Effect of Catalase or Alpha Lipoic Acid Supplementation in the Vitrification Solution of Ovine Ovarian Tissue

Abstract

Aim:

The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue.

Methods:

This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL⁻¹) or ALA (25, 50, or 100 μM mL⁻¹). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels.

Results:

The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 μM mL⁻¹ than those observed for the treatments INC, CAT (40 IU mL⁻¹), or ALA (25 or 50 μM mL⁻¹). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL⁻¹), ROS levels (10 and 20 IU mL⁻¹), as well as DNA damage revealed by ©H2AX (40 IU mL⁻¹).

Conclusions:

Although 100 μM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.

Introduction

In recent years the treatments for combating cancer have resulted in high rates of healing and survival. However, the patients who undergo chemotherapy and/or radiotherapy are likely to suffer premature ovarian failure and, consequently, early menopause, reducing the production of hormones. This leads to osteoporosis, cardiovascular diseases, psychosexual dysfunction,^1,2 and infertility.

With the purpose to restore reproductive function after cancer treatment, compared with embryo and oocyte cryopreservation, the ovarian tissue cryopreservation is the best option, since it is independent of age of the patient, that is, the material to be cryopreserved can be collected from both adult women and prepubertal girls. This strategy does not cause any delay in the start of anticancer treatments, and it may save not only fertility but also hormonal gonadal function.³ Ovarian tissue cryopreservation enables preservation of a large numbers of oocytes enclosed in ovarian follicles, before, and the structural and functional integrity of the ovary, thus enabling the restoration of endocrine and exocrine functions, with the advantage of being able to retrieve ovarian tissue regardless of the stage of the estrous or menstrual cycle.^4,5

Over the last decade, several researchers have reported the birth of healthy individuals after transplantation of cryopreserved mammalian ovarian tissue, mainly in women and farm species (e.g., ovine). However, the vast majority of these studies have used the slow freezing strategy (human⁶ and ovine^7–10). In contrast, the success of vitrification techniques has also been reported in recent years, including the birth of healthy individuals in ovine^11,12 and human.¹³ Despite these results, ovarian cryopreservation is considered an innovative treatment,^14–16 and the rate of pregnancy from the ovarian tissue cryopreserved transplant is only 20%–30%.¹⁷ Independent of the method (slow freezing or vitrification) used, the cryopreservation process can cause an imbalance in the production of reactive oxygen species (ROS) and, consequently, DNA damage. These are due to injuries in the cell membrane and mitochondrial dysfunction.¹⁸ Such injuries compromise the potential of oocyte development and, consequently, the competence of a future embryo.^19–22

To minimize the generation of ROS during the process of cryopreservation, antioxidants are added during slow freezing^23,24 or in vitrification^25,26 solutions. Catalase (CAT) is an antioxidant capable of decomposing hydrogen peroxide into water and oxygen.²⁴ Luz et al. showed that the addition of 20 IU mL⁻¹ of CAT in the freezing solution of preantral goat follicles decreased lipid peroxidation. When the same concentration of CAT was added in the vitrification solution (VS), it was observed that ROS levels in the caprine ovarian tissue remained unchanged compared to the fresh control (CTR), thereby reducing its deleterious effects.²⁶ Another prominent antioxidant agent is alpha lipoic acid (ALA), although it has not yet been used in ovarian tissue cryopreservation procedures. Some studies have shown that this antioxidant is favorable to the follicular and oocyte structures when manipulated in vitro. ALA added to the culture medium of fresh or vitrified isolated preantral follicles of mice decreased levels of ROS and increased the total antioxidant capacity.^27–29 In a study with goat oocytes, Zhang et al., reported that 25 μL of ALA, when added to maturation medium, significantly increased the oocyte maturation rate (69.8%) compared to nontreated oocytes (CTR: 57.8%).

It has been shown that the use of antioxidants minimizes the cryoinjuries during cryopreservation of ovarian tissue.²⁴ However, there are no reports on the use of these antioxidants on the VS of sheep ovarian preantral follicles. Therefore, the main objective of this study was to evaluate the effects of different concentrations of ALA and CAT on the VS of sheep ovarian tissue, on the following parameters: morphology and follicular activation, DNA damage, levels of ROS, and mitochondrial function.

