti-PTX). After incubation, the samples were washed again and thereafter mounted in ProLongGold containing 4′-6-diamidino-2-phenylindole (DAPI) (Invitrogen, 1:1000, 5 min, room temperature, in the dark) or Sytox Orange [Invitrogen, 5-mM Sytox Orange, 10 min, room temperature, in dark, according to Martinelli [33]]. For the detection of H3, NE, MPO, and PTX in NET structures, the following antibodies were used: anti- H3 (mouse monoclonal H11–4, 1:500, Merck Millipore), anti-NE (AB68672, 1:200, Abcam), anti-MPO (ORB11073, 1:500, Biorbyt), and anti-PTX (SAB2104614-50UG, 1:500, Sigma-Aldrich) antibodies. The samples were examined using an inverted Olympus IX81 fluorescence microscope being equipped with a digital camera (Olympus, Hamburg, Germany XM10). Scanning electron microscopy Bovine PMNs were co-cultured with sperm sample preparations (60 min, 37 °C, 5% CO2 atmosphere) on poly-L-lysine (Sigma-Aldrich) pre-coated coverslips (10 mm in diameter, Nunc, Dreieich, Germany). Samples were then fixed in 2.5% glutaraldehyde (Merck) and post-fixed in 1% osmium tetroxide (Merck). After washing in distilled water and dehydrating, they were dried by CO2-treatment to critical point and sputtered with gold. Samples were examined with a Philips XL30 scanning electron microscope (SEM) at the Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Germany, as described in Muñoz-Caro [25, 26]. Quantitative analysis of PMN-derived oxygen consumption and proton efflux rates PMN activation was monitored using a Seahorse XF analyzer (Agilent, Rathingen, Germany). Briefly, after isolation, 1 × 106 PMNs were centrifuged at 500 × g for 10 min. After removal of SN, the cells were suspended in 0.5 mL of XF RPMI assay medium (XF assay medium, Agilent) supplemented with 2 mM of L-glutamine, 1-mM pyruvate, and 10-mM glucose. About 1 × 105 PMNs, corresponding to 50 μL of the cell suspension, were gently placed in eight-well XF analyzer plates (Agilent) pre-coated for 30 min with 0.001% poly-L-lysine. About 50 μl of XF assay medium were added to blank wells (no cells), and then, 130 μL of XF assay medium were added to all wells (180-μL total volume) and incubated at 37 °C without CO2 for 45 min prior to XF assay. Purified SC were pelleted, suspended in XF assay medium and placed at 25.000 cells/20 μL in one of the four injection ports of the Seahorse XF analyzer. In case of SN stimulation, 20 μL of SN was placed in the injection port. For controls (PMN only condition), only XF assay medium was added. The real-time metabolic assay included the basal measurement of three readings, injection of SC or SN, and then 30 readings over time. The total run time was 240 min. Background subtraction, determination of oxygen consumption rate, proton efflux rate, and the area under the curve of obtained registries were performed in Wave software (Desktop Version, Agilent). Experimental set-up Experiment 1A–F (SEM): PMNs from three blood donors were incubated with SCs from three sperm donors. For each experiment, 1 × 106 PMN and 1 × 106 SCs (SCP) were used. Experiment 1G (dose-dependency): PMN from three blood donors were incubated with SCs (SCP) from three sperm donors (2 straws per bull, 1–2 ejaculates). Concentrations were adjusted as follows: 1 + 1 = 0.5 million PMN + 0.5 million SCs; 1 + 3 = 0.5 million PMN + 1.5 million SCs; and 1 + 6 = 0.5 million PMN + 3 million SCs. A number of replicates were 18. Experiment 1H (fresh/frozen-thawed sperm samples): PMN from three blood donors were incubated with fresh or frozen-thawed SCs (SCP) from one sperm donor (1 straw from 1 ejaculate). A number of replicates were 6. For each replicate, 200 000 PMN and 200 000 SCs (SCP) were used. Experiment 2A and B (time dependency): PMN from three blood donors were incubated with SCs (SCP) from one sperm donor (1 straw from 1 ejaculate) for short time span (0, 15, 30, 45, and 60 min) or for prolonged time (60, 120, and 180 min). A number of replicates were 3 for each experiment. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 2C (motile/immotile SCs): PMN from three blood donors were incubated with motile (>50% motility) or immotile (<3% motility) SCs (SCP) from three sperm donors (1 straw from 1 ejaculate). A number of replicates were 9. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 2D (different sperm fractions): PMN from 21 blood donors were incubated with SC, SCP, or SN from three sperm donors (3–4 straws per bull from 1–2 ejaculates). A number of replicates were 21. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 3A–C (oxygen consumption rate, O2 consumption, proton efflux): PMN from six blood donors were incubated with SC or SN from one sperm donor (2 straws from 1–2 ejaculates). A number of replicates were 3. For each replicate, 105 PMN and 25 000 SC (respectively, equivalent volume of SN) were used. Experiment 4A–F (fluorescence microscopy): PMN from three different blood donors were incubated with SCs (SCP) from three sperm donors (1 straw, 1 ejaculate). For each experiment, 125 000–250 000 PMN and 31 250–750 000 SCs (SCP) were used. Experiment 5A (DNase): PMN from six blood donors were pre-incubated with DNase/without DNase and incubated with SCs (SCP) from two sperm donors (1 straw from 1 ejaculate). A number of replicates were 6. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 5B (DPI): PMN from 12 blood donors were pre-incubated with DPI/without DPI and incubated with SCs (SCP) from three sperm donors (1–2 straws per bull from 1 ejaculate). A number of replicates were 12. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 5C (Cl-amidine): PMNs from five blood donors were pre-incubated with Cl-amidine/without Cl-amidine and incubated with SCs (SCP) from three sperm donors (1–2 straws per bull from 1 ejaculate). A number of replicates were 9. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 6A (BAPTA, EGTA): PMN from three blood donors were pre-incubated with BAPTA or EGTA/without BAPTA or EGTA and incubated with SCs (SCP) from one sperm donor (1 straw from 1 ejaculate). A number of replicates were 3. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 6B (2-APB): PMNs from three blood donors were pre-incubated with 2-APP/without 2-APB and incubated with SCs (SCP) from one sperm donor (1 straw from 1 ejaculate). A number of replicates were 3. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 6C (ABAH): PMNs from three blood donors were pre-incubated with ABAH/without ABAH and incubated with SCs (SCP) from three sperm donors (1 straw from 1 ejaculate). A number of replicates were 6. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Experiment 6D (LY294, U0126): PMNs from three blood donors were pre-incubated with LY294 or U0126/without LY294 or U0126 and incubated with SCs (SCP) from two sperm donors (1 straw from 1 ejaculate). A number of replicates were 6. For each replicate, 250 000 PMN and 62 500 SCs (SCP) were used. Figure 1 Open in new tabDownload slide Bovine spermatozoa trigger NET formation. Bovine PMN and sperm samples were co-cultured and processed for SEM analysis showing that PMN performed NETosis in relation to these gametocytes by either attacking single specimen (A: stars, B–F) mainly via spread NETs at any region of the SC (C: head; D–F: tail; E and F: middle part) and via aggregated NET formation (A: circles, B). NET formation was quantified revealing that sperm-triggered NETosis was dose-dependent (G) and that fresh sperm samples induced stronger NETosis than frozen ones (H). 1 + 1 = 0.5 million PMN + 0.5 million SCs; 1 + 3 = 0.5 million PMN + 1.5 million SCs; 1 + 6 = 0.5 million PMN + 3 million SCs; **P < 0.01. Figure 1 Open in new tabDownload slide Bovine spermatozoa trigger NET formation. Bovine PMN and sperm samples were co-cultured and processed for SEM analysis showing that PMN performed NETosis in relation to these gametocytes by either attacking single specimen (A: stars, B–F) mainly via spread NETs at any region of the SC (C: head; D–F: tail; E and F: middle part) and via aggregated NET formation (A: circles, B). NET formation was quantified revealing that sperm-triggered NETosis was dose-dependent (G) and that fresh sperm samples induced stronger NETosis than frozen ones (H). 1 + 1 = 0.5 million PMN + 0.5 million SCs; 1 + 3 = 0.5 million PMN + 1.5 million SCs; 1 + 6 = 0.5 million PMN + 3 million SCs; **P < 0.01. Figure 2 Open in new tabDownload slide Influence of incubation time, sperm motility, and sperm preparation on NET induction. Bovine PMN and sperm samples were co-cultured for a (A) short- and (B) long-term period and assayed for NET formation. Furthermore, the NET formation induced by immotile (<3% motility) or motile (>50% motility) SCs was quantified (C). Finally, different fractions of SC preparations [SCP (frozen insemination portion), SC, SN obtained from SC portion] were tested for their capacity of NET induction (D). In all experimental settings, plain PMN served as negative controls. n. s. = not significant; **P < 0.01; ***P < 0.001. Figure 2 Open in new tabDownload