What’s Known

Despite the considerable development made in the sperm cryopreservation techniques, it has numerous harmful effects on sperm quality. The exact mechanism of this damage is not clear; however, the reduction in the sperm motility, increase in the mortality rate, and production of reactive oxygen species are the common damages incurred during cryopreservation and calcium overload conditions.

What’s New

Intracellular calcium content decreased after sperm cryopreservation, and it was found that the cryoinjury was not related to calcium overload. Calcium chelation with EGTA confirms this conclusion. EGTA showed no protective effect on fresh or cryopreserved human sperm. Adenosine triphosphate was decreased, while reactive oxygen species production increased in the EGTA-treated sperm.

Introduction

Calcium ion plays an important role in sperm motility, acrosome reaction, and fertilization. ^{1 - 3} Sperm exposure to progesterone or zona pellucid proteins induces hyperactivation, which is accompanied by intracellular Ca²⁺ (Ca²⁺_i) elevation. ⁴ An increase in Ca²⁺_ioccurs due to the entrance of calcium through its channels and/or its release from intracellular stores. ^{5 , 6} Ca²⁺_ielevation affects mitochondria and produces more adenosine triphosphate (ATP). ⁷ It also activates many signaling cascades and increases reactive oxygen species (ROS) production by mitochondria and membrane oxidizing enzymes. ^{8 , 9} In addition to the beneficial effects of calcium, its overload can potentiate a pathological condition by producing excessive amounts of ROS and oxidative stress, reducing ATP synthesis, and forming permeability transition (PT) pores in the mitochondrial inner membrane. ^{10 , 11} PT pores are permeable to solutes of <1500 Dalton. ¹² Mitochondrial swelling and its outer membrane rupture are the consequences of PT pore opening. Furthermore, calcium overload causes cytochrome C release from mitochondria, which is a key event in apoptosis. ¹³ Some researchers, such as Treulen, reported that PT pore formation and cell apoptosis were associated with an increment in Ca²⁺_i, mitochondrial membrane potential (MMP) dissipation, ATP level reduction, ROS production, and deterioration of plasma membrane integrity. ¹⁴

Sperm cryopreservation is used in infertility treatment clinics as a therapeutic option for various conditions, including storage and maintenance of the donor’s sperm for future usage, preservation of fertility following chemotherapy, radiotherapy, or various surgical procedures such as vasectomy. Despite the considerable development made, cryopreservation has numerous harmful effects on the structure and function of sperm, namely cryoinjury. ¹⁵ A reduction in sperm motility and viability, acrosomal loss, the increase in ROS production, damage to the mitochondria, and DNA fragmentation were observed in semen samples that thawed after cryopreservation. ^{16 , 17} Cryoinjury changes the membrane permeability to some ions such as calcium, ^{14 , 18} which leads to their entrance. Although some studies have revealed other aspects of this issue, they have shown that after thawing, Ca²⁺_idecreases significantly. ^{19 , 20} Calcium overload has been reported as a postulated mechanism for sperm damage. ^{14 , 18} As a result, and due to these harmful effects, some researchers believe that using calcium chelators would be effective in maintaining sperm viability and function. ²¹ Calcium chelation by agents such as ethylene glycol-bis (2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA) inhibited the zona pellucida-induced acrosome reaction and apoptosis, and increased sperm motility. ^{22 , 23} It has been shown that adding both EDTA and EGTA to freeze-drying medium protected rabbits’ sperm DNA. ²⁴ Adding EGTA and EDTA to cryopreservation medium improved the developmental ability of oocytes and decreased boar sperm DNA fragmentation. ²⁵ The protective effects of EDTA on cryopreserved human sperm have also been reported. ²⁶

There are several studies with conflicting results, suggesting that the use of calcium chelators cannot be effective since spermatozoa require calcium boost to succeed in reproduction. ^{27 , 28} Studies have shown that calcium chelating by these chelators could cause harmful effects, reduce sperm motility, disrupt sperm capacitation, decrease tyrosine phosphorylation, and block Ca²⁺_istores release. ^{14 , 28 , 29} A decrement on boar sperm motility was reported after the use of 6 mmol/L EDTA. ²⁵