Materials and Methods

This experiment was approved and performed under the guidelines of the Ethics Committee for Animal Use of the State University of Ceará (N˚6004631/2015). The cryoprotectants (ethylene glycol [EG] and dimethyl sulfoxide [DMSO]) were obtained from Dinâmica (Dinâmica Química, Diadema, SP, Brazil), and the other chemicals were purchased from Sigma (Sigma Chemical Co., St. Louis, MO), unless otherwise stated.

Ovaries

A total of 16 ovaries were collected from eight adult nonpregnant sheep at a local slaughterhouse. Immediately postmortem, the ovaries were washed once in 70% ethanol for 10 seconds and then washed twice in HEPES-buffered (25 mM) minimum essential medium (MEM) supplemented with 100 μg mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. Ovaries were transported to the laboratory in MEM at 20°C within 1 hour after harvesting.²⁶ At the laboratory, ovaries were stripped of the surrounding fat and fibrous tissue, and the ovarian cortex from each ovarian pair was cut into small fragments (approximately 3 × 3 × 0.5 mm, L × W × H, respectively) using the Thomas Scientific-Riggs Tissue Slicer (Thomas Scientific^®, Swedesboro, NJ) under sterile conditions.

Experimental design

This experiment was carried out in two steps. In the first step, 18 ovarian fragments (n = 18) from each ovine ovarian pair were used. Of these, two fragments were immediately fixed in paraformaldehyde 4% and considered the fresh tissue or CTR group, and two fragments were submitted to incubation (INC) within 24 hours and later fixed in as described above. The 14 remaining fragments were distributed into seven vitrification conditions: vitrification without antioxidant (VWA) or vitrification with different concentrations of CAT (10, 20, or 40 IU mL⁻¹) or ALA (25, 50, or 100 μM mL⁻¹). After vitrification, all fragments (two fragments/vitrification condition) were warmed, incubated for 24 hours,³⁰ and fixed. The tested concentrations of both antioxidants were selected based on previous studies (CAT^24,28; ALA^27,31). All fragments proceeded to morphological analysis and evaluation of follicular development. After INC time, the spent media were saved for ROS analysis. Every treatment was repeated four times.

In the second step, 15 ovarian fragments (n = 15) from each ovine ovarian pair were used. Based on the best treatments (higher percentage of morphologically normal, activated preantral follicles and lower ROS levels) obtained in the first step, only five treatments were repeated. Therefore, three fresh fragments were immediately fixed (CTR), while three others were only incubated (INC). The nine remaining fragments were distributed into three vitrification conditions (VWA, CAT40, or ALA100). All fragments were submitted to robust analyses such as ovarian stromal cell density, DNA damage, mitochondrial activity, and intracellular ROS levels on ovarian tissue. Every treatment was repeated four times.

Vitrification/warming procedures

The vitrification was performed using the Ovarian Tissue Cryosystem (OTC), a closed-system solid surface vitrification technique described previously by our team.²⁷ Briefly, the fragments were exposed to two VS. The VS1 consisted of MEM supplemented with 10 mg mL⁻¹ bovine serum albumin (BSA), 0.25 M sucrose, 10% EG, and 10% DMSO. The VS2 had a similar composition as VS1, but with higher concentration of cryoprotectants (20% EG and 20% DMSO). Both solutions (VS1 and VS2) were prepared without or with antioxidant agents (CAT or ALA) in different concentrations as mentioned above. The ovarian fragments were initially exposed to VS1 for 4 minutes followed by VS2 exposure for 1 minute. Both exposures were performed using the OTC. The VS was removed, and the OTC containing the ovarian tissue was closed and immediately immersed vertically into liquid nitrogen.

After cryostorage for up to 7 days, OTCs containing the vitrified ovarian tissue were warmed in air at room temperature (RT ∼25°C) for 1 minute, followed by immersion in a water bath (37°C) for 30 seconds. After warming, the cryoprotectants were removed by three-step washing solutions (WS; 5 minutes each) in WS1: MEM + 3 mg mL⁻¹ BSA + 0.5 M sucrose, WS2: MEM + 3 mg mL⁻¹ BSA + 0.25 M sucrose, and WS3: MEM + 3 mg mL⁻¹ BSA.^27,28 These three WS did not contain antioxidants.

