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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Cryobiology. 2020 Apr 24;94:66–72. doi: 10.1016/j.cryobiol.2020.04.007

Impacts of different synthetic polymers on vitrification of ovarian tissue

Mohammad Hamed Shahsavari a, Kele Amaral Alves b, Benner Geraldo Alves c, Laritza Ferreira de Lima c, Diego Alberto Montano Vizcarra c, Deysi Juana Dipaz Berrocal c, Luciana Mascena Silva c, Yago Pinto da Silva c, Mary B Zelinski d, José Ricardo de Figueiredo c, Gholamali Moghaddam a,*, Ana Paula Ribeiro Rodrigues c
PMCID: PMC7660932  NIHMSID: NIHMS1625468  PMID: 32339491

Abstract

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.

Keywords: Ovarian tissue, Synthetic polymers, Vitrification, Cryoprotective agents, In vitro culture

1. Introduction

Although the ovarian follicular reserve (primordial follicles) is depleted throughout the reproductive life of the female [26], it can be preserved using many laboratory technologies [13,35]. Some techniques are still considered experimental, like the cryopreservation of ovarian tissue associated with in vitro culture of preantral follicles. However, in the future these techniques could be used as a strategy for in situ conservation of genetic material of wild and/or valuable domestic species, such as bovine. Moreover, studies in bovine, caprine and ovine ovarian tissue cryopreservation have become important experimental models for human, due to their similarities in ovary size, fibrotic tissue structure and folliculogenesis length [17,30].

Vitrification process has become an effective alternative for the cryopreservation of oocytes and embryos [32,34] and has proven to be a very attractive method for the cryopreservation of primordial follicles within ovarian tissue [9,11]. As it is well known, many factors influence the likelihood of a successful vitrification outcome, such as the type and concentration of cryoprotective agents (CPAs) and carrier system [2]. In this technique, the exposure of biological samples to CPAs in high concentration associated with rapid cooling and warming rates can cause injuries in the tissue [8]. Because of their chemical nature, these compounds can cross the plasma membrane, but they can also reach the intracellular space through water channels, such as Aquaporins. Sales et al. [29] reported that both AQP3 and AQP9 mRNAs were down-regulated following vitrification and IVC of ovine ovarian tissue and speculated that these negative effects are caused during the vitrification process.

In this way, some studies had tried formulating new successful cryoprotective agents by designing vitrification solutions that are non-toxic but allow for vitrification at realistic cooling and warming rates [4], by adding synthetic polymers that act like the antifreeze (glyco) proteins (AF(G)Ps) [14,37]. In vivo, The AF(G)Ps have evolved to modulate ice formation/growth, enabling polar fish and cold weather insects to survive in extreme cold environments [22]. Several synthetic polymers were added by Fahy et al. [15], including a copolymer of PVA (polyvinyl alcohol, super cool X-1000), polyvinylpyrrolidone (PVP) K12, and polyglycerol (super cool Z-1000) to be used in the cryopreservation. Moreover, these polymers have been used to supplement vitrification solutions in a variety of different living systems such as mouse embryos, mouse oocytes [4,15], rat ovarian tissues [12,15]. However, to the best of our knowledge, there is no study evaluated the effect of synthetic polymers (super cool X-1000, Z-1000 and K12) on vitrification of bovine preantral follicle enclosed in ovarian tissue. The aim of the present study was to investigate the protective effects of different synthetic polymers on cellular morphology and viability (Ethidium homodimer-1 coloration & ROS levels) of bovine preantral follicle enclosed in ovarian tissue vitrified in a closed system [ovarian tissue cryosystem (OTC)] and in vitro cultured for 5 day.

2. Materials and methods

This experiment was approved and performed under the guidelines of Ethics Committee for Animal Use of University of Tabriz. Except where otherwise stated, all chemicals were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

2.1. Collection of ovaries and experimental groups

Ovaries (n = 10) were collected from five adult cross-bred cows at a local abattoir. Immediately postmortem, ovaries were washed once in 70% (v/v) ethanol and then washed twice in HEPES-buffered minimum essential medium (MEM) supplemented with 100 μgmL-1 penicillin and 100 μgmL-1 streptomycin. The ovaries were then transported to the laboratory in MEM at 20 °C within 1 h (h). At the laboratory, ovaries were stripped of surrounding fat and fibrous tissue and the ovarian cortex from each ovarian pair was cut into 60 small fragments (∼3 × 3 × 0.5 mm) using a Tissue Slicer (Thomas Stadie-Riggs Tissue Slicer/Blades) under sterile conditions.

