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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Biotechnology and Germplasm Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705
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Abstract |
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Key Words: 5,5',6,6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide • cryopreservation • flow cytometry • hydroethidine • motility
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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), the use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen (Johnson, 1985; Waberski et al., 1994). Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS; Pena et al., 2003; Roca et al., 2004, 2005). Mitochondria are a major site of intracellular ROS formation, which results in a disruption of electron transport (Halliwell and Gutteridge, 1999). The coupling of electron transport to oxidative phosphorylation maintains a high mitochondrial transmembrane potential (MMP) required for mitochondrial ATP production in somatic cells (Cramer and Knaff, 1990). The coupling of electron transport with oxidative phosphorylation in sperm could be disrupted by ROS formation and result in a decrease in the number of sperm that maintain a high MMP required for mitochondrial ATP production and sperm motility.
Chemiluminescence measurements of superoxide (·O2) and hydrogen peroxide (H2O2) are problematic because of poor specificity (Fridovich, 1997, 2003). As an alternative approach, flow cytometric procedures have been used to identify ROS in leukocytes by utilizing the dyes hydroethidine (HE) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) as ROS substrates (Bass et al., 1983; Bucana et al., 1986; Carter et al., 1994). The percentage of cells with a high MMP can be estimated flow cytometrically using the mitochondrial probe, 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cossarizza et al., 1993). The objective of this study was to validate the use of flow cytometry as a method to measure ROS and MMP in viable fresh and frozen-thawed sperm.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Sperm Processing
The management of the boars (GPK5/EB5 boar line or derived from this line and female line GPK2/GPK1, Monsanto Choice Genetics, St. Louis, MO) used in this study, semen collection, and determination of sperm concentration were conducted as previously described (Guthrie and Welch, 2005). Before initiation of incubation treatments, fresh and thawed sperm were extended to a concentration of 150 x 106/mL in Beltsville Thawing Solution (BTS; Guthrie and Welch, 2005) and isolated from semen constituents and extenders using a Percoll (P-1644, Aldrich, Milwaukee, WI) 2-step, discontinuous gradient centrifugation, consisting of 35 and 70% isotonic Percoll (Guthrie and Welch, 2005). The sperm pellets were washed and diluted to a suitable concentration (30 to 60 x 106 sperm/mL) in a modified noncapacitating Tyrode’s medium (TYR; Guthrie and Welch, 2005). The Percoll isolation procedure is required because constituents of the freezing extenders interfere with the use of JC-1 for determination of MMP in boars (H. D. Guthrie, unpublished data) and bull sperm (Garner et al., 1999).
Cryopreservation
The boar semen cryopreservation protocol (Guthrie and Welch, 2005) was based on one published previously (Almlid and Johnson, 1988). Sperm (1 x 109 sperm/mL) were frozen in 0.5-mL French straws (13408/0010, Minitube of America, Verona, WI) in a freezing extender composed of 11% lactose (wt/vol; L2643, Sigma-Aldrich, St. Louis, MO) and 20% chicken egg yolk (vol/vol, isolated from whole eggs), with final concentrations of 3% glycerol (vol/vol; G6279, Sigma, St. Louis, MO) and 0.5% Equex STM Paste (vol/vol; Nova Chemical Sales, Scituate, MA). The contents of the straws were frozen in liquid N2 vapor in a programmable freezer (IceCube 1810, Minitube of America, Verona, WI), and the contents of the straws were thawed as previously described (Guthrie and Welch, 2005).
Sperm Motion Analysis
The percentage of motile sperm was determined in 2-chamber MicroCell glass counting chambers (Conception Technologies, San Diego, CA) using a Hobson Sperm Tracker (Hobson Tracking Systems Ltd., Sheffield, England) set for boar sperm analysis (Holt et al., 1996). Sperm were extended to 30 x 106/mL in BTS containing 0.3% BSA (A2153, Sigma-Aldrich, Cohn Fraction V) and warmed in a heating block at 38°C for 30 min before analysis. The sperm motion variables recorded for statistical analysis were percent motile (motility); curvilinear velocity (VCL), µm/sec; cross-beat frequency (BCF), Hz; and percent linear motion (LIN).
