The Effect of Astaxanthin on Motility,Viability,Reactive Oxygen Species,Apoptosis,and Lipid Peroxidation of Human Spermatozoa During the Freezing

Abstract

Cryopreservation of spermatozoa is a general procedure to preserve viable sperm for an indefinite period. Despite the efficiency of sperm cryopreservation, excessive reactive oxygen species (ROS) production during cryopreservation can induce structural and functional changes in spermatozoa. Also, cryopreservation has been shown to decrease the spermatozoa's antioxidant activity inducing them to be more sensitive to damage caused by ROS. Experimental evidence suggests that astaxanthin (AXT) has essential activities such as antioxidant, antibacterial, and antithrombotic properties. Therefore, this study aimed to evaluate the effect of AXT on the sperm quality of healthy men during freezing–thawing. In the first phase, 10 semen samples with different concentrations of AXT (0.0, 0.5, 1, and 2 μM) were cryopreserved to achieve an optimal dose of AXT. Then, motility, viability, and phosphatidylserine (PS) externalization were evaluated. In the second phase, 25 samples were collected and divided into 3 groups: fresh group, control group (untreated frozen–thawed samples), and AXT group (treated frozen–thawed with AXT). Then, samples were cryopreserved in freezing media supplemented with or without the optimal concentration of AXT (1 μM). After thawing, the levels of sperm parameters, including motility (using a computer-assisted sperm analyzer), viability (eosin–nigrosin), early apoptotic change (annexin V/propidium iodide), ROS (flow cytometry), and lipid peroxidation (LPO) (using enzyme-linked immunosorbent assay), were evaluated. Our results showed that the addition of 1 μM AXT to sperm freezing media improved all parameters of sperm motility and viability (p ≤ 0.05). Furthermore, it could reduce the levels of ROS parameters (intracellular hydrogen peroxide and superoxide) compared with the control group (p ≤ 0.05). Also, AXT significantly decreased the level of PS externalization (p ≤ 0.05) and LPO (p ≤ 0.05) after the freezing–thawing process. In conclusion, our findings demonstrated that human semen treatment with 1 μM AXT before the freezing–thawing process has protective effects against oxidative stress and could diminish the destructive effects of this process on sperm quality.

Introduction

Cryopreservation of gametes and embryos has broad importance among assisted reproductive techniques.¹ Semen freezing is currently used for pre-radiation and chemotherapy treatments, for men undergoing vasectomy, cancer, and storage of donor semen for HIV and hepatitis patients.^2–4 However, the results of studies prove that the cryopreservation of sperm leads to the induction of damage such as osmotic stress, ice crystals formation, destruction of plasma membrane function, mitochondrial dysfunction, and DNA fragmentation, which may eventually lead to death or severe damage of sperm by reducing motility, survival, and fertilization capacity.^5,6 Furthermore, studies have indicated that the cryopreservation of human semen causes an increase in reactive oxygen species (ROS), which induces substantial sperm damage.⁷

Besides, scanty cytoplasm in sperm reduces the defensive activity of antioxidants. Moreover, sperm cells are significantly more sensitive to lipid peroxidation (LPO) because their membranes contain a high proportion of polyunsaturated fatty acids.^8,9 In addition, the freezing–thawing process has been shown to decrease the antioxidant activity of the spermatozoa, making them more sensitive to damage caused by ROS.¹⁰ The disruption of mitochondrial and plasma membranes resulting from exposure to high ROS concentrations causes chromosomal damage and DNA fragmentation and reduces sperm motility.^11–13

Much research has focused on the effects of antioxidants on female and male gametes, as well as embryonic development.^14–16 Indeed, many antioxidants are beneficial in treating male fertility.^17–19 The main antioxidant enzymes present in seminal plasma are superoxide dismutase (SOD), which is responsible for the exposure of superoxide radicals, producing H₂O₂ and oxygen, and catalase (CAT), which works by extracting H₂O₂ from water and O₂.^20–22

