Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells

Abstract

Cryopreservation of spermatogonial stem cells is considered as a useful procedure for preserving fertility in children with testis cancer. SSCs were isolated from testes mice, and then antioxidant was added to the freezing medium. The Bax expression level in antioxidant groups was significantly (P ≤ 0.05) lower than the control group and a significant rise of Bcl2 expression was detected in the antioxidant groups. ROS production with antioxidant was significantly lower compared with the control group. Cryopreservation with the addition of the antioxidants can help increase the number of SSCs and improve the quality and viability of these cells after cryopreservation.

Keywords:

• Catalase
• cryopreservation
• spermatogonial stem cell
• α-tocopherol

Introduction

Spermatogenesis is a continuous process during male reproductive period and has the ability to generate chains of differentiating spermatogonia (Conrad et al. 2008). The tissues of testes are unique that their performance depends on the continued proliferation and differentiation of spermatogonial stem cells (SSCs); therefore, for a normal spermatogenesis, a pool of SSCs is required in testis (Brinster et al. 1998). These cells are located on the basement membrane of seminiferous tubules and surrounded by Sertoli cells (Abbasi et al. 2015, Clermont 1972). In cancer patients who are treated with radiotherapy and chemotherapy, spermatogenesis may be damaged, followed by infertility (Cokkinides et al. 2007). Freezing is one of the most important methods for preserving fertility and long-term storage and availability of SSCs (Aliakbari et al. 2016, Time 1985). SSCs from mature spermatozoa are more resistant against possible damage caused by the freezing process because relatively, SSCs have less metabolic activity than mature spermatozoa. However, recent findings about SSCs cryopreservation are not satisfactory (Kenney et al. 2001, van Casteren et al. 2009). SSCs incur large volume changes due to the various osmotic pressure during freezing (Attaran et al. 1966). The rapid changes in osmotic pressure affect the structure of cell membranes and intracellular structures (Gittes et al. 1972). Freezing can increase the reactive oxygen species (ROS) formation (Bilodeau et al. 2000) and this can cause oxidative stress on the tissues of the cells. Oxidative stress is evaluated by the balance between the generation and degradation of ROS within a tissue or cell suspension (Roca et al. 2005, Swi Chang 2007).

Oxygen, a very active molecule, can produce ROS that can cause damages in vital tissues and cell functions and thus, endangering cell viability. Superoxide and hydrogen peroxide are known as the most active free radicals of oxygen and they are active workers in their surrounding environment (Baumber et al. 2000, Weissig and Guzman-Villanueva 2015). Hydrogen peroxide is widely used as an oxidant. H₂O₂ is produced during the oxidation of biological molecules (e.g. glucose) by dissolved oxygen in the presence of corresponding oxidase (Rhee 2006). To prevent oxidative stress in cellular structures such as membranes, structural proteins, enzymes and nucleic acids, the presence of a complex network of antioxidants and enzymes are necessary in an organism. Oxidation of antioxidants can remove free radicals by terminating the reaction chain and protecting molecules from oxidative damage (Kothari et al. 2010). Until now, two different kinds of antioxidants are known: enzymatic type (such as catalase, superoxide dismutase and a few super-type peroxidases) and non-enzymatic type (such as vitamin E, vitamin C). Both kinds can neutralize excessive ROS formation and prevent cellular structure damage (Fernández‐Santos et al. 2007).

In this study, we used two different types of antioxidants: (i) α-Tocopherol, a water-soluble analogue of vitamin E that promotes the peroxidation of unsaturated lipids in cell membrane and protects intracellular phospholipid. During cryopreservation, vitamin E analogue has positive effects on membrane integrity of cryopreserved cells (Dad et al. 2006). (ii) Catalase is a common enzyme found in all living organisms exposed to oxygen. Catalase, an enzymatic antioxidant exists in peroxisomes, which can convert hydrogen peroxide metabolism to water and oxygen and neutralize the toxic effects of free radicals (Teke 2014). Scientists believe that catalase is an important enzyme that helps protect cells from oxidative damage by ROS, and it has also been found that a molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen (Bilodeau et al. 2002, Câmara et al. 2011).

Our finding shows a novel innovative cryomedium for SSCs’ cryopreservation that prevents the release of free radicals during the process and enhances SSCs survival rate after thawing, by using catalase and α-tocopherol as two important antioxidants in the cryomedium.

Methods

Animals

SSCs were obtained from 3- to 6-day-old male National Medical Research Institute (NMRI) mice, (day of birth is 0). They were maintained under standard laboratory conditions. Animal experiments were approved by the Ethics Committee of Tehran University of Medical Sciences and performed in accordance with the University guidelines.