ROS-Generating Systems
Effects of ROS formation on motility, viability, and MMP were investigated utilizing the following treatments: xanthine/xanthine oxidase (XA/XO) treatment to generate ·O2 and H2O2 extracellularly (Britigan et al., 1990), menadione (2-methyl-1, 4-napthoquinone) that generates ·O2 intracellularly as a consequence of disruption of mitochondrial NADH-ubiquinone oxidoreductase (Floreani et al., 2002), and direct treatment with H2O2.
A 2-step procedure was used to expose the sperm to various levels ROS through the use of ROS-generating systems and ROS scavengers. For the XA/XO oxidase system, a set of tubes containing the 0.5-mL sperm suspensions were prewarmed for 5 min at 38°C at a concentration of 60 x 106 sperm/mL with or without the ROS scavengers superoxide dismutase (SOD; S-8160, Sigma) and catalase (CAT; 0, 200, and 600 U/mL; C-1345, Sigma). A second set of tubes containing 0 or 2 mM XA (X0626, Sigma) or 0, 0.02, 0.06, or 0.2 U/mL of XO (X4500, Sigma), or both XA and the various doses of XO, were also warmed for 5 min. After warming, 0.5-mL volumes of the XA/XO system components from the second set of tubes were mixed with 0.5-mL volumes of sperm, providing the following final concentrations: sperm at 30 x 106/mL; SOD and CAT at 0, 100, or 300 U/mL; XA at 1 mM; XO at 0, 0.01, 0.03, or 0.1 U/mL; or XA and the various doses of XO. The mixtures were allowed to incubate an additional 30 min at 38°C before their preparation for motion and flow cytometric analysis.
A 2-step procedure was also used for generation of ROS by menadione (M5625, Sigma). Aliquots containing 0, 20, 60, or 200 µM of menadione were removed from a 200 mM working solution in DMSO, transferred into of 0.5 mL of TYR, and prewarmed to 38°C for 5 min. After warming, 0.5-mL suspensions containing 60 x 106 sperm were added and incubated at 38°C for 30 min.
Staining Sperm for Flow Cytometric Analysis of ROS and MMP
The dye H2DCFDA is freely permeable across cell membranes and is incorporated into hydrophobic regions of the cell (Bass et al., 1983). The acetate moiety is cleaved by cellular esterases, leaving impermeant, non-fluorescent, 2',7'-dichlorodihydrofluorescein (H2DCF). The H2DCF is oxidized by H2O2 to dichlorofluorescein (DCF), which emits fluorescence at 530 nm in response to 488 nm excitation (Carter et al., 1994).
Hydroethidine is the sodium borohydride-reduced form of ethidium (Bucana et al., 1986). Hydroethidine is freely permeable to cells and is oxidized by ·O2 to ethidium (Rothe and Valet, 1990; Carter et al., 1994) and other products (Fridovich, 2003; Zhao et al., 2003). Ethidium binds to DNA with fluorescent emission at 610 nm in response to 488-nm excitation.
The probe JC-1 is freely permeable to cells and undergoes reversible transformation from a monomer to an aggregate form (Jagg) when it binds to membranes having an MMP of 80 to 100 mV (Cossarizza et al., 1993). The Jagg form fluoresces at 590 nm in response to 488-nm excitation. A flow cytometric JC-1 protocol for sperm (Garner et al., 1997) was modified to enumerate viable sperm with high MMP by electronically gating on viable sperm that were negative for propidium iodide (PI).