Scientists have recently tried to restrict the effects of ROS produced during the freezing–thawing process by the addition of antioxidant supplements in a freezing media.^6,21,23 Tocopherol, albumin, vitamin C, vitamin E (α-tocopherol), taurine, and hypotaurine are referred to as antioxidants that reduce ROS.²⁴ Between them, vitamin E plays its antioxidant role by existing within the cell wall and suppressing LPO.²⁵

Astaxanthin (AXT) is a keto-carotenoid extracted from the algae Haematococcus pluvialis, a type of Chlorophyta. AXT is known to have antioxidant effects against various forms of oxidative stress.^26–28 It has a better ability to remove O₂ compared with vitamin E²⁹ and β-carotene.³⁰

Therefore, this study was conducted to evaluate the effect of AXT on the most critical functional parameters of human sperm, including levels of motility, viability, ROS, phosphatidylserine (PS) externalization, and LPO during the process of cryopreservation.

Materials and Methods

Semen collection

The ethics committee (IR.TUMS.MEDICINE.REC.1397.742) of Tehran University of Medical Sciences (Tehran, Iran) approved this study. All subjects were informed concerning to this study, and written consent was acquired from them. Semen samples were collected from 25 normozoospermic patients aged 20–40 years, by masturbation after 3–5 days of ejaculatory abstinence, while participating in a male infertility investigation at the Noor laboratory and andrology center of Shariati Hospital in Tehran (Tehran, Iran). All participants were nonsmokers and did not use any drugs or alcohol. Samples were collected into sterile vials and were left to be liquefied at 37°C for 30 min. We excluded any samples that did not liquefy during this period. Semen samples were analyzed according to the World Health Organization (WHO) guidelines for concentration, motility, and morphology.³¹

Experimental design

After proving the normality of the samples, to determine the effective dose of AXT (SML0982-50MG; Sigma, China), we evaluated sperm motility, viability, and PS externalization during cryopreservation.³² AXT was dissolved in dimethyl sulfoxide (DMSO; M81802; Sigma, Temecula, CA), and a concentration of 100 μM was prepared and stored in the freezer at −20°C, as a pink solution. In our previous study, we analyzed the effect of DMSO alone on spermatozoa viability and progressive and total motility, and the results showed that DMSO did not have additional side effects on spermatozoa parameters compared with fresh untreated samples.³³

Also, in this study, we analyzed the effect of DMSO in the low sample of semen at a final concentration of 0.1% of DMSO that was used to dilute the AXT, and the results showed no additional side effects in spermatozoa viability and any motility (data not shown). Ten semen samples were collected, and each sample was divided into four groups: control group and AXT groups at different doses (0.5, 1, and 2 μM). The control group and AXT groups were cryopreserved, and after thawing, the total motility (using computer-assisted sperm analyzer [CASA]), viability (using eosin–nigrosin staining), and early apoptotic change (using annexin V/propidium iodide [PI] assay) were assessed.

After selecting the effective AXT dose, an additional 25 semen samples were collected, and each sample was divided into 3 equal aliquots: fresh group, control group (untreated frozen–thawed samples), and AXT group (treated frozen–thawed samples with the effective dose of AXT).

The motility (using CASA), viability (eosin–nigrosin staining), ROS (using flow cytometry with 2′,7′-dichlorofluorescein diacetate [DCFH-DA] and dihydroethidium [DHE] assays), early apoptotic change (using annexin V/PI assay), and LPO (using enzyme-linked immunosorbent assay and malondialdehyde [MDA] assay) in each group were evaluated.

Freezing and thawing

For the freezing–thawing experiment, the samples of control and AXT groups were then diluted by dropwise addition of a prewarmed sperm freezing medium (FertiPro, Beernem, Belgium) in a proportion of 1:1. Cryopreservation of samples was performed as described previously³³ after balancing with room temperature for 10 min, and the cryotubes were suspended in the liquid nitrogen vapor (10–15 cm above the level of liquid nitrogen at −80°C for 15 min). Eventually, they were plunged into liquid nitrogen (−196°C) and kept for 2 weeks. After the cryostorage duration, the samples were thawed at room temperature for 15 min. Then, an equal amount of Ham's F-10 medium (Life Technology, Carlsbad, CA) supplemented with 10% human serum albumin (HSA; Life Global) was added to the samples and then centrifuged at 200 g for 5 min. After removing the supernatant, the pellet was suspended in Ham's F-10 medium, and associated tests were performed to assess the motility, viability, ROS, PS externalization, and LPO in the two groups.