The incubation of bovine cryopreserved sperm with EDTA containing medium could not prevent the mitochondrial permeability damage. ¹⁴ In addition, EDTA has time and dose-dependent spermicidal activity. ^{24 , 30}

EGTA is known as a specific calcium chelator, which has a lower affinity for other cations, whereas EDTA function is not specific for calcium and chelates several other metal ions such as Mg, Mn, Co, Zn, Pb, Cu, and Fe. It has a potential inhibitory activity on metal cation-dependent enzymes.

With regard to these controversies and to elucidate the exact mechanism of EGTA function on fresh and cryopreserved-thawed human sperm, we assessed the sperm kinematics, Ca²⁺_i, apoptosis rate, ROS production, membrane integrity, mitochondrial membrane potential, and ATP content in the EGTA-incubated fresh and thawed sperms.

Materials and Methods

Samples Preparation and Cryopreservation

Semen samples were obtained randomly from 20-40-year-old fertile men who had referred to Shiraz Infertility Center from March 2017 to 2018 to undergo a routine semen analysis. The local Ethics Committee of Shiraz University of Medical Sciences approved this research (IR.sums.REC.1391-6159). All donors had signed a written informed consent. They were healthy, not drug addict or alcohol and dietary supplement consumers. A routine semen analysis was performed and oligozoospermic, azoospermic, teratozoospermic, and leukocytospermia samples, as well assemen with abnormal appearance, pH, and viscosity, were excluded from the study based on the guidelines from World Health Organization, 2010.31 The semen characteristics of fertile men were considered as: semen volume, more than 1.5 mL; total sperm number, at least 39 million per ejaculate; sperm concentration, at least 15 million per mL; vitality, more than 58% live; progressive motility, at least 32%; total (progressive ‏and non-progressive) motility, at least 40%; and morphologically normal forms, 4.0%. ³¹ After the usual routine semen analysis, the samples were transferred to our laboratory in less than one hour.

Forty samples were washed with Ham’s F-10 medium (N6633, Sigma, Germany) and incubated at 37 °C, 5% CO₂, for one hour. The swim-upped sperm were counted and sperm viability and kinematics were assessed. All procedures were done according to the guidelines by World Health Organization, 2010. ³¹ The samples were diluted to 10×10⁶ sperm/ml and, randomly, divided into fresh (F) and cryopreserved-thawed (CT) groups.

For sperm cryopreservation, the samples were mixed (1:1) gradually with cryoprotective medium (11010010, ORIGIO, Denmark) for 30 minutes, loaded into a cryotube, placed on nitrogen vapor for 30 minutes, then, plunged into liquid nitrogen for at least one month. Thawing procedure was done at 37 °C.

The F and CT samples were divided into control and EGTA-treated subgroups. The sperm were incubated with Ham’s F10 and 1 mM EGTA (E3889, Sigma, Germany), in control and EGTA subgroups, respectively. In CT group, EGTA was added to the cryoprotective medium. All assessments were performed after 30 minutes of incubation in F group and after 30 minutes of thawing in washed CT sperm group.

Assessing Sperm Kinematics

Sperm kinematics was recorded by a Video Test (VT) sperm analyzer 3.1 software (VT sperm 3.1, Russia), using an Olympus CX41 (nimax GmbH, Germany) microscope. The percentage of progressive, non-progressive, immotile sperm, and motility parameters, such as straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), and curvilinear velocity (VCL, μm/s) of at least 200 sperms were evaluated for all samples. ³¹

Assessing Membrane Integrity

Hypo-osmotic swelling test (HOS test) was used to assess membrane integrity. ³¹ Hypo-osmotic medium contained 0.735 g of sodium citrate dehydrate (W302600, Sigma, Germany) and 1.351 g of D-fructose (F0127, Sigma, Germany) in 100 mL of purified water. This solution was mixed 1:1 with sperm samples. Spermatozoa with intact membranes were swollen within five minutes and the shape of all flagella were stabilized after 30 minutes. At least, 100 sperms were evaluated after five and 30 minutes.

