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Vitrification of Mouse Oocytes in Ethylene Glycol-Raffinose Solution: Effects of Preexposure to Ethylene Glycol or Raffinose on Oocyte Viability
Cryobiology 42, 103–111 (2001) doi:10.1006/cryo.2001.2310, available online at http://www.academicpress.com on
Vitrification of Mouse Oocytes in Ethylene Glycol–Raffinose Solution: Effects of Preexposure to Ethylene Glycol or Raffinose on Oocyte Viability E. C. dela Peña, Y. Takahashi,1 E. C. Atabay, S. Katagiri, and M. Nagano Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan We investigated the effects of preexposure to ethylene glycol (EG) or raffinose on the viability of vitrified mouse oocytes. Ovulated oocytes at the metaphase II stage were preexposed either to 2 M EG for 0, 2, or 5 min or to ascending concentrations (0.15 followed by 0.3 M ) of raffinose solution for 2, 5, or 10 min each (here referred to as 2-2, 5-5, and 10-10 min, respectively). The oocytes were then exposed to a vitrification solution (VS), 6 M EG ⫹ 0.3 M raffinose, for 0.5, 1, 2, or 5 min and then vitrified or immediately diluted. After warming, the developmental capacity of oocytes was determined after in vitro fertilization. Volume changes in oocytes during preexposures and exposure to the VS were also investigated. The results demonstrated that preexposure to 2 M EG allowed shorter exposure times of oocytes to the VS and that predehydration in raffinose solutions for 5-5, but not 2-2 or 10-10 min, allowed a wider range of exposure times to the VS. Experiments on volume change suggested that the optimum time of exposure to the VS depends on the amount of EG permeation after preexposure to 2 M EG or to raffinose solutions. Preexposures to 2 M EG or raffinose under optimized conditions increased the viability of vitrified-warmed oocytes compared to direct exposure to VS without preexposures. © 2001 Academic Press Key Words: oocytes; preexposure; dehydration; raffinose; ethylene glycol; vitrification.
Successful vitrification requires the combination of a high concentration of permeable cryoprotectant agent (CPA) and a very rapid cooling rate; however, high concentrations of CPA cause cell damage due to osmotic and cytotoxic effects (12, 13). Partial replacement of permeable CPAs by nonpermeable compounds such as sugar, polyvinylpyrrolidone, dextran, and Ficoll has been attempted in an effort to improve vitrification (6, 15). The critical cooling rate required to avoid ice crystallization in solutions with penetrating cryoprotectants was altered by the addition of raffinose, trehalose, sucrose, or glucose (16). Recently, raffinose has been found to modify the vitrification properties of ethylene glycol (EG) by raising the glass transition point comparable with other sugars (6). In addition, it had been found equally as effective as sucrose Received November 16, 2000; accepted March 19, 2001; published online May 21, 2001. This work was supported by Grant-in-Aid for Scientific Research 10556058 from the Japan Society for the Promotion of Science. 1 To whom correspondence should be addressed. 103
and glucose in the quick freezing of mouse morulae using glycerol (21) and beneficial in vitrifying mouse preimplantation-stage embryos in combination with dimethyl-sulfoxide and propylene glycol (18, 19). Attempts to vitrify oocytes and embryos in EG–raffinose solutions, however, have not been reported. Preexposure of embryos to a low concentration of permeable CPA before exposure to a high CPA concentration is also a well-known effective approach to improve embryo viability after vitrification (5, 12). However, the preexposure to sugar solutions before vitrification to effect dehydration has not been widely used and studied. It has been suggested that preexposure to sugar unfavorably affects the subsequent survival of mouse embryos (17); however, recent studies on preexposure in ascending concentrations of sucrose before exposure to a high concentration of CPAs showed increased survival and development of mouse blastocysts (8) and oocytes (9, 20). Despite the previous studies on preexposure before vitrification, detailed studies of the conditions during preexpo0011-2240/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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sure, especially during preexposure to sugar and subsequent exposure to the vitrification solution (VS), have not been well demonstrated. In the present study, we treated the oocytes with a low concentration of EG or with raffinose before exposure to a VS, a mixture of EG and raffinose, and investigated the interaction between the effects of preexposure and VS exposure on the viability of mouse oocytes. The present study also examined the volume changes of oocytes during both preexposure to EG or raffinose and exposure to the VS. MATERIALS AND METHODS
Source of Mouse Oocytes Female C57BL/6J and male CBA mice purchased from Japan SLC Inc. (Shizuoka, Japan) were housed in the animal housing facilities of the Graduate School of Veterinary Medicine following the guidelines set by the university to produce F1 offspring. They were kept under light- and temperature-controlled conditions (12 h light: 12 h dark photoperiod, 22°C) and given chow pellets and water ad libitum. Female F1 mice, 6–8 weeks of age, were induced to superovulate by intraperitoneal injection of 5 IU of equine chorionic gonadotropin (Teikoku Hormone Mfg. Co. Ltd., Tokyo, Japan) and 5 IU of human chorionic gonadotropin (hCG, Teikoku Hormone) given 48 h apart. They were killed by cervical dislocation 13 h after the hCG injection. The cumulus cell-enclosed oocytes were released from the ampulla into Dulbecco’s phosphate-buffered saline (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 4 mg/ml BSA, 75 mg/ml penicillin, and 50 mg/ml streptomycin (DPBS ⫹ BSA). Cumulus cells were removed by exposing them to DPBS containing 150 U/ml hyaluronidase (bovine testis type I-S, Sigma Chemical Co., St. Louis, MO, U.S.A.) for 2 to 3 min at 22 to 25°C. Cumulus-free oocytes were washed three times in fresh DPBS ⫹ BSA medium and pooled in a 35-mm plastic petri dish (Nalge Nunc Int. Kamstrup, Denmark) until they were used. Only oocytes with normal morphological appearance and with a visible polar body were used in the experiments.