Isolation of SSCs

In order to isolate SSCs, two-step enzymatic digestion was used. Testes of NMRI mice were collected and washed in phosphate-buffered saline (PBS; Sigma, Steinheim, Germany). The tunica albuginea was removed and remaining part of the testes were cut into small pieces and transferred to the digestion medium consisting of collagenase type IV (1 mg/mL; Sigma), DNase (10 μg/ml; Sigma), hyaluronidase (0.5 mg/mL; Sigma) in Minimum Essential Medium Eagle solution (MEMα; Sigma) for 20 min by shaking at 37 °C in a 5% CO₂ until the tubules were separated. After that, cells were centrifuged at 1500g for 5 min and washed twice with PBS. Finally, the second step digestion was performed for testis cells suspension with the same enzymes as above (15 min). Then, the digested cells were washed with PBS (Guan et al. 2009).

Cryopreservation

Immediately after cell isolation, the following freezing media were added to the cell suspension: the basic freezing medium (control group) consisted of dimethyl sulfoxide (DMSO: 1.4 M; Sigma), 10% fetal bovine serum (FBS; Sigma) and MEM-α (Sigma) (Izadyar et al. 2002). In the treatment groups, Catalase (20 and 40 μl/ml) (Sigma) and α-tocopherol (100 and 200 μl/ml) (Sigma) were added to the basic freezing media. Cells were then placed in a freezing container at −80 °C for at least 1 day. After overnight storage, cryovials containing frozen cells were immersed into liquid nitrogen for 1 week.

Thawing procedure

After removal from liquid nitrogen, samples were maintained at room temperature for 30 s, then, cells were held in water bath 37 °C for 2 min. The cryovial contents were transferred to a tube with pre-warmed medium (MEM+%10 FBS).The cells were washed two times with medium by centrifuging at 3200g for 5 min (Izadyar et al. 2002). After removing the supernatant solution, the pellet cells were then taken for viability assessment, intracellular ROS measurement, flow cytometry and gene expression analysis.

Flow cytometry

Flow cytometry technique was performed using testicular cells before cryopreservation by applying standard procedures. Among them, 10⁶ were centrifuged at 1200g for 7 min and the cell pellet was re-suspended in 100 μl of PBS/FBS. To identify Stra8 positive cells, 10 μl primary antibody (Anti-Stra8 antibody; Abcam, Cambridge, MA) was added to the cells (1 h, at room temperature). Cells were washed in 1 ml of PBS/FBS and 10 μl secondary antibody (Donkey Anti-Rabbit; Abcam) conjugated with fluorescein isothiocyanate (FITC) was added (1 h, 4 °C). Cells used as controls were not treated with antibodies. Cells were kept on ice in the dark until analysis by flow cytometry (Baazm et al. 2013).

Cell viability

Cell viability was measured by methylthiazoltetrazolium (MTT; Sigma) assay before and after cryopreservation. Briefly, 400 μl MEM and 40 μl MTT were added to at least 400 cells and incubated for 4 h at 37 °C after which the media was replaced with 400 μl DMSO. The cells were kept at room temperature for 30 min, and then the optical density (OD) at 570 nm was measured using a microplate reader.

Intracellular ROS measurement

CM-H2DCFDA (General Oxidative Stress Indicator) can detect intracellular H₂O₂ and was used for intracellular ROS measurement before and after cryopreservation. DCFDA (10 μl, Sigma, Life Technologies C6827) was added to cells and incubated at 37 °C for 25 min. The cells were washed two times with PBS by centrifuging at 2500g for 5 min. Green fluorescence was evaluated by flow cytometry between 500 and 530 nm (Rhee et al. 2010).

Real-time polymerase chain reaction (real-time PCR)

After cryopreservation, expression levels of BAX and BCL2 were studied by real-time PCR. The primer sequences that were used in this study have been shown in Table 1. Total RNA was extracted by RNease Kit (Ready Mini Kit; Qiagen, Valencia, CA), according to the manufacturer’s catalog. cDNA synthesis was performed by reverse transcription kit (Transcriptor First Strand cDNA Synth, Roche, Indianapolis, IN) with 1 μg of total RNA according to the manufacturer’s catalog. Real-time PCR was performed using Applied Bioscience 7500 fast with SYBR Green detection for analysis. Melt curve analysis was performed after each run to check for the presence of non-specific PCR products and primer dimers. All samples were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal control) using the comparative CT method (ΔΔCT).

Table 1. The primers sequence of GAPDH, BCL2 and BAX genes.

