Polyamines protect boar sperm from oxidative stress in vitro

Abstract

Sperm are susceptible to excessive reactive oxygen species (ROS). Spermine and spermidine are secreted in large amounts by the prostate and potent natural free radical scavengers and protect cells against redox disorder. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. Seven mature and fertile Duroc boars (aged 15 to 30 mo) were used in this study. In experiment 1, spermine and spermidine (3.6 ± 0.3 and 3.3 ± 0.2 mmol/L, respectively) were abundant in seminal plasma, and the content of polyamine decreased (P < 0.05) after preservation at 17 °C for 7 d or incubation at 37 °C for 6 h. In experiment 2, using labeling of spermine or spermidine by conjugation with fluorescein isothiocyanate and ultra-high-performance liquid chromatography, we found that the accumulation of spermine or spermidine in sperm was inhibited by quinidine and dl-tetrahydropalmatine (THP, organic cation transporters [OCT] inhibitors, P < 0.05), but not mildronate and l-carnitine (organic cation/carnitine transporter [OCTN] inhibitors, P > 0.05). In experiment 3, the addition of spermine or spermidine (0.5 mmol/L) in the extender resulted in higher motility, plasma membrane and acrosome integrity, and lower ROS level after preservation in vitro at 17 °C for 7 d (P < 0.05). In experiment 4, in the condition of oxidative stress (treatment with H₂O₂ at 37 °C for 2 h), the addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) in extender increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase; reduced glutathione and oxidized glutathione ratio (P < 0.05); and alleviate oxidative stress-induced lipid peroxidation, DNA damage, mitochondrial membrane potential (ΔΨ_m) decline, adenosine triphosphate depletion, and intracellular calcium concentration ([Ca²⁺]_i) overload (P < 0.05), thereby improving boar sperm motility, the integrity of plasma membrane and acrosome (P < 0.05) in vitro. These data suggest that spermine and spermidine alleviate oxidative stress via the antioxidant capacity, thereby improving the efficacy of boar semen preservation.

Lay Summary

Boar semen preservation and artificial insemination are widely used in the pig industry. Although preservation in vitro prolongs sperm lifespan, reactive oxidative species (ROS) also accumulate in sperm with the increased preservation period. ROS over-accumulation would impair motility, the integrity of plasma membrane and acrosome, mitochondrial function, and eventually lead to infertility. Spermine and spermidine are secreted in large amounts by the prostate and are potent natural free radical scavengers. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. We found for the first time that organic cation transporters mediated polyamines uptake in sperm cells, and that extracellular polyamines decreased during preservation in vitro. The addition of polyamines increased the activities of glutathione-related antioxidant enzymes and reduced glutathione and oxidized glutathione ratio, and alleviate oxidative stress-induced mitochondrial dysfunction, lipid peroxidation, and DNA damage, thereby maintaining sperm quality in vitro. These data suggest that spermine and spermidine alleviate oxidative stress, thereby improving the efficacy of boar semen preservation.

Spermine and spermidine that are secreted in large amounts by the prostate alleviate oxidative stress, thereby improving the efficacy of boar semen preservation.

Introduction

Boar semen preservation and artificial insemination (AI) are widely used in the pig industry. Due to the biological characteristics of boar sperm, semen was generally diluted with the extender and appropriately preserved at 16~18 °C in vitro. Although preservation in vitro prolongs sperm lifespan, reactive oxidative species (ROS) also accumulate in sperm with the increased preservation period (Ren et al., 2020; Sun et al., 2020). Boar seminal plasma has a relatively finite antioxidant capacity, which is further diminished after dilution (Sun et al., 2020) and will not be enough to restore redox homeostasis in sperm during the preservation (Tian et al., 2019).

Oxidative stress caused by redox unbalance surpassing a physiological range could undermine the structural and functional integrity of sperm (Aitken and Drevet, 2020). ROS over-accumulation in sperm produces oxidation of lipids, proteins, and DNA that lead to lipid peroxidation (LPO), oxidation of essential structural proteins and enzymes, and mutations of 
DNA, which would impair sperm motility, the integrity 
of plasma membrane and acrosome, mitochondrial function, and eventually lead to infertility (McPherson and Lane, 2015; McPherson et al., 2019; Nikitaras et al., 2021). Boar sperm are highly susceptible to oxidative stress due to the high polyunsaturated fatty acids (PUFAs) content in the plasma membrane (Awda et al., 2009). As a result, redox homeostasis management that prevents ROS over-accumulation and oxidative damage may help maintain the integrity of sperm structure and function (Scarlata and O’Flaherty, 2020).

Polyamines, including spermine and spermidine, are secreted in large amounts by the prostate (Lefevre et al., 2011). They are aliphatic amines with three or four methylene carbon chains connecting the amino or imino groups (Murray et al., 2018). A positive correlation between polyamine concentrations in seminal plasma and sperm quality has been demonstrated in human, dog, ram, and bull (Vanella et al., 1978; Shohat et al., 1990; Melendrez et al., 1992; Setyawan et al., 2016; Singh et al., 2017; Saraf et al., 2020). Spermine and spermidine are potent natural free radical scavengers in the nucleus and mitochondria (Ha et al., 1998). It has been reported that spermine prevents the generation of destructive hydroxyl radicals in human granulocytes (Lovaas and Carlin, 1991). Spermine and spermidine protect mouse fibroblast against oxidative damage (Rider et al., 2007). Furthermore, spermine reduces ROS levels in frozen–thawed canine sperm (Setyawan et al., 2016). However, it is unclear how polyamines are transported into sperm and the role of polyamines.

Therefore, we used boar sperm as a model to study the polyamines uptake in sperm and elucidate whether polyamines maintain sperm functionality in vitro and the underlying mechanism.

Material and Methods

All experimental procedures involving the care and use of animals were approved by the Northwest A&F University Institutional Animal Care and Use Committee (ethical approval code: H17-09).