Vitrification Procedures After preexposure to EG (Kanto Chemical Co. Inc., Tokyo, Japan) or raffinose (Sigma) solutions, groups of 20 oocytes were initially rinsed in a 100-l drop of VS which was composed of 6 M EG with 0.3 M raffinose in DPBS ⫹ 10% fetal calf serum (FCS, Gibco Laboratories, Grand Island, NY, U.S.A.). The oocytes were finally transferred to a 40-ml droplet of the VS at room temperature (22 to 25°C). The exposed oocytes were drawn into 0.25-ml French straws (I.M.V., L’Aigle, France). The straws were heat sealed and cooled in liquid nitrogen (LN2) vapor for at least 1 min. Finally, the straws were plunged into LN2 and stored for 1 to 120 days. Exposure times of 0.5, 1, 2, and 5 min elapsed from the time the oocytes were initially rinsed in a 100-ml drop of VS until they were cooled in LN2 vapor. For loading the straw, approximately 100 ml of DPBS ⫹ BSA with 1 M sucrose (Kanto) was put into the straw followed by a short column of air and approximately 10 ml of the VS. Then 40 ml of the VS containing the oocytes was aspirated and separated by an air space on each side. The remainder of the straw was filled with DPBS ⫹ BSA with 1 M sucrose. Warming and Dilution Procedures Straws containing the oocytes were held in air for 10 s, and then for an additional 20 s in 20°C water. The contents of the straw were then expelled and gently mixed into 1 ml of 1 M sucrose in DPBS ⫹ BSA for 10 min at room temperature (22 to 25°C). Thereafter, the oocytes were rehydrated in about 4 ml of fresh DPBS ⫹ BSA kept at 37°C for 5 min. To assess the survival rate, all of the recovered oocytes were transferred into a 0.4-ml drop of TYH medium (22) under paraffin oil and incubated for 1 h in a humidified atmosphere of 5% CO2 in air at 37°C before fertilization in vitro. The oocytes without apparent abnormal morphology were judged as survived. In Vitro Fertilization Treated (vitrified and exposed) and nontreated oocytes underwent insemination in
VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL
vitro adapting the procedure described by Toyoda et al. (22). Sperm collected from the cauda epididymis of 3- to 6-month-old F1 (C57BL/6JxCBA) mice were allowed to disperse in 0.4 ml of TYH medium under paraffin oil and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Thereafter, the sperm concentration was determined using a hemocytometer, and the incubation was continued further for 1 to 1.5 h for the induction of sperm capacitation. The insemination drops containing oocytes were supplemented with 5 to 10 ml of preincubated sperm suspension, providing a final sperm concentration of 1.5 ⫻ 105 cells/ml, and were incubated. The oocytes with apparent abnormal morphology were excluded. In Vitro Culture and Determination of in Vitro Viability After 5 h of co-incubation with sperm, the oocytes were washed four times and cultured in 25-ml drops of KSOM (1) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37°C for 120 h. The in vitro developmental ability of the oocytes was assessed by determining the number of oocytes that cleaved to the two-cell stage and developed to the expanded/hatching blastocyst stage at 24 and 120 h postinsemination, respectively. The cell number of blastocysts obtained after 120 h of culture was determined by an air-dry procedure described elsewhere (4). Volumetric Measurement during Exposures Oocytes were initially placed in DPBS ⫹ BSA. One oocyte was pipetted into 25 ml of 2 M EG or raffinose (0.15 then 0.3 M) in DPBS ⫹ 10% FCS for 10 min each at room temperature (22 to 25°C). The oocytes were finally exposed to VS for 10 min. Before and during preexposure to EG or raffinose and exposure to VS, oocytes were observed under an inverted microscope with an attached CCD camera (Dxc-108, Sony, Tokyo, Japan). Images were recorded and stored using a digital video system (DHR-1000, Sony). The volume of oocytes was determined from the cross-sectional diameter of projected oocytes as described by Jackowski et al. (3) at
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time points of 0.5, 1, 2, 5, and 10 min. Only oocytes that assumed a spherical shape and retained the circular cross section during exposures were analyzed. Experimental Design To study the effects of preexposure to 2 M EG solution and subsequent exposure to VS, groups of 20 oocytes were initially exposed to 2 M EG solution for 2 or 5 min. The pretreated oocytes were exposed to the VS for varying periods of 0.5, 1, 2, and 5 min and then vitrified or immediately placed in 1 M sucrose solution without vitrification. The preexposed and nonvitrified or vitrified oocytes as well as similar groups of vitrified oocytes without preexposure underwent insemination and culture in vitro. Another set of experiments was conducted to investigate the volumetric changes of oocytes during sequential exposure to 2 M EG and the VS. To study the effects of preexposure to raffinose solution, the oocytes were sequentially preexposed to 0.15 and 0.3 M raffinose solutions for 2, 5, and 10 min each, hereafter referred to as 2-2, 5-5, and 10-10 min, respectively. They were then exposed to VS for 0.5, 1, 2, or 5 min with or without vitrification. Morphologically normal nonvitrified and vitrified-warmed oocytes were fertilized in vitro and their developmental capacity was assessed. Another set of experiments was conducted to determine the volumetric changes of oocytes during sequential exposure to raffinose solutions and VS. After determining the preexposure treatments that gave the best results in the above-mentioned experiments, the viability of vitrified oocytes with or without preequilibration was compared with that of untreated fresh oocytes. All groups were inseminated and cultured in vitro and cell counts of blastocysts were determined following the procedures described above. Statistical Analysis The interaction between the effects of preexposure to EG or raffinose and subsequent exposure to the VS was analyzed using the two-way analysis of variance (ANOVA) using StatView software (Abacus Concepts, Inc., Berkeley, CA,
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U.S.A.). When a significant F ratio was found, the data were analyzed further by one-way ANOVA with Fisher’s protected least significant difference (PLSD) as a posthoc test. Comparison between the vitrified and nonvitrified fresh oocytes was made using one-way ANOVA followed by Fisher’s PLSD. RESULTS
Effects of Preexposure to a 2 M EG Solution There were no differences in the recovery rates of oocytes among the treatment groups without vitrification (they ranged from 95.0 ⫾ 4.1 to 97.5 ⫾ 2.9%). A significant interaction was revealed between the periods of preexposure to 2 M EG and exposure to the VS on the survival rate and ability of oocytes to cleave (P ⬍ 0.001), as shown in Table 1. Preexposure periods did not affect the survival and cleavage rates when 0.5- to 2-min exposures to the VS were used, whereas 2- and 5-min preexposures exhibited higher rates than the directly exposed group when the oocytes were exposed to the VS for 5 min. No significant interaction was revealed between the effects of preexposure to raffinose solutions and exposure to the VS on the development of oocytes to the blastocyst stage
(P ⬎ 0.05). Shorter exposure periods to VS showed higher developmental rates to the blastocyst stage regardless of the preequilibration procedures (P ⬍ 0.05). The recovery rates of vitrified-warmed oocytes did not differ among the treatment groups (ranged from 95.0 ⫾ 7.1 to 97.5 ⫾ 2.9%). Neither crack nor breakage of zona pellucida was observed after vitrification and warming. A significant interaction between the effects of period of preexposure to 2 M EG and exposure to the VS on survival, cleavage, and blastocyst formation were revealed (P ⬍ 0.001), as shown in Table 2. When oocytes were directly exposed to the VS without preexposure to 2 M EG, high survival, cleavage, and blastocyst development rates were obtained after 1- and 2min exposures to VS (P ⬍ 0.05). When oocytes were preexposed to 2 M EG for 2 and 5 min, survival and development rates were highest at 1 and 0.5 min exposure to the VS, respectively (P ⬍ 0.05). As shown in Fig. 1, oocytes rapidly shrank upon direct exposure to the VS, exhibiting a maximum shrinkage within 30 s of exposure and gradually increasing in size thereafter. On the other hand, oocytes preexposed to 2 M EG
TABLE 1 Viability of Mouse Oocytes Preexposed to Ethylene Glycol (EG) and Exposed to Vitrification Solution (VS) without Vitrification Parameters
Preexposure to 2 M EG (minutes)
Percentage of oocytes after exposure to VS at different periods (minutes) 0.5
1
2
5
Survival
0 2 5
93.3 ⫾ 6.1a 95.0 ⫾ 4.1a 95.0 ⫾ 4.1a
92.5 ⫾ 6.9a 97.5 ⫾ 2.9a 96.3 ⫾ 4.8a
95.0 ⫾ 6.3a 96.3 ⫾ 2.5a 93.8 ⫾ 4.8a
70.8 ⫾ 8.6Ab 88.8 ⫾ 2.5Bb 86.3 ⫾ 2.5Bb
Cleavage
0 2 5
88.3 ⫾ 4.1a 87.5 ⫾ 2.9a 86.3 ⫾ 2.5a
85.0 ⫾ 4.5a 87.5 ⫾ 2.9a 90.0 ⫾ 4.0a
88.3 ⫾ 8.2a 86.2 ⫾ 2.5a 81.3 ⫾ 2.5b
61.7 ⫾ 5.2Ab 75.0 ⫾ 4.1Bb 71.3 ⫾ 2.5Bc
Blastocyst
0 2 5
79.2 ⫾ 3.8 80.0 ⫾ 4.1 78.8 ⫾ 2.5
73.3 ⫾ 6.0 78.8 ⫾ 2.5 76.3 ⫾ 6.3
71.7 ⫾ 11.2 76.3 ⫾ 2.5 72.5 ⫾ 2.9
38.3 ⫾ 6.0 45.0 ⫾ 4.1 42.5 ⫾ 2.9
Mean
79.3 ⫾ 3.3a
75.7 ⫾ 5.5ab
73.2 ⫾ 7.5b
41.4 ⫾ 5.3c
Note. Percentage values represent means ⫾ SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes treated (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
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VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL TABLE 2 Viability of Mouse Oocytes Preexposed to EG and Exposed to VS with Vitrification Parameters
Preexposure to 2 M EG (minutes)
Percentage of oocytes after exposure to VS at different periods (minutes) 0.5
1
2
5
Survival
0 2 5
72.5 ⫾ 6.5 87.5 ⫾ 2.3Bab 91.3 ⫾ 6.3Ba
81.3 ⫾ 6.3 91.3 ⫾ 4.8Ba 83.8 ⫾ 4.8Aa
78.8 ⫾ 10.3 81.3 ⫾ 4.8b 68.8 ⫾ 8.5b
45.0 ⫾ 9.1b 40.0 ⫾ 8.2c 32.5 ⫾ 6.5c
Cleavage
0 2 5
56.3 ⫾ 4.8Aa 80.0 ⫾ 4.0Ba 83.8 ⫾ 4.8Ba
73.8 ⫾ 9.5Ab 86.3 ⫾ 4.8Ba 75.0 ⫾ 4.1Aa
71.3 ⫾ 8.5b 66.3 ⫾ 4.8b 63.8 ⫾ 8.5b
32.5 ⫾ 9.6c 33.7 ⫾ 4.8c 22.5 ⫾ 6.5c
Blastocyst
0 2 5
38.8 ⫾ 4.8Aa 62.5 ⫾ 6.5Ba 70.0 ⫾ 4.1Ba
50.0 ⫾ 4.1Ab 76.3 ⫾ 2.5Bb 55.0 ⫾ 4.1Ab
55.0 ⫾ 4.1Ab 52.5 ⫾ 6.5Ac 40.0 ⫾ 4.1Bc
13.8 ⫾ 9.5c 16.3 ⫾ 7.5d 6.3 ⫾ 4.8d
Aa
Aa
a
Note. Percentage values represent means ⫾ SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes vitrified (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–d) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
and then exposed to VS demonstrated two cycles of shrink and swell response. The maximum shrinkage upon initial exposure to 2 M EG was achieved within 30 s, after which the oocytes started to increase in volume. The oocytes shrank again upon exposure to VS and then gradually reexpanded thereafter. Effects of Preexposure to Raffinose Solutions There were no differences in the recovery rates of oocytes among the treatment groups
FIG. 1. Volume changes of oocytes during preexposure to 2 M EG (䊊) and exposure to the vs (䊉). (䊐) Isotonic volume. (a) Direct exposure to vitrification solution, (b) 2 min, (c) 5 min preexposure to 2 M EG. Each point represents the mean ⫾SD of five oocytes.