For each animal, 10 fragments were used as fresh control samples and 10 fragments were used as fresh cultured samples (without vitrification) followed by in vitro culture (IVC) for 1 (D1) or 5 (D5) days. The remaining 40 fragments were randomly distributed across the following vitrified groups (n = 10 in each group): (1) Sucrose; (2) X-1000; (3) Z-1000 and (4) PVP. After warming, thawed 2 fragments (vitrified control) in each cryopreserved group were immediately submitted to analysis and the remained fragments (n = 8) were cultured in vitro for 1 or 5 days (D1 or D5). Fresh and vitrified control, as well as vitrified groups samples were immediately intended for histological, viability analysis [Reactive oxygen species (ROS) production, degenerate cells assay (Ethidium homodimer-1)] and immunohistochemistry analysis [Aquaporin (AQP) 3 and 9] (Fig. 1).

Fig. 1.

Fig. 1.

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Experimental design to assess the effect of different synthetic polymers to vitrify bovine preantral follicles enclosed in ovarian tissue.

2.2. Vitrification and warming procedures

All procedures for exposure to cryoprotectant agents (CPAs) and vitrification were performed by using the Ovarian Tissue Cryosystem (OTC), as described by our team previously [7]. Shortly, equilibrium of the samples in the vitrification solution (VS) was performed gradually in two steps: (i) VS1 contained MEM supplemented with 10 mg/mL BSA, 10% ethylene glycol (EG) (Dinâmica - Dinâmica Química, Diadema, Brazil) and 10% dimethyl sulfoxide (DMSO) (Dinâmica) with 0.25 M sucrose (only in vitrified Sucrose group) or polymers (0.2% [v/v] Supercool X-1000, 0.4% Supercool Z-1000 or 0.2% PVP K-12; 21st Century Medicine, Fontana, CA, USA) [33]. Similarly, (ii) VS2 was composed of MEM supplemented with 10 mg/mL BSA, 20% EG and 20% DMSO with 0.25 M sucrose (only in vitrified Sucrose group) or polymers (0.2% [v/v] Supercool X-1000, 0.4% Supercool Z-1000 or 0.2% PVP K-12; 21st Century Medicine, Fontana, CA, USA). Initially, the ovarian fragments were exposed to VS1 for 4 min at 20 °C followed by an exposition to VS2 for 1 min at 20 C. The vitrification solution was then removed and the OTC containing the ovarian tissue was closed and immediately immersed vertically into liquid nitrogen (−196 °C).

After 1 week, the OTCs containing the vitrified ovarian fragments were warmed in air at room temperature (RT ∼25 °C) for 1 min, followed by immersion in a water bath (37 °C) for 30 s. After this, the CPAs were removed using a three-step exposure to washing solutions (5 min each) at 20 °C. These washing solutions (WS) were composed of: WS1 (MEM +3 mg/mL BSA+0.5 M sucrose); WS2 (MEM +3 mg/mL BSA+0.25 M sucrose) and WS3 (MEM +3 mg/mL BSA). The three WS did not contain antioxidants (catalase) nor synthetic polymers [29].

2.3. In vitro culture

For the in vitro culture, the cortex tissue samples were transferred to 24-well culture dishes containing 1 mL of the culture medium per well. The culture was performed at 38.5 °C in 5% CO2 in a humidified incubator. Fresh media were incubated for 1 h prior to use, and the culture media were replenished every other day. The culture medium consisted of the McCoy medium with bicarbonate supplemented with HEPES (20 mM; Invitrogen Ltd), glutamine (3 mM; Invitrogen Ltd), BSA (Fraction V 0.1%), penicillin G (0.1 mg/mL), streptomycin (0.1 mg/mL), transferrin (2.5 mg/mL), selenium (4 ng/mL), insulin (10 ng/mL) and ascorbic acid (50 mg/mL). Fragments were cultured for 1 (D1) or 5 (D5) days [8,25].

2.4. Histological analysis

All samples were fixed in Millonig’s solution (phosphatebuffered 40% vol/vol formaldehyde in water) for 2 h, dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin wax, and serially sectioned into 7 μm sections. Every fifth section was mounted on a glass slide, stained with Periodic acid–Schiff (PAS), and evaluated using a light microscope (Nikon, Tokyo, Japan) at magnification of 400 ×. Only preantral follicles with visible oocyte nuclei were counted. The developmental stages of follicles have been defined previously [8] as primordial (one layer of flattened and cuboidal granulosa cells) or growing [one (primary) or more (secondary) layers of cuboidal granulosa cells around the oocyte and without an antrum] follicles. The preantral follicles were also classified as morphologically normal if they presented intact oocytes and granulosa cells or degenerated (atretic) if they contained a pyknotic oocyte nucleus or shrunken ooplasm, with or without disorganized granulosa cells and/or detachment of the basement membrane [8]. 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 [7].

2.5. Assessment of tissue viability by fluorescence microscopy

2.5.1. Ethidium homodimer-1 coloration

The Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes an increase in fluorescence 40 fold by binding to the nucleic acids, producing a bright red fluorescence in dead cells (ex/em ∼ 495 nm/∼635 nm), while not permeate intact cell membranes of live cells (Sofoudis and Koufioti, 2015). Those fragments cultured for 5 days were incubated in 1000-mL drops containing 100 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) at 37 °C for 15 min. Then, the fragments were washed in MEM HEPES and analyzed using an epifluorescence microscope (Nikon, Tokyo, Japan) at magnification × 400. The fluorescence signals emitted by ethidium homodimer-1 were monitored at 568 nm, respectively and program Zen converts the fluorescence intensity of each sample in numbers and these data were statistically evaluated and expressed in mean fluorescence for each probe [10].

