Boar spermatozoa are very susceptible to reactive oxygen species (ROS), but ROS involvement in damage and/or capacitation is unclear. The impact of exposing fresh boar spermatozoa to an ROS-generating system (xanthine/xanthine oxidase; XA/XO) on sperm ROS content, membrane lipid peroxidation, phospholipase (PL) A activity, and motility, viability, and capacitation was contrasted to ROS content and sperm function after cryopreservation. Exposing boar sperm (n = 4–5 ejaculates) to the ROS-generating system for 30 min rapidly increased hydrogen peroxide (H2O2) and lipid peroxidation in all sperm, increased PLA in dead sperm, and did not affect intracellular O2˙− (flow cytometry of sperm labeled with 2′,7′-dichlorodihydrofluorscein diacetate, BODIPY 581/591 C11, bis-BODIPY-FL C11, hydroethidine, respectively; counterstained for viability). Sperm viability remained high, but sperm became immotile. Cryopreservation decreased sperm motility, viability, and intracellular O2˙− significantly, but did not affect H2O2. As expected, more sperm incubated in capacitating media than Beltsville thawing solution buffer underwent acrosome reactions and protein tyrosine phosphorylation (four proteins, 58–174 kDa); which proteins were tyrosine phosphorylated was pH dependent. Pre-exposing sperm to the ROS-generating system increased the percentage of sperm that underwent acrosome reactions after incubation in capacitating conditions (P < 0.025), and decreased capacitation-dependent increases in two tyrosine-phosphorylated proteins (P ≤ 0.035). In summary, H2O2 is the major free radical mediating direct ROS effects, but not cryopreservation changes, on boar sperm. Boar sperm motility, acrosome integrity, and lipid peroxidation are more sensitive indicators of oxidative stress than viability and PLA activity. ROS may stimulate the acrosome reaction in boar sperm through membrane lipid peroxidation and PLA activation.
Sperm cryopreservation is not common in swine artificial insemination programs. Frozen-thawed boar spermatozoa are less fertile than fresh or cooled semen , for reasons not yet completely understood. Boar spermatozoa are susceptible to cold shock , possibly relating to the lipid composition of the membrane, which contains a high concentration of polyunsaturated fatty acids (PUFA) [3, 2, 4]. Cryopreservation significantly increases production of reactive oxygen species (ROS)  in bovine  and equine  sperm. ROS have dual effects on sperm function, at low concentrations inducing sperm capacitation [8, 9], hyperactivation , acrosome integrity , and sperm-oocyte fusion , while, in contrast, excessive amounts of ROS damage DNA [13, 14], inhibit sperm-oocyte fusion , and reduce sperm motility (human , equine , and porcine ).
Sperm capacitation in many mammals involves protein tyrosine phosphorylation (human , cattle , and mouse ), and in boar spermatozoa, capacitation is associated with tyrosine phosphorylation [21–23], specifically of 32-kDa proteins [24, 25]. Various specific signaling pathways mediating such phosphorylation during sperm capacitation include protein kinase (PK) A and PKC , cAMP/PKA (of fibrous sheath protein) and protein tyrosine kinase (PTK) [27, 28]. ROS at physiological concentrations induce sperm capacitation, acrosome reaction, and tyrosine phosphorylation in human [10, 29], equine , and bovine  spermatozoa, but their role in porcine capacitation is unknown. ROS can induce cellular tyrosine phosphorylation in association with receptor or nonreceptor tyrosine kinases . ROS, acting in concert with such factors as bicarbonate, loss of membrane cholesterol, and increasing intracellular Ca2+, can increase intracellular superoxide, which activates adenyl cyclase in rat sperm , increasing cAMP concentrations. Increased cAMP activates PKA, activating tyrosine kinase and inhibiting tyrosine phosphatase. Hydrogen peroxide (H2O2) can directly activate the kinase and inhibit the phosphatases, and indeed, H2O2 can replace bicarbonate in activating the cyclase in bull sperm . Lipid peroxidation, resulting from this low concentration of ROS, promoted sperm binding to the zona pellucida . However, H2O2 at high concentration in bull spermatozoa inhibits tyrosine phosphorylation , and ROS have been implicated in damaging sperm .
DNA is very susceptible to ROS damage, as oxidants like HO˙ can cause hydroxylation, ring opening, fragmentation , transitory crosslinking of protein-DNA , and strand breaks that may be mutagenic or lethal for the cell , and certainly decrease fertilization rates . ROS also cause sperm to become refractory to calcium signals and inhibit ionophore-induced sperm-oocyte fusion , perhaps by stimulating peroxidation that reduces plasma membrane fluidity and interfering with membrane-bound enzymes and/or ion channels . ROS impair motility, perhaps depleting ATP by H2O2 inhibiting oxidative phosphorylation and/or glycolysis, thereby limiting ATP replenishment , by depressing glycolytic flux , by decreasing phosphorylation of axonemal proteins required for sperm motility , or via such products of lipid peroxidation as malondialdehyde and 4-hydroxynonenol (4HN), inhibiting anaerobic glycolysis, DNA, RNA and protein synthesis .