Assessing ROS Production

ROS production was assessed using the chemiluminescent method. ³¹ Freshly prepared luminol (A8511, Sigma, Germany) with a concentration of 250 μM and 12 U/mL horseradish peroxidase (P6782, Sigma, Germany) were added to 300 μL of treated sperm in 96 black well plates. Luminol reacts with hydrogen peroxide and emits energy as a photon; horseradish peroxidase enhances the oxidation of luminal. Light emissions were recorded using a microplate reader with the ability to assess luminescence (Synergy HT, Bio Tek, Germany) every 10 seconds for 30 minutes and was reported as the relative light unit (RLU).

Assessing Apoptotic Rate

Apoptotic rate was assessed using an Annexin V–FITC, PI kit (phosphatidyl serine detection kit, IQ Products®, Netherlands). According to the manufacturer’s instruction, the cells (1.0×10⁶ cell/mL) were washed by cold calcium buffer. The suspensions (100µL) were incubated with 10 µL of FITC-conjugated Annexin-V for 20 minutes and 10 µL of propidium iodide (PI) for at least 10 minutes on ice in dark. At least, 50,000 sperm were analyzed using a BD FACSCaliberTM flow cytometer (BD Biosciences, USA). The data were analyzed using FlowJo® (version 10.4.1) software. The cells were classified into the following four categories according to staining with Annexin and/or PI (stained +, and unstained -): (Q1) necrotic sperm (Annexin-V (-)/PI (+)), (Q2) late apoptotic cells (Annexin-V (+)/PI (+)), (Q3) early apoptotic cells (Annexin-V (+)/PI (-)], and (Q4) viable cells (Annexin-V (-)/PI (-)).

Assessing Ca2+i

The suspension containing 1×106 sperm was loaded with 4 μM fluo-3/AM (73881, Sigma, Germany) and 0.08% pluronic acid F-127 (p2443, Sigma, Germany) for 30 minutes. The samples were washed and 5 μM PI (p4170, Sigma, Germany) was added to the medium. The incubation was done in darkness at 37 ˚C. ³² At least, 50,000 sperm were analyzed using a BD FACSCaliberTM flow cytometer and FlowJo® software. The cells were classified into a quadrant according to their Ca²⁺_icontent and viability. Q1-Q4 represent the dead sperm with low Ca²⁺_i(Fluo3-AM^-, PI⁺), dead sperm with high Ca²⁺_i(Fluo3-AM⁺, PI⁺), live sperm with high Ca²⁺_i(Fluo3-AM⁺, PI^-), and live sperm with low Ca²⁺_i(Fluo3-AM^-, PI^-), respectively. Mean Fluorescence intensity (MFI) of fluo-3/AM was calculated by the software.

Assessing MMP

MMP was assessed using MitoProbeTM JC1 assay kit (M34152, Molecular Probes, USA). According to the manufacturer’s instruction, 1×10⁶ sperm were suspended in 1 mL warm phosphate-buffered saline, and 2 μM tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was added to the medium. After 30 minutes incubation in 5% CO₂ at 37 °C, the assessment was performed by a flow cytometer using 488 nm excitation with 530 nm and 585 nm bands pass emission filters. JC-1 dye exhibits potential-dependent accumulation in mitochondria. Its aggregation at high mitochondrial membrane potential yields a red to orange colored emission at 585 nm. At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence with the emission of 530 nm. Changing fluorescence from red to green means that the membrane potential has decreased. Fifty μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an MMP disrupter concurrently with JC-1 to confirm that JC-1 was sensitive to mitochondrial membrane changes (positive control). The cells, which were concurrently treated with JC-1 and CCCP showed green fluorescence.

