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ISSN : 1738-2432(Print)
ISSN : 2288-0151(Online)
Reproductive & developmental Biology Vol.36 No.4 pp.255-260

Successful In Vitro Development of Preantral Follicles Isolated from Vitrified Mouse Whole Ovaries

Dong-Hoon Kim, Jin-Gu No, Jong-Ju Park, Jin-Ki Park, Jae-Gyu Yoo
Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon
(Received: 17 November 2012/ Accepted: 14 December 2012)


The purpose of this study was to assess follicular viability and competence through in vitro culture of preantralfollicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified-warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro maturedoocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution ofwhole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposuregroups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups (69.4±2.8 and 67.8±3.1)when compared to that of control group (71.7±2.1). There was no statistical significant difference in blastocystdevelopment rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cellsper blastocyst was significantly lower (p<0.05) in the vitrification group (41.9±20.2) than in the fresh control group(55.1±22.5). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovariescan achieve full maturation, normal fertilization and embryo development.



Cryopreservation of ovaries is a promising technique for long-term fertility preservation for female. This technique would enable to preserve and restore ovarian function in cancer patients who subjected to radiotherapy or chemotherapy. In addition, the technique might be useful for protection of the reproductive potential in valuable and endangered species (Oktay et al., 1998).

Cryopreserved ovarian tissues could be used in the autologous transplantation and the production of mature oocytes derived from preantral follicles within ovarian tissues after culture. Pioneering studies on the cryopreservation of ovarian tissue were carried out in rodents. These experiments resulted in the birth of offspring after orthotopic transplantation of frozen-thawed ovarian tissue (Parrott, 1960). Over recent years, significant improvements of cryopreservation technique has been demonstrated successful cryopreservation of ovarian tissues in rat (Aubard et al., 1998), rabbit (Daniel et al., 1983), sheep (Gosden et al., 1994) and human(Gook et al., 1998; Oktay et al., 1998). In addition, preantral follicles isolated from frozen-thawed mouse tissue could be successfully grown to antral follicles and produced mature oocytes (Newton and Illingworth, 2001).

Slow freezing method was used in most previous studies on cryopreservation of ovarian tissue (Candy et al., 1997; Gunasena et al., 1997; Newton and Illingworth, 2001). However, it is a time-consuming procedure and requires special equipment like a programmable freezer. Vitrification is an alternative cryopreservation technique might be effective organ cryopreservation method. The vitrification technique is also a convenient tool for cryopreserving small specimens. High concentrations of cryoprotectants are necessary to solidify without the formation of ice crystals during rapid cooling process of vitrification. However, cryoprotectants are very toxic to both oocytes and cells. Cryoprotectant toxicity can be minimized by increasing the cooling rate. Furthermore, the high rates of cooling prevent chilling damage (Rall and Fahy, 1985). The traditional vitrification system using cryovial or cryotube could not achieved good result with mouse ovaries (Newton and Illingworth, 2001). Direct contact between the ovary containing medium and liquid nitrogen could increased cooling rate (Chen et al., 2006) and cryotop safeguard the ovarian tissues during rapid cooling process (Hasegawa et al., 2004; 2006).

The purpose of this study was to evaluate the developmental potential of preantral follicles in vitrified mouse ovaries. In vitro growth of preantral follicles isolated from vitrified ovaries followed by in vitro maturation, fertilization and culture were performed. In addition, these findings were compared to those of fresh controls.


Collection of Ovaries

Ovaries from 12-day-old ICR mice were dissected free of fat and mesentery. Intact ovaries were transferred into Leibovitz L-15 medium (Gibco-BRL, Grand Island. NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL).

Vitrification and Thawing

Open straw was made by cut at the cotton plug part of 0.25 mL straws with a razor blade.

For vitrification, whole ovaries were exposed to Leibovitz L-15 medium supplemented with 20% FBS, 10% (v/v) dimethyl sulfoxide (DMSO; Sigma, St Louis, MO, USA) and 10% (v/v) ethylene glycol (EG; Sigma) for 5 minutes. The ovaries were subsequently moved to Leibovitz L-15 medium supplemented with 20% FBS, 0.5M sucrose (Sigma), 20% DMSO and 20% EG and equilibrated for 5 or 10 min. Loading of ovaries into these straws was performed using the capillary effect by simple touching a 3- to 5-μl droplet containing the ovaries with the end of the straw. The straws were submerged into liquid nitrogen immediately after loading.

Thawing was performed by placing the end of the straw directly into the 0.5 M sucrose solution (prepared in Leibovitz L-15 medium containing 20% FBS). The straw into the 0.5 M sucrose solution. And then recovered ovaries were sequentially transferred to 0.5, 0.25, 0.125 and 0 M sucrose solution at interval of 5 min. Each step was carried out at room temperature.