Gene name	Sequence
GAPDH	For: 5'-AGCAAGGACACTGAGCAAGAG-3'
	Rev: 5'-GCAGCGAACTTTATTGATGGT-3'
BCL2	For: 5'-GGGGTCATGTGTGTGGAG-3'
	Rev: 5'-TCACTTGTGGCCCAGGTA–3'
BAX	For: 5'-CTGGATCCAAGACCAGGGTG-3'
	Rev: 5'-CCTTTCCCCTTCCCCCATTC-3'

Statistical analysis

Results were expressed as mean ± standard deviation (SD). The statistical significance between the mean values was determined by analysis of variance (ANOVA) followed by Tukey’s post hoc test with P≤0.05 as the statistically significant criterion. A total of three independent experiments were performed.

Results

Purification of SSCs

After enzymatic digestion, stra8 antibody was used to determine the purity of the isolated cells by flow cytometry. Flow cytometry analysis showed that 46.84% of all cells expressed stra8 (Figure 1).

Figure 1. Flow cytometry analysis for the detection of Stra8 marker in testis isolated cells. Of testicular cells, 46.84% expressed Stra8 marker.

Effect of cryopreservation on viability SSCs

The results from MTT assay showed that the majority of the cells (89.85 ± 6%) (P≤0.05) were viable immediately after enzymatic digestion. One week after cryopreservation under the different conditions, SSCs were thawed. The survival of cryopreserved cells (70.65 ± 6%) was significantly lower than fresh cells (89.85 ± 6%) (P≤0.05). Furthermore, the viability studies showed that adding catalase and α-tocopherol to the freezing medium exert significant and dose dependent effect on survival rate of frozen cells. Cell viability was significantly (P≤0.05) increased by the addition of 40 μl catalase (81.45 ± 5%) to the freezing medium compared with 20 μl catalase (77.65 ± 4%) and the control group (70.65 ± 6%). The survival rate of cryopreserved cells in the presence of 200 μl α-tocopherol (79.31 ± 4%) was significantly (P≤0.05) higher than the cells treated with 100 μl α-tocopherol (76.41 ± 4%) and the control group (70.65 ± 6%). Among the different conditions studied in the present work, the best results were obtained using 40 μl catalase and 200 μl α-tocopherol. These two concentrations were used for further studies (Figure 2).

Figure 2. MTT analysis for assessment of viability in different treatment groups. Data show means ± SD; ^****P≤0.0001.

Gene expression

In this study, for the evaluation of optimal condition for SSCs cryopreservation, we examined the level of Bax and Bcl2 expression. These results indicated that the level of Bax expression in the catalase and α-tocopherol groups was significantly (P≤0.05) lower than the control group and the lowest Bax expression was found when 40 μl catalase was added to the freezing medium. A significant (P≤0.05) rise of Bcl2 expression was detected in the catalase and α-tocopherol groups, and the cells treated with catalase had the highest Bcl2 expression (Figure 3A and B).

Figure 3. (A) BCL2, BAX and GAPDH1 RT-PCR. (1)100 bp DNA ladder, (2) negative control of RT-PCR (H2o), (3) control, (4) α-tocopherol and (5) catalase. (B) Expression pattern of apoptotic (Bax) and antiapoptotic (Bcl2) genes after cryopreservation analyzed by real-time PCR. Levels of Bax and Bcl2 were significantly decreased and increased respectively after adding 40 μl/ml catalase to the basic freezing medium. Data show mean ± SD; ****P≤ 0.0001.

Intracellular ROS measurement in thawed cells

Measurement of cellular ROS production before and after cryopreservation was done by flow cytometry using DCF-DA as a specific probe for H₂O₂ detection. ROS production in the fresh group (27.25 ± 3) was significantly (P≤0.05) lower compared with the control group (69.89 ± 6%), α-tocopherol (56.34 ± 4%) and catalase (45.38 ± 8%) (Figure 4A and B).

Figure 4. (A) Flow cytometry analysis for the detection of ROS in different treatment groups. a group (fresh), b group (control), c group (catalase 40 μl/ml)), d group (α-tocopherol 200 μl/m. M1: without DCF-DA, M2: with DCF-DA. (B) ROS production of SSCs before and after cryopreservation analyzed by flow cytometry. Note that the significant lower production of ROS after cryopreservation in 40 μl/ml catalase compared to control. Data show mean ± SD; ****P≤0.0001.

Discussion

In the present study, an optimal cryopreservation protocol was developed for mouse SSCs with antioxidants. Cryopreservation of SSCs in the presence of catalase or α-tocopherol can increase cell viability and reduce ROS production and apoptosis.

The cryopreservation method should be optimized to have functional survival rates of SSCs. Studies show that cryopreservation of SSCs is feasible but there are limited studies on this method of preservation (Avarbock et al. 1996). The main problem with SSCs is the rare amount of this particular cell type in testis (Kubota et al. 2004). On the other hand, after cryopreservation, the number of these cells that undergo apoptosis increase (Hermann et al. 2007). In the current study, we obtained SSCs from the testes of 3–6-day-old mice. The purity of the SSCs preparation was evaluated using the Stra8 surface marker by flow cytometry.