slide Influence of incubation time, sperm motility, and sperm preparation on NET induction. Bovine PMN and sperm samples were co-cultured for a (A) short- and (B) long-term period and assayed for NET formation. Furthermore, the NET formation induced by immotile (<3% motility) or motile (>50% motility) SCs was quantified (C). Finally, different fractions of SC preparations [SCP (frozen insemination portion), SC, SN obtained from SC portion] were tested for their capacity of NET induction (D). In all experimental settings, plain PMN served as negative controls. n. s. = not significant; **P < 0.01; ***P < 0.001. Statistics The data are expressed as mean ± standard deviation. Experiments 1G, 1H, 2C, 2D, 5A, 5B, 5C, 6A, 6B, and 6C: The statistical significances were determined by one factorial analysis of variance (ANOVA) and Student–Newman–Keuls test. Experiments 2A and 2B: The statistical significances were determined by two factorial ANOVA and a multiple t-test with Bonferroni–Holm correction. Experiments 3A, 3B, and 3C: Statistical significances were determined by one factorial ANOVA followed by a Tukey post-test for multiple comparisons over area under the curve values. Significant differences were observed at levels of: P < 0.05 (*); P < 0.01 (**); and P < 0.001 (***). Results Bovine sperm preparations induce PMN activation and the formation of different phenotypes of NETs. Ultrastructural SEM analyses unveiled that the exposure of bovine PMN to sperm resulted in the formation of a delicate network composed of thin strands of PMN-derived fibers being attached to these gametes seemingly entrapping them (Figure 1). During NETosis, some sperm-exposed PMN exhibited the morphology of intact cells, while others obviously died while performing NETosis (Figure 1). Based on literature [25, 34–36], NETs present at different phenotypes, i.e., as diffuse NETs, spread NETs, and/or aggregated NETs can be observed. Therefore, we, here, analyzed if SCP induced different NET phenotypes and found that all types of NETs were detected in PMN/sperm co-cultures (Figures 1 and 4). Thus, using SEM analyses, SCs were found to be attacked by NETs either as single specimen via spread NETs or in groups via aggregated NETs-mediated agglutination (Figure 1A–F). As depicted in immunofluorescence analyses, the formation of diffuse NETs was also detected (Figure 4). Moreover, no obvious preference of NET-mediated contact for distinct sperm areas was observed since SCs were entrapped by their tails, heads, and middle parts (Figure 1C–F). In terms of entrapped spermatozoa, numerous SCs were found to be captured by aggregated NETs. Overall, bovine spermatozoa seemed to be firmly immobilized by NETs, but neither surface damage nor morphological sperm alterations were detected up to 180 min of PMN exposure. Quantitative assessment of sperm-triggered NETosis revealed this process as dose-dependent (Figure 1G). Thus, an enhanced level of NET formation was estimated at increasing PMN: sperm ratios. Furthermore, fresh sperm revealed as a significantly more potent NET inducer when compared with frozen sperm (fresh vs. frozen sperm: P < 0.01). Given that frozen sperm is routinely used for artificial insemination, we restricted all other experiments to this sperm formulation. Bovine sperm-triggered NETosis is a SC motility- and time-independent process and, as PMN activation, mainly relies on factors present in the SN of sperm samples. Applying short- (0–60 min) and long-term (1–3 h) kinetics, we could show that sperm-triggered NETosis occurred independent of exposure time. Thus, no significant differences were observed within 0–60 and 60–180 min of PMN: sperm co-cultures (Figures 2A and B). Interestingly, NET formation occurred very fast and almost immediate upon confrontation, as visualized by NET formation at “0” min (analysis immediately after confrontation). However, due to technical restrictions which include a minimal incubation period of 15 min for micrococcal nuclease supplementation, this time point rather reflects 15 min of exposure than 0 min. In order to evaluate whether SC viability influenced NETosis, PMNs were co-cultured with SCs showing motility at more than 50% (“motile”) or less than 3% (“immotile”). Given that no significant differences could be determined in this experimental setting, sperm-triggered bovine NETosis is proved independent on SC vitality (Figure 2C). Given that frozen SC preparations used for artificial insemination always contain extender and traces of