INC conditions

Incubated (INC) or vitrified ovarian fragments (of the seven vitrification conditions) were incubated in 1 mL of culture medium for 24 hours³⁰ in a humidified incubator at 39°C with 5% CO₂. The culture medium consisted of α-MEM supplemented with ITS (10 μg mL⁻¹ Insulin, 5.5 μg mL⁻¹ Transferrin, and 5 ng mL⁻¹ Selenium), Glutamine (2 mM), Hypoxanthine (2 mM), BSA (1.25 mg mL⁻¹), LIF - Leukemia Inhibitory Factor (50 ng mL⁻¹), KL -Kit Ligand Kit (50 ng mL⁻¹), and FSH - Follicle Stimulating Hormone (100 ng mL⁻¹), adapted of the Lunardi et al. (2017).⁵

Follicular morphology and activation

Fresh or vitrified ovarian tissue was fixed in 4% paraformaldehyde (PAF) for 2 hours at 37°C, dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin wax, and sectioned (7 μm thick). The histological sections were stained with Periodic Acid Schiff—hematoxylin. For morphological evaluation, the slides were coded and examined under a microscope (Nikon, Japan) with a magnification of 400 × , and the follicles were classified according to the integrity and development stage.

The follicle development stages were defined as: primordial, one layer of flattened pregranulosa cells surrounding the oocyte; transitional, one layer of flattened and cuboidal granulosa cells; primary, one layer of cuboidal granulosa cells; and secondary, two or more layers of cuboidal granulosa cells.³² The last three stages were classified as activated follicles. To avoid evaluating and counting the same follicle more than once, preantral follicles were analyzed only in the sections in which an oocyte nucleus was observed and matched with the same follicle on adjacent sections, thereby ensuring that each follicle was counted only once.

Preantral follicles were morphologically classified as (i) normal if they contained an intact oocyte and intact granulosa cells and (ii) atretic if they contained a pyknotic oocyte nucleus, shrunken ooplasm, accompanied or not by disorganized granulosa cells (e.g., increase in volume with or without detachment from the basement membrane). The presence of at least one of the aforementioned features was indicative of atresia.²⁸

ROS levels after INC time

The ROS levels were determined by a spectrofluorimetric method,³³ using 2′,7′-dihydrodichlorofluorescein diacetate (DCHF-DA, D6883; Sigma-Aldrich) assay. A sample aliquot (50 μL of the spent medium) was incubated with 5 μL of DCHF-DA (1 mM) at room temperature. The oxidation of DCHF-DA to fluorescent dichlorofluorescein was measured for the detection of ROS. The 2′,7′-dichlorofluorescein (DCF) fluorescence intensity emission was recorded at 520 nm (with 480 nm excitation) 2 hours after the addition of DCHF-DA to the medium, using a spectrofluorometer (Shimadzu model RF-5301PC, Tokyo, Japan).

Ovarian stromal cell density

Ovarian stromal cell density was evaluated by calculating the number of stromal cells in an area of 100 × 100 μm. For each treatment, 10 fields per animal were assessed, resulting in a total of 40 fields per treatment, and the mean number of stromal cells per field was calculated.²⁸ All evaluations and measurements were performed by a single operator.

Gamma H2AX staining for detection of damage of DNA

For this analysis, we used a mouse monoclonal primary antibody for the detection of H2AXph139 (1:200 ab26350; Abcam, Inc., Cambridge, MA) protein, which is involved in DNA damage. The secondary antibody was Alexa Fluor^® 488 anti-rabbit IgG (ab150113; Abcam, Inc.) diluted 1:500 for detection of H2AXph139. The negative CTR was obtained by omitting the primary antibodies. Samples from CTR, as well as from INC and vitrified fragments (VWA, CAT40, and ALA100), were fixed in 4% paraformaldehyde in PBS (pH 7.2) and, subsequently, dehydrated and embedded in paraffin wax. Tissue sections (5 μm) mounted on Superfrost Plus slides (Knittel Glass, Bielefeld, NW, Germany) were deparaffinized with CitriSolv (Fisher Scientific, Ottawa, Ontario, Canada) and rehydrated in a graded ethanol series. Antigen retrieval was performed by incubating the tissue sections in 0.01 M sodium citrate buffer (pH 6.0) for 5 minutes, in a pressure cooker. After cooling, tissue sections were washed in PBS and blocked for 1 hour at room temperature using PBS containing 1% (w/v) BSA. Following antigen retrieval, slides were incubated overnight at 4°C with primary antibodies (H2AXph139). Then, slides were incubated with the secondary antibody Alexa Fluor^® 488 for 1 hour at RT and stained with Evans Blue (1:10,000). The slides were mounted with VECTASHIELD Mounting Medium^® (Vector Laboratories, Inc., Burlingame, CA). Immunostaining was evaluated using a confocal laser scanning microscope (LSM 710, Zeiss, Oberkochen, Germany). All analyses were performed using the same configurations.³⁴