preexposed to raffinose without vitrification (they ranged from 96.3 ⫾ 4.8 to 98.8 ⫾ 2.5%). As shown in Table 3, a significant interaction between the effects of preexposure to raffinose solution and to the VS on cleavage and development to blastocysts was observed without vitrification (P ⬍ 0.001). When oocytes were exposed to the VS for 0.5 to 2 min, high rates of survival, cleavage, and development to blastocysts were obtained in the oocytes preexposed to raffinose for 2-2 and 5-5 min compared to those of 10-10 min. With prolonged exposure to the VS for 5 min, development to blastocysts was reduced regardless of the preexposure periods (P ⬍ 0.05). After vitrification, the recovery rates of oocytes did not differ among the treatment groups (they ranged from 96.0 ⫾ 4.2 to 98.0 ⫾ 2.7%). Vitrification of oocytes after preexposure to raffinose solutions did not cause zona crack or breakage. There was a significant interaction between the exposure periods to the VS and preexposure to raffinose solutions on survival, cleavage, and development to blastocyst (P ⬍ 0.001), as shown in Table 4. A higher proportion of oocytes survived, cleaved, and developed to blastocysts when the oocytes were sequentially preexposed to raffinose solutions for 5-5 min compared to 2-2 and 10-10 min after 0.5 to 2 min exposure to the VS (P ⬍ 0.05). With 5-5
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DELA PEÑA ET AL. TABLE 3 Viability of Mouse Oocytes Preexposed to Raffinose and Exposed to VS without Vitrification
Parameters
Preexposure to 0.15–0.3 M raffinose (minutes)
Percentage of oocytes after exposure to the VS at different periods (minutes) 0.5
1
2
5
Survival
2-2 5-5 10-10
97.5 ⫾ 2.9 97.5 ⫾ 2.9Aa 83.8 ⫾ 4.8Ba
95.0 ⫾ 4.1 96.3 ⫾ 2.5Aa 81.3 ⫾ 6.3Ba
95.0 ⫾ 4.1 95.0 ⫾ 4.1Aa 78.8 ⫾ 2.5Bab
80.0 ⫾ 10.8b 75.0 ⫾ 4.1b 71.3 ⫾ 8.5b
Cleavage
2-2 5-5 10-10
88.8 ⫾ 2.5Aa 87.5 ⫾ 6.5Aa 56.3 ⫾ 9.5B
83.8 ⫾ 2.5Ab 91.3 ⫾ 2.5Aa 61.3 ⫾ 8.5B
86.3 ⫾ 4.8Aab 90.0 ⫾ 4.1Aa 51.3 ⫾ 6.3B
48.8 ⫾ 2.5c 46.3 ⫾ 4.8b 46.3 ⫾ 2.5
Blastocyst
2-2 5-5 10-10
80.0 ⫾ 4.1Aa 78.7 ⫾ 2.5Aa 38.8 ⫾ 6.3Ba
76.3 ⫾ 2.5Aa 81.3 ⫾ 2.5Aa 38.8 ⫾ 6.3Ba
75.0 ⫾ 4.1Aa 77.5 ⫾ 6.5Aa 33.8 ⫾ 4.8Ba
33.8 ⫾ 2.5Ab 31.3 ⫾ 6.3Ab 17.5 ⫾ 2.9Bb
Aa
Aa
Aa
Note. Percentage values represent means ⫾SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes treated (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
min preexposure, the exposure times to the VS of 0.5 to 2 min showed similar high rates of development to blastocysts. Prolonged exposure to VS for 5 min reduced viability in each treatment group (P ⬍ 0.05). Stepwise exposure to raffinose solutions caused a stepwise shrinkage of oocyte, as shown in Fig. 2. The osmotic response during final equilibration in the VS was similarly character-
ized by initial rapid shrinkage, with a subsequent gradual increase in the cell volume as the exposure time progressed. Comparison between Vitrified and Nonvitrified Fresh Oocytes As shown in Table 5, both preexposed groups showed higher development rates to blastocysts than the directly vitrified oocytes, but lower
TABLE 4 Viability of Mouse Oocytes Preexposed to Raffinose and Exposed to VS with Vitrification Parameters
Preexposure to 0.15–0.3 M raffinose (minutes)
Percentage of oocytes after exposure to the VS at different periods (minutes) 0.5
1
2
5
Survival
2-2 5-5 10-10
72.0 ⫾ 5.7 87.0 ⫾ 2.7Ba 61.0 ⫾ 4.2Ca
83.0 ⫾ 2.7 91.0 ⫾ 2.2Aa 65.0 ⫾ 10.6Ba
83.0 ⫾ 2.7 89.0 ⫾ 4.2Aa 67.0 ⫾ 2.7Ba
42.0 ⫾ 5.7Ac 57.0 ⫾ 8.4Bb 12.0 ⫾ 9.0Cb
Cleavage
2-2 5-5 10-10
56.0 ⫾ 6.5Aa 83.0 ⫾ 5.7Ba 49.0 ⫾ 4.2Aa
66.0 ⫾ 4.2Ab 84.0 ⫾ 8.2Ba 58.0 ⫾ 4.5Cb
71.0 ⫾ 2.2Ab 86.0 ⫾ 4.2Ba 41.0 ⫾ 5.5Cc
25.0 ⫾ 7.9Ac 40.0 ⫾ 3.5Bb 0Cd
Blastocyst
2-2 5-5 10-10
48.0 ⫾ 4.5Aa 73.0 ⫾ 5.7Ba 26.0 ⫾ 2.2Ca
55.0 ⫾ 5.0Ab 75.0 ⫾ 3.5Ba 29.0 ⫾ 5.5Ca
46.0 ⫾ 2.2Aa 73.0 ⫾ 2.8Ba 20.0 ⫾ 3.5Cb
10.0 ⫾ 7.1Ac 30.0 ⫾ 3.5Bb 0Cc
Aa
Ab
Ab
Note. Percentage values represent means ⫾SD of five replicates. The percentage in each replicate was calculated based on the number of oocytes vitrified (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
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VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL
FIG. 2. Volume changes in oocytes during preexposure to 0.15 (䉭) and 0.3 M (䉱) raffinose solutions and exposure to the VS (䊉). (䊐) Isotonic volume. (a) Preexposure to raffinose of 2-2, (b) 5-5, and (c) 10-10 min. Each point represents the mean ⫾ SD of five oocytes.
rates than the nontreated fresh group (P ⬍ 0.05). The mean cell numbers of blastocysts derived from the preexposed/vitrified and nonvitrified fresh oocytes were significantly higher than those of the directly vitrified ones (P ⬍ 0.05). DISCUSSION
The present results showed that the optimum time of exposure to VS was affected by periods of preexposure to a lower concentration of cryoprotectant, which is in agreement with the previous findings on preexposure of mouse blastocysts to 10–20% EG or glycerol before exposure to a mixture of EG or glycerol, sucrose, and Ficoll (22, 23).