Assessing Intracellular ATP

The ATP contents of spermatozoa were determined using the bioluminescence assay kit CLS II (Roche Diagnostics GmbH, Germany). According to the manufacturer’s instruction, 25 µL of the samples (1×10⁶ sperms per mL) were added to 225 µL boiling extraction medium, which consisted of 100 mM Tris–HCl and 4 mM EDTA (pH 7.75). After boiling for two minutes at 100 ºC, samples were centrifuged at 1,000 g for 60 seconds and their supernatant was frozen in -20 ºC until further assessment. In the assessment day, a serial dilution of an ATP standard solution (ranging between 10^-5 to 10^-10 M) was prepared, and 50 µL of luciferase reagent was added to 50 µL of the samples and standards. After a one-second delay, The luminescence was measured using a microplate reader (Synergy HT, Bio Tek, Germany) for 10 seconds. ATP standard curve was plotted and the ATP concentrations of all samples were calculated. The data were expressed as nM of ATP per 10⁶ sperms.

Statistical Analysis

Statistical analyses were performed using SPSS software, version 16.0. Data normality was checked using Shapiro–Wilk test. Sperm motility, membrane integrity, and ROS production had a normal distribution, and their comparisons were done by ANOVA. Other data were statistically analyzed using non-parametric test (Kruskal Wallis). The data are presented as mean±SEM, and P<0.05 was considered statistically significant.

Results

Sperm Kinematics

Sperm progressive and non-progressive motility reduced significantly (P=0.0001) after thawing, and the percentage of immotile sperms increased (P=0.0001) (table 1). EGTA decreased the percentage of progressive motile sperms in F and CT samples, and this reduction was greater in F group (36.5%) than the CT group (16.9%). Velocities of motile sperms were recorded and analyzed. The results showed that the VSL and VAP reduced after thawing. EGTA decreased both VSL and VAP in F sperm (P=0.0001), whereas VCL reduced significantly in CT group (P=0.01) (table 1).

Membrane Integrity

Assessing sperm membrane integrity by HOS test showed severe damage to sperm membrane integrity after thawing, and using EGTA did not affect the cell membrane integrity (figure 1).

Figure 1. The graph represents the effect of EGTA on membrane integrity of fresh and cryopreserved-thawed sperm. ANOVA test was used to compare the membrane integrity between the experimental groups; #Significant difference between fresh and cryopreserved-thawed sperm (P<0.0001). Results are expressed as mean±SEM.

ROS Production

Chemiluminescence signals are indicators of ROS production and are recorded as RLU. ROS production increased after thawing and EGTA augmented the ROS generation in both CT and F groups (P=0.0001) (figure 2).

Figure 2. The graph represents the effect of EGTA on the ROS production offresh and cryopreserved-thawed sperm. ANOVA test was used to compare ROS production between the experimental groups; *Significant difference with the related control group (P<0.0001), #Significant difference between the fresh and cryopreserved-thawed sperm (P=0.01). Results are expressed as mean±SEM.

Sperm Apoptosis

Cryopreservation reduced the percentage of live cells and increased the apoptosis rate. EGTA decreased sperm viability in F group significantly (P=0.009), but it did not affect CT group apoptosis (figure 3).

Figure 3. The graph represents the effect of EGTA on the apoptotic rate of fresh and cryopreserved-thawed sperm. Kruskal-Wallis test was used to compare the viability and mortality rate between experimental groups. Flow cytometric analysis of EGTA-treated and untreated fresh and cryopreserved-thawed sperm stained with AnnexinV-FITC and PI; (Q1) necrotic cells (Annexin-V (-)/PI (+)), (Q2) late apoptotic cells (Annexin-V (+)/PI (+)), (Q3) early apoptotic cells (Annexin-V (+)/PI (-)), and (Q4) viable cells (Annexin-V (-)/PI (-)). (B) Stacked bar of sperm apoptosis in fresh and cryopreserved-thawed sperm and the effect of EGTA on them. *Significant difference with the related control group (P=0.05), #Significant difference between the fresh and cryopreserved-thawed sperm, (P=0.05). Results are expressed as mean±SEM.

