Sublethal Xanthine Oxidase Stress Prefreezing of Bull Sperm Improves the Post-Thaw Functionality and Fertility Potential Parameters

Abstract

Oxidative stress during cryopreservation causes mechanical, biochemical, and structural damage to the sperm, leading to lower viability and fertility potential. In recent years, a novel method based on the use of mild stress for preconditioning of sperm before cryopreservation has been applied to improve the quality of thawed sperm, although its molecular mechanism remains unknown. In this study, we investigated the protective effects of sublethal oxidative stress by xanthine oxidase (XO) on thawed bull sperm performance through modulations of mitochondrial uncoupling protein 2 (UCP2) expression. Semen samples were collected from six bulls, then mixed and divided into four aliquots: frozen control (XO-0) and frozen groups treated with different concentrations of XO, 0.01 μM (XO-0.01), 0.1 μM (XO-0.1), and 1 μM (XO-1). Thawed sperm were evaluated for motion parameters, viability, acrosome integrity, mitochondria activity, membrane integrity, and UCP2 expression. A significant increase of total motility and viability rate was observed in XO-0.1 compared with other frozen groups (p < 0.05). The highest percentage of progressive motility was in XO-0.01 and XO-0.1 compared with other groups (p < 0.05). Moreover, a significantly higher level of sperm mitochondrial membrane potential and membrane integrity was observed in XO-0.1 (p < 0.05). We also found the lowest percentage of sperm mitochondria activity in XO-1 (p < 0.05). In addition, the highest expression of UCP2 was observed in XO-1 (p < 0.05). Our findings suggest that stress preconditioning of bull sperm before cryopreservation can improve thawed sperm functions, which might be mediated through an increase of UCP2 expression.

Introduction

Semen cryopreservation has long been seen as a beneficial approach for domestic animals reproductive biotechnology. It also is recognized as a contributing factor of jeopardized species maintenance.^1–3 Frozen bull semen plays an important role in artificial insemination for both commercial aims and genetic resources preservation.⁴ The cryopreservation process has damaging effects on the sperm, which leads to reducing its cryosurvival.⁵ Cryodamage is mostly related to ice crystal formation, production of reactive oxygen species (ROS), injuries to the mitochondria, and plasma membrane integrity.

Therefore, an improved technique is required to avoid these side effects.⁶ In recent years, several studies have reported that preconditioning of sperm with sublethal oxidative stress before cryopreservation could improve the quality of thawed sperm.^7–9 Recently, preconditioning of bull sperm with xanthine oxidase (XO) improved the sperm cryosurvival rate,¹⁰ although the mechanisms underlying the increased cryotolerance are unknown. It has been reported that increased expression of uncoupling protein 2 (UCP2) in the preconditioned sperm may be responsible for improved cryosurvival in sperm.⁹

Mitochondrial UCPs are located in the mitochondrial inner membrane and activated by environmental stimuli including exposure to cold and chemical compounds such as 2,4-dinitrophenol (DNP) and 4-hydroxynonenal (HNE).^11,12 UCPs have an important role in cellular hemostasis and preventing mitochondrial ROS production by proton gradient manipulation and regulating adenosine triphosphate (ATP) production.^13,14 The higher levels of ROS increase UCP2 activity in mitochondria.¹² It has been reported that inducing UCP2 by DNP decreases cryoinjury in the yellow catfish sperm.¹¹ The aim of this study was to investigate the preconditioning effects of bull sperm before cryopreservation with XO, through modulation of activated UCP2 protein.

Materials and Methods

Chemicals and ethics considerations

All chemicals used in the study were obtained from Sigma (St. Louis, MO). XO (Sigma, CN. 1.17.3.2.) was used for induction of sublethal oxidative stress. This study was approved by the ethics committee of the Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran (IR.ACECR.ROYAN.REC.1398.13).

Animal and semen collection

Semen samples of six Holstein bulls were collected using an artificial vagina, two times a week during 1 month (six replicates). There were six replicates for each bull in this study. A total of 36 samples were collected. Each ejaculate was evaluated for volume, color, concentration, morphology, and percentage of motility after collection. Selected samples had the following standards: normal morphology of >85%, concentration of >1 × 10⁹ sperm/mL, and progressive motility of >60%. To eliminate individual variability, samples were pooled together and then divided into four equal aliquots.