Preliminary testing was conducted to optimize the temperature and duration of the staining protocols for maximum fluorescence development (data not shown). Working solutions for HE (20 mM; D-1168, Molecular Probes Inc., Eugene, OR), H2DCFDA (20 mM; D-399, Molecular Probes), Yo Pro-1 (0.1 mM; Y-3603, Molecular Probes), and JC-1 (0.15 mM; T-3168, Molecular Probes Inc.) were prepared in DMSO. The exclusion of the DNA dye Yo Pro-1 from plasma membrane intact sperm identifies them as viable cells. The working solution for PI (2.4 mM; P-4170, Sigma Chemical Co., St. Louis, MO) was prepared in TYR.
After the incubation treatments, aliquots of 1 x 106 sperm (33 µL) were transferred to 12 x 75-mm polypropylene tubes containing 467 µL of TYR prepared for each stain combination at the following final concentrations: 1) HE and Yo Pro-1, 4 and 0.05 µM, respectively; 2) H2DCFDA and PI, 200 and 9.6 µM, respectively; and 3) JC-1 and PI, 0.3 and 9.6 µM, respectively. Staining times and temperatures for the stain combinations were as follows: HE and Yo Pro-1, 40 min and 25°C; H2DCFDA and PI, 60 min and 25°C; and JC-1 and PI, 10 min and 38°C.
Flow Cytometric Analysis
A Beckman-Coulter Epics XL-MCL Analyzer (Beckman-Coulter, Hialeah, FL) equipped with a single 488 nm excitation source was used for all flow cytometric analyses. A forward and side scatter gate was used to select single sperm from clumps and debris. Fluorescence from ethidium- and Yo Pro-1-stained sperm was collected in fluorescence detector 3 with a 620 nm bandpass (BP) filter and fluorescence detector 1 with a 525 nm BP filter, respectively. Statistical regions were set manually to enumerate the total viable sperm population (Yo Pro-1-negative), as shown in Figure 1 (quadrants 3 and 4 in panels A and B), and the viable subpopulation containing ROS, designated ethidium-positive (E+), as shown in quadrant 4 of panels A and B. Data in quadrants 1 and 2 represented nonviable sperm penetrated and stained by Yo Pro-1. The log of the mean ethidium fluorescence intensity/sperm of the viable sperm population was recorded from the fluorescence detector 3 output. For example, in Figure 1 sperm were incubated in the absence (panel A) or in the presence (panel B) of 1 mM XA and 0.1 U/mL XO. In the absence of XA/XO, only 1.4% of this population contained sufficient ROS to oxidize HE to ethidium (as shown in quadrant 4 of Figure 1), whereas in the presence of XA/XO, 94.4% of the viable sperm contained the oxidized form.
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(quadrants 3 and 4 in panels A and B) and the viable subpopulation containing ROS, designated DCF-positive (DCF+), shown in quadrant 4 of panels A and B. Data in quadrants 1 and 2 of Figure 2 represent nonviable sperm penetrated and stained with PI. The mean log of DCF fluorescence intensity/sperm was recorded for viable sperm from the fluorescence detector 1 output. In the absence of XA/XO, only 0.1% of this population contained sufficient ROS to convert H2DCFDA to DCF (as shown in quadrant 4 of Figure 2), whereas in the presence of XA/XO, 73.3% of the viable sperm contained the oxidized form.
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, panels A and D, for fresh and frozen-thawed sperm, respectively. The percentage of viable sperm (panels A and D) was 84.1 and 43.3%, respectively.
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, panels B, C, E, and F) was delineated by a statistical marker set just above the dead sperm Jagg fluorescence level (see panels C and F). The dot-plots (panels B and E), showing the Jagg fluorescence (vertical axis) distribution of viable sperm, were derived by gating on the PI- populations (panels A and D), respectively. The dot-plots in panels C and F, showing the Jagg fluorescence (vertical axis) distribution of nonviable sperm, were derived by gating on the PI+ populations in panels A and D, respectively. The percentage of viable sperm and the percentage of Jag+-viable sperm, and the log of the mean Jagg fluorescence detector intensity/sperm, were recorded from the fluorescence detector 2 output. The percentage of Jagg+ sperm shown in panels B, C, E, and F of Figure 3 was 99, 30, 93, and 9%, respectively.