Assessment of motility

The sperm motility parameters were determined by a CASA device with the Sperm Class Analyzer software (SCA motility module, Version 4.2; Microptic, Barcelona, Spain). To do this, 10 μL of the sample was located on a glass slide under a square glass cover (22 × 22 mm) and the image was taken with 400 × magnification. Total motility, progressive motility, and velocity parameters, including VCL (velocity of curvilinear) in μm/s, VSL (velocity of straight line) in μm/s, VAP (velocity of average path) in μm/s, LIN % (linearity = VSL/VCL × 100), STR % (straightness = VSL/VAP × 100), and WOB % (wobble VAP/VCL), were measured.³⁴

Assessment of sperm viability parameter

Viability was assessed using eosin–nigrosin staining.³⁵ To do this, 20 μL of semen was mixed with 20 μL of 1% eosin (w/v). Then, 20 μL of nigrosin (10% w/v) was added. The sperm smear was placed on a glass slide and allowed to air-dry. A minimum of 200 spermatozoa on each slide was counted under oil immersion light microscopy at 1000 magnification. They included unstained (intact) and red (with damaged membranes) spermatozoa. The sperm viability was defined as the percentage of membrane intact spermatozoa.

Assessment of H₂O₂ and O₂⁻ by flow cytometry

For detecting intracellular H₂O₂ and O₂⁻, we used specific probes, DCFH-DA (Sigma, St Louis, MO) and DHE (Sigma), respectively.^35,36 Briefly, levels of H₂O₂ and O₂⁻ were evaluated in fresh, control, and AXT spermatozoa after incubating samples with DCFH-DA (25 μM) and DHE (1.25 μM) at 25°C for 40 min for DCFH-DA and for 20 min for DHE in the dark. Green fluorescence color emitted from DCFH-DA was assessed at wavelengths between 500 and 530 nm (in the FL-1 channel). Red fluorescence color emitted from DHE was evaluated at wavelengths between 590 and 700 nm (in the FL-2 channel). Samples were analyzed by the flow cytometer of Becton Dickinson FACScan.

Detection of PS externalization

For the assay of annexin V to detect the translocation of PS from the inner to the outer leaflet of the plasma membrane of spermatozoa,³⁷ the Phosphatidylserine Detection Kit (IQ Products, Netherlands) was used according to the manufacturer's recommendations. Briefly, an aliquot containing 1 × 10⁶ spermatozoa/mL was washed in calcium buffer, and the cells were readjusted in 1 mL of calcium buffer. Then, 10 μL of annexin V PE apoptosis detection kit was incubated with a 100 μL of cell suspension for 20 min on ice in the dark. After washing again with calcium buffer and the addition of 10 mL of PI, the cells were incubated on ice for 10 min.

Signals were identified through FL-1 and FL-3 channels, which could detect green fluorescence (for annexin V) and red fluorescence (for PI), respectively. Four different fractions of spermatozoa were distinguished by flow cytometric analysis, namely (1) AnV⁻/PI⁺ (dead spermatozoa without PS externalization), (2) AnV⁺/PI⁺ (dead spermatozoa with PS externalization), (3) AnV⁺/PI⁻ (vital spermatozoa with PS externalization), and (4) AnV⁻/PI⁻ (vital spermatozoa without PS externalization).

Measurement of LPO levels

The level of LPO in spermatozoa was measured by reaction of thiobarbituric acid with MDA³⁸ by using the MDA Assay Kit (ZellBio GmbH, Germany) according to the manufacturer's protocol. To accomplish this, reagents were added to samples and the mixture was heated for 1 h in a boiling water bath (pink color formation). After that, the pink color supernatant was pipetted into the microplate, and the absorbance was read at 535 nm to determine the MDA levels.