Intracellular Calcium

Fluo3/Am and PI staining were used to assess Ca²⁺_iin live and dead cells (table 2). Cryopreservation-thawing process significantly decreased the percentage of live cells with high Ca²⁺_i(Fluo3-AM⁺, PI^-) and increased the percentage of dead cells with low Ca²⁺_i(Fluo3-AM^-, PI⁺) (P=0.0001). MFI of Fluo-3 AM was lower in the CT group than the F group (table 2). EGTA reduced the count of live cells with high Ca²⁺_i(Fluo3-AM⁺, PI) and increased the live cells with low Ca²⁺_i(Fluo3-AM^-, PI^-) in F group. EGTA decreased MFI in both F and CT groups.

Mitochondrial Membrane Potential

MMP was assessed using JC-1 staining via flow cytometric method (table 3). Red and green florescent intensity indicated the mitochondria with high and low membrane potential, respectively. Red/green ratio was decreased by 59.8% in CT group compared to F group. EGTA did not have any significant effect on MMP of both F and CT groups.

Intracellular ATP

ATP content did not change after the cryopreservation-thawing process, but adding EGTA reduced ATP in both F and CT sperm (P=0.02) ( figure 4).

Figure 4. The graph represents the effect of EGTA on intracellular ATP of fresh and cryopreserved-thawed sperm. Kruskal Wallis test was used to compare intracellular ATP between the experimental groups. *Significant difference with related control group (P=0.01). Results are expressed as mean±SEM.

Discussion

A decrease in motility and an increase in cell ROS production, accompanied by Ca²⁺_ireduction, were the damages caused by the cryopreservation-thawing procedure. Cryopreservation thawing also decreased sperm viability and disrupted MMP. The damaging effects of cryopreservation on sperm motility, viability, cell apoptosis, PT-pore formation, MMP disruption, antioxidants reduction, and ROS production are the well-known damages happening after the cryopreservation-thawing procedure, ^{16 , 17} but the mechanism(s) of these destructive effects and their correlation with Ca²⁺_ihave not been conclusively investigated or proven yet. In the present study, in order to clarify the role of calcium in cryoinjury and its relation to mitochondrial function and finding the importance of membrane integrity, all aspects of sperm function were evaluated and considered together.

The elevation of Ca²⁺_iafter cryopreservation has been reported by some researchers. ^{14 , 18} In contrast, some other studies have shown the reduction of Ca²⁺_iafter thawing. ^{20 , 33} Kumaresan et al. showed that the proportion of sperm with high calcium decreased during the initial five minutes of incubation. ³³ Based on the findings of these studies, it is possible that Ca²⁺_iis increased promptly after a cryopreservation-thawing process, but this increase cannot be sustained more than a few minutes. Our study showed that the rate of live-high Ca²⁺_isperm was reduced after cryopreservation, the percentage of dead-low Ca²⁺_isperm increased, and significant damage to membrane integrity was observed (figure 2). It is possible that Ca²⁺_ireduction was relative to calcium extrusion from the disintegrated cell membranes, induced by a cryopreservation-thawing process. We assessed the calcium levels after 30 minutes of incubation, while it might be expected that a time-dependent calcium measurement could have provided us with more precise information.

Extracellular calcium chelating with EGTA reduced the MFI of Fluo3-AM in F and CT groups. The percentage of live sperm, containing a higher level of calcium, was reduced significantly in EGTA-treated fresh sperm, but the same results were not obtained in thawed sperm. EGTA chelated the extracellular calcium; therefore, it was expected that calcium entrance would be inhibited and Ca²⁺_ireduced in the fresh sperm. Using calcium-imaging method and tracing the calcium entrance could confirm our suggestion. However, in cryopreserved sperm, the cell membrane was severely damaged and calcium homeostasis was disrupted in a considerable number of sperms.