Experimental design

Experiment 1 was devised to investigate the content of spermine and spermidine in fresh boar seminal plasma and the changes in the polyamines content during preservation in vitro. Semen was diluted by Modena and preserved at 17 °C for 7 d or incubated at 37 °C for 6 h. The immediately extended raw semen samples (0 d or 0 h), preserved samples (7 d), and incubated samples (6 h) from seven ejaculates, with each from different boars, were centrifuged to separate the supernatant containing seminal plasma and Modena from the sperm. The supernatant was then stored at −80 °C until further polyamines content analysis via ultra-high-performance liquid chromatography (UHPLC).

Experiment 2 was designed to investigate whether organic cation transporters (OCT) or organic cation/carnitine transporters (OCTN) mediate the polyamines uptake in boar sperm. To this end, the OCT or OCTN inhibitors, including quinidine (inhibitor of OCT1/2/3), dl-tetrahydropalmatine (THP, inhibitor of OCT1/3), l-carnitine (inhibitor of OCTN1), and mildronate (inhibitor of OCTN2), were used in the present study. First, in order to find the optimal concentration and assure sperm viability and membrane stability, the semen samples from four ejaculates, with each from different boars, were diluted by Modena in the presence or absence of different concentrations (0, 30, 60, 90, and 120 μmol/L) of the OCT or OCTN inhibitors and incubated at 37 °C for 6 h, and sperm motility and plasma membrane integrity were evaluated after the incubation. Next, we investigated the effect of specific concentration inhibitors on polyamines uptake in sperm. The raw semen from these six ejaculates were diluted with the Modena with 60 μmol/L quinidine, 60 μmol/L THP, 60 μmol/L l-carnitine, or 60 μmol/L mildronate. After incubation at 37 °C for 6 h, semen samples were centrifuged to remove the supernatant. The sperm (precipitation) were then stored at −80 °C until further intracellular polyamines content analysis via UHPLC. Moreover, the effect of specific concentration inhibitors on the accumulation of fluorescein isothiocyanate-labeled spermine (SM-FITC) or spermidine (SD-FITC) in boar sperm was evaluated. The fresh semen samples from three ejaculates, with each from different boars, were centrifuged to remove seminal plasma, and the sperm samples were resuspended with Modena containing SM-FITC or SD-FITC. Before the incubation, 60 μmol/L quinidine, 60 μmol/L THP, 60 μmol/L l-carnitine, or 60 μmol/L mildronate were added to the Modena. After incubation at 37 °C for 6 h, the accumulation of SM-FITC or SD-FITC in boar sperm was observed by epifluorescence microscope.

Experiment 3 sought to investigate whether exogenous spermine or spermidine could maintain boar sperm functionality during preservation at 17 °C in vitro. The diluent used for exogenous spermine and spermidine stock dilution is double distilled water. Sperm samples from five ejaculates, with each from different boars, were preserved in Modena extender with different concentrations (0, 0.25, 0.5, 1, and 2 mmol/L) of spermine or spermidine for 1 or 7 d. Sperm motility, plasma membrane integrity, acrosome integrity, and cellular ROS level were detected after the preservation.

Experiment 4 cast about investigating whether spermine or spermidine also protected boar sperm at 37 °C and exerted their role through enhancing antioxidant properties. Sperm samples from five ejaculates, with each from different boars, were preserved in Modena extender at 37 °C for 2 h with 1 mmol/L spermine or 0.5 mmol/L spermidine (pretreatment for 30 min) in the presence or absence of 0.1 mmol/L H₂O₂. Sperm motility, membrane integrity, mitochondrial membrane potential (ΔΨ_m), cellular adenosine triphosphate (ATP) levels, intracellular Ca²⁺ concentration ([Ca²⁺]_i), ROS levels, LPO levels (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene [BODIPY] 581/591C11 and 4-hydroxylnonenal [4-HNE] assay), DNA oxidative damage (8-hydroxy-2ʹ-deoxyguanosine [8-OHdG] assay), and antioxidant enzyme activities including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), γ-glutamate cysteine ligase (GCL), peroxidase (POD), superoxide dismutase (SOD), NAD(P)H: quinone oxidoreductase 1 (NQO1), and reduced glutathione and oxidized glutathione (GSH/GSSG) ratio were evaluated after the preservation.

Reagents and media

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (USA). quinidine, THP, l-carnitine, and mildronate were obtained from MedChemExpress (China). Modena solution was used as the basic medium, which contained 152.8 mmol/L d-glucose, 26.7 mmol/L trisodium citrate, 11.9 mmol/L sodium hydrogen carbonate, 15.1 mmol/L citric acid, 6.3 mmol/L ethylenediaminetetraacetic acid disodium (EDTA-2Na), and 46.6 mmol/L tris (hydroxymethyl) aminomethane (Tris) (pH = 7.2). The Modena solution was supplemented with penicillin G sodium salt (1,000 IU/mL; Solarbio, China), streptomycin sesquisulfate (1 mg/mL; Solarbio, China), and polymyxin B (400 IU/mL; Amresco, USA), and filtered with a 0.22 μm filter to prevent bacterial contamination.

Semen collection and processing

Seven mature and fertile Duroc boars (aged 15 to 30 mo) were used in this study. The boars were housed individually, maintained under natural daylight, and provided with free access to food and water. The sperm-rich fraction was collected with the gloved hand technique twice a week, with fresh semen placed in a 37 °C bath and delivered to the laboratory within 15 min for the evaluation of sperm motility and concentration. Only semen samples with over 80% total motility were used for the present study. The ejaculated semen was diluted by a Modena solution containing spermine or spermidine at a final concentration of 5 × 10⁷ sperm per mL for the following processes.

Sperm motility, integrity of plasma membrane and acrosome

Sperm motility parameters were evaluated using computer-assisted sperm analysis (CASA; HVIEW, China), the settings and methods were performed as previously described (Li et al., 2020). Based on our previous study (Li et al., 2020), sperm plasma membrane integrity and acrosome were determined with an epifluorescence microscope (Nikon 80i, Japan) using Live/Dead Sperm Viability Kit (Invitrogen, USA) and FITC-peanut agglutinin (FITC-PNA)/prodium iodide (PI) probes, respectively. A minimum of 1,000 sperm were observed from at least five randomly selected fields for each sample. At least three technical replicates were evaluated for each sample.