Assay for the detection of DNA fragmentation (TUNEL staining)

The DNA fragmentation was analyzed by TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling) assay, using the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Mannheim, BW, Germany), according to the manufacturer's instructions. The ovarian tissues derived from CTR, as well as from INC and vitrified fragments (VWA, CAT40, and ALA100), were prepared as described for the gamma H2AX staining. To block endogenous peroxidase the slides were incubated with 3% H₂O₂ in methanol and then blocked for 1 hour at RT using PBS containing 1% BSA. After washing, the slides were incubated with the TUNEL reaction mixture (50 μL) for 1 hour at 37°C. Converter POD was added, and the location of the protein expression was demonstrated by INC with DAB (0.05% DAB in Tris/HCl, pH 7.6, 0.03% H₂O₂). Finally, the sections were counterstained with hematoxylin. The follicles were considered with fragmented DNA when the oocytes were detected having dark brown stained nuclei.³⁵ As an internal positive CTR, the sections were treated with 10 U mL⁻¹ DNase I (Invitrogen™, Carlsbad, CA) in 1 mg mL⁻¹ BSA, for 10 minutes at RT, before INC with the TUNEL reaction mixture to induce the nonspecific breaks in the DNA. The negative CTR sections omitted the terminal deoxynucleotidyl transferase enzyme.³⁴

Mitochondrial activity and intracellular ROS levels

This analysis was performed according to Fabbri et al., with some modifications. Briefly, the quantification of MitoTracker Orange CMTMRos and DCF fluorescence intensities, which indicate apparent energy status (mitochondrial activity) and intracellular ROS levels, was evaluated. The ovarian tissue samples from CTR, as well as from INC and vitrified fragments (VWA, CAT40, and ALA100), were incubated for 30 minutes in 998 μL of PBS with 2 μL of MitoTracker Orange CMTMRos (M7510; Molecular Probes, Eugene, OR) at 39°C under 5% CO₂ to detect and localize actively respiring mitochondria. After INC with the mitochondria specific probe, ovarian samples were incubated for 15 minutes in 999 μL of PBS with 1 μL of DCF to detect and localize intracellular sources of ROS. The samples were fixed in 4% paraformaldehyde for 12 hours at 4°C, dehydrated in sucrose for 72 hours, inserted in Tissue-Tek, and subsequently stored at −80°C. After that, the slides were fixed in 3% formaldehyde for 15 minutes, washed in PBS for 5 minutes and mounted with Fluoroshield Mounting Medium with Propidium Iodide (ABCAM-ab104129), and evaluated using a confocal laser scanning microscope (LSM 710; Zeiss, Oberkochen, Germany).

Statistical analyses

All statistical analyses were performed using SigmaPlot 11 (Systat Software, Inc.). Data that were not normally distributed (Shapiro–Wilk test) were transformed to natural logarithms. Comparison of means was performed between treatments by Kruskal–Wallis test. The proportion of morphologically normal follicles and follicle class distribution (primordial and activated) among treatments were analyzed by chi-square or Fisher's exact test. Pearson correlation coefficient was performed to evaluate the association between MitoTracker and DCHF-DA fluorescence intensity. In addition, the odds ratio and 95% confidence interval (CI) were calculated to determine the effect of treatments performed in presence of normal follicles. Data were presented as mean (±standard error of mean) and percentage, unless otherwise indicated. Statistical significance was defined as p < 0.05 (two sided).

Results

Follicular morphology

A total of 888 preantral follicles being CTR (n = 99); INC (n = 92); VWA (n = 120); CAT10 (n = 109); CAT20 (n = 90); CAT40 (n = 78); ALA25 (n = 120); ALA50 (n = 90); and ALA100 (n = 90) were analyzed. Morphologically normal or atretic follicles were observed in the fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with antioxidants (CAT or ALA), as shown (Fig. 1).

FIG. 1.

Representative images of the morphology of ovine preantral follicles after staining with periodic acid Schiff-hematoxylin. Normal follicles are shown in CTR (A), INC (B), CAT40 (F), and ALA100 (I), while degenerate follicles are represented in VWA (C), CAT10 (D), CAT20 (E), ALA25 (G), and ALA50 (H). O, oocyte; Nu, oocyte nucleus; GC, granulosa cells; r, retraction (400 × ). Arrows indicate normal follicles; Arrowheads indicate degenerate follicles. Scale bars = 50 μm. ALA, alpha lipoic acid; CAT, catalase; CTR, control; INC, incubation; VWA, vitrification without antioxidant.