Boar spermatozoa are sensitive to peroxidative damage, potentially through their high concentration of PUFAs [2–4, 41], which serve as preferred substrates for ROS and HO˙ generation in membranes , which would be exacerbated by the relatively low antioxidant capacity of boar seminal plasma (reviewed in ). Lipid peroxidation disrupts membrane structure and function [44, 45] by disordering membrane phospholipid structure  and changing membrane fluidity. Extracellular Ca2+ binding to membrane phospholipids decreases lipid bilayer fluidity, further damaging bilayer structure through inducing lipid-phase separation, formation of heterogeneous structural domains, and membrane fusion ([47, 48], reviewed in ), increasing membrane permeability and affecting ion transport . Lipid peroxidation also disrupts mitochondrial enzymes, eventually uncoupling respiration associated with oxidative phosphorylation .
Boar sperm contain phospholipase (PL) A2 , and increasing its extra- or intracellular activity by the coupled action of lipid peroxidation and Ca2+ on membrane phospholipids ultimately alters membrane structure and initiates membrane degradation . Membrane lysis and cell death could result from excessive enzyme activity .
Despite knowing how ROS affect sperm function in different species, there is very little known about the influence of ROS on boar sperm function. The current study hypothesizes that: 1) the process of freezing and thawing boar spermatozoa increases production of one or more ROS; and 2) altered ROS levels affect motility, viability, capacitation/acrosome reaction, lipid peroxidation, and PLA activity of fresh sperm. The specific goals were to evaluate relative O2˙− and H2O2 in fresh and frozen-thawed boar sperm and determine how generating ROS in fresh boar spermatozoa would affect sperm function (motility, viability, and acrosome integrity), sperm intracellular levels of O2˙− and H2O2, lipid peroxidation, and PLA activity.
Materials and Methods
Boars of proven fertility were housed and handled according to the requirements of the Canadian Council on Animal Care. The sperm-rich fraction was collected using the gloved-hand method into a 35°C Thermos, ejaculates with >70% motility (visual estimate) were selected, and sperm concentration determined by a calibrated spectrophotometer (Spectronic 20; Milton Roy, Rochester, NY).
Sperm freezing and thawing
The sperm-rich fraction (one ejaculate from each of four boars) was assessed for motility (computer-assisted sperm analysis [CASA]) and viability (SYBR-PI). Each ejaculate was extended, cooled, frozen in 0.5-ml straws (IMV Technologies, France), stored in liquid nitrogen, and subsequently thawed as previously described . Briefly, sperm were cooled at 0.1°C/min to 25°C, centrifuged (800 × g, 10 min, 25°C), resuspended in BF5 buffer A containing 20% egg yolk , cooled to 5°C (0.1°C/min), diluted in BF5-B with glycerol (final concentration, 3% glycerol, 6 × 108 spermatozoa/ml), sealed in straws, and the straws frozen (−30°C/min from 5 to −80°C, plunged and stored in liquid nitrogen). Straws were thawed (60°C, 5 sec) and their contents emptied into 37°C tubes and either immediately mixed with Beltsville thawing solution (BTS) (unwashed; 205.37 mM glucose [dextrose], 20.4 mM sodium citrate dihydrate, 14.88 mM NaHCO3, 3.36 mM EDTA, 10.01 mM KCl, and dihydrostreptomycin [770mg/g activity]; pH 7.4; ) or centrifuged (washed; 300 × g, 3 min, 37°C) to remove the egg yolk extender and the pellet mixed with BTS (final concentration, 3 × 107 spermatozoa/ml). Motility, progressive motility, viability, and intracellular levels of O2˙− and H2O2 were then assessed on the washed and/or unwashed sperm.
Motility and progressive motility of 200 sperm were assessed by CASA (IVOS System Software; Hamilton Thorn Bioscience) sampled from 1 × 107 spermatozoa/ml in BTS: BF5 fraction-A (3:1, vol:vol; 177.62 mM Glucose [dextrose], 16.51 mM Tris [hydroxymethyl] amino methane, 52.35 mM TES-Tris [hydroxymethyl] methyl 2 amino ethane sulphonic acid, 20% egg yolk [vol:vol], 0.33% Orvus ES Paste, streptomycin [770 mg/g activity], pH 7.3–7.4; ) using a four-chamber standard count analysis slide (Leja products B.V. Luzernestraat 10, 2153 GN Nieuw-Vennep, The Netherlands), at 37°C. CASA settings were: frames acquired, 100 at 30 Hz; minimum contrast, 50 pixels; minimum cell size, 7 pixels; nonmotile head size, 11 pixels; nonmotile head intensity, 118; medium average path velocity, 45 μm/sec; low path velocity, 20 μm/sec; and threshold straightness 45 μm/sec.