Sperm ΔΨ_m, ATP content, and [Ca²⁺]_i

Here, ΔΨ_m and ATP content were evaluated using a mitochondrial membrane potential detection kit with JC-1 (Beyotime, China) and an ATP assay kit (Beyotime, China), as per Li et al. (2020). The [Ca²⁺]_i was assessed after loading spermatozoa with Fluo-4 AM (Beyotime, China) as a marker of [Ca²⁺]_i. Briefly, 500 μL sperm sample (5 × 10⁷ cells per mL) was loaded by the addition of 0.5 μL Fluo-4 AM stock solution (2 mmol/L) followed by incubation in the dark for 45 min at 37 °C. The fluorescence intensity of samples was detected by a multi-detection microplate reader (BioTek, Synergy H1, USA) at Excitation/Emission = 488/520 nm. Sperm counts were performed for each sample to normalize [Ca²⁺]_i to sperm number. At least three technical replicates were evaluated for each sample.

Quantification of spermine and spermidine

Briefly, each 1 mL sample was treated with 50 μL perchloric acid (70%), vortexed for 2 min, and centrifuged at 12,000 × g for 30 min at 4 °C, resulting in 500 μL supernatant that was transferred to a new tube (5 mL) and mixed with 500 μL deionized water. Benzoyl chloride (7 μL) and sodium hydroxide solution (2 mol/L, 1 mL) were added, vortexed for 2 min, and incubated at 37 °C for 30 min. Subsequently, sodium chloride solution (360 g/L, 2 mL) was added, vortexed for 1 min, and incubated at 37 °C for 5 min. The reaction mixtures were extracted with 2 mL ether at RT for 10 min and centrifuged at 2,000 × g for 5 min, resulting in 1 mL supernatant transferred to a new tube (1.5 mL). The ether was cleaned by a vacuum concentrator (Christ, RVC 2-18 CDplus, Germany) and precipitates were dissolved by 200 μL methanol. The sample was filtered through a 0.22 μm filter before injecting into the C18 column (75 mm × 2 mm, 1.6 μm, Shim-pack XR-ODSIII, Shimadzu). The content of spermine and spermidine in the seminal plasma samples was detected by a Nexera UHPLC system (Shimadzu, LC-30A, Japan). The sample injection volume was 5 μL, the flow rate was 0.2 mL/min, column temperature was 30 °C; mobile phase A (42%) was 6% acetonitrile, mobile phase B (58%) was methanol. Detection (UV-detector) was performed at 230 nm. At least six technical replicates were evaluated for each sample.

Conjugation of spermine and spermidine with FITC and spermine-FITC and spermidine-FITC uptake

According to the manufacturer’s instructions, the conjugation of spermine and spermidine with FITC was performed using the FITC conjugation kit (Fast)-Lightning-Link (Abcam, UK). Aliquots of 100 μL (1 × 10⁷) semen samples were centrifuged at 800 × g for 10 min at RT to remove seminal plasma, and the sperm was resuspended by (100 µL) Modena alone or with the specific inhibitors. The sample was supplemented with FITC-conjugated spermine or spermidine (final concentration 3 mmol/L) and incubated at 37 °C for 6 h in the dark. After the incubation, the suspension was centrifuged for 5 min at 800 × g, and the residual FITC-conjugated spermine or spermidine was removed by washing with 200 µL of phosphate-buffered saline (PBS). The samples were thus fixed with 4% paraformaldehyde (PFA) at RT for 10 min, followed by washing by PBS twice. Subsequently, the samples were re-stained by PI (4.8 μmol/L) at RT for 10 min and washed by PBS twice. To remove FITC fluorescence from the sperm, the samples were incubated in 0.4% trypan blue solution for 10 min on ice and washed twice with PBS. The samples were immediately visualized under an epifluorescence microscope (Nikon 80i, Japan) adjusted at 488 and 525 nm for fluorescein excitation. The sets (acquisition time, brightness, and contrast) for acquiring and treating all epifluorescence images are homogeneous. At least three technical replicates were evaluated for each sample.

Detection of oxidative stress indicators

The intracellular ROS level was measured using the probe of 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, China), as per Lv et al. (2021). The probe BODIPY 581/591C11 (Molecular Probes) is a sensitive fluorescent probe for early LPO. The intact probe fluoresces red when it is intercalated into the membrane (λ excitation = 590 and λ emission = 635 nm), and it shifts to green (λ excitation = 485 and λ emission = 535 nm) after the oxidative radical attack. Briefly, samples (1 mL) containing 2 × 10⁷ spermatozoa were washed twice by centrifugation for 5 min at 800 × g. The BODIPY 581/591C11 probe (working solution: 10 μmol/L) was added to the resuspended spermatozoa suspensions to make the probe a final concentration of 2 μmol/L and incubated at 37 °C for 30 min in the dark. The samples were washed by centrifugation at 800 × g for 5 min to remove the unbound probe. The fluorescence intensity of red and green was analyzed by a multi-detection microplate reader (BioTek, Synergy H1, USA).

For determination of late LPO (4-HNE positive cells) and DNA oxidative damage (8-OHdG positive cells), samples (1 mL) containing 2 × 10⁷ spermatozoa in Modena were washed and then fixed with 1,000 µL of 4% PFA in PBS for 10 min at RT, washed in PBS. The samples were permeabilized with 0.25% (v/v) Triton-X 100 in PBS for 10 min at RT and washed again in PBS. The samples were stored in 10% (v/v) donkey serum in PBS for 2 h at RT. Samples (5 × 10⁶ spermatozoa per mL) in 100 μL of PBS were incubated with one of the primary antibodies for 4-HNE (Abcam, 1:50) or 8-OHdG (Santa Cruz, 1:50) overnight at 4 °C. The samples were washed and resuspended with secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG from YEASEN, 1:100) for 2 h at 37 °C. Finally, the samples were washed and re-stained with PI (Thermo Fisher, 1:1,000) for 10 min. Cells were then washed in PBS, and the samples were immediately analyzed via flow cytometry (BD, FACS Aria III). The geometric mean of fluorescence intensity of 4-HNE or 8-OHdG (green fluorescence, FL-1) was used to indicate the LPO level or DNA oxidative damage measured after sperm (red fluorescence, FL-2) were gated in the analysis plot. The controls consisted of unstained, single-stained, and secondary-only antibody staining controls to set gates and compensations properly. The positive controls for 4-HNE or 8-OHdG were samples incubated for 1 h at 37 °C in the presence of 2 mol/L of H₂O₂. At least three technical replicates were evaluated for each sample.

