- Email: [email protected]
Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification
ELSEVIER
EFFICIENT CRYOPRESERVATION OF BOVINE BLASTOCYSTS FROM NUCLEAR TRANSFER WITH SOMATIC CELLS USING DEHYDRATION AND VITRIFICATION B.X. Nguyen,’ Laboratory
DERIVED PARTIAL
Y. Sotomaru, T. Tani, Y. Kato, Y. Tsunoda’
of Animal Reproduction, College of Agriculture Kinki University,Nara, 63 I-8505, Japan.
Received for publication: Apri 1 6, 1999 Accepted: October 8, 1999
ABSTRACT Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification. 8 20K1 by Elswier Science Inc. Key words: bovine embryos, nuclear transfer, cumulus cells, vitrification
Acknowledgments This work was supported by grants from the Program for Promotion of Basic ResearchActivities for Innovative Biosciences(PROBRAIN). ‘Present address: Laboratory of Biology of Reproduction & Development, National Center for Sciencesand Technology, Nghia Do, Ha Noi, Vietnam. E-mail: [email protected] ‘Correspondence and reprint requests: E-mail: [email protected]
Theriogenology 0 2000 Elaevier
63: 143~1446,2000 Sciince Inc.
0093-691X/OO&eee front matter PII %093-691X(00)00266-7
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Theriogenology
INTRODUCTION The development of somatic cell nuclear transfer technology has been progressing rapidly (6, 9, 20, 21, 22). As the production of a large number of identical bovine clones from somatic cells is becoming more feasible, we set ourselves the task of developing a simple and efficient freezing method for banking bovine blastocysts cloned from somatic cells, either on the laboratory or large industrial scale. To our knowledge, until now there have been no reports on the cryopreservation of somatic nuclear transferred embryos. The only publication concerning the cryopreservation of embryos cloned from blastomeres used slow freezing in a mixture of propanediol and sucrose(17). Generally, it is considered that nuclear transferred embryos are more sensitive to freeze-thawing damage than in vitro fertilized embryos or in vivo produced embryos. This may be due to some of their specific properties, such as the low number of cells per blastocyst (4, 17), or to the loss of the integrity of the zona pellucida caused by the micromanipulation during enucleation and cell injection. Conflicting results have been published concerning the role of cell number and of the zona pellucida in cryopreservation of embryos, Gustafsson et al. (7) and Tsunoda et al. (18) reported that the in vitro survival rates of bovine or goat embryos frozen after biopsy or bisection was significantly reduced compared with those of embryos frozen without biopsy, those biopsied only or those protected with agar. A loss of survival was also reported for mouse embryos bisected at the 2-cell stage and frozen slowly in dimethylsulphoxide after in vitro culture to the 16 -cell stage (1.5). In contrast, Vajta et al. (19), in experiments using vitrification of Day 7 and Day 8 in vitro-fertilized and zona-dissected bovine blastocysts in a mixture of ethylene glycol and dimethylsulphoxide, reported that an intact zona pellucida was not required to obtain high survival and hatching rates. In the present study, we examined the viability of frozen-thawed bovine blastocysts obtained after nuclear transfer with cumulus cells. The studies were carried out in comparison with bovine blastocysts produced by in vitro fertilization and by using 2 principal approaches: 1) vitrification in a solution containing a high concentration of ethylene glycol; 2) vitrification in combination with partial dehydration using a freezing solution containing ethylene glycol plus sucrose and Ficoll.