2.5.2. ROS levels

The 2-,7-dichlorodihydrofluorescein diacetate (DCFH2-DA) method was used as described previously [1]. The DCFH2-DA dye (Invitrogen, Life Technologies GmbH Karlsruhe, Germany), added at the beginning of the measurements (at 10 mM), is taken up by cells and in the presence of intracellular ROS converted to the highly fluorescent compound DCF (dichlorofluorescein). After 15 min incubation at 37 °C, cells were examined under a confocal microscope equipped with an argon laser (488 nm, 200 mW) and program Zen converts the fluorescence intensity of each sample in numbers and these data were statistically evaluated and expressed in mean fluorescence for each probe [1].

2.6. Immunolocalization of aquaporins (AQPs)

Fresh or vitrified ovarian fragments were fixed in 4% paraformaldehyde for 18 h, dehydrated, and embedded in paraffin. The blocks were sectioned at a thickness of 5 μm and the sections were mounted on glass slides. Immunohistochemical reactions were performed according to the protocol described previously by our team [28, 29]. Briefly, epitopes were activated by incubation in citric acid at 98–100 °C for 7 min, while non-specific binding was blocked by incubation in 5% normal goat serum diluted in phosphate-buffered saline (PBS). Subsequently, sections were incubated for 18 h at 4 °C with anti-AQP3 and -AQP9 antibodies (AQP3, 1:1000; AQP9, 1:100), with all antibodies from Alomone Laboratories. Afterwards, the sections were incubated for 45 min with the secondary biotinylated antibody anti-rabbit IgG (Santa Cruz Biotechnology, USA) diluted 200 times in PBS containing 5% normal goat serum. The sections were then incubated for 45 min with avidin-biotin complex (1:200; ABC kit, Vector Laboratories, Burlingame, Calif., USA). The location of the protein was demonstrated with 3,3′-diaminobenzidine tetrahydrochloride. Finally, sections were counterstained with hematoxylin. In the negative control samples the primary antibodies were omitted. The immunostaining was classified as absent (−), weak (+), moderate (++) or strong (+++) according to Sales et al. [29].

2.7. Statistical analysis

In this study, data that were not normally distributed (Kolmogorov-Smirnov test) were submitted to logarithmic transformation. Comparisons of means (morphologically normal follicles, follicular activation, ethidium homodimer, and dichlorofluorescein) were analyzed by Kruskal-Wallis test and Mann-Whitney test, when appropriate. All statistical tests were performed using Sigma Plot 11 (Systat Software Inc., USA). Differences were considered significant when P < 0.05.

3. Results

3.1. Histological evaluation of preantral follicles

For this analyze a total of 6865 follicles (8273 histological sections) were evaluated and morphological features of normal and atretic preantral follicles found are shown in Fig. 1, while the percentage of normal follicles is shown in Table 1. Immediately after vitrification/warming or after all vitrification/IVC (D1 and D5) samples, the percentages of morphologically normal follicles were decreased (P < 0.05) in the Z-1000 samples in comparison with fresh control. However, there were no significant differences in the percentage of normal follicles among IVC Control (D1 and D5), post-thawed samples (sucrose, X-1000 and PVP) and fresh control samples. Moreover, the percentage of morphologically normal follicles in IVC Control (D1) was similar to vitrified/IVC after day 1, when sucrose was used such as non-penetrating CPA. Immediately after vitrification/warming, the percentages of morphologically normal follicles in the Z-1000 samples was the lowest (P < 0.05) among all treatments. After in vitro culture for 1 day, although, there were no significant differences in the percentage of normal follicles among sucrose group and X-1000 samples, the percentage of morphologically normal follicles were increased (P < 0.05) in the sucrose group in comparison with Z-1000 and PVP samples. Furthermore, in groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples, lower in the PVP samples and the lowest in Z-1000 samples on day 1 (Fig. 1). Considering all vitrified and cultured treatment on day 5, the percentage of morphologically normal follicles was the highest in the IVC Control and in the mean time sucrose samples had more percentage of morphologically normal follicles than groups which contain polymer. Furthermore, the percentages of morphologically normal follicles were increased (P < 0.05) in the Z-1000 samples in comparison with PVP samples. On the other hand, the percentages of normal follicles in all vitrified and cultured treatment (D1 and D5) were decreased (P < 0.05) in comparison with post-thawed ones. Although, sucrose maintained a similar percentage of morphologically normal follicles from D1 to D5, all other cultured samples on day 5 experienced a decrease in the percentage of normal follicles when compared to those of cultured samples on day 1.

Table 1.