Vitrification of ovarian tissue in the presence of ALA at all tested concentrations (25, 50, 100 μM mL⁻¹) maintained the percentage of morphologically normal follicles similar (p > 0.05) to the fresh CTR and nonvitrified ovarian tissue cultured (INC) for 24 hours. In contrast, after culture, vitrified tissue without antioxidant (VWA), as well as in the presence of CAT, regardless of the concentration resulted in lower (p < 0.05) percentage of normal follicles than the CTR or INC treatment (Fig. 2A).

FIG. 2.

Percentage of morphologically normal follicles (A) and follicular development (B) in fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with antioxidants in different concentrations of catalase (CAT 10, 20, or 40 IU mL⁻¹) or alpha lipoic acid (ALA 25, 50, or 100 μM mL⁻¹). *Differ from CTR. ^a,bDiffer among follicular categories (p < 0.05) in activated follicles. ^A,B,CDiffer among treatments (p < 0.05) in primordial follicles.

Association analyses among treatments on percentage of normal preantral follicles

Data concerning the concentration and type of antioxidants used in this study were assessed using the odds ratio approach (Table 1). CAT (all combined data from three concentrations) and VWA treatments had a similar odds ratio (p > 0.05) to the percentage of normal preantral follicles after vitrification. In contrast, the likelihood of having morphologically normal preantral follicles in ALA (all combined data from three concentrations) was 1.6 and 1.8 times higher compared with the VWA (p < 0.05) and CAT (p < 0.01) groups, respectively. Moreover, the INC had a superior (p < 0.01) odds ratio to normal preantral follicles than VWA (2.6 times) and CAT (3.1 times) groups. Finally, ALA and INC showed a similar (p > 0.05) probability to sustain normal preantral follicles.

Table 1.

Association Analysis Between Treatments (Incubation only × Vitrification; Without × With Antioxidant and CAT × ALA) on Percentage of Normal Morphologically Preantral Follicles

Comparisons	Normal preantral follicles (%)	Odds ratio	CI 95%	p-Value
VWA	59.1 (71/120)	1.1	0.7–1.8	0.53
CAT^a	55.2 (153/277)	1.1	0.7–1.8	0.53
VWA	59.1 (71/120)	1.6	1.0–2.4	0.05
ALA^b	69.6 (209/300)	1.6	1.0–2.4	0.05
CAT	55.2 (153/277)	1.8	1.3–2.6	0.01
ALA	69.6 (209/300)	1.8	1.3–2.6	0.01
INC	79.3 (73/92)	2.6	1.4–4.9	0.01
VWA	59.1 (71/120)	2.6	1.4–4.9	0.01
INC	79.3 (73/92)	3.1	1.7–5.4	0.01
CAT	55.2 (153/277)	3.1	1.7–5.4	0.01
INC	79.3 (73/92)	1.6	0.9–2.9	0.09
ALA	69.6 (209/300)	1.6	0.9–2.9	0.09

Data of analyses with Catalase (CAT 10, CAT 20, and CAT 40 IU mL⁻¹) combinated.

Data of analyses with Alpha Lipoic Acid (ALA 25, ALA 50, and ALA 100 μM mL⁻¹) combinated.

ALA, alpha lipoic acid; CAT, catalase; CI, 95% confidence interval; CTR, control; INC, incubation; VWA, vitrification without antioxidant.

Follicular activation

The percentage of primordial and activated follicles is shown in Figure 2B. The vitrified ovarian tissue in VWA or in CAT10, ALA50, and ALA100 showed a higher (p < 0.05) activation (represented by high percentage of activated follicles) in comparison with CTR. Moreover, it was observed that the percentage of activated follicles was higher in the ALA100 than in the INC, as well as in the CAT40, ALA25, and ALA50 treatments (p < 0.05).

ROS levels in spent incubated medium

The data showed that INC and ALA100 were the only two treatments in which ROS levels did not differ from each other (p > 0.05) and were significantly lower than the CAT10 and CAT20 concurrently (Fig. 3).

FIG. 3.

Mean (± standard error of the mean) reactive oxygen species (relative fluorescence units) produced by ovine ovarian tissue fragments after vitrification and incubation for 24 hours. ^A,BUppercase letters indicate difference among treatments (p < 0.05).