Statistical analysis

Results are expressed as the mean ± standard deviation. The GraphPad Prism software (version 8) was used for statistical analysis. The Kolmogorov–Smirnov test was used to verify the normal or non-normal distribution of values. Statistical significance was assessed using analysis of variance (ANOVA) with Student–Newman–Keuls and was considered statistically significant if p < 0.05.

Results

Determination of effective dose of AXT

The results of Table 1 show the effects of AXT at concentrations of 0.5, 1, and 2 μM on viability, total motility, and PS externalization after the freezing–thawing process. Therefore, due to the significant difference between the 1 μM AXT dose compared with the control group, it was selected as the effective dose for the freezing process (Table 1).

Table 1.

Determination of Effective Dose of Astaxanthin by Cryopreservation and Thawing of Samples

Concentration	Viability	Total motility	Early apoptotic change
Control	31.60 ± 3.10	23.40 ± 5.00	9.90 ± 2.31
0.5 μM	38.52 ± 6.43	25.20 ± 4.50	9.40 ± 1.80
1 μM^*	54.00 ± 4.28^*	31.60 ± 3.90^*	7.98 ± 2.70^*
2 μM	43.80 ± 4.10	25.4 ± 3.20	8.54 ± 0.50

p < 0.05.

Sperm motility test (CASA)

As shown in Table 2, the freezing–thawing process effectively reduces progressive and total motility compared with the fresh group (20.64 ± 3.8 vs. 49.52 ± 0.53, p < 0.05, and 45.21 ± 6.56 vs. 61.11 ± 0.47, p < 0.05, respectively). In the AXT group, both parameters were significantly increased compared with the control group (25.25 ± 3.4 vs. 20.64 ± 3.8, p < 0.05, and 51.77 ± 2.69 vs. 45.21 ± 6.56, p < 0.05, respectively). Also, the sperm velocity parameters including VCL, VSL, VAP, and LIN in the control group were significantly decreased compared with the fresh group. In the AXT group, VCL, VSL, VAP, and LIN were significantly increased compared with the control group (p < 0.05). About the STR parameter, AXT decreased it but was not significant (p > 0.05). However, no significant difference was observed between the two groups in the WOB parameter (Table 2).

Table 2.

Mean (±Standard Deviation) of Spermatozoa Velocity Parameters Using Computer-Assisted Sperm Analyzer in the Fresh, Control, and Astaxanthin Groups

Group	Total motility (%)	Progressive motility (%)	VCL (μm/s)	VSL (μm/s)	VAP (μm/s)	LIN (%)	STR (%)	WOB (%)
Fresh	61.11 ± 0.47^a	49.52 ± 0.53^a	99.39 ± 0.8^a	60.17 ± 0.47^a	69.83 ± 0.78^a	60.53 ± 0.59^a	86.16 ± 0.47	70.25 ± 0.68
Control	45.21 ± 6.56^b	20.64 ± 3.8^b	56.74 ± 5.25^b	32.80 ± 6.07^b	36.69 ± 7.08^b	57.80 ± 4.59^b	89.39 ± 3.76	64.66 ± 4.29
AXT	51.77 ± 2.69^c	25.25 ± 3.4^c	60.32 ± 2.89^c	37.67 ± 5.13^c	42.34 ± 5.14^c	62.45 ± 3.46^c	88.97 ± 4.28	70.19 ± 3.91

Within a single column, values with different superscripts (a, b, and c) differ significantly (p ≤ 0.05).

AXT, astaxanthin; LIN, linearity (VSL/VCL × 100); STR, straightness (VSL/VAP × 100); VAP, velocity of average path; VCL, velocity of curvilinear; VSL, velocity of straight line; WOB, wobble VAP/VCL.

Post-thawing viability

Eosin–nigrosin staining was used to assess the sperm viability. As shown in Figure 1, the freezing–thawing process significantly decreased the sperm viability in the control group compared with the fresh group (70.09 ± 5.30 vs. 45.76 ± 5.29, p ≤ 0.0001, respectively). Adding 1 μM of AXT increased the sperm viability after the freezing–thawing process in the AXT group compared with the control group (52.98 ± 7.23 vs. 45.76 ± 5.29, p ≤ 0.004, respectively).