SP besides SCs, we also tested these different fractions for their ability to induce NETosis. Even though pure SCs significantly triggered NETosis, albeit to a lower extent, (PMN control vs. PMN + pure SCs: P < 0.01), the main reaction was based on SN (sperm extender + traces of SP)-induced NETosis (PMN controls vs. SN: P < 0.01; Figure 2D). However, SEM-based analyses revealed that these NETs were also clearly directed against SCs (Figure 1A–F). Effective NETosis requires PMN activation We therefore additionally analyzed whether the confrontation of bovine PMN with bovine sperm would lead to PMN activation. This was assessed by means of PMN-derived oxygen consumption and proton efflux rate measurements using Seahorse XF Analyzer (Agilent). We here tested the reactions of PMN upon two different fractions, SC and SN, which were added by injection into the system via instrument-given injection ports during the assay (see Figure 3A, arrow). Supplementation of SN led to a rapid and significant increase of the oxygen consumption (PMN vs. PMN + SN: P = 0.007) and proton efflux (P = 0.001) in PMN, while the supplementation of SC had no effect on these indicators of PMN activation (Figure 3). Figure 3 Open in new tabDownload slide SN induces oxygen consumption rate and proton efflux in bovine PMN. After basal measurements, XF Seahorse medium (20 μL), SC (25.000), or SN (20 μL) fractions were added to 105 PMN, and the oxygen consumption rate and proton efflux were measured over 240 min (A). The area under the curve of oxygen consumption rate (B) and proton efflux (C) of the registries was calculated and plotted as bar graph (mean ± SD). n. s. = not significant; **P < 0.01. Figure 3 Open in new tabDownload slide SN induces oxygen consumption rate and proton efflux in bovine PMN. After basal measurements, XF Seahorse medium (20 μL), SC (25.000), or SN (20 μL) fractions were added to 105 PMN, and the oxygen consumption rate and proton efflux were measured over 240 min (A). The area under the curve of oxygen consumption rate (B) and proton efflux (C) of the registries was calculated and plotted as bar graph (mean ± SD). n. s. = not significant; **P < 0.01. Bovine sperm-triggered NETs contain histones (H3), neutrophil elastase (NE), myeloperoxidase (MPO), and pentraxin (PTX). In order to analyze classic components being present in sperm-triggered extracellular structures, (immuno-) fluorescence-based analyses were performed. Thus, DAPI staining proved the DNA nature of extracellular NET-like structures (Figure 4B, D, and F). Furthermore, antibody-based staining revealed the presence of histones (H3, Figure 4B) and of the granular proteins NE (Figure 4D), MPO (not shown), and PTX (Figure 4F) in DNA-positive sperm-triggered NET structures. Interestingly, bovine SCs themselves showed a positive reaction for PTX (Figure 4F). Furthermore, these experiments also proved the presence of aggregated NETs and diffuse NETs phenotypes (Figure 4B, D, and F). Figure 4 Open in new tabDownload slide Detection of H3, NE, and PTX in sperm-triggered NETs. Bovine PMN and sperm samples were co-cultured, fixed, stained for DNA (DAPI, blue, B, D, and F) and processed for antibody-based detection of H3 (B, green), NE (D, green), and PTX (F, green). In parallel, phase contrast microscopy (A, C, and E) was performed to better visualize the different structures and cell types. Figure 4 Open in new tabDownload slide Detection of H3, NE, and PTX in sperm-triggered NETs. Bovine PMN and sperm samples were co-cultured, fixed, stained for DNA (DAPI, blue, B, D, and F) and processed for antibody-based detection of H3 (B, green), NE (D, green), and PTX (F, green). In parallel, phase contrast microscopy (A, C, and E) was performed to better visualize the different structures and cell types. Functional inhibition experiments reveal sperm-triggered NETosis as NOX-, PAD4-, and Ca++-dependent but independent of ERK 1/2- and PI3K-signaling pathways. Via functional inhibition experiments, we aimed to analyze the dependency of sperm-induced NETosis on different molecule and signal transduction activities. In a first setting, we confirmed that NETosis was indeed based on DNA extrusion. Thus, digesting DNA via DNase I treatments entirely deleted sperm-triggered NETosis when compared with non-treated PMN (P < 0.01, Figure 5A). As expected, blockage of NOX via DPI treatments significantly inhibited sperm-induced NETosis when compared with non-treated controls (P < 0.05, Figure 5B) proving it as an NOX-dependent