Detection of antioxidant properties

The activities of GPx, GR, GST, GCL, POD and SOD, and GSH/GSSG ratio in sperm were determined using total GPx assay kit with NADPH (Beyotime, China), GR assay kit with DTNB (Beyotime, China), total SOD assay kit with WST-8 (Beyotime, China), GST assay kit (Solarbio, China), GCL assay kit (Solarbio, China), total POD assay kit (Solarbio, China), and GSH and GSSG assay kit (Beyotime, China), according to the manufacturer’s protocols. The activity of NQO1 was determined using dichloroindophenol (DCIP) as the two-electron acceptor (Benson et al., 1980). Briefly, the reaction mix contained in 50 mmol/L Tris–HCl, pH 7.5, 0.08% Triton X-100, 0.25 mmol/L NADPH, and 80 μmol/L of DCIP in the presence or absence of 60 μmol/L dicumarol. The reaction was started by adding sample (5 μL), and the reduction of DCIP was monitored at 600 nm, 25 °C for 3 min. The total protein concentration was practiced as the BCA protein assay kit (TaKaRa, Japan). GPx, GR, GST, GCL, POD, SOD, and NQO1 activities in sperm were represented as mU/mg or U/mg protein (prot.). All experiments were carried out in quadruplicate (n = 4).

Statistical analysis

All values are presented as mean ± standard error of the mean (SEM). All data were tested for normality and variance homogeneity before statistical analysis. Data were transformed by arc-sin square root transformation when necessary. Data were analyzed by one-way ANOVA (with repeated measures) and the Duncan test was used to perform post hoc analyses. Statistical analysis was determined using the paired Student’s t-test for Figure 1. All analyses were performed using SPSS v23.0 for Windows (SPSS Inc., USA). Significant differences among treatments were set at *: P < 0.05 and **: P < 0.01.

Figure 1.

Changes in polyamine content of seminal plasma diluted with Modena overtime during preservation in vitro. Semen samples were preserved for incubation for 6 h at 37 °C (A) or 7 d at 17 °C (B). Spermine and spermidine concentrations of seminal plasma diluted with Modena were determined by UHPLC (n = 7). Graph bars represent mean ± SEM. Asterisks represent a significant difference from control. *P < 0.05, **P < 0.01 determined by paired Student’s t-test. SM, spermine; SD, spermidine.

Results

Spermine and spermidine content of seminal plasma decrease after preservation in vitro

Spermine and spermidine content of seminal plasma were determined by UHPLC. Spermine and spermidine in eight boar fresh seminal plasma samples were determined at the level of 3.6 ± 0.3 and 3.3 ± 0.2 mmol/L, respectively. Compared with the control (0 h), significant decreases in spermine and spermidine content of seminal plasma were observed after incubation at 37 °C for 6 h (P < 0.05; Figure 1A). After 7 d of preservation at 17 °C, the spermine and spermidine content exhibited significant decreases in comparison with the control (0 d; P < 0.05; Figure 1B). Thus, we speculated that boar sperm could utilize and consume the spermine and spermidine in the seminal plasma.

OCTs mediate polyamines uptake in sperm

We found that 60 μmol/L quinidine, 60 μmol/L THP, 60 μmol/L l-carnitine, or 60 μmol/L mildronate did not significantly increase or decrease sperm motility and plasma membrane integrity compared with control (P > 0.05; Supplementary Figure S1, Supplementary Table S1), and thus was the optimal concentration for subsequent experiments. The accumulation of spermine or spermidine in boar sperm was inhibited by 60 μmol/L quinidine or 60 μmol/L THP, but neither 60 μmol/L mildronate nor 60 μmol/L l-carnitine (P < 0.05; Figure 2A). Moreover, SM-FITC or SD-FITC in the sperm was significantly decreased after the supplementation of 60 μmol/L quinidine or 60 μmol/L THP (Figure 2B). However, no difference was detected between the control and the group supplemented with 60 μmol/L l-carnitine or 60 μmol/L mildronate (Figure 2B). Therefore, we speculate that OCT1 and OCT3 could mediate the uptake of spermine and spermidine in boar sperm.

Figure 2.

Effects of OCT and OCTN inhibitors on polyamines uptake of boar sperm. Effects of OCT or OCTN inhibitors on the spermine and spermidine content in sperm (A). The polyamine contents in sperm were determined by UHPLC (n = 6). Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. Effects of OCT or OCTN inhibitors on uptake of SM-FITC or SD-FITC (B). Sperm nuclei were stained with PI. White arrow indicates spermatozoa enriched with SM-FITC or SD-FITC (n = 3). The images were visualized using an epifluorescence microscope. Bars = 20 μm. SM-FITC, FITC-labeled spermine; SD-FITC, FITC-labeled spermidine.