The epifluorescence images of the 3 fluorescent labeling patterns of JC-1 and PI in 3 unfixed boar sperm are shown in Figure 4. The staining pattern for the 3 sperm from left to right are a viable sperm with a high MMP (nonred fluorescent nucleus with a Jagg+ "red-orange" fluorescent midpiece), a nonviable sperm with a low MMP (red fluorescent nucleus with no Jagg+ "red-orange" midpiece), and a viable sperm with a low MMP (nonred fluorescent nucleus with no Jagg+ "red-orange" midpiece).
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Experiment 1. XA/XO ROS-Generation Studies
Preliminary experiments were conducted to establish the concentrations of XA and XO and incubation times that increased intracellular ROS formation and decreased motility without decreasing viability (data not shown). Incubation of sperm with up to 300 U of SOD/mL had no effect (P = 0.43) on ROS formation in the presence or absence of XA/XO (data not shown).
Experiment 1 was conducted to determine the effects of XA/XO on intracellular ROS content and the effects of ROS on motion variables after a 30-min aerobic incubation in the absence or presence XA/XO and CAT. One ejaculate was collected from each of 5 boars, and each sperm sample was divided into 6 portions. Two XA/XO-free portions served as detached controls, one without CAT and the other with CAT at 300 U/mL. The remaining 4 parts received XA at a dose of 1 mM and were in a 2 x 2 factorial arrangement of XO at 0.03 and 0.1 U/mL and CAT at 0 and 300 U/mL. The first statistical model was a randomized complete block, with boar as the blocking variable and the 6 treatment groups as fixed effects that included the 2 detached controls. The second statistical model was randomized complete block, with a 2 x 2 factorial arrangement of XO and CAT without the detached controls.
Experiment 2. Effects of H2O2 on Fresh Sperm
The oxidation of HE was selected to determine ROS accumulation in subsequent experiments because in preliminary experiments both dyes were oxidized by 300 µM H2O2, and HE required a shorter incubation period than H2DCFDA for maximum fluorescence development. In addition, results from Exp. 1 showed that CAT inhibited ROS formation generated by XA/XO, measured by either ethidium or DCF fluorescence detection.
Experiment 2 was conducted to confirm that HE could be used for the measurement of H2O2, and to confirm that CAT and not SOD would attenuate H2O2-induced oxidation of HE and inhibition of motility. One ejaculate was collected from each of 6 boars, and each sperm sample was divided into 7 portions for aerobic incubation with 7 treatment combinations. Treatments 1 through 7 consisted of the following dosages for H2O2 (µM), CAT (U/mL), and SOD (U/mL), respectively: 1) 0, 0, 0; 2) 0, 0, 300; 3) 0, 300, 0; 4) 300, 0, 0; 5) 300, 0, 300; 6) 300, 300, 0; and 7) 300, 300, 300. The experimental design was a randomized complete block with the 7 treatments as fixed effects. The error variances for 2 variables, percentage E+ sperm and motility, were partitioned into 2 variance groups to compensate for heterogeneity of treatment variance; one group contained the 2 treatment combinations incubated with 300 µM H2O2 and no CAT, and the other group contained the other 5 treatment combinations.
Experiment 3. ROS in Fresh and Thawed Sperm
This experiment was conducted to determine the intracellular amounts of ROS in fresh and frozen-thawed sperm in the absence and presence of XA/XO. Fresh semen was analyzed on the day of collection, and cryopreserved semen was thawed and analyzed during the following week. Eight ejaculates were used, and each sperm sample, fresh or frozen-thawed, was divided into 3 parts and assigned to one of three 30-min incubation treatments: 25°C, no XA/XO; 38°C, no XA/XO; and 38°C, XA at 1 mM and XO at 0.1 U/mL. The experimental design was a randomized complete block with semen treatment (fresh or frozen-thawed) as the whole plot and the 3 incubation treatments as the subplots. The error variances of 2 variables, percentage of E+ sperm and ethidium fluorescence intensity, were partitioned into 2 variance groups; one group incubated in the absence of XA/XO, and the other group in the presence of XA/XO. The error variance of percentage of Jagg+ sperm was partitioned into 2 variance groups: the fresh and frozen-thawed treatments.