The isolated cells were frozen in different conditions and their viability was evaluated by MTT assay. As was previously demonstrated, freezing process can decrease the number of viable cells (Kanatsu‐Shinohara et al. 2003). This reduction in the survival rate of SSCs may be caused by the accumulation of toxic products of metabolism and ROS formation (Izadyar et al. 2002). In this study, for increasing the number of viable cells after cryopreservation, we added catalase and α-tocopherol to the basic freezing medium. These two antioxidants could increase cell viability up to ∼80%. Previous studies indicated that ∼70% of SSCs were viable after cryopreservation (Koruji et al. 2007). Izadyar et al. (2002) reported that by adding glycerol or DMSO to the freezing medium, type A spermatogonia can be cryopreserved successfully. Frederickx et al. (2004) evaluated different cryopreservation protocols on the basis of cell survival and recovery in mice. They compared two different cryoprotective agents (DMSO, ethylene glycol) in order to evaluate the survival of a testicular cell suspension. They showed that cell survival was possible after cryopreservation of the testicular germ cell suspensions.

The most common ROS molecule produced during cryopreservation are O₂ and H₂O₂. In order to reduce the toxic effects of these molecules, ROS scavenging enzymes and antioxidants can be used (Zhang et al. 2012). In the current study, we used catalase and α-tocopherol for this purpose. Catalase has the ability to convert H₂O₂ to H₂O and O₂ and eliminate potential ROS toxicity (Koepke et al. 2008). Fernandez-Santos et al. (2009) showed the effect of catalase supplementation during in vitro culture of frozen ⁄ thawed bull spermatozoa. They indicated that catalase can protect thawed bull spermatozoa against oxidative stress.

α-Tocopherol, a lipid soluble antioxidant can break the covalent links that ROS forms between fatty acid chains in lipid membranes and can protect membrane components from ROS damaging effects (Ford 2004). Fernandez-Santos et al. 2007 showed potential protective effects of enzymatic and non-enzymatic antioxidants against cryopreservation injuries of red deer epididymal spermatozoa.

One week after freezing, we analyzed Bax and Bcl2 gene expression. Our study clearly showed that by using catalase and α-tocopherol, the apoptotic rate of cells were decreased and catalase (40 μl) had a high impact on decreasing apoptosis. Proteins of the Bcl-2 family with either pro (Bax) or anti apoptotic (Bcl-2) activity have crucial roles in regulation of apoptosis (Iannolo et al. 2008). BCL-2 gene can suppress the apoptotic death of some cells such as lymphocytes and is essential for survival in a cell-type-specific manner (Coultas et al. 2005, Jagani et al. 2015). In addition, BCL-2 can regulate death of primordial germ cells and oogonia stem cells during e embryonic period (Aitken et al. 2011). BAX gene is critical for activation of the downstream events that result in cell death (Iannolo et al. 2008).

On the other hand, we analyzed ROS formation during SSCs cryopreservation. The ROS production of SSCs frozen/thawed with catalase and α-tocopherol was less than control the group. In this study, by using catalase and α-tocopherol, we could decrease ROS formation during freezing process. Our results showed that these two antioxidants, especially catalase (40 μl/ml), have high effect on decreasing ROS formation. Anti-oxidants either scavenge ROS directly or prevent propagation of lipid peroxidation in cell membranes and can protect SSCs from the damaging effects of cryopreservation (Bian and Chang 2015, Upreti et al. 1998). A number of studies have demonstrated the positive effect of adding antioxidants to freezing medium of other cells such as sperm and hematopoietic cells. Lalita et al. added a combination of catalase and trehalose to freezing medium for human hematopoietic cells. Their study showed that this combination can help protect various functions of hematopoietic cells after freezing (Sasnoor et al. 2005). Franco et al. reported that by using superoxide dismutase and catalase supplementation in semen cryopreservation procedure, the human sperm recovery was improved. These results suggest that catalase supplementation can contribute greatly to the prevention of sperm membrane lipid peroxidation by ROS (Rossi et al. 2001).

Conclusions

Our findings demonstrated that SSCs derived from neonate mouse testis can be successfully cryopreserved with antioxidants, and the method used for cryopreservation in this study can increase cell viability and reduce ROS production and apoptosis.

Declaration of interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. There were also no conflict of interest for designing and carrying out this current work. The authors alone are responsible for the content and writing of this article.

Funding information

We all want to say that this is an original article, which has been supported by Tehran University of Medical Sciences with the Grant No. 20163. We are grateful for the funding support by the University. The results described in this article were part of student thesis. In the present work, we used animal model with considering all the rights based on Ethical Committee of Medical Faculty of Tehran University.