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MATERtALS
AND
METHODS
Embryo Production by In Vitro Fertilization (IVF) The cumulus oocyte complexes were aspirated from follicles of slaughter housederived bovine ovaries. Immature oocytes were matured in TCM-199 (Gibco, Tokyo, Japan) medium containing 1OW (v/v) heat-treated fetal bovine serum (FBS) for 22 to 24 h at 39°C in a water-saturated CO, incubator with 5% CO, in air. Frozen semen was thawed in a water bath at 37°C. Spermatozoa were washed twice by centrifugation at 600 x g for 5 min in bovine serum albumin (BSA, Sigma, St.Louis, MO)-free modified Brackett-Oliphant medium (BO) supplemented with caffeine (Sigma; 5). The sperm suspensionwas diluted with BO medium supplemented with BSA (20 mg/mL) and heparin (5 IU/mL). Aliquots of the suspensionwere incubated for 30 min in SO-uL drops. Matured oocytes were partially separated from cumulus cells by pipetting in hyaluronidase (300 B-J/ mL, Sigma) and incubated with capacitated spermatozoa at a concentration of 5x 105/mL. At 6 h after sperm-oocyte incubation, the cumulus cells were removed by pipetting and the oocytes were cultured in CRl-aa (13) for 48 h in 5% CO,, 5% 0, and 90% N,. Then the embryos were transferred to CRI-aa medium supplemented with 10% FBS and mouse fetal fibroblasts in 5% CO, in air. On Day 7 or 8 of in vitro culture (Day 0 was the day of fertilization), visually normal blastocysts were frozen. Embryo Production by Nuclear Transfer (NT) Oocytes with a first polar body were enucleated mechanically at 22 to 24 h after maturation, and chromosome removal was confirmed by staining with Hoechst 33342 dye (Aldrich, Milwaukee, WI) under ultraviolet light using the methods previously reported (17). Cumulus cells with oocytes obtained from ovaries were cultured in modified D-MEM (Dulbecco’s Modified Eagle’sMedium; Nissui, Tokyo, Japan) supplemented with 10% FBS, as described in a previous study (9). After 5 to 7 d, cumulus cells were disaggregated for subculture using Dulbecco’s phosphate buffered saline (PBS; Gibco) containing 0.1% trypsin and 0.05% EDTA. Cells were passagedevery 3 to 4 d and cells passagedfor 2 to 7 times were cultured in modified D-MEM supplemented with 0.5% FBS for 10 to 17 d. Cells cultured in 0.5% or less FBS for more than 3 d were previously found to enter a quiescent state(9). A single cumulus cell was introduced into an enucleated oocyte through a slit in the zona pellucida and then electrically fused with two direct current (DC) pulses of 150 v/mm for 25 us in Zimmerman fusion medium (23). The fusion was verified by observation 15 min later (0 h for development), and fused oocytes were activated by two DC pulses at 20 v/mm for 20 us repeated twice at an interval of 15 min. The fused oocytes were cultured in CR1 -aa medium containing cycloheximide ( 10 ccg/mL) for 6 h. They were then cultured in cycloheximide-free medium for a further 48 h in
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5% CO,, 5% 0, and 90% N2. On Day 3, the nuclear transplant embryos were coculutured with mouse fetal fibroblasts in CR-laa medium supplemented with IOoh FBS under 5% CO, in air. On Day 7 or 8 of in vitro culturing (Day 0 was the day of nuclear transfer), visually normal blastocysts were selected for further investigation. Vitrification The embryos were scored according to their stage of development and morphological appearance. Day 7 and Day 8 expanded blastocysts were used for the experiments. Although we did not measure the diameter of the embryos, the developmental stagesof IVF and NT embryos were considered to be similar on the sameday. Embryos were equilibrated at room temperature in a solution consisting of 3.75, 7.5 and 20% ethylene glycol in PBS with 20% calf serum (17) for 1 mm/step. They were then transferred to vitrifying solution kept at 4°C box prior to utilization, and then loaded into 0.25mL straws. The straws were heat-sealed and held on ice for 2 to 3 min, then plunged into liquid nitrogen. Vitrified embryos were stored for 1 h to 1 to 2 d in liquid nitrogen. In Experiment 1, embryos were vitrified in a solution consisting of 60% ethylene glycol (EC; Sigma) in PBS containing 20% CS. In Experiment 2, a modification of the medium described by Kasai et al. (8) was used. The embryos were vitrified in a solution of 39% ethylene glycol + 0.7M sucrose + 8.6% Ficoll. Warming and Dilution of Cryoprotectants The straws were removed from liquid nitrogen, kept for 5 set in air and warmed by gently agitating in a 35 to 38°C water bath for 30 sec. The contents of the straw (0.2 mL) were expelled into a culture dish, diluted with 0.3 mL of PBS-20% CS and left for 2 min at room temperature. Then embryos were washed twice in PBS-20% CS, and the morphology of the embryos was judged under an inverted microscope ( x 200).
Embryo Culture and Assessmentof Viability Embryos were co-cultured with a feeder layer of mouse fibroblast cells under paraffin oil at 39°C in 5% CO, in air for 3 d. The cell culture consisted of ES-DMEM (12) supplemented with 10% FBS. The blastocoele re-expansion which means the full recovery of the original volume of frozen-thawed blastocysts were examined at 6, 12 and 24 h after thawing. The blastocysts with the original volume of the blastocoele within 24 h after thawing were considered to have survived. The hatching rate of surviving embryos was examined at 12 h intervals for 3 d after thawing.