Experiment 4. Menadione Studies
Preliminary experiments were conducted by incubating sperm for 5 min to 4 h using doses of menadione ranging from 30 µM to 3 mM (data not shown). Incubation for 30 min with a dose of 100 µM was chosen as the greatest concentration in subsequent experiments because it did not decrease (P = 0.37) viability compared with no menadione and reduced motility to a range of 40 to 60% compared with a total inhibition of motility after a 30- to 60-min exposure to doses of >100 µM menadione.
Experiment 4 was conducted to determine the effects of menadione dose on ROS formation, MMP, and motion variables. Each semen sample was divided into 4 portions for aerobic incubation with menadione at 0, 10, 30, or 100 µM at 38°C for 30 min. The experimental design was a randomized complete block with dose of menadione as the fixed effect. Data for the variable percentage of E+ sperm were transformed to arcsine square roots to compensate for a positive correlation between the means and variances.
Statistical Analysis
Data were analyzed using the MIXED model procedure of SAS (release 8.02, SAS Inst. Inc., Cary, NC), as described for each experiment. The model diagnostics included testing for a normal distribution of error residuals and for homogeneity of treatment variance (Little et al., 1996). Comparison of least squares means was made by the LSMEANS statement using the DIFF option.
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RESULTS |
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The effects of XA/XO and CAT on sperm ethidium fluorescence intensity are shown in Table 1. Basal, spontaneous ROS formation in the absence XA/XO was low in viable boar sperm; only 3.8 and 1% of the sperm were E+ or DCF+, respectively. Compared with the detached controls (no XA/XO), XA/XO treatment increased (P < 0.01) the percentage of E+ and DCF+ sperm and the mean ethidium and DCF fluorescence intensity per sperm. The presence of 0.03 and 0.1 U/mL of XO increased (P < 0.05) the percentage E+ sperm to 90.1 and 95.5% and increased (P < 0.05) ethidium fluorescence intensity by 3- and 4.6-fold, respectively.
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). The presence of CAT during incubation with XA/XO attenuated the XA/XO-induced increases in ethidium and DCF fluorescence intensity for both XO concentrations.
The effects of XO and CAT concentration on motion variables are shown in Table 2. Motility in the presence of 0.1 U/mL XO (33%) was decreased (P < 0.05) compared with 0.03 U/mL of XO (62%) and restored when CAT was present with 0.1 U/mL of XO (72%). Compared with no CAT, the values of VCL, BCF, and LIN were increased (P < 0.01) when CAT was present with either 0.03 or 0.1 U/mL of XO. The percentage of viable sperm present after treatment, as estimated by negative PI and Yo Pro-1 staining, was 60.3 and 61.0%, respectively, and in neither case was viability affected by incubation treatment (data not shown).
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Effects of H2O2, CAT, and SOD concentrations on sperm ethidium fluorescence intensity and motility are shown in Table 3. Incubation with H2O2 for 30 min increased (P < 0.05) the percentage of E+ sperm, increased (P = 0.041) ethidium fluorescence intensity, and decreased (P < 0.01) motility. Presence of CAT during the incubation with H2O2 completely blocked the H2O2 stimulatory effect on ethidium fluorescence and the H2O2 inhibitory effect on motility. By contrast, the incubation of SOD with H2O2 had no significant effect on the development of ethidium fluorescence or the inhibitory effect on motility. Percentage of viable sperm estimated by Yo-Pro1-negative staining was 89.6% and was not affected by incubation treatment (data not shown).