Freezing and thawing process

The basic extender used was composed of 27.1 g/L Tris, 14 g/L citric acid, and 10 g/L fructose. The osmolarity and pH were set at 320–330 mOsm/kg H₂O and 7.2, respectively. For preparing the freezing extender, 4% glycerol and 20% centrifuged egg yolks were added to the basic extender. Semen samples were divided into the following four aliquots: frozen control (without treatment, XO-0) and frozen groups exposed to different concentrations of XO, 0.01 (XO-0.01), 0.1 (XO-0.1), and 1 μM (XO-1) before cryopreservation.

Diluted semen samples were aspirated into 0.25 mL straws (IMVTechnologies, L'Aigle Cedex, France), and then equilibrated at 5°C for 2 hours. The straws were frozen in liquid nitrogen vapor, 5 cm above the liquid nitrogen, for 12 minutes, and then immersed directly into the liquid nitrogen for storage. After at least 1 week, the frozen straws were thawed at 37˚C for 30 seconds in a water bath and then sperm functional parameters and the expression level of UCP2 of frozen–thawed sperm were evaluated.

Semen evaluation after thawing

Motility and velocity parameters

Sperm motility and velocity parameters were measured by computer-aided sperm analysis (version 5.1; Microptic, Barcelona, Spain). A diluted semen sample (5 μL) was placed on a prewarmed chamber (Leja Products Nieuw-Vennep, Netherlands). The chambers were placed on microscope plates (37°C) and analyzed for the following motility parameters: total motility (%), progressive motility (%), average path velocity (VAP, μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross frequency (Hz), straightness (%), and linearity (%).¹⁵

Viability

The viability of samples was assessed by the eosin–nigrosin stain method. Twenty microliters of sperm suspension was added to 20 μL of the working solution (1% eosin [w/v] and nigrosin [10% w/v]) and incubated at 37°C for 10 minutes. Then, a prepared smear of the stained sperm was dried at room temperature. Lastly, the percentage of live sperm, that is, unstained sperm, was assessed by counting 200 cells under a light microscope (CKX41; Olympus, Tokyo, Japan) at 400 × magnification.¹⁶

Plasma membrane functionality

The plasma membrane functionality was analyzed using the hypo-osmotic swelling test. The hypo-osmotic solution (100 mOsm/kg) consisted of 9 g/L fructose and 4.9 g/L sodium citrate in distilled water. In brief, 5 μL of each sample with 50 μL of HOS solution was pooled and incubated at 37°C for 20 minutes. Then, 200 sperm cells with swollen and nonswollen tails were recorded under a light microscope (CKX41; Olympus, Tokyo, Japan) at 400 × magnification.¹⁷

Mitochondrial activity

The percentage of sperm with active mitochondria was determined by the lipophilic cation JC-1 (T4069; Sigma-Aldrich). In total, 1 μL of JC-1 was added to 300 μL of the sperm suspension (1–2 × 10⁶ sperm/mL). The mixture was incubated at 37°C for 15 minutes in the dark. Then, the sperm suspension was washed and analyzed by a flow cytometer. The sperm containing high mitochondrial activity was identified by a positive signal for JC-1 (red/orange fluorescence).¹⁷

Acrosome integrity

Acrosome integrity was assayed by the pisum sativum agglutinin (PSA) test (FITC-PSA; L0770; Sigma-Aldrich). In brief, 5 μL of each sample was fixed with ethanol (96% purity) at 4°C for 20 minutes. Then, 10 μL of the sperm suspension was mixed with 30 μL of PSA on a glass slide. At least 200 sperm were counted by a fluorescent microscope (BX51, Olympus, Tokyo, Japan; excitation: 455–500 nm and emission: 560–570 nm) at 400 × magnification. Sperm with a green fluorescent head was considered as an “intact acrosome.”¹⁶

Western blotting

The sperm total protein was extracted using lysis buffer (7 M urea, 2 M thiourea, 4% 3-cholamidopropyl dimethylammonio 1-propanesulfonate [w/v], 75 mM dithiothreitol, 1% ampholyte [w/v], and 40 mM Tris-HCl). Then, the protein concentration was assessed using the Bradford procedure.¹⁸ A maximum of 40 μg of protein was analyzed by 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene fluoride membrane (Millipore). Then, blots were blocked with 5% BSA (Biorad, blotting-grade blocker) in Tris-buffered saline containing 0.1% tween 20 (TBST) (Biorad, 10 × buffered saline) for 1 hour at room temperature.