Ovarian stromal cell density

Images of stromal cells of CTR groups, INC, VWA, CAT40, and ALA100 are shown in Figure 4. There was significant reduction in cell density in all treatments compared with CTR with the exception of the VWA treatment. In addition, within only the vitrified treatments, the cell density was significantly reduced in CAT40 compared with VWA and ALA100 treatments (Table 2).

FIG. 4.

Representative images of ovarian stromal cell densities (A, B, C, D, and E) and images of cell death analysis by apoptosis in ovine preantral follicles enclosed in ovarian tissue using the TUNEL technique (F, G, H, I, and J) in the fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with antioxidants in different concentrations of catalase (CAT 40 IU mL⁻¹) or alpha lipoic acid (ALA 100 μM mL⁻¹). (K) and (L) represent negative and positive control, respectively. Arrow indicates TUNEL-positive reaction; arrowheads indicate TUNEL-negative reaction. Scale bars = 50 μm.

Table 2.

Mean (± SEM) Stromal Cell Density and Percentage of Preantral Follicles with γH2AX foci and TUNEL Signal Positive in Fresh (CTR), Incubated (INC), or Vitrified Tissue Without or With Antioxidants (CAT or ALA)

Treatments	Stromal cell density	Preantral follicles with γH2AX foci (%)	TUNEL-positive preantral follicles (%)
CTR	170.6 ± 4.8^A	62.5 (10/16)^AB	2.8 (2/71)^A
INC	145.3 ± 5.6^B	45.2 (33/73)^A	42.1 (24/57)^B
VWA	162.2 ± 6.8^AB	82.1 (23/28)^BC	5.4 (4/73)^A
CAT40	126.0 ± 5.0^C	92.3 (24/26)^C	5.4 (2/37)^A
ALA100	145.7 ± 5.4^B	80.0 (4/5)^AC	0.0 (0/19)^A

A,B,C

Uppercase letters indicate difference among treatments (p < 0.05).

Gamma H2AX foci detection in preantral follicles

Immunofluorescence analysis was performed to localize a protein involved in signaling (γH2AX) of DNA damage (Table 2) in preantral follicles enclosed in ovarian tissue (Fig. 5). The percentage of follicles positively immunostaining for γH2AX in CAT40 treatment was higher than CTR and INC treatments. The immunostaining for γH2AX was also higher in vitrified tissue without antioxidant (VWA) than the INC group. It should be noted that although the ALA100 group was similar to the other cryopreserved groups only ALA100 maintained similar damage signaling (p > 0.05) to those found in the INC and CTR groups.

FIG. 5.

Representative images of gamma - H2AX (A, D, G, J, M, and P), DAPI (B, E, H, K, N, Q), and the overlapping of both (C, F, I, L, O, and R) in the fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with antioxidants in different concentrations of catalase (CAT 40 IU mL⁻¹) or alpha lipoic acid (ALA 100 μM mL⁻¹). Scale bars = 50 μm.

Assessment of DNA fragmentation by TUNEL staining

The TUNEL test revealed the presence of DNA fragmentation in preantral follicles (Fig. 5). The percentage of sheep preantral follicles positive for TUNEL showed that fragments from INC treatment presented significantly higher percentage of TUNEL-positive cells than the CTR and all other vitrified treatments (Table 2).

Mitochondrial activity and intracellular ROS levels

The mitochondrial activity and intracellular ROS levels were analyzed by fluorescence intensity probes MitoTracker Orange and DCHF-DA (Figs. 6 and 7A, B), respectively. Regarding the mitochondrial activity (Fig. 7A), only INC treatment was higher (p < 0.05) than CTR, but similar to the other treatment groups. In relation to the levels of intracellular ROS (Fig. 7B) the fluorescence intensity was significantly higher (p < 0.05) in INC, CAT40, and ALA100 compared to the CTR. Furthermore, INC was significantly higher (p < 0.05) than VWA and similar to CAT40 and ALA100.

FIG. 6.

Representative images of mitochondrial activity represented by the fluorescence intensity of MitoTracker (A, D, G, J, and M), intracellular ROS levels repre-sented by fluorescence intensity of DCHF-DA (B, E, H, K, and N), and the merge of the two probes (C, F, I, L, and O) in the fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with antioxidants in different concentrations of catalase (CAT 40 IU mL⁻¹) or alpha lipoic acid (ALA 100 μM mL⁻¹). Scale bars = 20 μm.

FIG. 7.