To determine percent live sperm without flow cytometry, 1 × 107 spermatozoa/ml in BTS extender were stained with the Live: dead sperm viability kit (Molecular Probes, Inc., Eugene, OR) and 2 × 100 spermatozoa viewed at 400× magnification with a fluorescence microscope (Laborlux S; Leitz, Germany), fitted with a blue filter (450–490 nm) and classified as live (green) or dead (red).
Capacitation and acrosome reaction
Where indicated, fresh sperm were incubated in capacitating conditions  or BTS. Briefly, 5 × 106 spermatozoa/ml were diluted into BTS or capacitating medium (4.8 mM KCl, 1.2 mM KH2PO4, 95 mM NaCl, 5.55 mM glucose [dextrose], 25 mM NaHCO3, 2 mM CaCl2, 2 mM pyruvate, 0.4% BSA [fatty acid free, fraction V]; pH 7.4). The pH was adjusted repeatedly with NaOH or HCl over 2 h of stirring. The medium's pH was then stable; without this lengthy adjustment period, the medium's pH rose within 5 min. The mixture was incubated for 4 h at 36°C, 5% CO2, and 100% humidity. Aliquots were taken at the indicated times and received either dimethyl sulfoxide (DMSO) or the Ca2+ ionophore A23187 (final concentration, 4 μM in DMSO; Molecular Probes), incubated for 30 min (36°C, 5% CO2, and 100% humidity), and capacitation status assessed.
To determine capacitation status microscopically, sperm were smeared on glass slides, air dried, fixed with 100% ethanol (−20°C, 20 sec), and again air dried. Smears were stained with Coomassie blue (1 g Coomassie blue, 50% methanol, 40% water, and 10% glacial acetic acid) for 2 min , washed thoroughly with distilled water, air dried, and the acrosomes of 100 sperm were visually determined to be intact or reacted (totally + partially) under bright-field microscopy (400×). All slides were coded to be blind to treatment and scored by one person.
To assess capacitation status through tyrosine phosphorylation, proteins were extracted by adding 20 μl of 10 mM Na2VO3 (Fisher, Mississauga, ON, Canada) to 1 ml sperm aliquot (5 × 106 spermatozoa/ml), centrifuging (13 000 × g, 10 min, room temperature), and resuspending sperm pellets with 50 μl of 0.2 mM Na2VO3 and 12.5 μl of 5× sample buffer containing 0.350 g dithiothreitol, 0.5 g SDS, 2.0 ml Tris (1 M, pH 6.8), 2.5 ml of 50% glycerol, and 150 μl of 2.5% bromophenol blue, and boiled for 5 min. The boiled sperm preparation was centrifuged (13 000 × g, 5 min, room temperature), and the supernatant kept on ice until assessed by SDS-PAGE, using 4% polyacrylamide stacking gel/10% running gel for 15 min at 75 V followed by 100 V at room temperature over 150 min or until the dye front reached the bottom of the gel. The gel was equilibrated in transfer buffer (Bio-Rad, Mississauga, ON, Canada; containing 25 mM Tris, 192 mM Glycine and 20% [vol:vol] methanol; pH 8.3) for 30 min at room temperature and then transferred electrophoretically (100 V) to Immobilon polyvinylidene difluoride (PVDV) transfer membrane (Millipore, ON, Canada) for 60 min at 4°C using transfer buffer. Nonspecific binding was blocked by incubating membranes with a 5% (wt:vol) solution of skim milk powder in Tris (20 mM, pH 7.8)-buffered saline (TBS) containing 1% Tween 20 (TTBS, 0.1% vol:vol; Fisher, Mississauga, ON, Canada) overnight at 4°C with shaking. After blocking, membranes were incubated with a monoclonal antibody (1:2000 in TTBS) developed in mouse against phosphotyrosine proteins (anti-phosphotyrosine [4G10], HRP conjugate; Upstate Biotechnology, Lake Placid, NY) for 2 h at room temperature with shaking. Membranes were then washed with TTBS three times for 10 min and twice with milliQ water, and positive immunoreactive bands were detected using the enhanced chemiluminescence detection system . Bands were detected and their volume, area, and molecular mass (kDa) were measured using Image Quant TL.lnk software (Amersham Bioscience), and then band intensities were calculated.