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Effects of freeze-thawing and incubation with XA/XO on sperm ethidium fluorescence and Jagg formation are shown in Table 4. Incubation treatment was a source of variation for the development of ethidium fluorescence (P < 0.01), whereas storage treatment and the interaction of storage and incubation treatments were not. In the absence of XA/XO, the percentage E+ viable sperm after 30-min incubation at 25°C was very low in fresh and frozen-thawed semen (1.1 and 1.9%, respectively). Compared with 25°C, incubation at 38°C increased (P < 0.05) the percentage of E+ viable sperm in both fresh and frozen-thawed semen (3.5 and 2.8%, respectively). Ethidium fluorescence intensity followed a pattern similar to that of the percentage E+ sperm; there was no significant difference between fresh or frozen-thawed semen at either incubation temperature. Compared with no XA/XO, the presence of XA/XO increased (P < 0.05) the percentage of E+ viable sperm and ethidium fluorescence intensity for both fresh and frozen-thawed semen (E+ sperm: 94.4 and 87.9%, respectively), and the effect did not differ significantly between fresh and frozen-thawed semen.
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). The incidence of Jagg+ viable sperm was >90% over all treatment combinations; however, values for frozen-thawed semen were less (P < 0.05) than those of fresh semen. The Jagg fluorescence intensity was increased (P < 0.05) in frozen-thawed sperm after incubation at 38°C compared with incubation at 25°C; Jagg fluorescence intensity did not differ (P = 0.11) between the absence and presence of XA/XO (Table 4).
The effects of freeze-thawing and incubation with XA/XO on sperm motion variables and viability are shown in Table 5. Motility, VCL, BCF, and viability decreased (P < 0.05) in frozen-thawed compared with fresh sperm. Compared with incubation at 25°C, incubation at 38°C did not affect motility (P = 0.22) and BCF (P = 0.10); however, VCL and viability were decreased (P < 0.05). The presence of XA/XO decreased motility and BCF compared with no XA/XO at 38°C in fresh sperm and compared with no XA/XO at 25°C in frozen-thawed sperm. Incubation at 38°C decreased (P < 0.05) VCL and viability compared with incubation at 25°C for both fresh and frozen-thawed sperm, but XA/XO at 38°C had no additional effect on either variable.
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The concentration of menadione was a significant source of variation for the percent of E+ viable sperm (P < 0.01), ethidium fluorescence intensity (P < 0.01), and Jagg fluorescence intensity (P < 0.01; Table 6). The percentage of E+ viable sperm was low for 0 and 10 µM menadione, approximately 1.5%. However, increasing the menadione concentration to 30 and 100 µM increased the percentage of E+ viable sperm to 24.6 and 92.1%, respectively. Ethidium fluorescence intensity tended to increase between 10 and 30 µM menadione, but only the increase between 30 and 100 µM menadione was significant (P < 0.05). The percentage of Jagg+ viable sperm after 30-min incubation averaged 95% and did not change significantly in the absence or presence of menadione (data not shown). However, Jagg fluorescence intensity decreased in a dose related fashion from 28.3 with 0 µM menadione to 11.6 with 100 µM of menadione.
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). Motion variables did not vary among menadione concentrations ranging from 0 to 30 µM, but all were decreased at 100 µM compared with the other menadione concentrations. Incubation of sperm with increasing concentrations of menadione for 30 min at 38°C had no effect (P = 0.44) on the percentage of viable sperm with a mean for the experiment of 78% (data not shown).
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DISCUSSION |
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; Zamzami et al., 1995; Haugland, 2005), whereas others have concluded that under certain conditions HE can serve as a substrate for oxidation by either H2O2 or ·O2 (Kobzik et al., 1990; Rothe and Valet, 1990).
