Cryopreservation of ovarian tissue is a promising approach for fertility preservation in human and animal species (Picton, 2018). In human, many studies on fertility preservation has been reported with a purpose to evade infertility caused from some kinds of malignant diseases (Donnez and Dolmans, 2017; Forman, 2018) and to restore from chemical therapies (Nichols-Burns et al., 2014). In animals, many studies has also been reported a purpose with preservation of genetic resource, preservation of fertility, and preliminary study for human in many species including pig (Borges et al., 2009) sheep (Fathi et al., 2011), goat (Rodrigues et al., 2004), cow (Kagawa et al., 2009), dog (Brandão et al., 2021), mouse (Salehnia et al., 2002; Terraciano et al., 2020), and non-human primate (Nyachieo et al., 2013; Lu et al., 2014).

Generally, cryopreservation of oocyte and ovarian tissue can be carried out by two different methods such as conventional slow freezing and vitrification method. These two methods showed comparable result on its post-warming and -thawing survival even different hurdles of cryoinjury (Hwang and Hochi, 2014). The main hurdles during slow freezing is a formation of ice crystal resulting physical damage within the cell and tissue (Hwang and Hochi, 2014), while the temperature falls ultra-rapidly during vitrification procedure, there is no time or energy for molecular rearrangement of water resulting no cryoinjury by formation of ice crystal (Leonel et al., 2019). Also, elimination of cellular water during vitrification procedure can help to prevent ice crystallization of water within the cell and tissue (Hwang and Hochi, 2014). On the other hands, higher concentration of cryoprotectant from vitrification method than conventional freezing method is not very helpful to revive the cell and tissue after thawing and warming procedure by generating chemical injury including premature cortical granule exocytosis, microtubule disorganization and multiple aster formation (Hwang and Hochi, 2014). However, vitrification method is already well established for cryopreservation of human and animal oocytes, ovarian tissue vitrification remains still a challenge because of its diversity of composition (Hwang et al., 2013; Leonel et al., 2019).

Although, the pigs can be an ideal research model for agricultural and biomedical study due to the similar physiology, development, and disease patterns as seen in humans, the fertility preservation by vitrification method in pig are very limited and less well studied than other species (Borges et al., 2009; Whyte and Prather, 2011). In pigs, similar to other species, many conditions can affect the efficiency of ovarian tissue vitrification, which include but are not limited to concentration of cryoprotectant, volume of specimens, type of device resulting speed of cooling and warming (Gandolfi et al., 2006). Additionally, a morphological assessment is the best way to examine the post-warming result of vitrification methods, the results can be finalized by analyzing cryoinjuries during vitrification procedure.

Therefore, the present study was conducted to investigate whether porcine ovarian tissue can be cryopreserved by vitrification method and to examine the possible cryoinjuries on its diversity composition during vitrification procedures.

Chemicals

All chemicals used in the present study were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA) unless otherwise stated.

Preparation of ovarian tissue slices

Ovaries were obtained from prepubertal gilts at a local slaughterhouse (Nonghyup Moguchon, Gimje, Korea) and transported to the laboratory within 1 h in saline at approximately 30 to 35℃. Ovaries were washed twice in saline containing antibiotics and large follicles (> 3 mm in diameter) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe. Then, the ovarian tissues were collected with size of 10 mm (width) × 10 mm (length) × 1 mm (thickness) each using a square measure (Kitazato, Shizuoka, Japan). For fresh control group, fresh ovarian tissue slices were washed twice in PBS and fixed in 10% buffered formalin solution (M961-20FW, Simport Scientific Inc., Quebec, Canada) for further experiment. Five individual slices were taken from five different ovaries in each group.

Vitrification procedures of ovarian tissues

Vitrification procedure of ovarian tissue of pig was conducted using commercial vitrification kit (VT301-CT, Kitazato) according to manufacturer’s protocol. This kit includes equilibration solution (ES) and vitrification solution (VS) prepared with medium 199 mixed with serum substitute supplement, ethylene glycol, gentamycin, sucrose, and polyvinylpyrrolidone. Briefly, each slice was treated firstly within 15 mL of ES in 60 mm dish for 25 min at room temperature. After treatment of ES, the slices were put on the surface of 15 mL of VS in 60 mm dish and let it down for 15 min at room temperature. Finally, the slice was mounted onto a device of Cryotissue after removal of excess solution by absorption with sterilized gauze and plunged into liquid nitrogen. The cooling rate of the Cryotissue system is -17,000℃ /min according to manufacturer’s protocol. The Cryotissue containing vitrified ovarian tissue slice were kept in the liquid nitrogen at least 2 weeks until warming. Additionally, for vitrification control group, fresh ovarian tissue slices were directly plunged into liquid nitrogen and kept at least 2 weeks until warming.