Polyamines improve sperm quality during preservation in vitro

No significant difference was detected in sperm motility parameters between the 0, 0.25, 0.5, 1, and 2 mmol/L of spermine or spermidine group (P > 0.05; Tables 1 and 2) when the samples were preserved at 17 °C for 1 d. After preservation at 17 °C for 7 d, higher progressive motility, straight-line velocity (VSL), and average path velocity (VAP) were observed in 0.5 mmol/L spermine group and higher progressive motility, VSL, curvilinear velocity (VCL), and VAP in 0.5 mmol/L spermidine group (P < 0.05, Tables 1 and 2). No difference was detected between the control and spermine or spermidine groups in total motility after preservation at 17 °C for 7 d (P > 0.05, Tables 1 and 2). Regarding plasma membrane and acrosome integrity, no difference was observed between the control and polyamines groups after preservation at 17 °C for 1 d. During preservation at 17 °C for 7 d, adding 0.5 mmol/L spermine or spermidine to the extender contributed to maintaining sperm plasma membrane and acrosome integrity (Figure 3A and B). Compared with control, polyamines addition did not affect the ROS level after preservation at 17 °C for 1 d (P > 0.05; Figure 3C). Furthermore, the addition of 0.5 mmol/L spermine or spermidine resulted in a lower ROS level after preservation at 17 °C for 7 d compared with the control (P < 0.05; Figure 3C). Thus, these results demonstrated that spermine and spermidine could maintain sperm motility, plasma membrane and acrosome integrity, and reduce sperm ROS accumulation during preservation in vitro.

Table 1.

Effect of spermine addition on sperm motility parameters after long-term preservation at 17 °C¹

	Preservation for 1 d
	Control	SM-0.25 mmol/L	SM-0.5 mmol/L	SM-1 mmol/L	SM-2 mmol/L
Total motility, %	91.67 ± 0.77	92.02 ± 0.53	89.23 ± 2.50	94.07 ± 0.38	90.35 ± 1.28
Progressive motility, %	77.40 ± 0.83	76.64 ± 1.91	75.71 ± 1.69	79.53 ± 1.33	76.42 ± 1.31
VSL, μm/s	65.19 ± 5.00	66.92 ± 4.48	72.08 ± 8.02	69.85 ± 1.16	72.21 ± 3.80
VCL, μm/s	136.90 ± 7.11	140.11 ± 6.33	142.96 ± 8.88	148.22 ± 6.1	153.90 ± 4.18
VAP, μm/s	78.20 ± 3.93	82.26 ± 3.81	80.69 ± 4.22	88.35 ± 3.71	91.23 ± 4.89

	Preservation for 7 d
Total motility, %	79.38 ± 0.74	83.33 ± 1.67	85.09 ± 2.51	84.38 ± 1.16	71.24 ± 5.66
Progressive motility, %	50.62 ± 2.16bc	57.43 ± 4.97ab	60.71 ± 2.47a	56.90 ± 2.67ab	35.86 ± 7.50c
VSL, μm/s	40.32 ± 2.35b	43.56 ± 2.20ab	45.36 ± 3.82a	40.12 ± 6.35abc	25.07 ± 10.33c
VCL, μm/s	97.46 ± 0.26	102.25 ± 3.54	103.34 ± 3.86	94.21 ± 4.69	59.99 ± 18.28
VAP, μm/s	33.37 ± 3.19bc	37.32 ± 1.35ab	40.30 ± 2.90a	41.96 ± 3.29a	23.54 ± 5.03c

Sperm motility parameters (total motility, progressive motility, VSL, VCL, and VAP) were determined using the CASA system (n = 5). Values are presented as mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine.

Table 2.

Effect of spermidine addition on sperm motility parameters after long-term preservation at 17 °C¹

	Preservation for 1 d
	Control	SD-0.25 mmol/L	SD-0.5 mmol/L	SD-1 mmol/L	SD-2 mmol/L
Total motility, %	91.67 ± 0.77	91.74 ± 1.80	90.47 ± 1.19	90.90 ± 0.39	89.88 ± 1.93
Progressive motility, %	77.40 ± 0.83	77.78 ± 1.88	74.78 ± 2.77	73.16 ± 1.71	74.66 ± 2.30
VSL, μm/s	65.19 ± 5.00	68.95 ± 4.52	75.90 ± 6.24	75.96 ± 6.71	69.54 ± 3.82
VCL, μm/s	136.90 ± 7.11	153.02 ± 6.89	168.20 ± 15.06	161.55 ± 14.28	146.82 ± 6.08
VAP, μm/s	78.20 ± 3.93	90.75 ± 4.35	97.45 ± 7.94	92.86 ± 10.85	88.53 ± 4.34

	Preservation for 7 d
Total motility, %	79.38 ± 0.74	81.26 ± 3.20	82.30 ± 2.25	80.80 ± 2.40	82.27 ± 0.33
Progressive motility, %	50.62 ± 2.16b	56.30 ± 2.54ab	61.27 ± 2.39a	55.21 ± 2.88ab	55.26 ± 4.66ab
VSL, μm/s	40.32 ± 2.35b	46.40 ± 4.85ab	49.30 ± 5.14a	40.72 ± 7.92ab	37.07 ± 5.43b
VCL, μm/s	97.46 ± 0.26b	106.61 ± 2.73ab	108.75 ± 3.13a	93.97 ± 5.36b	84.53 ± 8.45b
VAP, μm/s	30.88 ± 1.85b	34.68 ± 2.82ab	36.34 ± 2.88a	32.55 ± 0.90b	34.51 ± 1.87ab

Sperm motility parameters (total motility, progressive motility, VSL, VCL, and VAP) were determined using the CASA system (n = 5). Values are presented as mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SD, spermidine.

Figure 3.

Effects of spermine or spermidine on sperm quality after preservation for 1 or 7 d at 17 °C. Sperm plasma membrane integrity (A), acrosome integrity (B), and ROS level (C) were evaluated using SYBR-14/PI, FITC-PNA, and ROS assay kits after preservation at 17 °C for 1 or 7 d, respectively (n = 3). MFI, mean of fluorescence intensity. Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine; SD, spermidine.