FIG. 1.

The effect of ATX on sperm viability after the freezing–thawing process. *p ≤ 0.05. ATX, astaxanthin.

Post-thaw cytosolic ROS

DCFH-DA and DHE were used to assess the total H₂O₂ and O₂⁻ generation. As shown in Figure 2, the freezing–thawing process led to an increase in intracellular H₂O₂ and O₂⁻ in the control group compared with the fresh group (41.42 ± 7.22 vs. 63.05 ± 7.82, p ≤ 0.01, and 38.59 ± 5.34 vs. 62.75 ± 4.34, p ≤ 0.01, respectively). However, H₂O₂ and O₂⁻ in the AXT group were significantly lower (56.72 ± 6.55 vs. 63.05 ± 7.82, p ≤ 0.02, and 56.51 ± 5.52 vs. 62.74 ± 4.34, p ≤ 0.01, respectively) compared with the control group (Fig. 2).

FIG. 2.

The effect of AXT on ROS change by measuring intracellular hydrogen peroxide (DCFH-DA) and superoxide (DHE) following the sperm freezing–thawing process. *p ≤ 0.05. DCFH-DA, 2′,7′-dichlorofluorescein diacetate; DHE, dihydroethidium; ROS, reactive oxygen species.

PS externalization

To analyze the occurrence of early apoptosis in different groups, evaluation of PS in the sperm cell membrane, which is a diagnostic marker of apoptosis, was used. The addition of 1 μM of AXT significantly reduced PS externalization (AnV⁺/PI⁻) in the AXT group (3.95 ± 1.74 vs. 8.56 ± 2.06) compared with the control group, and the percentage of viable cells (AnV⁻/PI⁻) significantly increased in the treatment group (31.60 ± 4.88 vs. 21.39 ± 3.48) compared with the control group (Fig. 3).

FIG. 3.

The effect of AXT on early apoptotic sperm following freezing–thawing. Each column represents the mean ± SD. AnV⁻/PI⁺ only shows the percentage of necrotic spermatozoa. AnV⁺/PI⁺ shows the percentage of apoptotic and necrotic spermatozoa. AnV⁺/PI⁻ only shows the percentage of early apoptotic spermatozoa. A⁻/PI⁻ A⁺/PI⁻ shows the percentage of neither apoptotic nor necrotic spermatozoa (viable spermatozoa). *p ≤ 0.05. PI, propidium iodide; SD, standard deviation.

Post-thaw LPO

To evaluate LPO in different groups, the amount of MDA was measured. Our results showed that the use of AXT significantly reduced the MDA in the AXT group compared with the control group (1.47 ± 0.15 vs. 2.18 ± 0.40, p ≤ 0.001) (Fig. 4).

FIG. 4.

The effect of AXT on the sperm LPO after the freezing–thawing process. *p ≤ 0.05. LPO, lipid peroxidation.

Discussion

This study was an investigation to determine the effects of AXT on frozen–thawed sperm. The present study showed that the addition of 1 μM of AXT to the freezing media can increase motility and viability and decrease ROS, PS externalization, and membrane LPO in sperm cells.

Although the freezing of sperm is one of the achievements of modern assisted reproductive technology science, this process can do some structural and functional damage in the sperm due to the excessive generation of ROS and LPO of the sperm membrane, which can result in a decrease in the viability and fertilizing ability of spermatozoa.^39–41

Various antioxidants have been tested to reduce the damaging effects of cryopreservation of sperm.^6,42–44 However, the antioxidant capacity of sperm cells is inconsiderable in forbidding oxidative stress during the freezing–thawing process.⁴⁵ Also, sperm membranes have unsaturated fatty acids and can be damaged by ROS and LPO.⁴⁶