Percentage of morphologically normal preantral follicles (fresh, vitrified, in vitro cultured or vitrified/in vitro cultured).

Fresh Control
 79.3 ± 1.9
Treatments Post-thawing IVC (Day 1) IVC (Day 5)
IVC Control – 75.2 ± 2.6 aA 85.2 ± 2.8 bA
Sucrose 80.8 ± 2.7 aA 69.2 ± 2.9 bAB* 70.3 ± 3.0 bB*
X-1000 80.2 ± 3.4 aA 66.1 ± 3.0 bB* 47.1 ± 4.1 cCD*
Z-1000 62.3 ± 3.3 aB* 28.3 ± 2.6 bD* 51.8 ± 4.7 cC*
PVP 76.0 ± 3.1 aA 46.6 ± 2.8 bC* 38.2 ± 3.9 cD*
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*

Differ from Control (P < 0.05).

a,b,c

indicate differences within a row (P < 0.05).

A,B,C

indicate differences within a column (P < 0.05).

For all results with the same letter, the difference between means is not statistically significant.

The mean (±SEM) percentages of primordial or developing follicles are presented in Table 2. Post-thawed Z-1000 samples resulted in a lower percentage of follicles when compared to fresh control (P < 0.05). Although the percentage of primordial or developing follicles in IVC Control on D1 was higher than that of fresh control (P < 0.05). Furthermore, the percentages of primordial or developing follicles for polymer treatment groups and IVC Control group on D5 were higher (P < 0.05) than that of the fresh control group. Considering IVC Control group on D1 and D5, the percentage of developing follicles did not differ between treatment groups and IVC Control group (P > 0.05). Immediately after warming (vitrified samples), the percentages of primordial or developing follicles in post-thawed PVP samples was higher than that of post-thawed Z-1000 samples. After in vitro culture for 1 day, the percentage of developing follicles did not differ between treatment groups. Although the percentage of primordial or developing follicles on D5 did not differ between treatment groups and IVC control samples, there was significant difference (P < 0.05) between treatment groups. In this case, PVP and sucrose cultured samples on day 5 had lower percentages of primordial or developing follicles than that of X-1000 cultured samples. After 1 and 5 days of culture, there was a significant reduction (p < 0.05) in the percentage of primordial follicles (data not shown) with concomitant increase (p < 0.05) in the percentage of developing follicles in IVC Control, indicating the follicular activation process. In the treatments vitrified and cultured, this activation happens in the 5 day of culture. In all treatment groups, the percentages of developing follicles on D5 were higher than those of post-thawed samples (P < 0.05). Although just X-1000 and the cultured control treatment increased the percentage of developing follicles with the progression of the culture period from D1 to D5 (Table 2).

Table 2.

Percentage (mean ± SEM) of developing follicles. Bovine ovarian tissues were non-vitrified (fresh control), cultured control (cultured control), vitrified–thawed (Post-thawing), or vitrified and culture in the presence of sucrose, X-1000, Z-1000 and PVP.

Fresh control
 47.6 ± 2.5
Treatments Post-thawing IVC (Day 1) IVC (Day 5)
IVC Control – 55.8 ± 3.3 aA* 68.9 ± 3.5 bAB*
Sucrose 43.4 ± 3.6 aAB 52.5 ± 3.8 abA 61.3 ± 3.9 bB*
X-1000 41.0 ± 4.6 aAB 52.6 ± 3.5 bA 75.5 ± 5.1 cA*
Z-1000 35.1 ± 3.8 aA* 53.5 ± 4.5 bA 61.9 ± 6.0 bAB*
PVP 47.5 ± 3.8 aB 51.5 ± 3.9 abA 60.2 ± 5.5 bB*
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*

Differ from Control (P < 0.05).

a,b,c

indicate differences within a row (P < 0.05).

A,B,C

indicate differences within a column (P < 0.05).

3.2. Assessment of follicular viability by ethidium homodimer-1

The viable and nonviable cell in fresh control, IVC control and vitrified/IVC for 5 days are represented in Fig. 2 and the rate can be seen in Table 3. The levels of degeneration and tissue damage in all treatments were higher (P < 0.05) compared to the fresh control group (Table 3). However, this damage was similar (P > 0.05) among post- thawed cultured (Sucrose, X-1000, Z-1000, and PVP) and fresh cultured groups (P > 0.05). Furthermore, the sucrose and PVP treatment had lower tissue damage (P < 0.05) in comparison with X-1000 group (see Fig. 3).

Fig. 2.

Fig. 2.

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Representative photomicrographs of preantral follicles. Fresh (a), in vitro cultured (b) or vitrified [Sucrose group (c), X-1000 group (d), Z-1000 group (e) and PVP group (f)]. Scale bar = 100 μm.

Table 3.

Fluorescence intensity assessment of follicle degeneration and ROS levels (mean ± SEM) on bovine ovarian fragments submitted to treatments. Bovine ovarian tissues were non-vitrified (fresh control), cultured control (cultured control) or vitrified and culture for 7 days in the presence of sucrose, X-1000, Z-1000 and PVP.