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Hatching meant that blastocysts at evaluation had escaped partly from the zonae pellucidae. Statistical Analysis Data were analyzed by 2 tests. RESULTS The percentage of blastocysts with intact morphology, the survival and the hatching rates, and the time course of hatching are presented in Table 1.
Table. 1 In vitro survival and hatching of bovine blastocysts after vitrification No.of embryos treated Experiment1 NT IVF Experiment2 NT IVF
No. of No.of embryos embryos with intact sulviving morphology(%) %
No.of hatchingembryos culturedfor (%) 2 days 3 days 1 &Y
43 60
43 (100) 58 (97)
19 (44) a 47 (78) b
2 (ll)a 3 (6) a
13 (68) a 19 (40) b
18 (95)a 26 (55) b
44 52
44 (100) 50 (96)
41 (93) c 24 (59) b 40 (77) b 12 (30) c
38 (93) c 28 (70) a
39 (95) a 34 (85) a
a,b,c Values with different superscripts within the same column are significantly different (PcO.05 to 0.001). NT = nuclear transfer.
In Experiment 1, a high rate of recovery of morphologically intact embryos was observed in both nuclear transfer and in vitro fertilized blastocysts. However, significant (PcO.001) reduction in the survival rate was observed for the nuclear transfer blastocysts compared with in vitro fertilized blastocysts. The hatching rate of surviving nuclear transfer blastocysts was significantly higher than the rate of in vitro fertilized blastocysts. In Experiment 2, nearly all of the recovered blastocysts were morphologically intact. The survival rate of nuclear transfer blastocysts was higher than that of in vitro fertilized blastocysts. The hatching rate was not significantly different between the 2 groups. However, the time coursesof the hatching of surviving blastocysts after thawing were different. The nuclear transfer blastocysts started to hatch earlier than in vitro fertilized blastocysts.
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Table 2 shows the kinetics of re-expansion of blastocoeles monitored at 6 and 12 h after thawing. In the first experiment, the proportions of re-expanded blastocysts at 6 and 12 h after thawing were considerably lower for nuclear transfer blastocysts than for IVF blastocysts (P
Table 2. The kinetics of the post-thawing expansion of bovine blastocysts after vitrification Experiment
No.of embryos examined
No.and % of embryosre-expandedat 13 h
Ah
Experiment 1 NT IVF
43 60
1 (2) 17 (28)
a b
15 (35) 41 (68)
a b
Experiment 2 NT IVF
42 45
26 (62) 2 (4)
c a
39 (93)
c
31 (69)
b
a,b,c Values with different superscripts within the samecolumn are significantly different (P
DISCUSSION Our results show that bovine blastocysts derived from nuclear transfer with cumulus cells could be frozen at - 196°C without lossof viability by a simple method combining of partial dehydration and vitrification. As shown in the experiment with embryos dehydrated and vitrified in freezing solution consisting of ethylene glycol-sucrose -Ficoll, nearly all frozen-thawed
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embryos were recovered morphologically intact. The survival rate and the proportion of hatching nuclear transferred embryos were high (93 and 95%, respectively). This survival rate was higher than that of 59% reported by Takano et al. (17) for bovine blastocysts derived from nuclear transfer with blastomeres and frozen slowly in 1.5M ethylene glycol. The efficiency of vitrification with partial dehydration in a solution of ethylene glycol was also demonstrated by the ability of frozen-thawed embryos to regain their original appearance, and by the developmental aspects observed during culturing after thawing. In nearly all surviving embryos, re-expansion to the original volume was observed from 6 to 12 h after thawing. Most surviving blastocysts started to hatch during the first 2 d of culture, and the hatched blastocysts attached to the bottom of the culture dish and increased in size from 7- to IO-fold compared with the size of expanding blastocysts when they were co-cultured with mouse fibroblasts in DMEM for a longer period after thawing. The survival rate of nuclear transfer blastocysts was significantly higher than that of blastocysts produced by in vitro fertilization and frozen by the same method (93 vs 77%). The re-expansion of nuclear transfer blastocysts after thawing started earlier than that of in vitro fertilized blastocysts. Hatching also started earlier in the nuclear transfer blastocysts, but this may have been due to the slit made in the zona pellucida. Our results strongly suggest that ethylene glycol is an efficient cryoprotectant for the cryopreservation of nuclear transferred embryos due to its properties of rapid permeability and low toxicity (1, 3, 8). These properties of ethylene glycol are important because they permit the minimization of the duration of treatment of embryos with cryoprotectant before freezing and, consequently, the minimization of the toxic effects of the cryoprotectant. These same properties also have important effects on the procedure of removal of cryoprotectant after thawing. Due to its rapid permeability, the dilution of ethylene glycol from embryos can be achieved without damaging osmotic and flow stressby addition of PBS to the vitrification solution. However, our results showed that the high concentration of ethylene glycol at which vitrification could be achieved (60%) exceeded the limit of concentration that nuclear transfer blastocysts can tolerate. The survival rate of nuclear transfer blastocysts vitrified in ethylene glycol alone was significantly lower than that of blastocysts frozen in the mixture of ethylene glycol-sucrose-Ficoll. This negative effect of the high concentration of ethylene glycol was observed also in the low hatching rate on Day 1 after thawing and the low re-expansion rate at 6 h after thawing.