The membranes were immunoblotted with primary antibody (anti-UCP2([(G-6): (sc-390189)] diluted in 5% bovine serum albumin in TBS-Tween (1:2500 dilution) and were stored overnight at 4°C. Membranes then were incubated with a secondary antibody (antimouse, 1:500; Beyotime, China) for 2 hours at room temperature. Western blot images were quantified with Image J software (NIH, Bethesda, MD), and anti-β-actin (1:200, cat. no. A2228; Sigma-Aldrich), which was used as a housekeeping protein.⁹

Statistical analysis

All data were checked for normal distribution by the Shapiro–Wilk test and analyzed using Proc GLM of SAS 9.1 (SAS Institute, version 9.1, 2002, Cary, NC). The mean of the treatments was compared using Tukey's test and the level of significance was p < 0.05. The results are expressed as mean ± standard error of the mean (SEM).

Results

Effects of XO on the sperm motility and velocity parameters

Table 1 gives the total motility in XO-0.1 (74.1% ± 1.85%) was significantly higher in comparison with other groups (p < 0.05). The percentage of progressive motility was significantly increased in the XO-0.01 and XO-0.1 groups (39.8% ± 2.25% and 36.6% ± 1.25%, respectively). VAP was observed to be significantly higher in the XO-0.01 and XO-0.1 groups (26.6 ± 1.18 μm/s and 24.5 ± 1.18 μm/s, respectively) compared with other groups.

Table 1.

The Effects of Different Concentrations of the Xanthine Oxidase on Bull Sperm Motility Parameters After Freezing–Thawing

Groups	TM (%)	PM (%)	VAP (μm/s)	VSL (μm/s)	VCL (μm/s)	LIN (%)	BCF (Hz)	ALH (m)	STR (%)
XO-0	64.9 ± 1.85^b	26.9 ± 1.25^b	19 ± 1.18^b	17.6 ± 2.02^a	32.5 ± 1.72	37.2 ± 1.97	19.45 ± 1.67	1.62 ± 0.08	74.6 ± 6.2
XO-0.01	62.8 ± 1.85^b	36.6 ± 1.25^a	26.6 ± 1.18^a	15.8 ± 2.02^a	30.2 ± 1.72	34.6 ± 1.97	20.75 ± 2.66	1.72 ± 0.07	76.7 ± 5.4
XO-0.1	74.1 ± 1.85^a	39.8 ± 1.25^a	24.5 ± 1.18^a	18.9 ± 2.02^a	30.9 ± 1.72	36.2 ± 1.97	19.59 ± 2.49	1.75 ± 0.12	73.9 ± 6.8
XO-1	56.3 ± 1.85^c	23.9 ± 1.25^b	10.6 ± 1.18^c	9.26 ± 2.02^b	29.4 ± 2.02	35.06 ± 1.97	20.06 ± 1.53	1.64 ± 0.09	75.2 ± 3.3

Data are expressed as mean ± SEM. Different letters within the same column show significant differences among the groups at p < 0.05.

ALH, amplitude of lateral head displacement; BCF, beat cross frequency; LIN, linearity; PM, progressive motility; SEM, standard error of the mean; STR, amplitude of straightness; TM, total motility; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight linear velocity; XO, xanthine oxidase.

Viability and plasma membrane functionality

Viability was significantly higher in the XO-0.1 group (56% ± 1.4%) compared with other groups (p < 0.05). No significant difference was seen in the sperm viability between the XO-0, XO-0.01, and XO-1 groups. XO-0.01 and XO-0.1 groups showed a significant increase in percentage of membrane functionality (59.7 ± 2.83 and 57.3 ± 2.83, respectively) compared with other groups (p < 0.05) (Table 2).