Treatments degeneration ROS
Fresh control 14.9 ± 0.3 34.3 ± 2.4
In vitro culture (Day 5)
Fresh cultured 37.8 ± 2.5 AB* 69.2 ± 3.0A*
Sucrose 31.7 ± 2.6A* 69.7 ± 3.2A*
X-1000 45.1 ± 4.1B* 47.5 ± 4.0B*
Z-1000 35.9 ± 1.4 AB* 40.9 ± 1.8B*
PVP 31.6 ± 1.5A* 37.1 ± 1.4B
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Fig. 3.

Fig. 3.

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Viability assessment of bovine ovarian tissue of fresh and vitrified samples cultured for 5 days using ethidium homodimer-1 (EthD-1). Staining with ethidium homodimer (red areas) reveals the nuclei of dead cells in the ovarian tissue. Representative photomicrographs of ovarian tissue from fresh control (a), fresh cultured samples (b), Sucrose group (c), X-1000 group (d), Z-1000 group (e) and PVP group (f). Scale bar = 100 μm.

3.2.1. Reactive oxygen species

PVP K-12 group was the only group in that ROS level was similar to fresh control (Table 3). In addition, the ROS levels did not differ (P > 0.05) among polymers (X-1000, Z-1000, and PVP). A higher (P < 0.05) ROS levels were observed in the IVC control and sucrose treatments in comparison with polymers (X-1000, Z-1000, and PVP).

3.3. Immunolocalization of aquaporins (AQPs)

Immunohistochemical analysis was performed using antibodies against two AQPs (3 and 9) and immunoreactivity was detected in three groups (fresh control, X-1000 and Z-1000) during post-thawing and in vitro culture for 1 day at various developmental stages. Data are presented in Fig. 4 and summarized in Table 4. In fresh control, we observed a moderate staining for AQP3 in the primordial, but weak in the developing follicles. The post-thawed X-1000 group, we observed staining similar the fresh control, except in the primary follicle (moderate staining). Moreover, the post-thawed Z-1000 group has an increase staining stage-dependent. However, the IVC control group had moderate staining in all the follicular stage. The post-thawed and cultured Z-1000 group presented similar staining to the fresh cultured, except in the intermediary category (moderate staining). In the post-thawed and cultured X-1000 the primordial and intermediary follicles presented a weak staining. Furthermore, secondary follicles showed strong labeling for AQP3 in cultured X-1000 group.

Fig. 4.

Fig. 4.

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ROS levels measurement of bovine ovarian tissue of fresh and vitrified samples cultured for 5 days using 2-,7-dichlorodihydrofluorescein diacetate (DCFH2-DA) method. Representative photomicrographs of ovarian tissue from fresh control (a), fresh cultured samples (b), Sucrose group (c), X-1000 group (d), Z-1000 group (e) and PVP group (f). Scale bar = 100 μm.

Table 4.

Relative intensity of immunostaining for aquaporin (AQP) 3 and 9 in bovine preantral follicles enclosed in ovarian tissue on different categories.

AQP-3
Post-thawing
 IVC (Day 1)
primordial transition primary secondary primordial transition primary secondary
Fresh control ++ + + NF ++ ++ ++ NF
X-1000 ++ + ++ NF + + ++ +++
Z-1000 + ++ +++ NF ++ + ++ NF
AQP-9
Fresh control + + +++ NF + ++ +++ ++
X-1000 +++ +++ + NF + + − −
Z-1000 ++ ++ − +++ − − − −
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(NF) No follicle observed, (−) Absent; (+) weak; (++) moderate; (+++) strong immunostaining.

The presence of AQP9 was not confirmed in all evaluated groups and different labeling patterns were recorded. Primordial and transitional follicles showed weak labeling for AQP9 in fresh control group, strong labeling in post-thawed X-1000 group and moderate labeling in post- thawed Z-1000 group. Primary follicles were strongly immunostained in fresh control group, whereas primary follicles of post-thawed X-1000 group showed only weak labeling and no labeling in post-thawed Z-1000 group for AQP9. However, secondary follicles showed strong labeling for AQP9 in post-thawed Z-1000 group. On day 1 of culture, the absence of AQP9 was confirmed in all follicles at various developmental stages of cultured Z-1000 group. Moreover, Primordial and intermediary follicles were weakly immunostained for AQP9 in post-thawed and cultured X-1000 group. In despite of this, the Fresh cultured group has an increase staining stage-dependent.

4. Discussion

The challenge in formulating successful cryoprotective agents is to design vitrification solutions that are non-toxic but allow for vitrification at realistic cooling and warming rates [4]. In an attempt to address this challenge, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue.