effector mechanism. Furthermore, treatments of PMN with Cl-amidine (PAD4 inhibitor) also led to a significant reduction of SCP-mediated NETosis revealing this process as PAD4-dependent (Cl-amidine treated vs. non-treated PMN: P < 0.05, Figure 5C). To unravel the role of Ca++ influx in sperm-triggered NETosis, we applied different inhibitors either indirect blocking Ca++ influx via chelation from the extracellular (EGTA) or intracellular (BAPTA) compartment, or store-operated Ca++ release (2-APB). Interestingly, both EGTA and BAPTA treatments significantly reduced sperm-triggered NETosis when compared with non-treated controls (EGTA: P < 0.01; BAPTA: P < 0.01, Figure 6A) thereby proving the key role of Ca++ disposability for effective NETosis. In contrast, 2-APB treatments failed to influence sperm-induced NET formation (Figure 6B), thereby indicating that other intracellular Ca++ sources than SOCE are necessary for proper sperm-induced NET formation. Figure 5 Open in new tabDownload slide Inhibition of sperm-triggered NETosis by treatments with DNase, DPI, and Cl-amidine. Bovine PMN and SCPs were co-cultured and treated with DNase I (A), DPI (NOX inhibitor) (B), or Cl-amidine (PAD4 inhibitor) (C). In all experimental settings, plain PMN served as negative controls. Thereafter, NET formation was quantified. n. s. = not significant; *P < 0.05; **P < 0.01. Figure 5 Open in new tabDownload slide Inhibition of sperm-triggered NETosis by treatments with DNase, DPI, and Cl-amidine. Bovine PMN and SCPs were co-cultured and treated with DNase I (A), DPI (NOX inhibitor) (B), or Cl-amidine (PAD4 inhibitor) (C). In all experimental settings, plain PMN served as negative controls. Thereafter, NET formation was quantified. n. s. = not significant; *P < 0.05; **P < 0.01. Figure 6 Open in new tabDownload slide Inhibition of sperm-triggered NETosis by treatments with BAPTA, EGTA, 2-APB, ABAH, LY294, and U0126. Bovine PMN and SCPs were co-cultured and treated with indirect inhibitors of intra- (BAPTA, A) and extra- (EGTA, A) cellular Ca++ influx, store-operated Ca++ entry (2-APB, B), MPO (ABAH, C), PI3K-mediated autophagy (LY294, D), or ERK1/2 signaling pathway (U0126, D). In all experimental settings, plain PMN served as negative controls. Thereafter, NET formation was quantified. n. s. = not significant; **P < 0.01. Figure 6 Open in new tabDownload slide Inhibition of sperm-triggered NETosis by treatments with BAPTA, EGTA, 2-APB, ABAH, LY294, and U0126. Bovine PMN and SCPs were co-cultured and treated with indirect inhibitors of intra- (BAPTA, A) and extra- (EGTA, A) cellular Ca++ influx, store-operated Ca++ entry (2-APB, B), MPO (ABAH, C), PI3K-mediated autophagy (LY294, D), or ERK1/2 signaling pathway (U0126, D). In all experimental settings, plain PMN served as negative controls. Thereafter, NET formation was quantified. n. s. = not significant; **P < 0.01. Furthermore, we, here, tested whether sperm-mediated NETosis requires MPO activity. Inhibition experiments using ABAH as an MPO blocker denied any importance of MPO since no significant differences were observed between ABAH-treated PMN and non-treated controls (Figure 6C). The same held true for treatments with U0126 (ERK 1/2 inhibitor) and Ly294 (PI3K inhibitor) thereby denying a pivotal role of these signaling pathways in sperm-triggered NETosis (Figure 6D). Discussion In the bovine system, the natural site of semen deposition is the vagina, and spermatozoa have to migrate to the uterus thereby leaving most SP behind. However, in case of artificial insemination, the insemination dose containing spermatozoa, semen extender, and variable amounts of SP is directly deposited into the uterus. PMNs were reported to migrate to the female reproduction tract in response to insemination [21, 23, 37–40]. Furthermore, the presence of PMN at times of semen deposition was demonstrated to impair fertility in several species [21, 41–45]. As an interesting finding, some studies have identified sperm samples from domestic animals [18, 19], and more recently from humans [4, 16], as potent NETosis inducers. To mimic the situation of artificial insemination, which is commonly used in cattle breeding, we here focused on PMN-derived reactions in response to thawed semen samples. For these experiments, we used peripheral PMNs which were recently proven to equally perform sperm-triggered NETosis as uterus-derived ones [19]. In the current study, DNA staining of sperm-induced NETs and resolution of extracellular fibers by