Polyamines alleviate H₂O₂-induced sperm quality decrease

Compared with the control, significant decreases in total motility, progressive motility, VSL, VCL, and VAP were observed in the H₂O₂ treatment group after incubation at 37 °C for 2 h (P < 0.05, Tables 3 and 4). However, the addition of spermine (0.5 and 1 mmol/L) attenuated the decrease of progressive motility induced by H₂O₂ (P < 0.05; Table 3), and spermidine (0.5 mmol/L) inhibited the decrease of total motility, progressive motility, VSL, VCL, and VAP induced by H₂O₂ (P < 0.05; Table 4). Compared with the control, significant decreases in the plasma membrane and acrosome integrity were observed in the H₂O₂ treatment group after incubation at 37 °C for 2 h (P < 0.05; Figure 4A and B). However, compared with H₂O₂ treatment group, the addition of spermine (0.5 and 1 mmol/L) or spermidine (0.5 mmol/L) resulted in higher plasma membrane integrity (P < 0.05; Figure 4A), and 0.25, 0.5, 1, and 2 mmol/L spermine or spermidine addition significantly improved acrosome integrity (P < 0.05; Figure 4B). Thus, these results demonstrated that spermine and spermidine can protect sperm motility, plasma membrane, and acrosome from oxidative damage.

Table 3.

Effects of exposure to H₂O₂ alone or with spermine on sperm motility parameters¹

	Control	H2O2 treatment
		SM-0	SM-0.25 mmol/L	SM-0.5 mmol/L	SM-1 mmol/L	SM-2 mmol/L
Total motility, %	81.20 ± 2.14a	62.85 ± 3.01bc	64.01 ± 3.73bc	73.92 ± 4.42ab	73.71 ± 2.12ab	60.03 ± 5.38c
Progressive motility, %	64.44 ± 2.34a	23.80 ± 5.35c	29.43 ± 5.90bc	45.14 ± 5.70b	44.07 ± 5.36b	26.74 ± 7.03c
VSL, μm/s	39.34 ± 2.37a	11.54 ± 3.91b	15.91 ± 4.36b	22.49 ± 5.46ab	22.40 ± 5.10ab	13.42 ± 5.49b
VCL, μm/s	80.31 ± 7.14a	26.67 ± 6.79b	37.08 ± 8.31b	49.41 ± 9.59b	51.22 ± 9.73b	40.69 ± 12.83b
VAP, μm/s	46.80 ± 5.63a	12.19 ± 4.20b	18.40 ± 5.12b	26.87 ± 6.26b	26.69 ± 6.97b	16.05 ± 6.10b

Sperm were pretreated with 0, 0.25, 0.5, 1, and 2 mmol/L spermine in Modena medium for 30 min and incubation with 100 μmol/L H₂O₂ for 2 h at 37 °C. Sperm motility parameters (total motility, progressive motility, VSL, VCL, and VAP) were determined using the CASA system (n = 5). Values are presented as mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine.

Table 4.

Effects of exposure to H₂O₂ alone or with spermidine on sperm motility parameters¹

	Control	H2O2 treatment
		SD-0	SD-0.25 mmol/L	SD-0.5 mmol/L	SD-1 mmol/L	SD-2 mmol/L
Total motility, %	81.20 ± 2.14a	62.85 ± 3.01bc	61.11 ± 6.73bc	77.84 ± 1.67a	69.73 ± 3.74ab	54.82 ± 4.45c
Progressive motility, %	64.44 ± 2.34a	23.80 ± 5.35c	34.48 ± 7.17bc	55.19 ± 2.99a	44.82 ± 3.22ab	18.26 ± 4.24c
VSL, μm/s	39.34 ± 2.37a	10.53 ± 2.76c	17.11 ± 4.56bc	28.67 ± 3.12ab	24.17 ± 3.15bc	10.86 ± 3.50c
VCL, μm/s	80.31 ± 7.14a	26.67 ± 6.79d	37.66 ± 4.19cd	61.10 ± 7.26ab	51.61 ± 8.40bc	37.06 ± 7.46cd
VAP, μm/s	46.80 ± 5.63a	12.19 ± 4.20d	18.86 ± 3.52cd	32.95 ± 4.79b	28.18 ± 4.61bc	15.96 ± 4.74cd

Sperm were pretreated with 0, 0.25, 0.5, 1, and 2 mmol/L spermidine in Modena medium for 30 min and incubation with 100 μmol/L H₂O₂ for 2 h at 37 °C. Sperm motility parameters (total motility, progressive motility, VSL, VCL, and VAP) were determined using the CASA system (n = 5). Values are presented as mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. N = 5. SD, spermidine.

Figure 4.

Effects of exposure to H₂O₂ alone or with polyamines on the integrity of sperm plasma membrane and acrosome. Sperm plasma membrane integrity (A) and acrosome integrity (B) were determined using SYBR-14/PI and PNA-FITC kits, respectively (n = 5). Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine; SD, spermidine.

Polyamines alleviate H₂O₂-induced sperm ATP depletion, ΔΨ_m decline, and [Ca²⁺]_i overload

H₂O₂ treatment led to a significant decrease in ΔΨ_m after incubation at 37 °C for 2 h, the addition of spermine (0.5 and 1 mmol/L) resulted in higher ΔΨ_m than H₂O₂ treatment group (P < 0.05), and no difference was detected between spermidine and H₂O₂ group (P > 0.05; Figure 5A). A significant decrease in ATP content was observed in the H₂O₂ treatment group after incubation at 37 °C for 2 h (P < 0.05). However, the spermine (1 mmol/L) or spermidine (0.5 and 1 mmol/L) treatment group led to a higher ATP content than H₂O₂ treatment group (Figure 5B; P < 0.05). Higher [Ca²⁺]_i were observed in the H₂O₂ groups after incubation for 2 h at 37 °C (P < 0.05; Figure 5C). However, the addition of spermine (0.25, 0.5, 1, and 2 mmol/L) or spermidine (1 mmol/L) attenuated [Ca²⁺]_i overload induced by H₂O₂ (P < 0.05; Figure 5C). These results demonstrated that spermine showed the capacity to maintain sperm ΔΨ_m, and both spermine and spermidine showed the capacity to alleviate ATP depletion and [Ca²⁺]_i overload in the condition of oxidative stress.

Figure 5.

Effects of exposure to H₂O₂ alone or with polyamines on ΔΨ_m, ATP content, and [Ca²⁺]_i. The sperm ΔΨ_m (A), ATP content (B), and [Ca²⁺]_i (C) were determined using JC-1 kit, ATP bioluminescence assay kit, and Fluo-4 AM, respectively (n = 3). The protein content of samples was executed to normalize ATP content. [Ca²⁺]_i (% of control) = the geometric mean of Fluo-4 fluorescence intensity of different concentration polyamines addition group/control group × 100%. Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine; SD, spermidine.