Table 2.

The Effect of Different Concentrations of the Xanthine Oxidase on Mitochondrial Activity, Acrosome Integrity, Viability, and Membrane Functionality of Post-Thawed Bull Sperm

Groups	Mitochondrial activity (%)	Acrosome integrity (%)	Viability (%)	Membrane functionality (%)
XO-0	47.1 ± 1.3^b	94.36 ± 2.6	50.2 ± 1.4^b	52.4 ± 2.83^b
XO-0.01	48.3 ± 1.3^b	90.25 ± 2.6	49.8 ± 1.4^b	59.7 ± 2.83^a
XO-0.1	57.1 ± 1.3^a	89.85 ± 2.6	56 ± 1.4^a	58.3 ± 2.83^a
XO-1	39.1 ± 1.3^c	90.26 ± 2.6	51.3 ± 1.4^b	54.56 ± 2.83^b

Different letters within the same column show significant differences among the groups (p < 0.05). Data are expressed as mean ± SEM.

Mitochondrial activity and acrosome integrity

The percentage of sperm mitochondrial activity was significantly increased in the XO-0.1 group (57.1 ± 1.30) compared with the control group (47.1 ± 1.3) (p < 0.05). Furthermore, the acrosome integrity of sperm was not significantly affected by the different concentrations of XO (p > 0.05) (Table 2).

Assessment of mitochondrial UCP2 expression

The highest expression of UCP2 protein was observed in the XO-1 group (3.5 ± 0.59) compared with the other groups (p < 0.05). Also, a significant increased expression of UCP2 was observed in the XO-0.01 and XO-0.1 groups (1.23 ± 0.29 and 1.35 ± 0.59, respectively) compared with the control group (0.63 ± 0.29) (Fig. 1).

FIG. 1.

(A) Role of mitochondrial UCP2 in the preconditioned sperm. Mitochondria produce ATP during OXPHOS. These organelles have large protein complexes that are necessary for ATP synthesis. Cofactors such as NADH+ and FADH2 give away electrons to a series of protein complexes locked in the inner mitochondrial membrane as the mitochondrial ETC. The respiratory chain uses energy released from electron transfer through complexes to transfer protons from the membrane to the intermembrane space. The energy from the proton leak is used to produce ATP. Some of the energy of the electrochemical gradient is not coupled to ATP production due to a proton leak, which consists of the return of protons to the mitochondrial matrix through alternative pathways that bypass ATP production. Several factors including ROS production and or DNP and HNE could cause proton leak and activate a family of mitochondrial proteins such as UCP2. Sperm membrane has high levels of PUFAs and are sensitive to oxidative stress. Therefore, these proteins minimize superoxide formation from O₂− and H₂O₂. (B) Western blot analysis. Bands for UCP2 were detected at 35 kDa (upper film). β-actin was used as the loading control (42 kDa) (lower film). (C) Relative expression (fold change) of the differential UCP2 expression by Western blot analysis between groups. Protein bands were quantified by using ImageJ software. Results are expressed as mean ± SEM. ATP, adenosine triphosphate; DNP, 2,4-dinitrophenol; ETC, electron transport chain; FADH2, flavin adenine dinucleotide; HNE, 4-hydroxynonenal; NADH, nicotinamide adenine dinucleotide; PUFA, polyunsaturated fatty acids; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; SEM, standard error of the mean; UCP2, uncoupling protein 2.

Discussion

Bull semen cryopreservation and artificial insemination are important factors for the management of cattle production.¹⁹ The sperm cryopreservation process is associated with physiological damage such as ice crystallization, and thermal and osmotic stresses that can decrease the fertility rate.²⁰ Recently, an induction of sublethal stress prefreezing has been introduced as a new strategy to increase sperm cryotolerance in bull,¹⁰ rooster,²¹ and human.²² Sharafi et al. reported that preconditioning with XO could improve thawed bull sperm quality, although molecular mechanisms underlying this approach have not been determined.¹⁰

It is known that sperm proteins have a key role in sperm homeostasis.^23,24 It has been reported that induced sublethal stress before cryopreservation could increase the expression of some specific proteins that are important in increasing sperm cryotolerance.^25,26 Accordingly, we hypothesized that preconditioning of bull sperm with XO may improve cryosurvival through activation of some mitochondrial proteins such as UCP2 in the thawed sperm. Our findings showed an XO dose-dependent increase in the UCP2 expression after exposure. However, total motility, membrane integrity, viability, and mitochondria activity significantly increased in the XO-0.1 treatment compared with the frozen control group.