In the present study, post-thawed and cultured Z-1000 samples on D1 resulted in a lower percentage of normal follicles in comparison with other groups. These results are consistent with that of previous study in rabbit [23], expressing that SuperCool Z-1000 included in vitrification media form a plasticised film and disrupt the embryonic implantation rate. Film formation from polymer solutions occurs as the solvent evaporates, since the polymer chains are intimately mixed [16]. In our study, probably, the Z-1000 could formed this film around the fragment and disrupted the CPA removal and perfusion of essential substances for in vitro culture. Furthermore, Wowk and Fahy [36] showed that Pseudomonas syringae INA was the only defined nucleator that PGL was effective against. Therefore, the ineffectiveness of Super cool Z-1000 (PGL) alone for reducing the number of ice nucleation events in large solution volumes suggests that the numerical majority of ice nucleating contaminants in the laboratory also do not resemble Pseudomonas syringae. Furthermore, no labeling for AQP9 of cultured Z-1000 group was observed. The strong expression of AQP9 in ovarian tissues could have an important implication in follicular development, because it is a GLP and therefore allows the passage of other electrolytes [29]. In this case, the protein could ensure a sufficient supply of estrogen to granulosa cells, making it essential for follicle development [31]. Therefore, the lower percentage of normal follicles in SuperCool Z-1000 group in comparison with other groups may also be due to the negative effects of this synthetic polymer on AQP9 presence after vitrification.

During almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. These results are consistent with that of previous study expressing that SuperCool X-1000 included in vitrification media has been beneficial in vitrification solutions for vitrified equine oocytes and similarly for tissue engineered bone [24]. PVA was found to be partially effective against almost all ice nucleating agents tested, including Pseudomonas syringae INA. Remarkably, considering its simple structure, the synthetic polymer PVA has been found to have ice recrystallization inhibition (IRI) activity comparable to the AF(G)P type 1 proteins without the detrimental effects of dynamic ice shaping (DIS) [18]. Immunohistochemistry confirmed the presence of AQP3 in all follicles at various developmental stages of post-thawed and cultured X-1000 group. This result is in accordance with the observations reported by Campos-Chillon et al. [ 6], who stated that during vitrification of bovine embryos, AQP3 assists in the permeability of ethylene glycol and also functions as a biomarker that indicates the quality of the embryos following cryopreservation [5]. Therefore, this wide presence of AQP3 may be correlated with the highest percentage of morphologically normal follicles in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. Sales et al. [29] reported that both AQP3 and AQP9 mRNAs were down-regulated following vitrification and IVC of ovine ovarian tissue and speculated that these negative effects are caused during the vitrification process. Thus, strong labeling for AQP9 in post-thawed X-1000 group might have been attributable to the higher capacity of preantral follicles to preserved normal morphology after vitrification, when compared with others.

Interestingly, the percentage of morphologically normal follicles in PVP K-12 group was the highest on post-warming day, lower on day 1 of culture and the lowest on day 5 when comparing to other group at the same day (Table 1). Hashimoto et al. [19] indicated that a cryoprotectant containing a combination of ethylene glycol and PVP supports follicle morphology in thin slices of monkey ovarian tissues after ultra-rapid vitrification compared with a cryoprotectant containing a combination of ethylene glycol and DMSO. However, Amorim et al. [3] showed that solution containing PVP were not superior to those obtained using Huang et al.’s [20] vitrification solution. It is possible these contradictory results may be due to the use of different kinds and concentrations of PVP. Furthermore, in this study the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group (Table 3) and only in PVP K-12 group, ROS level was similar to that of the fresh control group (Table 4). Isachenko et al. [21] reported that long exposure to SuperCool X-1000 could dramatically reduce the viability of human ovarian tissue after warming, possibly due to the toxicity of this product. On the other hand, the US Food and Drug Administration has approved PVP [27] and pure PVP shows high solubility and low toxicity [19]. Therefore, the unsatisfactory results obtained with vitrification of human ovarian tissue using an EG-based solution containing Ficoll or PVP [2] and contradictory results obtained with vitrification of bovine ovarian tissue using PVP K-12 in this study do not allow conclusions to be drawn about the impact of this synthetic polymer on the vitrification of ovarian tissue.

Taken together, we have shown that preantral follicles can be morphologically and functionally preserved following vitrification of bovine ovarian tissue using synthetic polymers. However, morphology after cryopreservation did not correlate with follicle viability and function (considering Supercool X-1000 and PVP K-12, respectively). We believe that further research to confirm the functionality of using these synthetic polymers in vitrification of bovine ovarian tissue is needed.

Acknowledgments

The authors gratefully acknowledge the entire staff of Department of Animal Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran and Laboratory of Manipulation of Oocytes and Ovarian Preantral Follicles (LAMOFOPA), Faculty of Veterinary Medicine of Ceará State University, Fortaleza, Ceara, Brazil.

Statement of funding

Funding for this work was provided by University of Tabriz and National Council of Technological and Scientific Development (CNPq: 433.262/2016-8). Ana Paula Ribeiro Rodrigues is the recipient of a grant (Number of the process: 308.071/2016-6) from CNPq. This work was also supported by a Fulbright Visiting Professor Scholarship to Ana Paula Ribeiro Rodrigues, NIH R01HD083930 to M.B. Zelinski and NIH P51OD011092 to the Oregon National Primate Research Center.