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These observations also explain the positive effect of partial dehydration in the vitrification of nuclear transfer blastocysts. Besidesthe general effect of protein and membrane stabilization of sucrose (2), some other mechanisms may also be exerting effects: 1) Addition of sucrose and Ficoll to the vitrification medium could allow reduction of the concentration of cryoprotectant needed to achieve vitrification to within the limit embryos could tolerate (39% instead of 60% in our case; 15). 2) Sucrose and Ficoll could also increase the capacity of embryos themselve to tolerate the cryoprotectant (8). The results of the present study showed that there was a difference between the abilities of nuclear transfer and in vitro fertilized blastocysts to tolerate the freezethawing procedures. Comparing the survival of nuclear transfer and in vitro fertilized blastocysts vitrified in the solution of ethylene glycol-sucrose -Ficoll, nuclear transferred embryos could survive the freezing-thawing procedures better than in vitro fertilized embryos. On the other hand, the low survival rate of nuclear transferred embryos frozen in a solution of 60% ethylene glycol showed that the in vitro fertilized blastocysts resisted the cryoprotectant toxicity better than nuclear transferred embryos. The present study demonstrated that the survival and hatching rates of in vitro fertilized blastocysts vitrified in a solution of ethylene glycol-sucrose-Ficoll were in the range of those reported by other authors (10, 11, 14, 16); however, the survival and hatching rates observed for nuclear transfer blastocysts were significantly higher. One of the important differences observed between vitrification of in vitro fertilized and nuclear transferred embryos concerned their ability to regain their original appearance after thawing. In our experiments with vitrification in the solution of ethylene glycol-sucrose-Ficoll, the re-expansion of blastocoeles in two-thirds embryos were completed within 6 h after thawing for nuclear transfer blastocysts. Taken together with the high survival rate after thawing, these findings allow us to suggest that the technique we have reported here is suitable for efficiently cryo-banking the blastocysts cloned from somatic ceils.
REFERENCES 1. Ali J, Shelton J N. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993; 99: 471-477. 2. Arakawa T, Timasheff S N. Stabilization of protein by sugars. Biochemistry 1982;21: 3.
6536-6544.
Bautista J A, Kanagawa H. Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice. Jpn J Vet Res 1998; 45: 13-19.
l3eriogenology 4.
5. 6.
7. 8.
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Bordignon V, Smith L S. Telephase encleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol Reprod Dev 1998; 49: 29-36. Brackett B G, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod Dev 1975; 40: 5-l 1. Cibelli J B, Stice S L, Golueke P J, I
9.
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11.
12. 13.
14.
15.
16.
17.