When the oocytes were placed directly into the VS, the oocytes showed rapid shrinkage due to dehydration with high extracellular osmotic pressure of the VS. The oocytes reached the minimum volume within 30 s, where intra- and extracellular osmotic pressures were equilibrated. Thereafter, the oocytes started to swell due to the influx of EG and water (11, 14 ). The longer exposure time to the VS up to 2 min with a resultant larger oocyte volume could have allowed sufficient permeation of EG, which resulted in high viability. With preexposure to 2 M EG and subsequent exposure to the VS, the oocytes showed shrinkage and swelling in a two-step manner due to the repeated dehydration and influx of EG and water. Preexposure to EG allowed initial permeation of EG and shorter time of the oocytes in the VS for sufficient permeation of EG compared to the time needed with the oocytes directly exposed to the VS, so that the shorter exposure to the VS with a correspondingly smaller oocyte volume resulted in high viability. With prolonged preexposure to 2 M EG for 5 min, a sufficient amount of EG could already have permeated the oocytes, so that a 0.5-min exposure to the VS is required to induce a small amount of EG influx and dehydration to concentrate the CPA. Based on results from the nonvitrified oocytes, a shorter period of exposure to VS better reduces the cellular toxicity of EG. Prolonged exposure to VS can lead to complete permeation of CPA, resulting in chemical toxicity and osmotic swelling during dilution (3, 5, 11).
TABLE 5 Viability of Fresh and Vitrified Oocytes Directly Exposed to VS or Preexposed to EG or Raffinose
a b
Vitrification procedures
Exposure to VS (minutes)
No. of oocytes (replicates)
Percentage of blastocysts
Direct exposure to VS Preexposure to 2 M EGa Preexposure to 0.15 and 0.3 M raffinoseb Nontreated fresh
2 1 1 —
80 (4) 80 (4) 80 (4) 80 (4)
53.8 ⫾ 6.3c 68.8 ⫾ 2.5d 67.5 ⫾ 6.5d 82.5 ⫾ 6.5e
Mean cell number of blastocysts 57.8 ⫾ 8.8c 81.2 ⫾ 8.6d 80.2 ⫾ 9.0d 78.2 ⫾ 13.7d
Preexposure to 2 M EG for 2 min. Preexposure to 0.15 and 0.3 M raffinose for 5 and 5 min, respectively. (c–e) Values (means ⫾ SD) with different superscripts within a column differ significantly (P ⬍ 0.05).
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DELA PEÑA ET AL.
In our preliminary experiment, direct exposure of oocytes to 0.3 M raffinose solutions resulted in poor postwarming viability, so that we applied the two-step preexposure in the present study. The volume change with preexposure to raffinose solutions was characterized by stepwise shrinkage due to dehydration in response to increasing extracellular osmotic pressures of solutions containing nonpermeable raffinose. After transfer to the VS, the oocytes shrank further due to the hyperosmolarity of the VS and then reexpanded with the influx of EG and water. The oocyte volume became larger when equilibrium was reached during exposure to the VS. This is attributed to the lower salt concentration in the VS compared to that of 0.3 M raffinose solution, since the VS was prepared by adding EG and raffinose to the isotonic DPBS ⫹ FCS. The period of preexposure to the raffinose solutions for dehydration also influenced the viability of the vitrified oocytes regardless of the period of exposure to the VS. There were no differences in the viability of nonvitrified oocytes after 2-2- and 5-5-min preexposures, but the viability of oocytes preexposed for 2-2 min was greatly reduced by vitrification. Although the reason for the differences between the 2-2- and 5-5-min preexposure groups after vitrification is not clear, it appears that the short period of exposure to the raffinose solutions could not produce the optimum dehydration of oocytes and permeation of EG to induce intracellular vitrification. The poor development of oocytes after prolonged exposure to the raffinose solutions for 10-10 min without vitrification clearly indicates that the predehydration procedure and subsequent exposure to VS is deleterious. A prolonged dehydration period could result in an increased concentration of intracellular salts, which causes injury to the membrane (7). The 5-5-min preexposure to the raffinose solutions could have produced the necessary degree of cellular dehydration (34%) while avoiding osmotic injury. The oocytes can shrink and swell to volumes of 30–290% without appreciable effects on their development (10). In addition, 5-5-min preexposure to raffinose allowed
oocytes a wider range of acceptable exposure times to the VS, which could be attributed in part to the slow rate of EG permeation into, and its rapid dilution out of, the shrunken ooplasm. Nevertheless, this improvement in oocyte survival with 5-5-min preexposure needs further clarification. This work is the first to report on predehydration with raffinose, and further investigations are therefore necessary to elucidate the mechanism of cryoinjury associated with preexposure to this sugar and subsequent exposure to the cryoprotectant. In conclusion, the present study showed that preexposure to a relatively low concentration of EG allowed shorter exposure times of oocyte in the VS, a mixture of EG and raffinose, and the reduced exposure time to VS, which minimizes the toxic effect of EG, improved oocyte viability. Likewise, predehydration with raffinose for 5-5, but not for 2-2 or 10-10 min, allowed a wider range of exposure times to the VS and increased the viability of vitrified oocytes, probably due to the reduction of EG needed relative to the small volume of oocytes after optimum cellular dehydration. REFERENCES 1. Erbach, G. T., Lawitts, J. A., Papaioannou, V. E., and Biggers, J. D. Differential growth of the mouse preimplantation embryo in chemically defined media. Biol. Reprod. 50, 1027–1033 (1994). 2. Hotamisligil, S., Toner, M., and Powers, R. D. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol. Reprod. 55, 161–168 (1996). 3. Jackowski, S., Leibo, S. B., and Mazur, P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J. Exp. Zool. 212, 329–341 (1980). 4. Kamiguchi, Y., and Mikamo, K. A new technique for chromosome study of murine oocytes. Proc. Jpn. Acad. 52, 316 (1976). 5. Kasai, M. Vitrification: Refined strategy for the cryopreservation of mammalian embryos. J. Mamm. Ova Res. 14, 17–28 (1997). 6. Kuleshova, L. L., MacFarlane, D. R., Trounson, A. O., and Shaw, J. M. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38, 119–130 (1999). 7. Mazur, P. The freezing of biological systems. Cryobiology 168, 939–949 (1970).
VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL 8. Nowshari, M. A., and Brem, G. Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure. Theriogenology 50, 1001–1013 (1998). 9. Nowshari, M. A., Nayudu, P. L., and Hodges, J. K. Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes. Theriogenology 42, 1193–1204 (1994). 10. Oda, K., Gibbons, W. E., and Leibo, S. P. Osmotic shock of fertilized ova. J. Reprod. Fertil. 95, 737–747 (1992). 11. Paynter, S. J., Fuller, B. J., and Shaw, R. W. Temperature dependence of kedem-katchalsky membrane coefficients for mature mouse oocytes in the presence of ethylene glycol. Cryobiology 39, 169–176 (1999). 12. Rall, W. F. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387–402 (1987). 13. Rall, W. F., and Fahy G. M. Ice-free cryopreservation of mouse embryos at ⫺196°C by vitrification. Nature 313, 573–575 (1985). 14. Schneider, U., and Mazur, P. Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos. Theriogenology 21, 68–79 (1984). 15. Shaw, J. M., Kuleshova, L. L., MacFarlane D. R., and Trounson, A. O. Vitrification properties of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology 35, 219–229 (1997). 16. Sutton, R. L. Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides. Cryobiology 29, 585–598 (1992).