Footnotes

Declaration of competing interest

There is no competing interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cryobiol.2020.04.007.

References

  • [1].Adam M, Saller S, Ströbl S, Hennebold JD, Dissen GA, Ojeda SR, Stouffer RL, Berg D, Berg U, Mayerhofer A, Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule, Hum. Reprod. 27 (2012) 3249–3258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Amorim CA, Curaba M, Van Langendonckt A, Dolmans MM, Donnez J, Vitrification as an alternative means of cryopreserving ovarian tissue, Reprod. Biomed. Online 2 (2011) 160–186. [DOI] [PubMed] [Google Scholar]
  • [3].Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, Donnez J, Van Langendonckt A, Vitrification and xenografting of human ovarian tissue, Fertil. Steril 98 (2012) 1–8. [DOI] [PubMed] [Google Scholar]
  • [4].Badrzadeh H, Najmabadi S, Paymani R, Macaso T, Azadbadi Z, Ahmady A, Super cool X-1000 and Super cool Z-1000, two ice blockers, and their effect on vitrification/warming of mouse embryos, Eur. J. Obstet. Gynecol. Reprod. Biol 151 (2010) 70–71. [DOI] [PubMed] [Google Scholar]
  • [5].Camargo LSA, Boite MC, Viana SW, Mota GB, Serapiao RV, Sa WF, Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro, Cryobiology 63 (2011) 256–262. [DOI] [PubMed] [Google Scholar]
  • [6].Campos-Chillòn LF, Walker DJ, Torre-Sanchez JF, Seidel GE, In vitro assessment of a direct transfer vitrification procedure for bovine embryos, Theriogenology 65 (2006) 1200–1214. [DOI] [PubMed] [Google Scholar]
  • [7].Carvalho AA, Faustino LR, Silva CM, Castro SV, Lopes CA, Santos RR, Báo SN, Figueiredo JR, Rodrigues AP, Novel wide-capacity method for vitrification of caprine ovaries: ovarian Tissue Cryosystem (OTC), Anim. Reprod. Sci 138 (2013) 220–227. [DOI] [PubMed] [Google Scholar]
  • [8].Castro SV, Carvalho AA, Silva CM, Santos FW, Campello CC, Figueiredo JR, Rodrigues AP, Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture, Cell Tissue Bank. 15 (2014) 591–601. [DOI] [PubMed] [Google Scholar]
  • [9].Corral A, Clavero M, Gallardo M, Balcerzyk M, Amorim CA, Parrado-Gallego Á, Dolmans MM, Paulini F, Morris J, Risco R, Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography, Cryobiology 81 (2018) 17–26. [DOI] [PubMed] [Google Scholar]
  • [10].da Silva Sampaio AM, Bruno JB, de Lima LF, Ribeiro de Sá NA, Lunardi FO, Ferreira AC, Vieira Correia HH, de Aguiar FL, Araújo VR, Lobo CH, de Alencar Araripe Moura A, Campello CC, Smitz J, de Figueiredo JR, Ribeiro Rodrigues AP, Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue, Theriogenology 85 (2016) 1457–1467. [DOI] [PubMed] [Google Scholar]
  • [11].Dalman A, Deheshkar Gooneh Farahani NS, Totonchi M, Pirjani R, Ebrahimi B, Rezazadeh Valojerdi M, Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: an evaluation of histological changes, WNT signaling pathway and apoptotic genes expression, Cryobiology 79 (2017) 29–36. [DOI] [PubMed] [Google Scholar]
  • [12].de Graaf IA, Draaisma AL, Schoeman O, Fahy GM, Groothuis GM, Koster HJ, Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification, Cryobiology 1 (2007) 1–12. [DOI] [PubMed] [Google Scholar]
  • [13].De Vos M, Smitz J, Woodruff TK, Fertility preservation in women with cancer, Lancet 384 (2014) 1302–1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Deller RC, Vatish M, Mitchell DA, Gibson MI, Synthetic polymers enable non- vitreous cellular cryopreservation by reducing ice crystal growth during thawing, Nat. Commun 5 (2014) 32–44. [DOI] [PubMed] [Google Scholar]
  • [15].Fahy GM, Wowk B, Wu J, Paynter S, Improved vitrification solutions based on the predictability of vitrification solution toxicity, Cryobiology 48 (2004) 22–35. [DOI] [PubMed] [Google Scholar]
  • [16].Felton LA, Mechanisms of polymeric film formation, Int. J. Pharm 457 (2013) 423–427. [DOI] [PubMed] [Google Scholar]
  • [17].Gandolfi F, Paffoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G, Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models, Fertil. Steril 85 (2006) 1150–1156. [DOI] [PubMed] [Google Scholar]
  • [18].Gibson MI, Barker CA, Spain SG, Albertin L, Cameron NR, Inhibition of ice crystal growth by synthetic glycopolymers: implications for the rational design of antifreeze glycoprotein mimics, Biomacromolecules 10 (2009) 328–333. [DOI] [PubMed] [Google Scholar]