Kato Y, Tani T, Sotomaru Y, Kurokawa I<, Kato J-Y, Doguchi H, Yasue H, Tsunoda Y. Eight calves cloned from somatic cells of a single adult. Science 1998; 282: 20952098. Mahmoudzadeh A R, Van Soom A, Bols P, Ysebaert M T, de Kruif A. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrosedilution on embryonic survival. J Reprod Fertil 1995; 103: 33-39. Martinez AG, Motos DG de, Furnus CC, Brogliatti GM. In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos. Theriogenology 1998; 50: 757-767. Robertson E J. Embryoderived stem cell line. In: Rodertson EJ fed), Teratocarcinomas and Embryonic Stem Cells. Oxford: IRL Press, 1987; 7 1- 1 12. Rose&ranks Jr C F, First NL. Culture of bovine zygotes to the blastocysts Theriogenology 1991; 35: 266. stage: effect of amino acids and vitamins. abstr. Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T. Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose and polyvinylpyrrolidone. Cryobiology 1996; 33: 29 I-299. Sotomaru Y, Kato Y, Tsunoda Y. Production of monozygotic twins after freezing and thawing of bisected mouse embryos. Cryobiology 1998; 37: 139145. Tachikawa S, Otoi T, I
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18. Tsunoda Y, Tokugawa T, Okubo Y, Sugie T. Beneficial effect of agar for the frozen storage of bisected goat embryos. Theriogenology 1987;28:3 17-322. 19. Vajta G, Holm P, Greve T, Callesen H. Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos. Theriogenology 1997; 47: 50 I-509. 20. Vignon X, Chesne P, Le Bourhis D, Flechon J E, Heyman Y, Renard J P. Developmental potential of bovine embryos reconstructed from enucleated maturated oocytes fused with cultured somatic cells. C R Acad Sci III 1998; 321: 735-745. 21.
22.
Wakayama T, Perry A C, Zuccotti M, Johnson K R, Yanagimachi R. Fullterm development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998; 394: 369-374. Wilmut I, Schnieke A E, McWhir J, Kind A J, Campbell K H S. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 310-313.
EFFICIENT CRYOPRESERVATION OF BOVINE BLASTOCYSTS FROM NUCLEAR TRANSFER WITH SOMATIC CELLS USING DEHYDRATION AND VITRIFICATION B.X. Nguyen,’ Laboratory
DERIVED PARTIAL
Y. Sotomaru, T. Tani, Y. Kato, Y. Tsunoda’
of Animal Reproduction, College of Agriculture Kinki University,Nara, 63 I-8505, Japan.
Received for publication: Apri 1 6, 1999 Accepted: October 8, 1999
ABSTRACT Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification. 8 20K1 by Elswier Science Inc. Key words: bovine embryos, nuclear transfer, cumulus cells, vitrification
Acknowledgments This work was supported by grants from the Program for Promotion of Basic ResearchActivities for Innovative Biosciences(PROBRAIN). ‘Present address: Laboratory of Biology of Reproduction & Development, National Center for Sciencesand Technology, Nghia Do, Ha Noi, Vietnam. E-mail: [email protected] ‘Correspondence and reprint requests: E-mail: [email protected]
Theriogenology 0 2000 Elaevier
63: 143~1446,2000 Sciince Inc.
0093-691X/OO&eee front matter PII %093-691X(00)00266-7
1440
Theriogenology
INTRODUCTION The development of somatic cell nuclear transfer technology has been progressing rapidly (6, 9, 20, 21, 22). As the production of a large number of identical bovine clones from somatic cells is becoming more feasible, we set ourselves the task of developing a simple and efficient freezing method for banking bovine blastocysts cloned from somatic cells, either on the laboratory or large industrial scale. To our knowledge, until now there have been no reports on the cryopreservation of somatic nuclear transferred embryos. The only publication concerning the cryopreservation of embryos cloned from blastomeres used slow freezing in a mixture of propanediol and sucrose(17). Generally, it is considered that nuclear transferred embryos are more sensitive to freeze-thawing damage than in vitro fertilized embryos or in vivo produced embryos. This may be due to some of their specific properties, such as the low number of cells per blastocyst (4, 17), or to the loss of the integrity of the zona pellucida caused by the micromanipulation during enucleation and cell injection. Conflicting results have been published concerning the role of cell number and of the zona pellucida in cryopreservation of embryos, Gustafsson et al. (7) and Tsunoda et al. (18) reported that the in vitro survival rates of bovine or goat embryos frozen after biopsy or bisection was significantly reduced compared with those of embryos frozen without biopsy, those biopsied only or those protected with agar. A loss of survival was also reported for mouse embryos bisected at the 2-cell stage and frozen slowly in dimethylsulphoxide after in vitro culture to the 16 -cell stage (1.5). In contrast, Vajta et al. (19), in experiments using vitrification of Day 7 and Day 8 in vitro-fertilized and zona-dissected bovine blastocysts in a mixture of ethylene glycol and dimethylsulphoxide, reported that an intact zona pellucida was not required to obtain high survival and hatching rates. In the present study, we examined the viability of frozen-thawed bovine blastocysts obtained after nuclear transfer with cumulus cells. The studies were carried out in comparison with bovine blastocysts produced by in vitro fertilization and by using 2 principal approaches: 1) vitrification in a solution containing a high concentration of ethylene glycol; 2) vitrification in combination with partial dehydration using a freezing solution containing ethylene glycol plus sucrose and Ficoll.