DNase I treatments confirmed the DNA backbone of these structures. It is well-known that DNases are also present in SP [18, 46] and these molecules were recently shown to block NETosis in the equine and the bovine system thereby improving semen fertility [21, 24]. However, this effect should obviously be restricted to fresh semen samples due to the high proportion of SP since insemination doses only bear small but varying volumes of this fluid due to the current extension/dilution protocols [47]. In contrast to equine spermatozoa [19, 24], we here showed that purified SC failed to cause PMN-derived oxygen consumption and proton efflux, which is a prerequisite of effective NETosis and likewise hardly induced NETosis. In contrast, also purified SC lead to significant NET induction (as detected via spectrofluorometric analysis), but most PMN activation and NET-triggering capacity could be attributed to the diluent of insemination doses consisting of semen extender and varying traces of SP. This agrees to recent findings of Alghamdi et al. who additionally showed that as little as 10% of SP being present in sperm samples was sufficient to trigger NETosis [19]. Considering the fact that commercially available insemination doses always contain some kind of semen extender and varying volumes of SP (depending on the density of original ejaculates) and that NETosis was linked to reduced sperm motility in vitro and ex vivo [16, 22, 23], the dilution protocols for insemination doses should be reconsidered. Besides DNA-related analyses, current co-localization experiments demonstrated the concomitant presence of H3, PTX, NE, and MPO in bovine sperm-induced NETosis thereby confirming classical molecular NET properties. These findings are in agreement to other reports on pathogen- [10, 11, 25, 27, 28, 32, 35, 48, 49] and spermatozoa [16]-mediated NETosis. Interestingly, PTX3 was previously shown to be expressed in the male genital tract and to be bound to human spermatozoa [50]. In line, we found that bovine SCs also stained positive for PTX. Even though we showed the presence of MPO in sperm-triggered NET structures, chemical blockage of this molecule via ABAH failed to modulate sperm-mediated reactions. This may indicate that conjoint actions of several NETosis-related molecules are needed for adequate and effective NETosis induction. However, ABAH treatments led to significant NETosis inhibition in other experimental settings [16, 25, 27, 32]. Current SEM analyses mainly indicated the presence of sperm-triggered suicidal NETosis leading to PMN cell death. In line, suicidal NETosis was also reported for several non-pathogenic [4, 18, 51] and infectious stimuli [6, 27, 28, 35, 48, 52, 53]. Additionally, sperm-mediated suicidal NETosis revealed as a dose-dependent process, which is in line to data on human sperm-induced NETosis [16]. In agreement to findings in the human and equine system [16, 18], bovine sperm-induced suicidal NETosis occurred very rapid upon PMN exposure to sperm samples, and a high level of NET extrusion was measured immediately after PMN/sperm confrontation (due to methodological requirements, this signifies 15 min of incubation). In contrast to equine and human data [16, 18], a significant time-dependency was not observed in the bovine system since equal levels of NETosis were detected within short- (0–60 min) and long-term (1–3 h) kinetics. Overall, we showed that sperm-induced NETs either attacked single spermatozoa without any preference for head or tail regions, or led to the capture of several specimens within clump-like structures thereby showing different NET phenotypes. Given that this phenomenon matched to findings on different parasite-triggered NET types [25, 35, 36, 54], we here described for the first time the presence of spread NETs, diffuse NETs, and aggregated NETs for SC preparations as NET inducers, all promoting firm and eventually massive sperm entrapment. Interestingly, these different NET phenotypes are mainly described in response to highly motile and large stages of parasite nematodes [25, 36, 54] which parallel typical characteristics of spermatozoa. However, viability of SCs was not essential for NETosis induction since sperm samples with low spermatozoa motility (<3%) equally induced NETosis as highly motile ones (>50% motility). Consequently, NETosis may contribute to impaired fertility even in cases of reduced sperm motility. Interestingly, a recent report on infertile human male patients showing leukocytospermia