In agreement with the results of others using the XA/XO ROS-generating system in human (Aitken et al., 1993; Griveau et al., 1995) and stallion (Baumber et al., 2000) sperm, and in other cell types (Zamzami et al., 1995; Halliwell and Gutteridge, 1999), results from the current study provide evidence that H2O2 is the primary source of oxidative damage in boar sperm. The presence of CAT during incubation with XA/XO or H2O2 decreased intracellular fluorescence of both DCF and ethidium, whereas SOD had no effect, indicating that both probes were measuring intracellular H2O2. Even in the absence of SOD, exposure of sperm mitochondria or contractile elements to ·O2 produced by XA/XO was probably limited because ·O2 is considered to be too short-lived and too polar to penetrate intact plasma membranes (Aitken, 1995; Halliwell and Gutteridge, 1999). In addition, H2O2 might have been the major source of oxidative damage in menadione-treated boar sperm because of the high levels of SOD activity present in boar sperm mitochondria and cytoplasm (Mennella and Jones, 1980).
Another important finding derived from the current study was that, in the absence of a ROS-generating system, intracellular ROS formation or accumulation was low in viable boar sperm of fresh and frozen-thawed semen, averaging less than 4%. In addition, the incidence of spontaneous ROS formation did not differ between fresh or frozen-thawed sperm. In the presence of XA/XO, viable sperm from fresh and cryopreserved semen did not differ with respect to their ability to oxidize HE. However, whereas the incidence of ROS formation in the absence of a ROS generator was low in boar sperm after a 30-min incubation of fresh or frozen-thawed semen, it is possible that incubation at 38°C for longer periods of time would increase ROS accumulation in frozen-thawed relative to fresh sperm.
Flow cytometric assays should be useful in identifying populations of sperm that may be dysfunctional because of intracellular ROS formation. Flow cytometric assays have an advantage over chemiluminescence methods for ROS measurement because intracellular measurements can be made exclusively in the viable portion of the sperm population, and chemiluminescence methods lack specificity (Fridovich, 1997; 2003). Specificity is reduced using lucigenin, luminol, and tetrazolium because they must be univalently reduced to the corresponding monocation before reacting with ·O2 and producing a luminescent signal. The lucigenin monocation is capable of autoxidation and produces ·O2 even in cases where no ·O2 was being produced in the absence of lucigenin. Luminal and tetrazoinyl radicals can also spontaneously reduce O2 to ·O2 in the presence of a variety of oxidants, and in all cases SOD has an inhibitory effect.
Membrane lipid peroxidation is one outcome of ROS formation in sperm (Aitken, 1995). Others have shown that freshly collected boar sperm are susceptible to FeSO4-ascorbate catalyzed lipid peroxidation as measured by malondialdehyde formation (Roca et al., 2004) and become more susceptible during hypothermic liquid storage (Cerolini et al., 2000) and with increased time after thawing (Breininger et al., 2005). Freeze-thawing increased boar sperm lipid peroxidation compared with fresh semen, but this effect was partially attenuated by the presence of butylated hydroxytoluene (Roca et al., 2004). However, the chemiluminescence methods for ROS measurement or the malondialdehyde method for lipid peroxidation measurement do not distinguish between viable and nonviable sperm. Thus, the formation of ROS and subsequent lipid peroxidation might have occurred only in a small portion of the population or primarily in nonviable sperm.
The effects of storage-associated or spontaneous accumulation of ROS in boar sperm is difficult to evaluate because in many studies lipid peroxidation was not detectable without the use of a catalyst such as Fe2+/ascorbate or a peroxidized fatty acid, or cold shock treatment (Mennella and Jones, 1980; Breininger et al., 2005). Another factor that could contribute to the low level of ROS formation in the current study is the composition of the cooling and freezing extenders. Dialyzed egg yolk was found to offer almost complete protection from Fe2+ catalyzed lipid peroxidation in ram sperm (Jones and Mann, 1977) and an egg yolk Tris-glycerol freezing extender scavenged ROS produced by XA/XO incubated with bull sperm (Chatterjee and Gagnon, 2001). An important constituent of BTS, EDTA (present at a concentration of 3.35 mM), was found to be inhibitory of Fe2+ catalyzed lipid peroxidation at 0.3 µM in ram (Jones and Mann, 1976) and at 0.28 µM in boar sperm (Comaschi et al., 1989).