Theriogenology
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MATERtALS
AND
METHODS
Embryo Production by In Vitro Fertilization (IVF) The cumulus oocyte complexes were aspirated from follicles of slaughter housederived bovine ovaries. Immature oocytes were matured in TCM-199 (Gibco, Tokyo, Japan) medium containing 1OW (v/v) heat-treated fetal bovine serum (FBS) for 22 to 24 h at 39°C in a water-saturated CO, incubator with 5% CO, in air. Frozen semen was thawed in a water bath at 37°C. Spermatozoa were washed twice by centrifugation at 600 x g for 5 min in bovine serum albumin (BSA, Sigma, St.Louis, MO)-free modified Brackett-Oliphant medium (BO) supplemented with caffeine (Sigma; 5). The sperm suspensionwas diluted with BO medium supplemented with BSA (20 mg/mL) and heparin (5 IU/mL). Aliquots of the suspensionwere incubated for 30 min in SO-uL drops. Matured oocytes were partially separated from cumulus cells by pipetting in hyaluronidase (300 B-J/ mL, Sigma) and incubated with capacitated spermatozoa at a concentration of 5x 105/mL. At 6 h after sperm-oocyte incubation, the cumulus cells were removed by pipetting and the oocytes were cultured in CRl-aa (13) for 48 h in 5% CO,, 5% 0, and 90% N,. Then the embryos were transferred to CRI-aa medium supplemented with 10% FBS and mouse fetal fibroblasts in 5% CO, in air. On Day 7 or 8 of in vitro culture (Day 0 was the day of fertilization), visually normal blastocysts were frozen. Embryo Production by Nuclear Transfer (NT) Oocytes with a first polar body were enucleated mechanically at 22 to 24 h after maturation, and chromosome removal was confirmed by staining with Hoechst 33342 dye (Aldrich, Milwaukee, WI) under ultraviolet light using the methods previously reported (17). Cumulus cells with oocytes obtained from ovaries were cultured in modified D-MEM (Dulbecco’s Modified Eagle’sMedium; Nissui, Tokyo, Japan) supplemented with 10% FBS, as described in a previous study (9). After 5 to 7 d, cumulus cells were disaggregated for subculture using Dulbecco’s phosphate buffered saline (PBS; Gibco) containing 0.1% trypsin and 0.05% EDTA. Cells were passagedevery 3 to 4 d and cells passagedfor 2 to 7 times were cultured in modified D-MEM supplemented with 0.5% FBS for 10 to 17 d. Cells cultured in 0.5% or less FBS for more than 3 d were previously found to enter a quiescent state(9). A single cumulus cell was introduced into an enucleated oocyte through a slit in the zona pellucida and then electrically fused with two direct current (DC) pulses of 150 v/mm for 25 us in Zimmerman fusion medium (23). The fusion was verified by observation 15 min later (0 h for development), and fused oocytes were activated by two DC pulses at 20 v/mm for 20 us repeated twice at an interval of 15 min. The fused oocytes were cultured in CR1 -aa medium containing cycloheximide ( 10 ccg/mL) for 6 h. They were then cultured in cycloheximide-free medium for a further 48 h in
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5% CO,, 5% 0, and 90% N2. On Day 3, the nuclear transplant embryos were coculutured with mouse fetal fibroblasts in CR-laa medium supplemented with IOoh FBS under 5% CO, in air. On Day 7 or 8 of in vitro culturing (Day 0 was the day of nuclear transfer), visually normal blastocysts were selected for further investigation. Vitrification The embryos were scored according to their stage of development and morphological appearance. Day 7 and Day 8 expanded blastocysts were used for the experiments. Although we did not measure the diameter of the embryos, the developmental stagesof IVF and NT embryos were considered to be similar on the sameday. Embryos were equilibrated at room temperature in a solution consisting of 3.75, 7.5 and 20% ethylene glycol in PBS with 20% calf serum (17) for 1 mm/step. They were then transferred to vitrifying solution kept at 4°C box prior to utilization, and then loaded into 0.25mL straws. The straws were heat-sealed and held on ice for 2 to 3 min, then plunged into liquid nitrogen. Vitrified embryos were stored for 1 h to 1 to 2 d in liquid nitrogen. In Experiment 1, embryos were vitrified in a solution consisting of 60% ethylene glycol (EC; Sigma) in PBS containing 20% CS. In Experiment 2, a modification of the medium described by Kasai et al. (8) was used. The embryos were vitrified in a solution of 39% ethylene glycol + 0.7M sucrose + 8.6% Ficoll. Warming and Dilution of Cryoprotectants The straws were removed from liquid nitrogen, kept for 5 set in air and warmed by gently agitating in a 35 to 38°C water bath for 30 sec. The contents of the straw (0.2 mL) were expelled into a culture dish, diluted with 0.3 mL of PBS-20% CS and left for 2 min at room temperature. Then embryos were washed twice in PBS-20% CS, and the morphology of the embryos was judged under an inverted microscope ( x 200).