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17. Szell, A., and Shelton, J. N. Role of equilibration before rapid freezing of mouse embryos. J. Reprod. Fertil. 78, 699–703 (1986). 18. Tada, N., Sato, M., Amann, E., and Ogawa, S. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriogenology 40, 333–344 (1993). 19. Tada, N., Sato, M., Amann, E., and Ogawa, S. Stage-dependent viability of mouse preimplantation embryos vitrified with sugar-containing solutions. J. Reprod. Dev. 39, 139–144 (1993). 20. Tada, N., Sato, M., Kasai, K., and Ogawa, S. Successful in vitro and in vivo development of cryopreserved mouse oocytes fertilized by cryopreserved mouse epididymal spermatozoa. J. Reprod. Dev. 40, 65–70 (1994). 21. Takahashi, Y., and Kanagawa, H. Quick freezing of mouse embryos by direct plunging into liquid nitrogen vapor: effect of sugars. Jpn. J. Vet. Res. 33, 141–144 (1985). 22. Toyoda, Y., Yokohama, H., and Hoshi, T. Studies on the fertility of mouse hamster eggs in vitro: in vitro fertilization of mouse eggs by epididymal sperm. Jpn. J. Anim. Reprod. 6, 129–141 (1971). 23. Zhu, S. E., Kasai, M., Otoge, H., Sakurai, T., and Machida, T. Cryopreservation of mouse blastocysts by vitrification in ethylene glycol-based solutions. J. Reprod. Fertil. 98, 139–145 (1993). 24. Zhu, S. E., Sakurai, T., Edashige, K., Machida, T., and Kasai, M. Optimization of the procedures for the vitrification of expanded mouse blastocyst in glycerolbased solution. J. Reprod. Dev. 40, 293–300 (1994).
Vitrification of Mouse Oocytes in Ethylene Glycol–Raffinose Solution: Effects of Preexposure to Ethylene Glycol or Raffinose on Oocyte Viability E. C. dela Peña, Y. Takahashi,1 E. C. Atabay, S. Katagiri, and M. Nagano Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan We investigated the effects of preexposure to ethylene glycol (EG) or raffinose on the viability of vitrified mouse oocytes. Ovulated oocytes at the metaphase II stage were preexposed either to 2 M EG for 0, 2, or 5 min or to ascending concentrations (0.15 followed by 0.3 M ) of raffinose solution for 2, 5, or 10 min each (here referred to as 2-2, 5-5, and 10-10 min, respectively). The oocytes were then exposed to a vitrification solution (VS), 6 M EG ⫹ 0.3 M raffinose, for 0.5, 1, 2, or 5 min and then vitrified or immediately diluted. After warming, the developmental capacity of oocytes was determined after in vitro fertilization. Volume changes in oocytes during preexposures and exposure to the VS were also investigated. The results demonstrated that preexposure to 2 M EG allowed shorter exposure times of oocytes to the VS and that predehydration in raffinose solutions for 5-5, but not 2-2 or 10-10 min, allowed a wider range of exposure times to the VS. Experiments on volume change suggested that the optimum time of exposure to the VS depends on the amount of EG permeation after preexposure to 2 M EG or to raffinose solutions. Preexposures to 2 M EG or raffinose under optimized conditions increased the viability of vitrified-warmed oocytes compared to direct exposure to VS without preexposures. © 2001 Academic Press Key Words: oocytes; preexposure; dehydration; raffinose; ethylene glycol; vitrification.
Successful vitrification requires the combination of a high concentration of permeable cryoprotectant agent (CPA) and a very rapid cooling rate; however, high concentrations of CPA cause cell damage due to osmotic and cytotoxic effects (12, 13). Partial replacement of permeable CPAs by nonpermeable compounds such as sugar, polyvinylpyrrolidone, dextran, and Ficoll has been attempted in an effort to improve vitrification (6, 15). The critical cooling rate required to avoid ice crystallization in solutions with penetrating cryoprotectants was altered by the addition of raffinose, trehalose, sucrose, or glucose (16). Recently, raffinose has been found to modify the vitrification properties of ethylene glycol (EG) by raising the glass transition point comparable with other sugars (6). In addition, it had been found equally as effective as sucrose Received November 16, 2000; accepted March 19, 2001; published online May 21, 2001. This work was supported by Grant-in-Aid for Scientific Research 10556058 from the Japan Society for the Promotion of Science. 1 To whom correspondence should be addressed. 103
and glucose in the quick freezing of mouse morulae using glycerol (21) and beneficial in vitrifying mouse preimplantation-stage embryos in combination with dimethyl-sulfoxide and propylene glycol (18, 19). Attempts to vitrify oocytes and embryos in EG–raffinose solutions, however, have not been reported. Preexposure of embryos to a low concentration of permeable CPA before exposure to a high CPA concentration is also a well-known effective approach to improve embryo viability after vitrification (5, 12). However, the preexposure to sugar solutions before vitrification to effect dehydration has not been widely used and studied. It has been suggested that preexposure to sugar unfavorably affects the subsequent survival of mouse embryos (17); however, recent studies on preexposure in ascending concentrations of sucrose before exposure to a high concentration of CPAs showed increased survival and development of mouse blastocysts (8) and oocytes (9, 20). Despite the previous studies on preexposure before vitrification, detailed studies of the conditions during preexpo0011-2240/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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sure, especially during preexposure to sugar and subsequent exposure to the vitrification solution (VS), have not been well demonstrated. In the present study, we treated the oocytes with a low concentration of EG or with raffinose before exposure to a VS, a mixture of EG and raffinose, and investigated the interaction between the effects of preexposure and VS exposure on the viability of mouse oocytes. The present study also examined the volume changes of oocytes during both preexposure to EG or raffinose and exposure to the VS. MATERIALS AND METHODS
Source of Mouse Oocytes Female C57BL/6J and male CBA mice purchased from Japan SLC Inc. (Shizuoka, Japan) were housed in the animal housing facilities of the Graduate School of Veterinary Medicine following the guidelines set by the university to produce F1 offspring. They were kept under light- and temperature-controlled conditions (12 h light: 12 h dark photoperiod, 22°C) and given chow pellets and water ad libitum. Female F1 mice, 6–8 weeks of age, were induced to superovulate by intraperitoneal injection of 5 IU of equine chorionic gonadotropin (Teikoku Hormone Mfg. Co. Ltd., Tokyo, Japan) and 5 IU of human chorionic gonadotropin (hCG, Teikoku Hormone) given 48 h apart. They were killed by cervical dislocation 13 h after the hCG injection. The cumulus cell-enclosed oocytes were released from the ampulla into Dulbecco’s phosphate-buffered saline (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 4 mg/ml BSA, 75 mg/ml penicillin, and 50 mg/ml streptomycin (DPBS ⫹ BSA). Cumulus cells were removed by exposing them to DPBS containing 150 U/ml hyaluronidase (bovine testis type I-S, Sigma Chemical Co., St. Louis, MO, U.S.A.) for 2 to 3 min at 22 to 25°C. Cumulus-free oocytes were washed three times in fresh DPBS ⫹ BSA medium and pooled in a 35-mm plastic petri dish (Nalge Nunc Int. Kamstrup, Denmark) until they were used. Only oocytes with normal morphological appearance and with a visible polar body were used in the experiments.