Polyamines alleviate H₂O₂-induced oxidative damage in sperm

Higher ROS level were observed in H₂O₂ groups after incubation for 2 h at 37 °C (P < 0.05; Figure 6A). However, the addition of spermine (0.5, 1, and 2 mmol/L) or spermidine (0.5, 1, and 2 mmol/L) in H₂O₂ treatments attenuated the increase of ROS level (P < 0.05). Higher LPO level was observed in H₂O₂ groups after incubation for 2 h at 37 °C (P < 0.05; Figure 6B). However, the addition of spermine (0.5 and 1 mmol/L) or spermidine (0.5 and 1 mmol/L) attenuated the increase of LPO level induced by H₂O₂ (P < 0.05; Figure 6B). H₂O₂ treatment led to significant increases in 4-HNE level and 8-OHdG level after incubation at 37 °C for 2 h. However, the addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) resulted in lower 4-HNE level and 8-OHdG level than H₂O₂ treatment group (P < 0.05; Figure 6C and D). Thus, the above results demonstrated that spermine and spermidine could counteract ROS over-accumulation and decrease LPO and 8-OHdG levels in sperm in the condition of oxidative stress.

Figure 6.

Effects of exposure to H₂O₂ alone or with polyamines on sperm ROS, LPO, 4-HNE, and 8-OHdG level. Sperm ROS and LPO levels were determined using the ROS assay Kit (A) and the BODIPY 581/591 C11 (B), respectively. Representative overlay histograms of changes in 4-HNE and 8-OHdG (C). 4-HNE and 8-OHdG levels are presented as the geometric mean of fluorescence intensity measured by flow cytometry (D). n = 3. Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P < 0.05) between treatments. SM, spermine; SD, spermidine.

Polyamines enhancing GSH-related antioxidant properties in sperm

H₂O₂ treatment decreased GSH/GSSG ratio after incubation at 37 °C for 2 h. The addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) resulted in a higher GSH/GSSG ratio than H₂O₂ treatment group (P < 0.05; Figure 7A). However, no significant difference in GCL activity of sperm was detected between the control and other treatments (P > 0.05; Figure 7B). Compared with control, addition of spermine in H₂O₂ treatment led to an increase in activities of GPx and GR (P < 0.05; Figure 7C and D), and addition of spermidine significantly increased GR activity (P < 0.05; Figure 7D). Compared with H₂O₂ treatment, addition of spermine significantly increased GPx and GST activities (P < 0.05; Figure 7C and E). However, no significant difference in SOD, POD, and NQO1 activities was detected between the control and other treatment groups (P > 0.05; Figure 7F–H). Therefore, these results showed that spermine and spermidine could enhance GSH-related antioxidant properties in sperm in the condition of oxidative stress.

Figure 7.

Effects of exposure to H₂O₂ alone or with polyamines on antioxidant properties of sperm. Cellular GSH/GSSG ratio (A), GCL (B), GPx (C), GR (D), GST (E), SOD (F), POD (G), and NQO1 (H) activities in sperm were measured (n = 4). Graph bars represent mean ± SEM. Different lower case letters indicate a significant difference (P<0.05) between treatments. SM, spermine; SD, spermidine.

Discussion

Preservation in vitro prolongs sperm lifespan, but physiological senescence occurs during preservation. Furthermore, ROS over-accumulation and oxidative stress can undermine the structural and functional integrity of sperm (Aitken and Drevet, 2020). In the present study, we observed that spermine and spermidine were abundant in seminal plasma, and the content of polyamine decreased during preservation. Polyamines were absorbed by sperm via OCTs, and reduced oxidative stress via its antioxidant capacity, thereby maintaining sperm functionality and improving the efficacy of semen preservation.

The content of polyamines in semen is much higher than other body fluids (Lefevre et al., 2011). In this study, we found that the concentration of spermine in boar seminal plasma was very close to that in human seminal plasma (Janne et al., 1973; Shohat et al., 1990; Oefner et al., 1992). Interestingly, the content of spermine and spermidine decreased in the diluted semen during preservation, suggesting that boar sperm consumed them during this period.

Polyspecific organic cation transporters mediate the transport of many endogenous low molecular weight organic cations and a series of drugs, such as choline, histamine, dopamine, or metformin (Courousse and Gautron, 2015; Koepsell, 2020). It was reported that OCT1, OCT2, OCT, OCTN1, and OCTN2 were responsible for spermine and spermidine uptake in mammalian somatic cells and Xenopus oocytes (Busch et al., 1996; Yabuuchi et al., 1999; Molderings et al., 2003; Sala-Rabanal et al., 2013; Higashi et al., 2014). Combined transporter inhibitors and FITC-labeled polyamines, we demonstrated that the redistribution of extracellular polyamines into sperm cells could be facilitated via OCTs-mediated transport, consistent with the previous reports in somatic cells (Busch et al., 1996; Sala-Rabanal et al., 2013; Higashi et al., 2014).

The motility is a prerequisite ability of sperm to move through the female reproductive tract and accomplish the mission of fertilization. The plasma membrane is the only barrier between sperm cells and the extracellular environment. The acrosome integrity is believed to be a precondition that enables the acrosome-reacted spermatozoa to penetrate the zona pellucida and fertilize the eggs. Therefore, sperm motility and integrity of plasma membrane and acrosome are essential indicators for assessing fertilizing potential. In the present study, we observed that addition of polyamines contributed to the maintenance of sperm motility and integrity of the plasma membrane and acrosome of boar sperm during storage at 17 °C, similar to that in canine sperm (Setyawan et al., 2016). We also found that addition of spermine and spermidine attenuated ROS stress in boar sperm, consistent with the report in frozen–thawed canine sperm (Setyawan et al., 2016).