Sperm Intracellular Levels of O2˙− and H2O2
Hydroethidine (HE; Molecular Probes, Inc.) and 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Inc.) detected O2˙− and H2O2, respectively, as described by Guthrie and Welch . Working solutions of 20 mM HE and H2DCFDA in DMSO were aliquoted and stored in the dark at −20°C. On the day of the experiment, 3 × 107 fresh or frozen-thawed sperm in BTS were mixed with HE or H2DCFDA (final concentrations: 1 × 106 sperm/ml; 4 μM HE or 200 μM H2DCFDA). To simultaneously differentiate living from dead cells , YO-PRO-1 (YO-PRO-1 iodide [491/509] in DMSO; Molecular Probes, Inc.; final concentration, 0.05 μM) was added to HE-treated sperm and propidium iodide (PI, Molecular Probes Inc.; final concentration, 9.6 μM) was added to H2DCFDA-treated sperm. Sperm were incubated for 60 min at 25°C and then fluorescence was assessed in a FACS Calibur flow cytometer (BD Bioscience, Mississauga, ON) with a 488-nm excitation source. A total of 10 000 individual sperm-sized events were selected based on forward and side scatter; HE and YO-PRO-1 detected with 650-nm long-pass (LP) and 530/30-nm filters, and H2DCFDA and PI detected with 530/30-nm and 650-nm LP filter, respectively. Using the statistical package of the FACS, evident sperm populations were gated and the mean fluorescence intensity and number of sperm in each population determined.
Effects of ROS on Boar Sperm
An ROS-generating system of xanthine and xanthine oxidase (Sigma, Oakville, ON, Canada) was prepared to generate O2˙− and H2O2 . Xanthine (20 mM in 1M NaOH) was diluted to 2 mM in water (pH 7.5) and 0.2 U xanthine oxidase was added to 0.5 ml of 2 mM xanthine. The pH was adjusted repeatedly to pH 7.4–7.5 with NaOH or HCl over 2 h of stirring using Accument pH Meter 925 (Fisher Scientific, ON, Canada). The medium's pH was then stable; without this lengthy pH adjustment, the medium's pH rose within 5 min. Then, 0.5 ml were mixed with freshly collected sperm (n = 1 ejaculate from each of five boars) in BTS ± ROS-generating system (final concentrations: 3 × 107 sperm; 0 or 1 mM xanthine + 0.1 U xanthine oxidase per ml) and incubated (30 min, 38°C, in air). Sperm were then cooled to room temperature over 10 min, stained with HE and YO-PRO-1 or H2DCFDA and PI, and assessed by flow cytometry for O2˙−, H2O2, and viability. Four separate ejaculates were similarly incubated ± ROS-generating system and assessed for motility (CASA) and viability (SYBR-PI).
To assess the effects of ROS on capacitation status, fresh sperm were similarly incubated ± ROS-generating system for 30 min, then identical aliquots were diluted into BTS or capacitating medium and incubated as described for capacitation. Immediately after the BTS/ROS incubation, and after the 4-h incubation, aliquots from each treatments (BTS or ROS treated; incubated in BTS or capacitating media) were assessed microscopically (n = 4) or for tyrosine phosphorylation (n = 4).
Lipid Modification by ROS
To assess the effect of ROS generation on lipid modification in fresh boar spermatozoa, sperm (n = 1 ejaculate from each of five different boars) were incubated with BTS or the ROS-generating system, as described above (final concentration, 2 × 107 spermatozoa/ml). To measure lipid peroxidation, one aliquot from each treatment (±ROS exposure) was then incubated (30 min, 25°C) with the fluorescent probe, BODIPY 581/591 C11 ; 2 μM (final concentration); 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; Molecular Probes), and then PI (24 μM, final concentration, for 15 min). Fluorescence was analyzed by flow cytometry (488-nm excitation, 650-nm emission LP filter for PI; 530/30-nm filter for BODIPY 581/591 C11).
To measure PLA activity, a second aliquot from each treatment was incubated with bis-BODIPY-FL C11 (Molecular Probes, Inc.) . Unlike previous studies that required incorporation of the probe into liposomes for delivery to the target cells , the probe here was directly incubated with boar spermatozoa after a preliminary experiment demonstrated that these conditions supported significant uptake of the probe by the cells (data not shown). Following the 30 min incubation ± the ROS-generating system, sperm were incubated with bis-BODIPY-FL C11 and PI (8 and 24 μM, final concentration; 20 min, 37°C) and sperm were then analyzed by flow cytometry (488-nm excitation, 650-nm emission LP filter for PI; 530/30-nm filter for bis-BODIPY-FL C11).