Preantral Follicle Isolation

Ovaries were immersed into Leibovitz L-15 medium containing 1 mg/ml collagenase (Type 1A, C-2674; Sigma) and 0.2 mg/ml DNase I (DN-25; Sigma) for 20 min at 37℃ and repeatedly drawn in and out of the pipette until the ovaries were dissociated into individual follicles. The follicles to be cultured were selected by the following criteria: i) intact round follicular structure with two to three layers of granulosa cells; ii) the oocyte had to be visible, round and centrally located within the follicle (Fig. 1).

Figure 1. Morphology of preantral follicle isolated from vitrified mouse ovary.

As a control, preantral follicles from fresh mouse ovary were isolated and cultured in an identical manner to those which had been isolated from vitrified-thawed ovary.

In Vitro Growth (IVG) of Preantral follicles

The IVG medium was αMEM medium supplemented with 100 mIU/ml FSH (Metrodin-HP; Sereno, Aubonne, Switzerland), 10 mIU/ml LH (L-5259; Sigma) and 5% FBS (Kim et al., 1999). The follicles were cultured on Transwell-COL membrane inserts (3.0 μm pore size, 24.5 mm diameter; Costar, Cambridge, MA, USA) in 6-well cluster dishes to prevent the loss of structural integrity between the oocyte and granulosa cells. The follicles were cultured for 10 days at 37℃ and 5% CO2 in air. Half of the medium was changed every 2 days (Fig. 2).

Figure 2. In vitro growth and maturation of preantral follicle isolated from vitrified mouse ovary. An preantral follicle after (A) 2 day, (B) 4 day, (C) 6 day, (D) 8 day and (E) 10 day of the in vitro growth. (F) An follicle stimulated with 1.5 IU/ml hCG for maturation.

In Vitro Maturation (IVM), Fertilization (IVF) and Subsequent Development

After 10 days of IVG, follicles were allowed to mature for 16~18 h in IVM medium supplemented with 1.5 IU/ml hCG (Profasi; Sereno) (Fig. 2). A part of matured follicles were selected randomly and adherent cumulus cells were removed. The maturation status and the diameter of the oocytes excluding the zona pellucida were examined with an inverted microscope and the ocular micrometer.

In vitro-matured oocyte-granulosa cell complexes were placed into 50 μl drops of Whittingham's T6 medium supplemented with 30 mg/ml bovine serum albumin (A-3311; Sigma) under mineral oil. The sperm collected from the cauda epididymis of mature ICR male mice were incubated for 2 h in fertilization medium before insemination. The oocytes were inseminated with spermatozoa with concentration of 1~2×106 / ml. Six hours after the insemination, the fertilized oocytes were cultured in 20 μl drops of KSOM medium supplemented with 10% (v/v) serum substitute supplement (SSS; Irvine, Santa Ana, CA, USA), 1% (v/v) essential amino acids (M-7145; Sigma) and 0.5% (v/v) non-essential amino acids (B-6766; Sigma) under mineral oil. The oocytes were examined the following day, and the number of 2-cell embryos was noted. The 2-cell embryos were further cultured for 4 days until the blastocyst stage.

Assessment of Cell Number of Blastocyst

The cell number of blastocysts (5 days after insemination) was evaluated by fluoresence staining. The blastocysts were fixed in 2% formaldehyde in PBS for 10 min at room temperature. The blastocysts were then placed on slides with a drop of mounting medium consisting of the 3:1 ratio of glycerol versus PBS containing 2.5 mg/ml sodium azide and 2.5 μg/ml Hoechst 33342 (B-2261; Sigma). A cover slip was placed on top of the oocytes and blastocysts, and the edge was sealed with nail polish. The stained blastocysts were examined under a fluorescence microscope.

Statistical Analysis

The statistical significance of the data was analyzed using a Student’s t-test and chi-square (χ2) test. A statistical significance was established at the p<0.05 level.


Recovery of Preantral Follicles from Vitrified Ovary

Fresh ovaries had three-fold more preantral follicles than vitrified-thawed ovaries (p<0.005) (Fig. 3). The periods of exposure to the vitrification solution did not significantly affect the number of harvested preantral follicles. Due to the death of granulosa and theca cells, the collapse of the oocyte within the follicle and the extrusion of denuded oocytes from the follicles were observed.

Figure 3. The number of preantral follicles harvested from vitrified mouse ovary. Values with different superscripts above each columnare significantly different (p<0.005).

In Vitro Growth and Maturation of Preantral Follicles Isolated from Vitrified Ovary

Five minutes exposure group (69.8%) in vitrification solution had significantly higher (p<0.05) survival rates of vitrified preantral follicles than 10 min exposure group (43.2%). The above value did not differ between the 5 min exposure and fresh group (62.1%) (Table 1).

Table 1. Survival and maturation rates of oocytes within preantral follicles of vitrified mouse ovaries after IVG and IVM.

After in vitro maturation, the proportion of oocytes that reached metaphase II stage were significantly higher (p<0.05) in 5 min exposure group (38.9%) in vitrification solution compared to the 10 min exposure group (26.0%). The proportion did not differ between the 5 min exposure and fresh group (37.9%) (Table 1). The diameter of metaphase II oocytes from vitrified groups was significantly smaller (p<0.05) than that of the fresh group (71.7±2.1 μm), and also there was significantly smaller in 10 min exposure group (67.8±3.1 μm) when compared to 5 min exposure group (69.4 ±2.8 μm) (Table 1).