Embryo Culture and Assessmentof Viability Embryos were co-cultured with a feeder layer of mouse fibroblast cells under paraffin oil at 39°C in 5% CO, in air for 3 d. The cell culture consisted of ES-DMEM (12) supplemented with 10% FBS. The blastocoele re-expansion which means the full recovery of the original volume of frozen-thawed blastocysts were examined at 6, 12 and 24 h after thawing. The blastocysts with the original volume of the blastocoele within 24 h after thawing were considered to have survived. The hatching rate of surviving embryos was examined at 12 h intervals for 3 d after thawing.
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Hatching meant that blastocysts at evaluation had escaped partly from the zonae pellucidae. Statistical Analysis Data were analyzed by 2 tests. RESULTS The percentage of blastocysts with intact morphology, the survival and the hatching rates, and the time course of hatching are presented in Table 1.
Table. 1 In vitro survival and hatching of bovine blastocysts after vitrification No.of embryos treated Experiment1 NT IVF Experiment2 NT IVF
No. of No.of embryos embryos with intact sulviving morphology(%) %
No.of hatchingembryos culturedfor (%) 2 days 3 days 1 &Y
43 60
43 (100) 58 (97)
19 (44) a 47 (78) b
2 (ll)a 3 (6) a
13 (68) a 19 (40) b
18 (95)a 26 (55) b
44 52
44 (100) 50 (96)
41 (93) c 24 (59) b 40 (77) b 12 (30) c
38 (93) c 28 (70) a
39 (95) a 34 (85) a
a,b,c Values with different superscripts within the same column are significantly different (PcO.05 to 0.001). NT = nuclear transfer.
In Experiment 1, a high rate of recovery of morphologically intact embryos was observed in both nuclear transfer and in vitro fertilized blastocysts. However, significant (PcO.001) reduction in the survival rate was observed for the nuclear transfer blastocysts compared with in vitro fertilized blastocysts. The hatching rate of surviving nuclear transfer blastocysts was significantly higher than the rate of in vitro fertilized blastocysts. In Experiment 2, nearly all of the recovered blastocysts were morphologically intact. The survival rate of nuclear transfer blastocysts was higher than that of in vitro fertilized blastocysts. The hatching rate was not significantly different between the 2 groups. However, the time coursesof the hatching of surviving blastocysts after thawing were different. The nuclear transfer blastocysts started to hatch earlier than in vitro fertilized blastocysts.