All data were analyzed with SAS 9.1 for Windows, TS Level 1M3 (The SAS Institute Inc., Cary, NC). Data regarding sperm function, intracellular ROS concentration, and lipid peroxidation were checked for normality and least-squares means were assessed by the general linear model procedure (GLM) and Tukey multiple comparison test to determine effects of cryopreservation, washing, and/or ROS pretreatment. To analyze the amount of protein detected by Western immunoblotting, Image Quant band intensities were log transformed prior to analysis using the Mixed Procedure as a split plot in which the four boars represented the blocks, and treatment (pre-exposure to BTS or ROS-generating system) was the main-plot factor. Incubation medium (capacitating medium or BTS) and time (immediately after pre-exposure and after 4 h) were the subplot factors. Specific differences among times, treaments, and media were analyzed with least-squares means and Tukey-Kramer test. Differences with values of P < 0.05 were considered to be statistically significant.
Fresh and Cryopreserved Sperm
Motility and viability
Compared with washed or unwashed frozen-thawed sperm, the fresh sperm group had more motile (23 ± 3.1% or 37.75 ± 4.5% vs. 83.75 ± 4.6%, respectively; P < 0.0001) and also more progressively motile sperm (19.5 ± 4.1 or 30 ±1.8% vs. 65.5 ± 6.6%; P < 0.0001); washing frozen-thawed sperm did not affect motility. As expected, more fresh sperm were alive than frozen-thawed spermatozoa (P < 0.0001); washing frozen sperm after thawing did not affect the percent live value (SYBR-PI and fluorescence microscopy; 84.25 ± 1.75% [fresh] vs. 24.75 ± 5.7% or 38 ± 2.35% [frozen-thawed washed and not washed]). Similar results were obtained with flow cytometric assessment of YO-PRO-1-stained sperm, although this stain identified a third subpopulation of presumably moribund sperm in addition to the live and dead sperm in fresh semen, while frozen-thawed sperm (whether washed or not) showed only live and dead subpopulations (Fig. 1, A–C).
Sperm intracellular levels of O2˙− and H2O2
Flow cytometry indicated that the average O2˙− content of all fresh sperm (ethidium fluorescence of live + dead combined) was similar to the average for frozen-thawed sperm, and dead fresh sperm had similar O2˙− to dead frozen-thawed sperm (data not shown). However, viable fresh sperm had more O2˙− than did viable frozen-thawed sperm (P < 0.0009; Fig. 1D). The DCF fluorescence demonstrated no significant difference between the H2O2 content of fresh or frozen-thawed sperm, either considering the average over the total sperm populations, or just the viable sperm (13.1 ± 2.3, 15.6 ± 2.4, 12.5 ± 2.1 fluorescence units for fresh, frozen-washed, and frozen-unwashed sperm, respectively; P > 0.05).
Effects of Exposure to an ROS Generator on Fresh Boar Sperm
Viability and motility
Exposing fresh boar spermatozoa to the ROS-generating system (30 min, 38°C) completely inhibited sperm motility (0% vs. 75.3 ± 4.3% motile; P < 0.01), but did not affect the percent of viable sperm as assessed microscopically (69.0 ± 2.9% vs. 72.3 ± 2.8% alive) or by flow cytometry, although the flow cytometer again detected an additional moribund population (81.7 ± 5.6% live, 14.8 ± 5.2% dead, and 3.5 ± 0.8% moribund for control sperm vs. 63.5 ± 13% live, 25.7 ± 13% dead, and 10.8 ± 5% moribund for ROS-exposed sperm; Fig. 2, A and B).
All live sperm had significantly more O2˙− than did the dead sperm (Fig. 2C; P < 0.05), but the ROS-generating system had no effect on the concentration of O2˙− in any sperm populations. The ROS-generating system did increase the H2O2 level of the average sperm population (P < 0.0004; Fig. 3). Interestingly, exposure to the ROS generator caused the live sperm to divide into three subpopulations (compare Fig. 3A with Fig. 3B), which differed in their H2O2 content. The most numerous of these subpopulations (86% of all viable sperm) contained significantly more H2O2 than did the single population of control sperm (Fig. 3C).
Sperm lipid modification
Exposure to the ROS generator significantly increased the intensity of BODIPY 581/591 C11 fluorescence, indicating a significantly greater extent of lipid peroxidation in the overall sperm population, and in each of the live and dead sperm populations (P < 0.004; Fig. 4A), with dead sperm showing the greatest extent of lipid peroxidation (P < 0.003).
PLA activity was greater in dead sperm than live (Fig. 4B; P < 0.0001), and exposure to the ROS generator increased this even further (P < 0.0002). However, the ROS generator did not affect PLA activity in live sperm.
The Ca2+ ionophore, A23187, did not affect the microscopically detectable acrosomal status of sperm at 30 min or 4 h in BTS or capacitation buffer (P > 0.05), so the data with and without ionophore were pooled. As expected, more sperm were acrosome reacted after 4-h incubation in capacitating medium than in BTS (P < 0.025; Fig. 5). Pre-exposure to ROS greatly increased the percent of sperm that acrosome reacted after 4-h incubation in capacitation medium compared with all other treatments (P < 0.0001; Fig. 5).