Quantification of active mitochondrial-specific fluorescence intensity of the MitoTracker stain (A) and intracellular ROS levels fluorescence intensity (B) of the DCHF-DA in the fresh (CTR), incubated (INC), or vitrified ovarian tissue without (VWA) or with the antioxidants in different concentrations of catalase (CAT 40 IU mL⁻¹) or alpha lipoic acid (ALA 100 μM mL⁻¹). Values are expressed as fluorescence units (mean ± SEM); A, B, C Different uppercase letters indicate a statistical difference (p < 0.05).

As shown in Figure 8, a positive correlation between mitochondrial activity (MitoTracker) and ROS levels (DCHF-DA) was observed.

FIG. 8.

Correlation between the mitochondrial activity and intracellular levels of ROS. Each point of the graph represents a sample of ovarian tissue (n = 30), r = 0.58; p = 0.001.

Discussion

Many reports have shown that the cryopreservation of ovarian tissue by slow freezing^7,36,37,38 or vitrification^11,14,39 provides great benefits to regenerative medicine, including the restoration of reproductive function. However, both cryopreservation methods cause a significant increase of oxidative stress, which can lead to degeneration of oocytes and follicles. Therefore, current efforts to improve ovarian tissue cryopreservation, in part, concentrate on establishment of an optimal protocol; it may be possible by supplementation of antioxidants into VS. In addition, after vitrification and warming, we considered it important to incubate the tissue for 24 hours, since according to Faustino et al., after this period there is a restoration of cellular metabolism. In this study, we investigated the effects of addition of CAT and ALA in the VS of sheep ovarian tissue on the follicular morphology and activation, stromal cell density, levels of ROS, DNA damage, as well as mitochondrial function of preantral follicles.

This study showed that vitrified follicles in ALA (25, 50, and 100 μM mL⁻¹) presented a percentage of morphologically normal follicles similar to CTR, INC, and VWA groups. Despite this, it was observed that the treatment with ALA was beneficial to maintain the follicular morphology after vitrification followed by INC in a short period. This is the first study showing the beneficial effect of ALA in the VS of sheep ovarian tissue. However, previous studies in mice have shown that this antioxidant maintained the normal follicular morphology, when added to the in vitro culture medium of fresh²⁹ or vitrified ovarian tissue.²⁷

Regarding the percentage of activated follicles, it was observed that although the VWA, CAT10, and CAT20 treatments were similar to ALA100 treatment, this last one was the only treatment that presented a percentage of activated follicles higher than INC treatment. It is known that during the cryopreservation procedure the endogenous protection system fails³⁰; therefore, we suggest that the use of exogenous antioxidants in the present study, especially ALA at 100 μM mL⁻¹, could constitute a helpful strategy to protect the function of endogenous cellular systems in the ovary. Comparing different concentrations (50, 100, 250, or 500 μM mL⁻¹) of ALA in the in vitro culture of fresh preantral follicles of mice ovarian tissue, Talebi et al., showed that the concentration of 100 μM mL⁻¹ was better than all others for follicular activation. Based on these findings, we believe that concentration of ALA lower than 100 μM mL⁻¹ was not able to eliminate a sufficient amount of free radicals to reduce injury to the cellular function.

In the present study, it was observed that the ROS levels in spent medium in INC and ALA100 treatments were similar to each other and lower than that found for CAT10 and CAT20. This result could be explained by the fact that the ALA synthesizes glutathione and increases the activity of the enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and CAT,⁴⁰ which are more effective than CAT. Previous studies carried out in mice also showed that ALA decreased the ROS levels, increased the total antioxidant capacity, and improved survival rate and developmental competence of vitrified-warmed or fresh isolated preantral follicles after long-term in vitro culture, compared to follicles cultured without this antioxidant.²⁸ In the present study, ALA addition at 100 mM mL-1 had a positive impact on ovine preantral follicles compared with CAT addition on morphology, follicular activation, and density of stromal cells. In addition, ALA reduced ROS levels over CAT 10 and CAT 20. According to Hatami et al. the ovarian tissue suffers morpho/functional deficits as a consequence of hypoxia or free radical generation during freezing/thawing procedures. Therefore, according to the type and concentration of antioxidant used in the VS, the follicular cryoinjuries can be attenuated.