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Table 2 shows the kinetics of re-expansion of blastocoeles monitored at 6 and 12 h after thawing. In the first experiment, the proportions of re-expanded blastocysts at 6 and 12 h after thawing were considerably lower for nuclear transfer blastocysts than for IVF blastocysts (P
Table 2. The kinetics of the post-thawing expansion of bovine blastocysts after vitrification Experiment
No.of embryos examined
No.and % of embryosre-expandedat 13 h
Ah
Experiment 1 NT IVF
43 60
1 (2) 17 (28)
a b
15 (35) 41 (68)
a b
Experiment 2 NT IVF
42 45
26 (62) 2 (4)
c a
39 (93)
c
31 (69)
b
a,b,c Values with different superscripts within the samecolumn are significantly different (P
DISCUSSION Our results show that bovine blastocysts derived from nuclear transfer with cumulus cells could be frozen at - 196°C without lossof viability by a simple method combining of partial dehydration and vitrification. As shown in the experiment with embryos dehydrated and vitrified in freezing solution consisting of ethylene glycol-sucrose -Ficoll, nearly all frozen-thawed
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embryos were recovered morphologically intact. The survival rate and the proportion of hatching nuclear transferred embryos were high (93 and 95%, respectively). This survival rate was higher than that of 59% reported by Takano et al. (17) for bovine blastocysts derived from nuclear transfer with blastomeres and frozen slowly in 1.5M ethylene glycol. The efficiency of vitrification with partial dehydration in a solution of ethylene glycol was also demonstrated by the ability of frozen-thawed embryos to regain their original appearance, and by the developmental aspects observed during culturing after thawing. In nearly all surviving embryos, re-expansion to the original volume was observed from 6 to 12 h after thawing. Most surviving blastocysts started to hatch during the first 2 d of culture, and the hatched blastocysts attached to the bottom of the culture dish and increased in size from 7- to IO-fold compared with the size of expanding blastocysts when they were co-cultured with mouse fibroblasts in DMEM for a longer period after thawing. The survival rate of nuclear transfer blastocysts was significantly higher than that of blastocysts produced by in vitro fertilization and frozen by the same method (93 vs 77%). The re-expansion of nuclear transfer blastocysts after thawing started earlier than that of in vitro fertilized blastocysts. Hatching also started earlier in the nuclear transfer blastocysts, but this may have been due to the slit made in the zona pellucida. Our results strongly suggest that ethylene glycol is an efficient cryoprotectant for the cryopreservation of nuclear transferred embryos due to its properties of rapid permeability and low toxicity (1, 3, 8). These properties of ethylene glycol are important because they permit the minimization of the duration of treatment of embryos with cryoprotectant before freezing and, consequently, the minimization of the toxic effects of the cryoprotectant. These same properties also have important effects on the procedure of removal of cryoprotectant after thawing. Due to its rapid permeability, the dilution of ethylene glycol from embryos can be achieved without damaging osmotic and flow stressby addition of PBS to the vitrification solution. However, our results showed that the high concentration of ethylene glycol at which vitrification could be achieved (60%) exceeded the limit of concentration that nuclear transfer blastocysts can tolerate. The survival rate of nuclear transfer blastocysts vitrified in ethylene glycol alone was significantly lower than that of blastocysts frozen in the mixture of ethylene glycol-sucrose-Ficoll. This negative effect of the high concentration of ethylene glycol was observed also in the low hatching rate on Day 1 after thawing and the low re-expansion rate at 6 h after thawing.
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These observations also explain the positive effect of partial dehydration in the vitrification of nuclear transfer blastocysts. Besidesthe general effect of protein and membrane stabilization of sucrose (2), some other mechanisms may also be exerting effects: 1) Addition of sucrose and Ficoll to the vitrification medium could allow reduction of the concentration of cryoprotectant needed to achieve vitrification to within the limit embryos could tolerate (39% instead of 60% in our case; 15). 2) Sucrose and Ficoll could also increase the capacity of embryos themselve to tolerate the cryoprotectant (8). The results of the present study showed that there was a difference between the abilities of nuclear transfer and in vitro fertilized blastocysts to tolerate the freezethawing procedures. Comparing the survival of nuclear transfer and in vitro fertilized blastocysts vitrified in the solution of ethylene glycol-sucrose -Ficoll, nuclear transferred embryos could survive the freezing-thawing procedures better than in vitro fertilized embryos. On the other hand, the low survival rate of nuclear transferred embryos frozen in a solution of 60% ethylene glycol showed that the in vitro fertilized blastocysts resisted the cryoprotectant toxicity better than nuclear transferred embryos. The present study demonstrated that the survival and hatching rates of in vitro fertilized blastocysts vitrified in a solution of ethylene glycol-sucrose-Ficoll were in the range of those reported by other authors (10, 11, 14, 16); however, the survival and hatching rates observed for nuclear transfer blastocysts were significantly higher. One of the important differences observed between vitrification of in vitro fertilized and nuclear transferred embryos concerned their ability to regain their original appearance after thawing. In our experiments with vitrification in the solution of ethylene glycol-sucrose-Ficoll, the re-expansion of blastocoeles in two-thirds embryos were completed within 6 h after thawing for nuclear transfer blastocysts. Taken together with the high survival rate after thawing, these findings allow us to suggest that the technique we have reported here is suitable for efficiently cryo-banking the blastocysts cloned from somatic ceils.
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Bordignon V, Smith L S. Telephase encleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol Reprod Dev 1998; 49: 29-36. Brackett B G, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod Dev 1975; 40: 5-l 1. Cibelli J B, Stice S L, Golueke P J, I
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