  • [19].Hashimoto S, Suzuki N, Yamanaka M, Hosoi Y, Ishizuka B, Morimoto Y, Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues, Reprod. Biomed. Online 21 (2010) 501–509. [DOI] [PubMed] [Google Scholar]
  • [20].Huang L, Mo Y, Wang W, Li Y, Zhang Q, Yang D, Cryopreservation of human ovarian tissue by solid-surface vitrification, Eur. J. Obstet. Gynecol. Reprod. Biol 139 (2008) 193–198. [DOI] [PubMed] [Google Scholar]
  • [21].Isachenko V, Isachenko E, Rahimi G, Krivokharchenko A, Alabart JL, Nawroth F, Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution, Cryo Lett. 23 (2002) 333–344. [PubMed] [Google Scholar]
  • [22].Makarevich AV, Kubovicova E, Popelkova M, Fabian D, Cikos S, Pivko J, Chrenek P, Several aspects of animal embryo cryopreservation: anti-freeze protein (AFP) as a potential cryoprotectant, Zygote 18 (2010) 145–153. [DOI] [PubMed] [Google Scholar]
  • [23].Marco-Jiménez F, Jiménez-Trigos E, Lavara R, Vicente JS, Generation of live offspring from vitrified embryos with synthetic polymers SuperCool X-1000 and SuperCool Z-1000, Cryo-Letters 35 (2014) 286–292. [PubMed] [Google Scholar]
  • [24].McGrath J, Liu BL, Vitrification solutions for the cryopreservation of tissue- engineered bone, Cell Preserv. Technol 2 (2004) 133–143. [Google Scholar]
  • [25].McLaughlin M, Telfer EE, Oocyte development in bovine pri- mordial follicles is promoted by activin and FSH within a two-step serum-free culture system, Reproduction 139 (2010) 971–978. [DOI] [PubMed] [Google Scholar]
  • [26].Mutlu MF, Erdem A, Evaluation of ovarian reserve in infertile patients, J. Turk. Ger. Gynecol. Assoc 13 (2012) 196–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Nair B, Final report on the safety assessment of polyvinylpyrrolidone (PVP), Int. J. Toxicol 17 (1998) 95–130. [Google Scholar]
  • [28].Sales AD, Brito IR, de Lima LF, Lobo CH, Duarte AB, Souza CE, Moura AA, de Figueiredo JR, Rodrigues AP, Expression and localization of Aquaporin 3 (AQP3) in folliculogenesis of ewes, Acta Histochem. 116 (2014) 831–837. [DOI] [PubMed] [Google Scholar]
  • [29].Sales AD, Duarte AB, Santos RR, Alves KA, Lima LF, Rodrigues GQ, Brito I, Lobo CH, Bruno JB, Locatelli Y, Figueiredo JR, Rodrigues AP, Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture, Cell Tissue Res. 365 (2016) 415–424. [DOI] [PubMed] [Google Scholar]
  • [30].Santos J, Rutigliano H, Sa Filho M, Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows, Anim. Reprod. Sci 110 (2009) 207–221. [DOI] [PubMed] [Google Scholar]
  • [31].Skowronski MT, Kwon TH, Nielsen S, Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system, J. Histochem. Cytochem. 57 (2009) 61–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Smith GD, Serafin PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, Alegretti J, Motta EL, Prospective randomized comparision of human oocyte cryopreservation with slowrate freezing or vitrification, Fertil. Steril 94 (2010) 2088–2095. [DOI] [PubMed] [Google Scholar]
  • [33].Ting AY, Yeoman RR, Lawson MS, Zelinski MB, Synthetic polymers improve vitrification outcomes of macaque ovarian tissue as assessed by histological integrity and the in vitro development of secondary follicles, Cryobiology 1 (2012) 1–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Ubaldi F, Anniballo R, Romano S, Baroni E, Albaricci L, Colamaria S, Capalbo A, Sapienza F, Vajta G, Rienzi L, Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program, Hum. Reprod. 25 (2010) 1199–1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Wang X, Gook DA, Walters KA, Anazodo A, Ledger WL, Gilchrist RB, Improving fertility preservation for girls and women by coupling oocyte in vitro maturation with existing strategies, Wom. Health 12 (2016) 275–278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Wowk B, Fahy GM, Inhibition of bacterial ice nucleation by polyglycerol polymers, Cryobiology 44 (2002) 14–23. [DOI] [PubMed] [Google Scholar]
  • [37].Wowk B, Leitl E, Rasch CM, Mesbah-Karimi N, Harris SB, Fahy GM, Vitrification enhancement by synthetic ice blocking agents, Cryobiology 3 (2000) 228–236. [DOI] [PubMed] [Google Scholar]

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