Fluorescence-activated flow cytometric assays of Jagg fluorescence have been used to identify sperm with high MMP in a number of experiments with sperm (Garner et al., 1997; Gravance et al., 2001; Love et al., 2003). Results of the current study are novel because Jagg fluorescence was measured in the viable portion of the sperm population. Viability or changes in viability may not be an issue in short-term studies of cultured or dispersed cells. However, the ability to distinguish between viable and nonviable sperm becomes particularly important for studies in which sperm are subject to long-term hypothermic storage or cryopreservation. In a study investigating mitochondrial function in frozen-thawed bovine sperm, the percentage of viable sperm estimated by SYBR-14 staining was positively correlated with the percentage of JC-1 fluorescent sperm in the total population, r = 0.99 (Garner et al., 1997). Similarly in the current study, the incidence of Jagg fluorescence was much less than in nonviable than in viable frozen-thawed sperm. Thus, the actual effect of an experimental treatment or cryopreservation on MMP in a sperm population could be greatly overestimated by the presence of dead cells.
Menadione-induced disruption of mitochondrial electron transport at complex I to produce ·O2 was used to examine the relationship between MPP and sperm motility. Hydrogen peroxide is likely a major source of oxidative damage in menadione-treated boar sperm because of the high levels of SOD activity in boar sperm mitochondria and cytoplasm (Mennella and Jones, 1980). Analysis of motion variables in Exp. 4 showed that 100 µM menadione increased ROS formation to include >90% of treated sperm and decreased motility and measures of sperm velocity compared with no menadione. Maintenance of high MMP (a high proton accumulation in the mitochondrial intramembrane space) is required to drive Fo/F1 ATPase catalyzed ATP production in somatic cells (Halliwell and Gutteridge, 1999). Others have shown that ATP content of sperm is decreased after XA/XO-induced ROS formation (de Lamirande and Gagnon, 1992b; Armstrong et al., 1999). Perhaps decreased MMP reduced ATP production to the point at which the amount of ATP produced is not sufficient to maintain motility. However, ROS damage of the sperm contractile apparatus (de Lamirande and Gagnon, 1992a) is also possible.
In conclusion, basal or spontaneous intracellular ROS formation was low in viable fresh and frozen-thawed boar sperm, and both fresh and thawed sperm were quite susceptible to external sources of hydrogen peroxide. In this study, it was expected that spontaneous ROS formation might increase and trigger a decrease in MMP, followed by decreased ATP production and motility. In the current study, the incidence of basal or spontaneous ROS formation in viable sperm in fresh and frozen-thawed semen was very low and did not vary greatly among boars. For this reason, the measurement of spontaneous ROS formation in fresh or frozen-thawed semen would not likely be predictive of semen quality or fertility independent of motility or viability. However, studies are being designed to investigate long-term hypothermic liquid storage of boar semen, and this is where an association between spontaneous or induced ROS formation and sperm quality and possibly fertility might be found.
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Footnotes |
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2 Corresponding author: dave{at}anri.barc.usda.gov
Received for publication February 28, 2005. Accepted for publication March 12, 2006.
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  R.J. Aitken and G.N. De Iuliis On the possible origins of DNA damage in human spermatozoa Mol. Hum. Reprod., January 1, 2010; 16(1): 3 - 13. [Abstract] [Full Text] [PDF]
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 B. J. Awda, M. Mackenzie-Bell, and M. M. Buhr Reactive Oxygen Species and Boar Sperm Function Biol Reprod, September 1, 2009; 81(3): 553 - 561. [Abstract] [Full Text] [PDF]
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 P. Cheng, C. A. Corzo, N. Luetteke, B. Yu, S. Nagaraj, M. M. Bui, M. Ortiz, W. Nacken, C. Sorg, T. Vogl, et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein J. Exp. Med., September 29, 2008; 205(10): 2235 - 2249. [Abstract] [Full Text] [PDF]
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 H. D. Guthrie and G. R. Welch Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid J Anim Sci, June 1, 2007; 85(6): 1402 - 1411. [Abstract] [Full Text] [PDF]
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