Cryopreservation reduced sperm motility significantly, and a similar reduction in motility was observed in fresh EGTA-treated sperm. The sperm motility was directly related to Ca²⁺_ilevel; ^{34 , 35} furthermore, intact flagella membrane was required for normal motility. ^{36 , 37} The inhibitory effect of EGTA on sperm motility has also been shown in other studies. ^{28 , 38} It has been shown that EGTA decreased sperm motility time-dependently. ³⁸ EGTA increased mouse sperm motility immediately after use, but the motility gradually decreased and, within 10 minutes, all sperms were immotile. ³⁹ The observed significant reduction of sperm motility in groups containing EGTA could have been induced by Ca²⁺_ilevel reduction. In addition, an intact membrane is also required for normal sperm motility.

Calcium overloads cause ROS production and PT pore generation, and inhibit ATP production. ^{7 - 9} Ca²⁺_ientrance to the mitochondria activates the rate-limiting enzymes of the tricarboxylic acid cycle, increases oxidative phosphorylation, and enhances ATP synthesis. Oxidative and nitrosative radicals arise from the respiratory chain and might lead to mitochondrial damage if the cell encounters calcium overload condition. Oxidative or nitrosative stress promotes the opening of PT pore, causes ATP depletion and apoptosis. The release of cytochrome C from the intermembrane space of mitochondria is a major trigger to activate the caspase cascade, initiating the programmed apoptotic cell death. ^{40 , 41} In some studies, a calcium chelator was used for the preservation of sperm quality after cryopreservation thawing and freeze-drying procedures. ^{25 , 42} To the best of our knowledge, no study has investigated the role of EGTA on ROS production or apoptosis of human sperm. This study showed that ROS production increased in CT group. EGTA did not reduce ROS, but it elevated ROS levels. ROS increased after thawing, and EGTA elevated the ROS level in thawing sperm as well as F sperm. The mitochondrial membrane disintegration and the leakage of the electron from the respiratory mitochondrial chain can be a reason for the excessive ROS generation after thawing. ⁴³ To clarify the mitochondrial activity, we assessed the MMP and cellular content of ATP. We showed that the MMP in CT group was impaired, and EGTA did not affect MMP in F and CT sperm groups. This observation elucidates the destructive effect of cryopreservation on mitochondria. ¹⁴

Cellular ATP content was reduced after EGTA treatment in both F and CT groups. Removed calcium from extracellular fluid produced a fast Na+-dependent human sperm depolarization. It seems that in the absence of calcium, sodium enters the cell through calcium channel, which could stimulate the Na+-K+ ATPase and increase cellular ATP consumption. ⁴⁴

The apoptotic rate increased after thawing, and EGTA could not inhibit this process. Although calcium overload has harmful effects on cells, sperm cryoinjury is mainly related to cell membrane disintegration. Most studies that used EGTA and EDTA in cryopreservation or freeze-drying media focused on their protective function on DNA and nucleases activity; however, there are some studies which have shown the inhibitory effect of EGTA and EDTA on viability. ^{14 , 25 , 38}

This study had some limitations. As previously described, using calcium-imaging methods and live calcium recording could have provided us with a better explanation for the role of calcium on sperm function in fresh and cryopreserved-thawed conditions. Furthermore, we did not assess the apoptotic indicators such as caspases activities. Actually, Annexin V only detects the exposed phosphatidylserine, which is translocated to the outer layer of the cell membrane. To come to a sound judgment about cell apoptosis, measuring apoptotic and anti-apoptotic factors seems to be necessary.

Conclusion

Cryopreservation reduced sperm motility and viability. It also increased ROS production, induced cell apoptosis disrupted MMP, and reduced Ca²⁺_i. According to our findings, EGTA did not have protective effects on human cryopreserved sperm. Therefore, we concluded that sperm cryoinjury was mainly related to the damage made to the membranes of intracellular organelles and the cell membrane. Thus, it could be suggested that improving the freezing and thawing methods and developing the new cryoprotective medium could reduce the sperm cryoinjury.

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