Vitrification Procedures After preexposure to EG (Kanto Chemical Co. Inc., Tokyo, Japan) or raffinose (Sigma) solutions, groups of 20 oocytes were initially rinsed in a 100-l drop of VS which was composed of 6 M EG with 0.3 M raffinose in DPBS ⫹ 10% fetal calf serum (FCS, Gibco Laboratories, Grand Island, NY, U.S.A.). The oocytes were finally transferred to a 40-ml droplet of the VS at room temperature (22 to 25°C). The exposed oocytes were drawn into 0.25-ml French straws (I.M.V., L’Aigle, France). The straws were heat sealed and cooled in liquid nitrogen (LN2) vapor for at least 1 min. Finally, the straws were plunged into LN2 and stored for 1 to 120 days. Exposure times of 0.5, 1, 2, and 5 min elapsed from the time the oocytes were initially rinsed in a 100-ml drop of VS until they were cooled in LN2 vapor. For loading the straw, approximately 100 ml of DPBS ⫹ BSA with 1 M sucrose (Kanto) was put into the straw followed by a short column of air and approximately 10 ml of the VS. Then 40 ml of the VS containing the oocytes was aspirated and separated by an air space on each side. The remainder of the straw was filled with DPBS ⫹ BSA with 1 M sucrose. Warming and Dilution Procedures Straws containing the oocytes were held in air for 10 s, and then for an additional 20 s in 20°C water. The contents of the straw were then expelled and gently mixed into 1 ml of 1 M sucrose in DPBS ⫹ BSA for 10 min at room temperature (22 to 25°C). Thereafter, the oocytes were rehydrated in about 4 ml of fresh DPBS ⫹ BSA kept at 37°C for 5 min. To assess the survival rate, all of the recovered oocytes were transferred into a 0.4-ml drop of TYH medium (22) under paraffin oil and incubated for 1 h in a humidified atmosphere of 5% CO2 in air at 37°C before fertilization in vitro. The oocytes without apparent abnormal morphology were judged as survived. In Vitro Fertilization Treated (vitrified and exposed) and nontreated oocytes underwent insemination in
VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL
vitro adapting the procedure described by Toyoda et al. (22). Sperm collected from the cauda epididymis of 3- to 6-month-old F1 (C57BL/6JxCBA) mice were allowed to disperse in 0.4 ml of TYH medium under paraffin oil and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Thereafter, the sperm concentration was determined using a hemocytometer, and the incubation was continued further for 1 to 1.5 h for the induction of sperm capacitation. The insemination drops containing oocytes were supplemented with 5 to 10 ml of preincubated sperm suspension, providing a final sperm concentration of 1.5 ⫻ 105 cells/ml, and were incubated. The oocytes with apparent abnormal morphology were excluded. In Vitro Culture and Determination of in Vitro Viability After 5 h of co-incubation with sperm, the oocytes were washed four times and cultured in 25-ml drops of KSOM (1) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37°C for 120 h. The in vitro developmental ability of the oocytes was assessed by determining the number of oocytes that cleaved to the two-cell stage and developed to the expanded/hatching blastocyst stage at 24 and 120 h postinsemination, respectively. The cell number of blastocysts obtained after 120 h of culture was determined by an air-dry procedure described elsewhere (4). Volumetric Measurement during Exposures Oocytes were initially placed in DPBS ⫹ BSA. One oocyte was pipetted into 25 ml of 2 M EG or raffinose (0.15 then 0.3 M) in DPBS ⫹ 10% FCS for 10 min each at room temperature (22 to 25°C). The oocytes were finally exposed to VS for 10 min. Before and during preexposure to EG or raffinose and exposure to VS, oocytes were observed under an inverted microscope with an attached CCD camera (Dxc-108, Sony, Tokyo, Japan). Images were recorded and stored using a digital video system (DHR-1000, Sony). The volume of oocytes was determined from the cross-sectional diameter of projected oocytes as described by Jackowski et al. (3) at
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time points of 0.5, 1, 2, 5, and 10 min. Only oocytes that assumed a spherical shape and retained the circular cross section during exposures were analyzed. Experimental Design To study the effects of preexposure to 2 M EG solution and subsequent exposure to VS, groups of 20 oocytes were initially exposed to 2 M EG solution for 2 or 5 min. The pretreated oocytes were exposed to the VS for varying periods of 0.5, 1, 2, and 5 min and then vitrified or immediately placed in 1 M sucrose solution without vitrification. The preexposed and nonvitrified or vitrified oocytes as well as similar groups of vitrified oocytes without preexposure underwent insemination and culture in vitro. Another set of experiments was conducted to investigate the volumetric changes of oocytes during sequential exposure to 2 M EG and the VS. To study the effects of preexposure to raffinose solution, the oocytes were sequentially preexposed to 0.15 and 0.3 M raffinose solutions for 2, 5, and 10 min each, hereafter referred to as 2-2, 5-5, and 10-10 min, respectively. They were then exposed to VS for 0.5, 1, 2, or 5 min with or without vitrification. Morphologically normal nonvitrified and vitrified-warmed oocytes were fertilized in vitro and their developmental capacity was assessed. Another set of experiments was conducted to determine the volumetric changes of oocytes during sequential exposure to raffinose solutions and VS. After determining the preexposure treatments that gave the best results in the above-mentioned experiments, the viability of vitrified oocytes with or without preequilibration was compared with that of untreated fresh oocytes. All groups were inseminated and cultured in vitro and cell counts of blastocysts were determined following the procedures described above. Statistical Analysis The interaction between the effects of preexposure to EG or raffinose and subsequent exposure to the VS was analyzed using the two-way analysis of variance (ANOVA) using StatView software (Abacus Concepts, Inc., Berkeley, CA,
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U.S.A.). When a significant F ratio was found, the data were analyzed further by one-way ANOVA with Fisher’s protected least significant difference (PLSD) as a posthoc test. Comparison between the vitrified and nonvitrified fresh oocytes was made using one-way ANOVA followed by Fisher’s PLSD. RESULTS
Effects of Preexposure to a 2 M EG Solution There were no differences in the recovery rates of oocytes among the treatment groups without vitrification (they ranged from 95.0 ⫾ 4.1 to 97.5 ⫾ 2.9%). A significant interaction was revealed between the periods of preexposure to 2 M EG and exposure to the VS on the survival rate and ability of oocytes to cleave (P ⬍ 0.001), as shown in Table 1. Preexposure periods did not affect the survival and cleavage rates when 0.5- to 2-min exposures to the VS were used, whereas 2- and 5-min preexposures exhibited higher rates than the directly exposed group when the oocytes were exposed to the VS for 5 min. No significant interaction was revealed between the effects of preexposure to raffinose solutions and exposure to the VS on the development of oocytes to the blastocyst stage