Boar spermatozoa in BTS for 30 min or 4 h consistently had two prominent tyrosine-phosphorylated (tyr-P) proteins at 39 and 35 kDa, but sperm incubated in capacitating medium for 4 h developed many more tyr-P proteins (Fig. 6). ROS pre-exposure reduced the intensity of the 174 and 58 kDa tyr-P proteins (P ≤ 0.035) that developed during capacitation, while high variability in the amount of the 105 and 94 kDa tyr-P proteins meant ROS pre-exposure resulted in a numerical, but not significant, decline in these capacitation-induced tyr-P proteins (Fig. 6C). Interestingly, different proteins became tyrosine phosphorylated if the pH of the capacitating medium was not very carefully controlled. The pH of this system readily rose to ∼7.9–8.0 unless the pH was thoroughly adjusted prior to use to keep it stable at ∼7.4 during the capacitating incubation. A distinctly different tyr-P protein profile was generated when pH rose compared with that generated when the pH was stabilized to ∼7.4 (compare Fig. 6A and Fig. 6B).
ROS were hypothesized to be agents of cryopreservation-induced changes in boar sperm, but, although the sperm did show the expected deterioration in viability and motility, cryopreserved boar sperm unexpectedly had less O2˙− than fresh sperm, and similar amounts of H2O2. Pursuing detailed examination of potential roles of ROS in capacitation determined that fresh boar sperm exposed to exogenous generation of ROS underwent minor changes in intracellular level of O2˙−, but significantly increased intracellular H2O2, essentially the opposite response generated by whole-sperm cryopreservation. We identified, for the first time, that these changes in intracellular ROS were accompanied by a great increase in membrane lipid peroxidation in live sperm, with no accompanying change in PLA activity; indeed, increased PLA was associated with dead sperm. Furthermore, the brief exposure to the ROS generator caused many more sperm to acrosome react—not immediately, nor in a simple buffer, but only after a capacitating incubation. This enhanced capacitation was accompanied by unique changes in tyr-P proteins, which were exquisitely sensitive to pH of the capacitating medium. Relating these ROS changes to sperm function will help elucidate fertilizing steps in boar sperm, and clarify cryopreservation damage.
As expected, motility, progressive motility, and viability of fresh spermatozoa were significantly higher than those of frozen-thawed sperm (whether the frozen-thawed sperm were washed free of extender or evaluated immediately after thawing, the latter being the condition they would be in at the time of insemination). This reduction in functional abilities could be due to cold shock , intracellular ice formation, and/or osmotic stress , which damage the sperm membrane, the mitochondria, the acrosome, and the sperm tail . Cryopreservation is known to reduce motility , with the loss of mitochondrial function  potentially interrupting ATP availability to the tail filaments  and elsewhere. Concomitant loss of membrane integrity  would both increase PI permeability and ultimately kill the cells.
Contrary to the hypothesis that cryopreservation would increase ROS, intracellular levels of H2O2 were unchanged and viable frozen-thawed sperm had significantly less intracellular O2˙− than did fresh spermatozoa. Somewhat similarly, Guthrie and Welch  reported that boar sperm had low basal concentrations of both of the ROS species, O2˙− and H2O2, although neither was observed to change after cryopreservation. The O2˙− concentration in the average and dead cryopreserved sperm did not differ from that of fresh sperm, and was significantly higher than that of the live frozen-thawed sperm, which might suggest that sperm that survived freezing and thawing had to have low amounts of O2˙−. The cryopreservation-associated decrease in O2˙− documented here could be related to the alteration of mitochondrial function. Oxidative phosphorylation is required for mitochondrial ATP production, and this process is coupled with electron transport and ROS formation (reviewed in ). Thus, cryopreservation could decrease oxidative phosphorylation, ATP synthesis, and ROS generation , causing both the reduction in O2˙− content and sperm motility. Since cryopreservation had limited effects on the boar sperm content of two well-known ROS, and yet, as expected, interfered with normal sperm function, it was important to directly explore the impact of ROS on boar sperm.