Warming procedures of ovarian tissues

Warming procedure of ovarian tissue of pig was conducted using commercial thawing kit (VT302-CT, Kitazato) according to manufacturer’s protocol. This kit includes thawing solution (TS), diluent solution (DS) and washing solution (WS) prepared with medium 199 mixed with serum substitute supplement, ethylene glycol, gentamycin, sucrose, and polyvinylpyrrolidone. Briefly, the Cryotissue containing vitrified ovarian tissue slice were immersed into TS warmed to 37℃ within 1 sec and treated for 1 min to be detached the slice by itself from the Cryotissue. Then, the vitrified-warmed ovarian tissue slices were treated at room temperature with 15 mL of DS, WS1 and WS2 in a stepwise manner for 3, 5, and 5 min respectively. The warming rate of the Cryotissue system is +32,000℃/ min according to manufacturer’s protocol. After treatment of WS2 ovarian tissue slices were washed twice in PBS and fixed in 10% buffered formalin solution (Simport Scientific Inc.) for further experiment. Additionally, for vitrification control group, frozen ovarian tissue slices were directly immersed into PBS warmed to 37℃ and fixed in 10% buffered formalin solution (Simport Scientific Inc.) for further experiment.

Histology of ovarian tissues

Vitrified-warmed, frozen, and fresh ovarian tissue slices in each of the three groups were fixed with 10% buffered formalin solution and subjected to histological evaluation. After fixation, all slices were washed three times in PBS and dehydrated with 50 to 100% of ethanol solutions in a stepwise manner. After paraffin embedding, slices were sectioned (4 μm thickness) and stained with hematoxylin and eosin for a general analysis of the tissue, follicle, and oocyte morphology.

TUNEL assay of ovarian tissues

The terminal deoxynucleotidyl transferase-mediated dUDP nick end labeling (TUNEL) assay with paraffin embedded ovarian tissue was performed with previous report by Hwang et al. (2020). Paraffin-embedded ovarian tissue slices on slides were deparaffinized by more than two treatments with xylene for 5 min each and then dehydrated by serial dilution in ethanol and distilled water. The slides were then washed twice in PBS supplemented with 0.1% polyvinylpyrrolidone and permeabilized with 0.5% of Triton X-100 for 30 min at room temperature. A TUNEL assay was applied to assess the presence of apoptotic cells using an In Situ Cell Death Detection Kit in accordance with the manufacturer’s instructions. After the TUNEL reaction, slides were covered in ProLong Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). The slides were maintained at -20℃, and the patterns of apoptotic cells were determined by under an epifluorescence microscope (Nikon, Tokyo, Japan).

Statistical analysis

The experiments were replicated at least five times in each group. The rates of damaged oocyte were analyzed using a one-way analysis of variance test (freely available at http://vassarstats.net) and the significance between the means of analyzed data was evaluated using Tukey’s multiple comparison test. p value less than 0.05 was considered statistically significant.

Histological evaluation of ovarian tissues

As shown in Fig. 1 and 2, histological evaluation was performed in 3 different groups. Similar to our expectation, some kinds of cryoinjuries in freezing control group was highly detected in comparison with fresh control and vitrification groups. In Fig. 1, overall shape change of ovarian tissue specimens was observed in freezing control group while the shapes were maintained in fresh and vitrification groups. As shown Fig. 2, damaged including inflated ball morphology (shrunken, red arrow) was discovered in freezing control group compared to fresh control and vitrification groups (intact, black arrow). In addition, another cryoinjury that form a wave pattern (red arrow heads) of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group (black arrow heads). In Fig. 3, the rates of damaged oocyte were examined between groups. The results showed that distorted morphology in oocytes was significantly (p < 0.05) lower in vitrification group (25.8 %) than freezing control group (67.7 %), while fresh control group (6.6 %) showed significantly (p < 0.05) lower rate than both groups.

Figure 1.Representative images of ovarian tissues from fresh control, freezing control, and vitrification groups after hematoxylin and eosin staining. The ovarian tissues were collected with size of 10 mm (width) × 10 mm (length) × 1 mm (thickness) each using a square measure. Scale bars = 2 mm.

Figure 2.Representative images of oocyte and tissues from fresh control, freezing control, and vit-rification groups after hematoxylin and eosin staining. Black and red arrows indicate intact and damaged oocytes respectively. Black and red arrow heads indicate normal and wave pattern cryoinjury respectively. Scale bars = 100 μm.