It has demonstrated that spermine against radical attack through a scavenging action, and this scavenging is due to the reaction of spermine with the hydroxyl radical and the production of spermine dialdehyde (1,12-bis-1,12-dioxo-4,9-diazododecane) (Ha et al., 1998). In the present study, polyamines alleviated H₂O₂-induced decline in sperm motility and damage of plasma membrane and acrosomes via enhancing antioxidant properties. We found that spermine or spermidine could potently induce an adaptive response to oxidative stress in GSH-related antioxidant properties, such as enhancing GR, GPx, and GST activities, and increasing GSH/GSSG ratio, consistent with the previous studies in rat primary cardiomyocytes and brain ventral striatum, piglet liver, and longissimus dorsi and mung bean (Farbiszewski et al., 1996; He et al., 2015; Nahar et al., 2015; Fang et al., 2018).

Excessive ROS could attack sperm membranes that are rich in PUFAs (Brouwers et al., 2005) and lead to LPO (Martinez et al., 2007). In the present study, LPO in sperm cells was assessed by fluorescence techniques using the C11-BODIPY (581/591; Brouwers et al., 2005) and 4-HNE (Ayala et al., 2014). We found that polyamines protect boar sperm to avoid LPO, which is in agreement with the previous reports in polymorphonuclear leukocytes and breast cancer cells (Chapman and Wallace, 1994; Belle et al., 2004). ROS attack at DNA molecules as well, which induces a mutagenic risk that can be transmitted to the next generations if the oocyte repair systems fail to correct the alterations in the paternal DNA (Drevet and Aitken, 2020). 8-OHdG is one of the more abundant forms of free radical-induced oxidative lesions, being widely used as a biomarker of oxidative DNA damage (Vorilhon et al., 2018). In the present study, we found that polyamines protected sperm DNA from oxidative damage, which is consistent with that in rat liver cells and human adipose stem cells (Ha et al., 1998; Douki et al., 2000; Minguzzi et al., 2019). These data indicate that polyamines prevent sperm from ROS stress.

In the present study, we found that spermine or spermidine alleviated ROS-induced intracellular calcium overload and ATP depletion in boar sperm. Polyamines regulated ΔΨ_m, the transport of Ca²⁺, oxidative phosphorylation, and enzymes of the Krebs cycle, and acted as free radical scavengers within mitochondria (Toninello et al., 1992; Clarkson et al., 2004; Hoshino et al., 2005; Sava et al., 2006; Pezzato et al., 2009). Spermine and spermidine are considered potent physiological inhibitors of the mitochondrial permeability transition pore (MPTP) in the isolated mitochondria (Chen et al., 2019), which is an indispensable target in regulating GSH levels in mitochondria under oxidative stress or mitochondrial swelling (Rigobello et al., 1993; Sava et al., 2006). Whether such an MPTP-dependent mechanism is also involved in the redox homeostasis in sperm by polyamines warrants further investigation.

Conclusion

In conclusion, OCTs mediate the polyamines uptake in sperm cells. Extracellular polyamines decreased during sperm preservation in vitro. Addition of polyamines increased GPx, GR, and GST activities and GSH/GSSG ratio, alleviated oxidative stress-induced LPO, DNA damage, ΔΨ_m decline, ATP depletion, and Ca²⁺ overload, thereby maintaining sperm motility, integrity of plasma membrane, and acrosome (Figure 8). Therefore, polyamines protect boar sperm from ROS stress in vitro, providing novel prospects for improving the efficacy of AI fertilization in pigs and possibly other animals.

Figure 8.

Proposed model of polyamines uptake by OCT maintain boar sperm redox homeostasis and functionality in vitro. Spermine and spermidine, transported into sperm by OCTs, contribute to lower ROS levels and oxidative damage and higher GSH/GSSG ratio and maintain redox homeostasis and integrity of plasma membrane, acrosome, ATP levels, ΔΨ_m, and [Ca²⁺]_i, thereby improving sperm functionality in vitro.

Glossary

Abbreviations

4-HNE

4-hydroxylnonenal

8-OHdG

8-hydroxy-2ʹ-deoxyguanosine

AI

artificial insemination

ATP

adenosine triphosphate

BODIPY

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

CASA

computer-assisted sperm analysis

DCF

2ʹ, 7ʹ-dichlorofluorescein

DCFH-DA

2ʹ, 7ʹ-dichlorodihydrofluorescein diacetate

DCIP

dichloroindophenol

EDTA-2Na

ethylenediamine tetraacetic acid disodium

FITC

fluorescein isothiocyanate

FITC-PNA

FITC-peanut agglutinin

GCL

γ-glutamate cysteine ligase

GPx

glutathione peroxidase

GR

glutathione reductase

GSH

reduced glutathione

GSSG

oxidized glutathione

GST

glutathione S-transferase

LPO

lipid peroxidation

MPTP

mitochondrial permeability transition pore

NQO1

NAD(P)H, quinone oxidoreductase 1

OCT

organic cation transporters

OCTN

organic cation/carnitine transporter

PBS

phosphate-buffered saline

PFA

paraformaldehyde

PI

prodium iodide

POD

peroxidase

ROS

reactive oxygen species

RT

room temperature

SD-FITC

FITC-labeled spermidine

SEM

standard error of the mean

SM-FITC

FITC-labeled spermine

SOD

superoxide dismutase

THP

dl-tetrahydropalmatine

Tris

tris (hydroxymethyl) aminomethane

UHPLC

ultra-high-performance liquid chromatography

VAP

average path velocity

VCL

curvilinear velocity

VSL

straight-line velocity

ΔΨ_m

mitochondrial membrane potential

Conflict of interest statement

The authors declare that they have no competing interests.

Author Contributions

R.N.L. and W.X.Z. conceived and designed the experiments. R.N.L., X.D.W., Z.D.Z., Y.H.L., and Y.Z. performed the experiments. R.N.L., H.Z.L., K.F.Z., and D.W. analyzed the data. R.N.L. and W.X.Z. wrote the paper. T.Z. and W.Z.D. reviewed and edited the paper. All authors have read and agreed to the published version of the manuscript.