Regarding the stromal cell density, it was observed that only CAT40 treatment reduced stromal cell density compared with all other treatments. A recent study by our team utilizing goat ovaries showed that vitrification in the presence of 20 IU mL⁻¹ of CAT did not reduce the cell density.²⁵ This could be due to the lower concentration used. Conversely, studies in humans,^41–43 as well as in goat and sheep,⁴⁴ have shown that cryopreservation without antioxidants also reduced the stromal cell density. This suggests that it is essential to define the type and concentration of antioxidants in VSs for preservation not only of the follicles but also of stromal cells because they are very important in the production of peptides and growth factors essential for cell growth and development.⁴⁵ Besides, stromal cells along with extracellular matrix components are important for the maintenance of cellular interactions thereby providing the necessary signaling for the formation, development, and migration of the follicles within the ovary.⁴⁶

Follicular DNA damage in ovarian tissues detected through ©H2AX staining revealed that CAT40 and VWA were similar; however, they showed more damage than the CTR and INC treatments, respectively. Beyond that, although the ALA100 was similar to the other cryopreserved fragments in relation to the damage signaling, it was the only one that maintained results similar to the INC and CTR, which did not suffer the stress of the vitrification procedure. In contrast, INC treatment had the highest and most expressive percentage of follicles positively marked by the TUNEL test. We believe that in the case of two treatments (VWA and CAT40), the damage was just beginning. However, it is possible that in both treatments, the cellular machinery was able to repair the damage that occurred and did not show a high percentage of TUNEL positive. γH2AX plays an important role in recruiting and maintaining DNA repair molecules at sites of damage until repair is complete.⁴⁷ Approximately 1% of these DNA breaks are converted into double-strand breaks, mainly during DNA replication, while the 99% remaining are repaired.⁴⁸ This may explain the low proportion of follicles marked for the TUNEL in the CTR, as well as in the other treatments (VWA, CAT40, and ALA100). In contrast, in INC treatment, at the time of analysis, the damage could already have been in an advanced stage. This could explain the decrease in the percentage of labeled γH2AX concomitantly to increase in the percentage of follicles that were TUNEL positive. In addition, phosphorylation of histone H2AX in serine139 is an early event in the process of damage,⁴⁹ while the TUNEL positive represents a late event in the process of apoptosis.^50,51

Due to the key role of mitochondria in the production of adenosine triphosphate, as well as in follicular development, cytoplasmic maturation, and oocyte competence,⁵² we analyzed the mitochondrial activity of preantral follicles present in the ovarian tissue. The mitochondrial activity of the tissue only incubated (INC) was significantly higher than for CTR. This may be due to hyperactivity of cells present in the tissue incubated, which could increase mitochondrial activity and consequently oxidative stress. These results are similar to that found by Fabbri et al. Interestingly the treatments submitted to vitrification (VWA, CAT40, and ALA100) were similar to CTR and INC. Although it is known that cryopreservation compromises the mitochondrial activity due to thermal shock,⁵³ osmotic forces, as well as physical and chemical conditions,³⁰ the vitrification by OTC system used in the present study did not affect the mitochondrial function. Mitochondria are the main sources of ROS⁵⁴ and are essential for many physiological processes.⁵⁵ Any incapacity of the antioxidant defense can increase the production of ROS and cause oxidative stress.^40,56,57 This may explain the increased production of ROS observed in this study after vitrification/warming. Similar results were observed after cryopreservation of oocytes of various species (cow:⁵⁸ porcine:^59,60 and mice:¹⁸) of mice embryos⁶¹ and in human ovarian tissue.³⁰ In the present study, there was a positive correlation between mitochondrial activity and intracellular levels of ROS. In frozen human ovarian tissue, Fabbri et al., also found that the increase in mitochondrial activity and elevated levels of ROS increased oxidative stress conditions. This confirms that damage to the mitochondria can cause an imbalance between production and removal of ROS, as well as excess ROS can lead to mitochondrial dysfunction.⁶²

In conclusion, the vitrification of ovine ovarian tissue in the presence of 100 μM mL⁻¹ of ALA preserved the morphology, promoted the activation, maintained the levels of ROS in the INC medium, maintained the mitochondrial activity, and was the only treatment that indicated early signs of DNA damage in preantral follicles similar to nonvitrified preantral follicles.

Footnotes

Acknowledgments

L.M.S. is a recipient of a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), and A.P.R.R. is recipient of a grant (Number of process: 457226/2013-7) from National Counsel of Technological and Scientific Development (CNPq).

Thanks to Analytics-UFC Central/CT-INFRA/MCTI-SISNANO/Pro Equipments CAPES.

This study was supported by CNPq and CAPES, Brazil.

Author Disclosure Statement

The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

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