(P ⬎ 0.05). Shorter exposure periods to VS showed higher developmental rates to the blastocyst stage regardless of the preequilibration procedures (P ⬍ 0.05). The recovery rates of vitrified-warmed oocytes did not differ among the treatment groups (ranged from 95.0 ⫾ 7.1 to 97.5 ⫾ 2.9%). Neither crack nor breakage of zona pellucida was observed after vitrification and warming. A significant interaction between the effects of period of preexposure to 2 M EG and exposure to the VS on survival, cleavage, and blastocyst formation were revealed (P ⬍ 0.001), as shown in Table 2. When oocytes were directly exposed to the VS without preexposure to 2 M EG, high survival, cleavage, and blastocyst development rates were obtained after 1- and 2min exposures to VS (P ⬍ 0.05). When oocytes were preexposed to 2 M EG for 2 and 5 min, survival and development rates were highest at 1 and 0.5 min exposure to the VS, respectively (P ⬍ 0.05). As shown in Fig. 1, oocytes rapidly shrank upon direct exposure to the VS, exhibiting a maximum shrinkage within 30 s of exposure and gradually increasing in size thereafter. On the other hand, oocytes preexposed to 2 M EG
TABLE 1 Viability of Mouse Oocytes Preexposed to Ethylene Glycol (EG) and Exposed to Vitrification Solution (VS) without Vitrification Parameters
Preexposure to 2 M EG (minutes)
Percentage of oocytes after exposure to VS at different periods (minutes) 0.5
1
2
5
Survival
0 2 5
93.3 ⫾ 6.1a 95.0 ⫾ 4.1a 95.0 ⫾ 4.1a
92.5 ⫾ 6.9a 97.5 ⫾ 2.9a 96.3 ⫾ 4.8a
95.0 ⫾ 6.3a 96.3 ⫾ 2.5a 93.8 ⫾ 4.8a
70.8 ⫾ 8.6Ab 88.8 ⫾ 2.5Bb 86.3 ⫾ 2.5Bb
Cleavage
0 2 5
88.3 ⫾ 4.1a 87.5 ⫾ 2.9a 86.3 ⫾ 2.5a
85.0 ⫾ 4.5a 87.5 ⫾ 2.9a 90.0 ⫾ 4.0a
88.3 ⫾ 8.2a 86.2 ⫾ 2.5a 81.3 ⫾ 2.5b
61.7 ⫾ 5.2Ab 75.0 ⫾ 4.1Bb 71.3 ⫾ 2.5Bc
Blastocyst
0 2 5
79.2 ⫾ 3.8 80.0 ⫾ 4.1 78.8 ⫾ 2.5
73.3 ⫾ 6.0 78.8 ⫾ 2.5 76.3 ⫾ 6.3
71.7 ⫾ 11.2 76.3 ⫾ 2.5 72.5 ⫾ 2.9
38.3 ⫾ 6.0 45.0 ⫾ 4.1 42.5 ⫾ 2.9
Mean
79.3 ⫾ 3.3a
75.7 ⫾ 5.5ab
73.2 ⫾ 7.5b
41.4 ⫾ 5.3c
Note. Percentage values represent means ⫾ SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes treated (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
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VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL TABLE 2 Viability of Mouse Oocytes Preexposed to EG and Exposed to VS with Vitrification Parameters
Preexposure to 2 M EG (minutes)
Percentage of oocytes after exposure to VS at different periods (minutes) 0.5
1
2
5
Survival
0 2 5
72.5 ⫾ 6.5 87.5 ⫾ 2.3Bab 91.3 ⫾ 6.3Ba
81.3 ⫾ 6.3 91.3 ⫾ 4.8Ba 83.8 ⫾ 4.8Aa
78.8 ⫾ 10.3 81.3 ⫾ 4.8b 68.8 ⫾ 8.5b
45.0 ⫾ 9.1b 40.0 ⫾ 8.2c 32.5 ⫾ 6.5c
Cleavage
0 2 5
56.3 ⫾ 4.8Aa 80.0 ⫾ 4.0Ba 83.8 ⫾ 4.8Ba
73.8 ⫾ 9.5Ab 86.3 ⫾ 4.8Ba 75.0 ⫾ 4.1Aa
71.3 ⫾ 8.5b 66.3 ⫾ 4.8b 63.8 ⫾ 8.5b
32.5 ⫾ 9.6c 33.7 ⫾ 4.8c 22.5 ⫾ 6.5c
Blastocyst
0 2 5
38.8 ⫾ 4.8Aa 62.5 ⫾ 6.5Ba 70.0 ⫾ 4.1Ba
50.0 ⫾ 4.1Ab 76.3 ⫾ 2.5Bb 55.0 ⫾ 4.1Ab
55.0 ⫾ 4.1Ab 52.5 ⫾ 6.5Ac 40.0 ⫾ 4.1Bc
13.8 ⫾ 9.5c 16.3 ⫾ 7.5d 6.3 ⫾ 4.8d
Aa
Aa
a
Note. Percentage values represent means ⫾ SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes vitrified (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–d) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
and then exposed to VS demonstrated two cycles of shrink and swell response. The maximum shrinkage upon initial exposure to 2 M EG was achieved within 30 s, after which the oocytes started to increase in volume. The oocytes shrank again upon exposure to VS and then gradually reexpanded thereafter. Effects of Preexposure to Raffinose Solutions There were no differences in the recovery rates of oocytes among the treatment groups
FIG. 1. Volume changes of oocytes during preexposure to 2 M EG (䊊) and exposure to the vs (䊉). (䊐) Isotonic volume. (a) Direct exposure to vitrification solution, (b) 2 min, (c) 5 min preexposure to 2 M EG. Each point represents the mean ⫾SD of five oocytes.
preexposed to raffinose without vitrification (they ranged from 96.3 ⫾ 4.8 to 98.8 ⫾ 2.5%). As shown in Table 3, a significant interaction between the effects of preexposure to raffinose solution and to the VS on cleavage and development to blastocysts was observed without vitrification (P ⬍ 0.001). When oocytes were exposed to the VS for 0.5 to 2 min, high rates of survival, cleavage, and development to blastocysts were obtained in the oocytes preexposed to raffinose for 2-2 and 5-5 min compared to those of 10-10 min. With prolonged exposure to the VS for 5 min, development to blastocysts was reduced regardless of the preexposure periods (P ⬍ 0.05). After vitrification, the recovery rates of oocytes did not differ among the treatment groups (they ranged from 96.0 ⫾ 4.2 to 98.0 ⫾ 2.7%). Vitrification of oocytes after preexposure to raffinose solutions did not cause zona crack or breakage. There was a significant interaction between the exposure periods to the VS and preexposure to raffinose solutions on survival, cleavage, and development to blastocyst (P ⬍ 0.001), as shown in Table 4. A higher proportion of oocytes survived, cleaved, and developed to blastocysts when the oocytes were sequentially preexposed to raffinose solutions for 5-5 min compared to 2-2 and 10-10 min after 0.5 to 2 min exposure to the VS (P ⬍ 0.05). With 5-5
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DELA PEÑA ET AL. TABLE 3 Viability of Mouse Oocytes Preexposed to Raffinose and Exposed to VS without Vitrification
Parameters
Preexposure to 0.15–0.3 M raffinose (minutes)
Percentage of oocytes after exposure to the VS at different periods (minutes) 0.5
1
2
5
Survival
2-2 5-5 10-10
97.5 ⫾ 2.9 97.5 ⫾ 2.9Aa 83.8 ⫾ 4.8Ba
95.0 ⫾ 4.1 96.3 ⫾ 2.5Aa 81.3 ⫾ 6.3Ba
95.0 ⫾ 4.1 95.0 ⫾ 4.1Aa 78.8 ⫾ 2.5Bab
80.0 ⫾ 10.8b 75.0 ⫾ 4.1b 71.3 ⫾ 8.5b
Cleavage
2-2 5-5 10-10
88.8 ⫾ 2.5Aa 87.5 ⫾ 6.5Aa 56.3 ⫾ 9.5B
83.8 ⫾ 2.5Ab 91.3 ⫾ 2.5Aa 61.3 ⫾ 8.5B
86.3 ⫾ 4.8Aab 90.0 ⫾ 4.1Aa 51.3 ⫾ 6.3B
48.8 ⫾ 2.5c 46.3 ⫾ 4.8b 46.3 ⫾ 2.5
Blastocyst
2-2 5-5 10-10
80.0 ⫾ 4.1Aa 78.7 ⫾ 2.5Aa 38.8 ⫾ 6.3Ba
76.3 ⫾ 2.5Aa 81.3 ⫾ 2.5Aa 38.8 ⫾ 6.3Ba
75.0 ⫾ 4.1Aa 77.5 ⫾ 6.5Aa 33.8 ⫾ 4.8Ba
33.8 ⫾ 2.5Ab 31.3 ⫾ 6.3Ab 17.5 ⫾ 2.9Bb
Aa
Aa
Aa
Note. Percentage values represent means ⫾SD of four replicates. The percentage in each replicate was calculated based on the number of oocytes treated (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
min preexposure, the exposure times to the VS of 0.5 to 2 min showed similar high rates of development to blastocysts. Prolonged exposure to VS for 5 min reduced viability in each treatment group (P ⬍ 0.05). Stepwise exposure to raffinose solutions caused a stepwise shrinkage of oocyte, as shown in Fig. 2. The osmotic response during final equilibration in the VS was similarly character-
ized by initial rapid shrinkage, with a subsequent gradual increase in the cell volume as the exposure time progressed. Comparison between Vitrified and Nonvitrified Fresh Oocytes As shown in Table 5, both preexposed groups showed higher development rates to blastocysts than the directly vitrified oocytes, but lower
TABLE 4 Viability of Mouse Oocytes Preexposed to Raffinose and Exposed to VS with Vitrification Parameters
Preexposure to 0.15–0.3 M raffinose (minutes)
Percentage of oocytes after exposure to the VS at different periods (minutes) 0.5
1
2
5
Survival
2-2 5-5 10-10
72.0 ⫾ 5.7 87.0 ⫾ 2.7Ba 61.0 ⫾ 4.2Ca
83.0 ⫾ 2.7 91.0 ⫾ 2.2Aa 65.0 ⫾ 10.6Ba
83.0 ⫾ 2.7 89.0 ⫾ 4.2Aa 67.0 ⫾ 2.7Ba
42.0 ⫾ 5.7Ac 57.0 ⫾ 8.4Bb 12.0 ⫾ 9.0Cb
Cleavage
2-2 5-5 10-10
56.0 ⫾ 6.5Aa 83.0 ⫾ 5.7Ba 49.0 ⫾ 4.2Aa
66.0 ⫾ 4.2Ab 84.0 ⫾ 8.2Ba 58.0 ⫾ 4.5Cb
71.0 ⫾ 2.2Ab 86.0 ⫾ 4.2Ba 41.0 ⫾ 5.5Cc
25.0 ⫾ 7.9Ac 40.0 ⫾ 3.5Bb 0Cd
Blastocyst
2-2 5-5 10-10
48.0 ⫾ 4.5Aa 73.0 ⫾ 5.7Ba 26.0 ⫾ 2.2Ca
55.0 ⫾ 5.0Ab 75.0 ⫾ 3.5Ba 29.0 ⫾ 5.5Ca
46.0 ⫾ 2.2Aa 73.0 ⫾ 2.8Ba 20.0 ⫾ 3.5Cb
10.0 ⫾ 7.1Ac 30.0 ⫾ 3.5Bb 0Cc
Aa
Ab
Ab
Note. Percentage values represent means ⫾SD of five replicates. The percentage in each replicate was calculated based on the number of oocytes vitrified (20 oocytes in each replicate). (A, B) Values with different superscripts within a column differ significantly (P ⬍ 0.05). (a–c) Values with different superscripts within a row differ significantly (P ⬍ 0.05).