Figure 3.Rates of damaged oocyte from fresh control, freezing control, and vitrification groups. (a-c) Different superscripts denote significant differences (p < 0.05).

Apoptotic patterns of ovarian tissues

As shown in Fig. 4, Apoptosis pattern was examined by TUNEL assay in 3 groups. In result, apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency.

Figure 4.Apoptotic cell patterns from fresh control, freezing control, and vitrification groups Black and red arrows indicate apoptotic cells of partial and whole around follicle respectively. Scale bar = 100 μm.

The pigs are very important animal for the research of agricultural and biomedical research field (Whyte and Prather, 2011), but the challenges to preserve genetic material from female is not sufficient to reflect its demands compared to other animal species, such as sheep (Fathi et al., 2011), goat (Rodrigues et al., 2004), cow (Kagawa et al., 2009), dog (Brandao et al., 2021), mouse (Salehnia et al., 2002; Terraciano et al., 2020), and non-human primate (Nyachieo et al., 2013; Lu et al., 2014). Successful establishment of cryopreservation system using female genetic material has many benefits in case of animal species especially valuable transgenic animal (Takeo et al., 2020) and endangered animal (Santos et al., 2010). In human differ from animal species, it has been made higher progress and well established because of their beneficial effects of successful cryopreservation of the oocyte, embryo, and ovarian tissue on fertility preservation against disease (Donnez and Dolmans, 2017).

In the present study, it was discovered that the vitrification method used present study could protect from oocyte morphology changes including shrunken, damaged and inflated form after warming by morphological analysis. Although morphological evaluation is very useful to assess before conducting further step, it is not correlated always to the survivability or developmental competence after warming (Hwang et al., 2018). Therefore, a comprehensive analysis of cryoinjury needed to be applied using other methods like viability tests, apoptosis assay, and in vitro culture to achieve growth and development (Ramezani et al., 2017). In addition, many studies have been described that xenografted ovarian tissue from some species could produce antral follicles with oocytes after cryopreservation (Gook et al., 2001; Gook et al., 2003; Bosch et al., 2004). In mice, it has been shown that generation of live young is possible using oocytes derived from xenografted ovaries tissue (Snow et al., 2002). Although the efficiency is very limited in pigs, whole ovarian tissue or fragmented ovarian cortex were cryopreserved and xenografted in an immunodeficient nude mice (Kaneko et al., 2003; Kikuchi et al., 2006; Lotz et al., 2015; Gabriel et al., 2017). Another alternative approach for fertility preservation was the xenograft of the whole ovary into ovariectomized nude rats (Nichols-Burns et al., 2014). Moreover, ovarian autograft in pigs is still under study for the preservation of fertility and hormonal function (Damásio et al., 2016).

To establish cryopreservation system, mainly two different types of method has been developed and applied including slow freezing and vitrification. As mentioned previously, the main hurdle during slow freezing is formation of ice crystal resulting physical cryoinjuries in cell and tissue (Hwang and Hochi, 2014). In the present study, we confirmed the physical cryoinjuries by ice crystal formation in freezing control group without dehydration procedure by vitrification resulting shape change of ovarian tissue specimens and distorted morphology of oocytes. Similarly, a previous report showed that the vitrification method is more reasonable than slow freezing to prevent cryoinjuries (Shi et al., 2017). However, the vitrification method also has negative side such as chemical cryoinjuries by high concentration of cryoprotectants (Hwang and Hochi, 2014). To restore fertility using oocyte, embryo and ovarian tissue, it is still required to overcome their sensitivity to cryoinjuries during cryopreservation procedures (Zhou and Li, 2009). Further optimization of vitrification procedure and treatment during and/or after warming could be a promising approach to reduce cryoinjury and to improve the viability of cell and tissue respectively (Hwang et al., 2016).

In conclusion, it was confirmed that vitrification method has a positive effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material and fertility preservation in pigs. Without dehydration procedure, physical cryoinjuries occurred in the tissue and oocytes from ovarian cortex caused by formation of ice crystal. Further study to restore fertility in pigs, in vitro culture, xenograft and allograft system is needed to be established.

None.

Conceptualization, I-S.H.; methodology, I-S.H.; Investigation, I-S.H.; data curation, I-S.H.; writing original draft preparation, I-S.H.; writing review and editing, I-S.H.; supervision, I-S.H.

This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ01335401), Rural Development Administration, Republic of Korea.

Not applicable.

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No potential conflict of interest relevant to this article was reported.