Our results confirmed that increasing and decreasing of UCP2 above physiological levels have a negative effect on the bull sperm quality. It seems that a particular physiological level of UCP2 is essential to accomplish optimal sperm function. Our results were in line with Sharafi et al., who reported that motility parameters were increased in preconditioned bull sperm by XO.¹⁰ However, the mechanisms of XO effects are not completely well known. It may be a result of improved mitochondrial activity and increased production of ATP in preconditioned sperm. Moreover, it has been reported that XO has nitrite reductase activity and can produce nitric oxide from nitrite.²⁷

Nitric oxide activates GMP/protein kinase G signaling and increases sperm motility.²⁸ Therefore, XO may improve sperm motility through the nitric oxide production. Previous studies showed enhanced viability in preconditioned sperm by oxidative agents such as NO,^7,8 H₂O₂,²⁹ and XO¹⁰ after freezing–thawing. We also observed that XO-0.1 significantly increased the viability of thawed sperm. It seems that upregulation of UCP2 may decrease ROS overproduction and cell death in the preconditioned sperm. During freezing–thawing, decreased mitochondrial membrane potential leads to the activation of apoptotic pathways in the sperm.³⁰ We observed that the mitochondrial activity percentage was increased in the XO-0.1 group.

Other studies have shown that mild sublethal stresses are able to increase the mitochondrial activity in the sperm of bull,^9,10 rooster,²¹ and human.²² Sperm mitochondria plays an important role in the ATP production. It seems that activation of UCP2 in the mitochondrial inner membrane could enhance mitochondrial activity by preventing dissociation of the proton gradient, whereas reduced expression of UCP2 may cause ROS accumulation and mitochondrial dysfunction.³¹ The sperm membrane has high levels of unsaturated fatty acids that are sensitive to oxidative stress. Previous studies demonstrated that mild stresses reduce the level of lipid peroxidation in the thawed sperm.^21,32

In this study, induction of sperm with mild sublethal stress by XO increased bull sperm membrane integrity. As mentioned by Wang et al., upregulation of UCP2 reduces malondialdehyde level in the zebrafish sperm exposed to cold stress.³ Therefore, it is suggested that enhanced expression of UCP2 in the preconditioned sperm can reduce membrane fatty acid oxidation, which leads to sperm plasma membrane stability.³³ Moreover, our results showed that preconditioning of bull sperm with XO had no effect on the acrosome integrity.

This finding was in agreement with a previous study, which reported that induced mild sublethal stress did not affect sperm acrosome integrity after thawing.⁷ Furthermore, our findings showed that membrane functionality, motility, and viability were the lowest in the XO-1 group. This finding was similar to Sharafi et al., who reported that increased oxidative stress at higher concentrations of the XO reduces sperm quality.¹⁰ Therefore, treatment with 0.1 μM XO before cryopreservation can improve sperm cryosurvival, which may be mediated through UCP2 protein activation.

Conclusion

Our results indicated that mild sublethal oxidative stress induced by 0.1 μM XO could increase cryotolerance in thawed bull sperm. However, the highest expression of UCP2 was observed in sperm exposed to 1 μM XO. It seems that an adequate abundance of USP2 protein is induced by 0.1 μM XO, which could protect sperm from cryoinjury. However, the intermediate signaling involved in sperm cryobiology by UCP2 should be further assessed.

Footnotes

Acknowledgments

The authors express their appreciation to Mrs. Sara Porazadi, Mrs. Masoumeh Azimi, and Mrs. Elham Yektadost without whom this study would have been impossible.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

No funding was received for this article.

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