proved spermatozoa elimination via NETosis in aseptic and septic conditions [4]. Overall, human sperm-triggered NETosis not only caused gametocyte entrapment but also diminished spermatozoa motility [4, 16]. Thus, former authors suggested that sperm-triggered NETosis might contribute to male infertility disorders. On a mechanistic level, suicidal NETosis is often linked to NOX and PAD4 activities. While NOX triggers the formation of reactive oxygen species, PAD4 mediates histone citrullination and stimulates nuclear chromatin decondensation and unfolding [55, 56] thereby permitting adequate NETs extrusion by activated PMN. By performing functional inhibition experiments, we here showed that the blockage of NOX and PAD4 significantly diminished NET formation proving sperm-triggered lethal NETosis as an NOX- and PAD4-dependent process. Considering that PMN-derived NOX-activation and subsequent reactive oxygen species production is known to be Ca++/SOCE-dependent [57], we here additionally analyzed different chemical blockers targeting intra- and extracellular Ca++ influx for their effects on sperm-triggered NETosis. Overall, sperm-mediated NET formation revealed as dependent on both extra- and intracellular Ca++ disposability since EGTA and BAPTA treatments both significantly reduced NETosis. Interestingly, treatments with 2-APB failed to inhibit sperm-triggered NETosis thereby denying a pivotal role of SOCE as intracellular Ca++ source. In line to the data on intra- and extracellular Ca++, a Ca++ dependency of NET formation was also reported for bovine NETosis being triggered by certain protozoan parasites (Eimeria bovis, Cryptosporidium parvum, Neospora caninum; [11, 26, 32, 54]) and for human NET formation in response to chemical stimuli [58]. However, the source of Ca++ partially differed for these NETosis inducers since SOCE was identified as a key Ca++ source in case of E. bovis-, C. parvum-, and N. caninum-triggered NETosis [11, 26, 32, 54]. Referring to cell signaling pathways, NETosis was recently described as Raf-MEK-ERK-dependent [26, 32, 59]. However, current inhibition assays using the ERK1/2 blocker U0126 failed to significantly influence bovine sperm-triggered NETosis. As such, ERK1/2-related cell signaling does not appear to represent the main signaling pathway. The same held true for PI3K/Akt kinase-related pathway, since inhibition thereof did not alter sperm-induced NET formation in the bovine system. The inhibitors may have had no or only a little effect under the experimental conditions used. Nevertheless, standardized and commonly accepted inhibitors and related concentrations according to literature were used [29–32]. Furthermore, data which prove inhibitor functions were not included since these need entirely different experimental settings. However, concerning the utilization of 2-APB, dose responses in the bovine system were explicitly characterized [29, 30] and reviewed [60] in the past. To summarize, we, here, show that bovine insemination doses induce PMN activation and the formation of different phenotypes of NETosis in a Ca++-, NOX-, and PAD4-dependent process. Hereby, NETosis is rather triggered by semen extender and SP remnants present in these formulations than by spermatozoa themselves. Since we speculate NETosis to impair fertilization, dilution protocols of commercially sold insemination doses should be reconsidered in the future. Acknowledgments We would like to thank M. Sparenberg and K. Failing for data analyses. Furthermore, we extend our gratitude to all staff members of the Oberer Hardthof [Large Animal Experimental Farm of the Justus Liebig University Giessen (JLU), Germany] during blood collection and A. Seipp (Institute of Anatomy and Cell Biology, JLU, Germany) for her excellent technical assistance in SEM analysis. Finally, we also thank T. Muñoz-Caro and L. M. R. Silva (Institute of Parasitiology, JLU Giessen) for sharing their knowledge on NET-related experiments. We would like to thank M. Sparenberg and K. Failing for data analyses, as well as T. Muñoz-Caro and L. M. R. Silva for sharing their knowledge on NETosis-related experiments. 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TI - Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4-, and Ca++-dependent process
JF - Biology of Reproduction
DO - 10.1093/biolre/ioaa003
DA - 2020-04-15
UR - https://www.deepdyve.com/lp/oxford-university-press/bovine-sperm-samples-induce-different-net-phenotypes-in-a-nadph-ctGxquJH5W
DP - DeepDyve
ER -