In Vitro Development of Oocytes within Preantral Follicles

After in vitro fertilization, developmental rate to blastocyst stage was not different between the vitrification group (8.6%) and the fresh group (12.0%) (Table 2).However, cell numbers of blastocysts derived from vitrification group (41.9±20.2) was significantly lower (p< 0.05) than those of the fresh group (55.1±22.5) (Table 3).

Table 2. Development of oocytes within preantral follicles derived from vitirified mouse ovaries.

Table 3. Number of cells per blastocyst derived from vitrified mouse ovaries.


Traditionally, ovarian tissues are cryopreserved using low concentration of cryprotectants and slow freezing techniques that dehydrate the cell during freezing to prevent intracellular crystallization (Cortvrindt et al., 1996; Cox et al., 1996; Gunasena et al., 1997; Sugimoto et al., 2000; Liu et al., 2001). By using orthotopic grafting after slow freezing, the birth of offspring has been reported in mice (Parrott, 1960; Cox et al., 1996; Gunasena et al., 1997) and sheep (Gosden et al., 1994. However, slow freezing method reduced the survival rates of follicles after culture in vitro (Newton and Illingworth, 2001).

Vitrification is an alternative freezing method of freezing in which cells is frozen in high concentrations of cryoprotectants, which upon rapid cooling imparts a glasslike state. The complete absence of ice crystal formation eliminates the risk of intracellular freezing and the build up of salts in the extracellular medium (Newton and Illingworth, 2001). In most previous studies, vitrification method using a cryovial or tube has been employed for the cryopresrvation of ovarian tissues (Newton and Illingworth, 2001).  However, this method has been reported that the percentages of morphologically normal or viable follicles were significantly lower than those of non-frozen ovary (Chen et al., 2006). The cause for this result may be the chilling injury by the insufficient cooling rates of ovarian tissues. Several methods such as use of small amounts of solution have been proposed to overcome this problem (Hasegawa et al., 2004; Chen et al., 2006; Kim et al., 2011; Bos-Mikich et al., 2012).

Using open pulled-straw for vitrification resulted in significant improvement in the cryopreservation of oocytes and blastocysts (Vajta et al., 1998). With a small amount of vitrification solution and direct contact with liquid nitrogen, the open pulled-straw method achieved high thermal change. However, the open pulled-straw is not appropriate for handling larger size of tissue, such as an ovary. Therefore, we used open straw method that can hold larger size of tissue for vitrification, with direct contact by liquid nitrogen.

It was showed that the birth of mouse pups after orthotopic transplantation of cryopreserved ovary by vitrification (Migishima et al., 2003). Mouse pups were also born after transfer of embryos derived from vitrified preantral follicles (dela Pena et al., 2002). Recently, it was reported that the birth of mouse pups from mouse oocytes in preantral follicles derived from vitrifiedwarmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization (Hasegawa et al., 2006; Wang et al., 2011).

The exposure time to the vitrification solution before vitrification is very important to achieve adequate permeation by the cryoprotectant without incurring toxic damage or the cells vulnerable to excessive volume changes during the return to isotonic conditions after thawing. Therefore, optimization of exposure time in vitrification solution needed for mouse ovaries vitrification procedure. Mouse preantral follicles that were vitrified using vitrification solution containing ethylene glycol and raffinose showed prolonged exposure of preantral follicles to the vitrification solution reduced their developmental capacity (dela Pena et al., 2002). In the present study, exposure of mouse whole ovaries to vitrification solution supplemented with 20% DMSO and EG for 5 min after 5 min exposure to 10% DMSO and EG yielded higher follicular survival rate and oocyte maturation rate than 10 min exposure vitrification condition. Therefore, pre-equilibartion in vitrification solution for 5 min was sufficient for the effective permeation of the cryoprotectant.

After in vitro fertilization, developmental rate to blastocyst stage was not different between the vitrification group and the fresh group. However, cell numbers of blastocysts derived from vitrification group was significantly lower than those of the fresh group. In the previous study, the blastocyst rates and the number of cells in the blastocyst from vitrified-warmed ovaries was not different from fresh ovaries (Hasegawa et al., 2006). In this study, although vitrified-warmed ovaries derived blastocysts had significantly less number than fresh ovary derived blastocyst, the proportion of developing to blastocysts from vitrified-warmed ovaries was as high as fresh ovaries. Although the exact reasons of lower cell number in the blastocyst from vitrified-warmed ovaries followed by in vitro growth and maturation is unknown, the in vitro culture conditions for preantral follicles and fertilized embryos in the present study is different those of previous studies. Further studies are required to improve the preantral follicle culture conditions for the in vitro growth and development of preantral follicles from vitrified-warmed ovaries.

In conclusion, our results indicate that mouse oocytes within preantral follicles isolated from vitrified-thawed whole ovaries can be successfully grown and matured to metaphase II and developed to blastocyst stage after fertilization.


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