The present study shows the antioxidative properties of AXT on sperm cells after the freezing–thawing process, in that the use of 1 μM of AXT in the freezing media improves the quality of human sperm compared with the control group. Abdi-Benemar et al.⁴⁵ also showed similar results on ram semen, but their optimal doses were 2 and 4 μM. Other studies have shown the positive effects of AXT on human and animal sperm, so that using it in a pre-freeze media increases motility and viability and decreases endogenous ROS and LPO production.^{22,28,47–50}

In 2014, Song et al.⁵¹ demonstrated that H₂O₂ produces ROS by inhibiting endogenous antioxidants such as SOD and CAT, while AXT, by inhibiting H₂O₂, inhibits the production of endogenous ROS. However, AXT suppresses H₂O₂-induced ROS generation by protecting these antioxidant enzymes from H₂O₂. In the present study, we showed that AXT could reduce endogenous H₂O₂ and O₂⁻ in the treated group compared with the control group. Annexin V/PI was carried out to detect an early apoptotic change,⁵² which means the translocation of PS to the cell membrane's outer surface. Balancing pro-apoptotic and antiapoptotic substances in cells can regulate apoptosis.⁵³ Bax is a protein regulator that induces cytochrome C secretion from mitochondria during cellular stress. By competing with this regulator, Bcl-2 inhibits cytochrome C secretion and thus inhibits apoptosis.⁵⁴

Some studies have demonstrated that H₂O₂ causes the translocation of pro-apoptotic members such as Bad and Bax to the mitochondria and that of antiapoptotic members to the cytosol. AXT can prevent apoptosis by reducing H₂O₂ generation.⁵¹ Also, the Nrf2/HO-1 signaling pathway controls the numbers of cytoprotective genes that can combat the detrimental effects of oxidative stress.⁵⁵ AXT, by inducing the Nrf2/HO-1 antioxidant pathway, decreases ROS production in human umbilical vein endothelial cells.⁵⁶ AXT also reactivates the Nrf2/HO-1 signaling pathway on mRNA and protein levels in sperm cells and reduces apoptosis.⁵⁷ The present study shows that AXT can reduce early apoptosis in the AXT group compared with the control group following the freezing–thawing process.

Heidari Khoei et al. showed that AXT could reduce MDA in mouse sperm cryopreservation. Also, our study results showed that AXT could reduce MDA in the treated group compared with the control group.⁵⁸ When ROS increases in the cell, it attacks the unsaturated fatty acids and produces LPO. MDA is an LPO product used in biochemical experiments to detect the extent of peroxidative damage produced in sperm.^59,60 LPO causes the loss of intracellular adenosine triphosphate, which can cause axonal damage, reducing sperm viability, increasing morphological defects of the sperm, and thus reducing sperm motility.^61,62 The presence of antioxidants in the sperm cell prevents LPO of sperm membranes.⁴⁵ Therefore, the sperm cell can maintain its metabolic activity, increasing sperm viability and maintaining its motility after freezing.⁶³

In conclusion, our findings showed that spermatozoa treatment with 1 μM of AXT before the freezing–thawing process has protective effects against oxidative stress and could diminish the destructive effects of this process on sperm quality.

Footnotes

Authors' Contributions

T.G., M.S.N., S.N., H.R., and F.A. are from Tehran University of Medical Sciences (Tehran, Iran), where education and research are the primary functions.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This article was funded by Tehran University of Medical Sciences.

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The Effect of Astaxanthin on Motility,Viability,Reactive Oxygen Species,Apoptosis,and Lipid Peroxidation of Human Spermatozoa During the Freezing–Thawing Process

Abstract

Introduction

Materials and Methods

Semen collection

Experimental design

Freezing and thawing

Assessment of motility

Assessment of sperm viability parameter

Assessment of H2O2 and O2− by flow cytometry

Detection of PS externalization

Measurement of LPO levels

Statistical analysis

Results

Determination of effective dose of AXT

Sperm motility test (CASA)

Post-thawing viability

Post-thaw cytosolic ROS

PS externalization

Post-thaw LPO

Discussion

Footnotes

Authors' Contributions

Author Disclosure Statement

Funding Information

References

Assessment of H₂O₂ and O₂⁻ by flow cytometry