The XA/XO ROS generation system employed here generates O2˙− and H2O2 outside the cell . Incubating boar spermatozoa for 30 min with this ROS-generating system significantly increased sperm intracellular content of H2O2 (Fig. 3), but not O2˙− (Fig. 2), confirming others' results with boar sperm , and also human  and equine spermatozoa . H2O2 has been suggested to be a major ROS responsible for oxidative damage in boar spermatozoa , produced by the substantial intracellular (mitochondrial and cytoplasmic) superoxide dismutase (SOD) in boar sperm  scavenging O2˙− and rapidly dismutating it to H2O2 . Since boar semen is extremely low in catalase [67, 68], which provides resistance to H2O2 , there would be insufficient catalase to convert the large amounts of XA/XO-induced H2O2, to water and oxygen. Certainly, adding catalase to the freezing extender alone or in combination with SOD was suggested to reduce post-thaw ROS generation, and thereby improve motility and viability of boar spermatozoa , supporting the contention that low catalase amounts in boar spermatozoa contributes to their sensitivity to H2O2. It also indirectly supports our suggestion that viable post-thaw sperm constitute a subpopulation having naturally low endogenous O2˙− levels, because, if O2˙− levels had risen and been removed by dismutation, H2O2 would have risen.
Such subpopulations are evident in the living sperm after 30-min exposure to ROS generators, which had self-segregated into three subpopulations having differing intracellular concentrations of H2O2 (Fig. 3). Since the most numerous population had the highest H2O2 content, it is tempting to speculate that there are minor subpopulations of sperm in an ejaculate that differ either in their endogenous content of SOD to create H2O2 (unlikely given the stability of O2˙− content), or in their amounts of catalase or other systems that would rapidly reduce any H2O2 created.
ROS and Boar Sperm Function
This brief incubation with the ROS-generating system completely inhibited boar sperm motility, as did similar incubations with human spermatozoa . However, Guthrie and Welch  found that 33% of boar sperm were motile after exposure to a similar XA/XO-generating system that also contained BSA, which has antioxidant properties . Indeed, we found that up to 10% of the spermatozoa diluted with capacitating medium containing 0.4% BSA remained motile after exposure to the ROS-generating system (data not shown). However, obviously most sperm quickly become immotile when exposed to the XA/XO-generating system. The H2O2 may oxidize intracellular sulfhydryl moieties, depressing glycolytic flux and decreasing ATP levels . Spermatozoal ability to generate ATP may also be limited by inhibiting one or more enzymes of oxidative phosphorylation and/or glycolysis , thereby decreasing phosphorylation of axonemal proteins.
Sperm motility could also be damaged by lipid peroxidation products releasing PUFAs from sperm plasma membrane to alter membrane fluidity, permeability, and cellular capacity to regulate intracellular ions . Lipid peroxidation of membrane phospholipids was significantly higher in all sperm exposed to the XA/XO ROS generator (Fig. 4), similar to that induced in boar sperm incubated with FeSO4/Na ascorbate ROS-generating system , which removed membrane PUFAs and produced cytotoxic aldehydes, such as malondialdehyde and 4HN, that could inhibit anaerobic glycolysis, in addition to altering membrane architecture .
ROS at low concentrations are known to induce sperm capacitation and acrosome reactions in sperm from other species [8, 11], and the current results demonstrate, for the first time, that there is a time-dependent, significant increase in the percentage of acrosome reactions seen in sperm exposed to an ROS-generating system and then incubated in capacitating medium (Fig. 5). However, despite the known association of increased protein tyr-P with capacitation of boar sperm [21–25], and the increased tyr-P of high-kDa proteins documented here with capacitation (particularly 174 kDa; Fig. 6), pre-exposure of sperm to the ROS generator reduced protein tyr-P, although there was still more tyr-P proteins than in sperm incubated in a noncapacitating medium. Either this degree of tyr-P was adequate to support the increased acrosome reactions detected microscopically, or the visual acrosomal removal could reflect membrane lipid peroxidation.
The proteins tyrosine phosphorylated with capacitation here had similar kDas to those identified in other studies of capacitated boar spermatozoa [21, 73]. Those studies only presented and discussed results from one individual gel of an individual ejaculate, whereas the much more rigorous approach taken here actually quantified protein profiles from multiple ejaculates from different boars, statistically analyzing the amount and kDa of each protein after unbiased Image Quant analysis of bands and their intensity. The current results, therefore, more closely represent the tyr-P protein profile of porcine sperm, with limited emphasis on possibly minor proteins unique to an individual boar.
An enhanced presence of the 32-kDa protein has been noted repeatedly in capacitated boar sperm [23–25, 74], but neither we (Fig. 6) nor Kalab et al.  found it to increase, and even when it does increase, it is not a prerequisite for capacitation in boar sperm . Furthermore, we could induce capacitation-dependent phosphorylation of an ∼32-kDa protein by not controlling pH and allowing it to rise above physiological levels (Fig. 6A). Also, sperm proteins here were extracted using nonreducing conditions that could allow proteins to retain more three-dimensional structure, affecting rate of movement through the gel, and thereby changing apparent kDa. However, the effect of pH, clearly documented here for the first time to impact the actual nature of protein phosphorylation and not just apparent molecular mass, cannot be underestimated.