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VITRIFICATION OF OOCYTES IN ETHYLENE GLYCOL
FIG. 2. Volume changes in oocytes during preexposure to 0.15 (䉭) and 0.3 M (䉱) raffinose solutions and exposure to the VS (䊉). (䊐) Isotonic volume. (a) Preexposure to raffinose of 2-2, (b) 5-5, and (c) 10-10 min. Each point represents the mean ⫾ SD of five oocytes.
rates than the nontreated fresh group (P ⬍ 0.05). The mean cell numbers of blastocysts derived from the preexposed/vitrified and nonvitrified fresh oocytes were significantly higher than those of the directly vitrified ones (P ⬍ 0.05). DISCUSSION
The present results showed that the optimum time of exposure to VS was affected by periods of preexposure to a lower concentration of cryoprotectant, which is in agreement with the previous findings on preexposure of mouse blastocysts to 10–20% EG or glycerol before exposure to a mixture of EG or glycerol, sucrose, and Ficoll (22, 23).
When the oocytes were placed directly into the VS, the oocytes showed rapid shrinkage due to dehydration with high extracellular osmotic pressure of the VS. The oocytes reached the minimum volume within 30 s, where intra- and extracellular osmotic pressures were equilibrated. Thereafter, the oocytes started to swell due to the influx of EG and water (11, 14 ). The longer exposure time to the VS up to 2 min with a resultant larger oocyte volume could have allowed sufficient permeation of EG, which resulted in high viability. With preexposure to 2 M EG and subsequent exposure to the VS, the oocytes showed shrinkage and swelling in a two-step manner due to the repeated dehydration and influx of EG and water. Preexposure to EG allowed initial permeation of EG and shorter time of the oocytes in the VS for sufficient permeation of EG compared to the time needed with the oocytes directly exposed to the VS, so that the shorter exposure to the VS with a correspondingly smaller oocyte volume resulted in high viability. With prolonged preexposure to 2 M EG for 5 min, a sufficient amount of EG could already have permeated the oocytes, so that a 0.5-min exposure to the VS is required to induce a small amount of EG influx and dehydration to concentrate the CPA. Based on results from the nonvitrified oocytes, a shorter period of exposure to VS better reduces the cellular toxicity of EG. Prolonged exposure to VS can lead to complete permeation of CPA, resulting in chemical toxicity and osmotic swelling during dilution (3, 5, 11).
TABLE 5 Viability of Fresh and Vitrified Oocytes Directly Exposed to VS or Preexposed to EG or Raffinose
a b
Vitrification procedures
Exposure to VS (minutes)
No. of oocytes (replicates)
Percentage of blastocysts
Direct exposure to VS Preexposure to 2 M EGa Preexposure to 0.15 and 0.3 M raffinoseb Nontreated fresh
2 1 1 —
80 (4) 80 (4) 80 (4) 80 (4)
53.8 ⫾ 6.3c 68.8 ⫾ 2.5d 67.5 ⫾ 6.5d 82.5 ⫾ 6.5e
Mean cell number of blastocysts 57.8 ⫾ 8.8c 81.2 ⫾ 8.6d 80.2 ⫾ 9.0d 78.2 ⫾ 13.7d
Preexposure to 2 M EG for 2 min. Preexposure to 0.15 and 0.3 M raffinose for 5 and 5 min, respectively. (c–e) Values (means ⫾ SD) with different superscripts within a column differ significantly (P ⬍ 0.05).
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DELA PEÑA ET AL.
In our preliminary experiment, direct exposure of oocytes to 0.3 M raffinose solutions resulted in poor postwarming viability, so that we applied the two-step preexposure in the present study. The volume change with preexposure to raffinose solutions was characterized by stepwise shrinkage due to dehydration in response to increasing extracellular osmotic pressures of solutions containing nonpermeable raffinose. After transfer to the VS, the oocytes shrank further due to the hyperosmolarity of the VS and then reexpanded with the influx of EG and water. The oocyte volume became larger when equilibrium was reached during exposure to the VS. This is attributed to the lower salt concentration in the VS compared to that of 0.3 M raffinose solution, since the VS was prepared by adding EG and raffinose to the isotonic DPBS ⫹ FCS. The period of preexposure to the raffinose solutions for dehydration also influenced the viability of the vitrified oocytes regardless of the period of exposure to the VS. There were no differences in the viability of nonvitrified oocytes after 2-2- and 5-5-min preexposures, but the viability of oocytes preexposed for 2-2 min was greatly reduced by vitrification. Although the reason for the differences between the 2-2- and 5-5-min preexposure groups after vitrification is not clear, it appears that the short period of exposure to the raffinose solutions could not produce the optimum dehydration of oocytes and permeation of EG to induce intracellular vitrification. The poor development of oocytes after prolonged exposure to the raffinose solutions for 10-10 min without vitrification clearly indicates that the predehydration procedure and subsequent exposure to VS is deleterious. A prolonged dehydration period could result in an increased concentration of intracellular salts, which causes injury to the membrane (7). The 5-5-min preexposure to the raffinose solutions could have produced the necessary degree of cellular dehydration (34%) while avoiding osmotic injury. The oocytes can shrink and swell to volumes of 30–290% without appreciable effects on their development (10). In addition, 5-5-min preexposure to raffinose allowed
oocytes a wider range of acceptable exposure times to the VS, which could be attributed in part to the slow rate of EG permeation into, and its rapid dilution out of, the shrunken ooplasm. Nevertheless, this improvement in oocyte survival with 5-5-min preexposure needs further clarification. This work is the first to report on predehydration with raffinose, and further investigations are therefore necessary to elucidate the mechanism of cryoinjury associated with preexposure to this sugar and subsequent exposure to the cryoprotectant. In conclusion, the present study showed that preexposure to a relatively low concentration of EG allowed shorter exposure times of oocyte in the VS, a mixture of EG and raffinose, and the reduced exposure time to VS, which minimizes the toxic effect of EG, improved oocyte viability. Likewise, predehydration with raffinose for 5-5, but not for 2-2 or 10-10 min, allowed a wider range of exposure times to the VS and increased the viability of vitrified oocytes, probably due to the reduction of EG needed relative to the small volume of oocytes after optimum cellular dehydration. REFERENCES 1. Erbach, G. T., Lawitts, J. A., Papaioannou, V. E., and Biggers, J. D. Differential growth of the mouse preimplantation embryo in chemically defined media. Biol. Reprod. 50, 1027–1033 (1994). 2. Hotamisligil, S., Toner, M., and Powers, R. D. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol. Reprod. 55, 161–168 (1996). 3. Jackowski, S., Leibo, S. B., and Mazur, P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J. Exp. Zool. 212, 329–341 (1980). 4. Kamiguchi, Y., and Mikamo, K. A new technique for chromosome study of murine oocytes. Proc. Jpn. Acad. 52, 316 (1976). 5. Kasai, M. Vitrification: Refined strategy for the cryopreservation of mammalian embryos. J. Mamm. Ova Res. 14, 17–28 (1997). 6. Kuleshova, L. L., MacFarlane, D. R., Trounson, A. O., and Shaw, J. M. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38, 119–130 (1999). 7. Mazur, P. The freezing of biological systems. Cryobiology 168, 939–949 (1970).
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