Unexpectedly, ROS pre-exposure, which enhanced the percentage of sperm undergoing the acrosome reaction after capacitating incubation, decreased capacitation-induced tyrosine phosphorylation of the high-kDa proteins. Although H2O2, the primary ROS generated in boar sperm (Fig. 3)  also inhibited tyr-P of proteins in bull spermatozoa , H2O2 did induce tyr-P in other species, in parallel to increasing sperm capacitation and the acrosome reaction [9, 31], consequent to an increase in cAMP, activation of tyrosine kinase and/or inhibition of tyrosine phosphatase [9, 75, 76]. ROS often affect target function by oxidizing sulfhydryl groups, forming a disulfide bridge (cystine) between two adjacent thiols in one protein, or between different proteins, resulting in changes of protein structure and function . This type of modification is reversible and specific, and could affect the transduction elements that are involved in sperm capacitation (reviewed in ), because a sulfhydryl/disulfide pair regulates the activity of tyrosine and Serine/Threonine protein phosphatase, PKA, PKC, PTK, and adenyl cyclase. Thus, H2O2 in the current study could be generated in sufficiently high concentrations to reverse the capacitation-associated inhibition or activation of tyrosine kinase and/or tyrosine phosphatase or both, which in turn inhibited tyrosine phosphorylation of the high-kDa proteins, particularly the 174 kDa protein. The H2O2 at this concentration could affect components of other pathways influencing tyrosine phosphorylation, such as the components of extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinase (MAPK) signaling pathway that participate in human sperm capacitation and tyrosine phosphorylation [79, 80], and/or cAMP-regulated tyrosine phosphorylation .
The increased capacitation detected histochemically could be related to the increased sperm membrane phospholipid peroxidation and PLA activity, also demonstrated here for the first time. Consequent disordering of membrane phospholipid structure  would change membrane fluidity, thereby increasing permeability and elevating intracellular Ca2+. The coupled action of lipid peroxidation and Ca2+ on membrane phospholipids may activate extra- or intracellular PLA2  that can create cis-unsaturated free fatty acids and lysophospholipids, facilitating the membrane instability and fusion  needed for fertilization  and the acrosome reaction (e.g., hamster ).
The bis-BODIPY-FL C11 probe measures PLA activity by the presence of endogenous PLA1 or PLA2, which cleaves off the BODIPY group and increases fluorescence emission at 530 nm, and therefore does not differentiate between cleavage of acyl chains at the 1 and 2 positions . Interestingly, viable sperm had the same PLA activity whether they were from control or XA/XO-exposed sperm; dead sperm from both groups had significantly higher PLA activity than the live sperm, and ROS generation resulted in dead sperm with more PLA activity than dead sperm in simple BTS. PLA2 is known to be involved in signal transduction pathways in animal cells , and, after being activated by either G proteins, phosphorylation or Ca2+ [84, 85], PLA hydrolyzes membrane phospholipids to lysophospholipids and free fatty acids; PLA2 preferentially targets phosphatidylcholine with arachidonic acid at the sn2 position, and so the most common PUFA released is arachidonic acid . The high levels of H2O2 documented to have been produced may have increased intracellular calcium concentration  through oxidative stress and lipid peroxidation, which could support PLA activation and help translocate it from the cytoplasm to the membrane . Other studies suggest that membrane lipid peroxidation increased activity of cPLA2 due to an increase in substrate availability for cPLA2, and implicated the high cPLA2 activity in cell death , and certainly sperm with excessive levels of PLA were dead. However, the brief ROS generation did not increase the numbers of dead sperm, so boar sperm do not appear to be highly sensitive to PLA-induced cell death.
In summary, the process of freezing and thawing significantly decreased sperm content of O2˙−. This phenomenon differs from pre-exposure to ROS, which caused fresh sperm subsequently incubated in capacitating conditions to increase intracellular H2O2 with no detectable increase in O2˙−, although O2˙− could have dismutated to H2O2. ROS pre-exposure also increased lipid peroxidation and PLA activity, which could have induced the greater incidence of acrosome reaction and loss of motility, although ROS inhibited capacitation-induced tyrosine phosphorylation of high-kDa sperm proteins. Boar sperm motility, acrosome integrity, and lipid peroxidation are more sensitive indicators of oxidative stress than viability and PLA activity. ROS and/or H2O2 may mediate capacitation of boar spermatozoa by various signaling pathways, such as the ERK pathway, which phosphorylates different proteins depending on their molecular mass. Thus, more studies are needed to investigate the physiological role of ROS in mediating signaling pathways that could regulate tyrosine phosphorylation of boar spermatozoa, such as the ERK family of MAPK pathway.
We thank Katie Hickey and Rachel Mixer for excellent technical assistance.