29, 493499
CRYOBIOLOGY
Viability
(1992)
of Nuclei of Two-Cell Fused with Blastomeres
Mouse Embryos Stored at 4°C and of Fresh Two-Cell Embryos
K. NAKAMURA”
AND
Y. TSUNODAt
*Sufety Evaluation Laboratory of Kaken Pharmaceutical Co., Ltd., 301 Gensuke Fujieda, Shizuoka 426 Japan: and fFaculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631 Japan This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0°C. Two-cell embryos were stored at 4’C for 24-96 h in PBl containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4°C fresh Fl was significantly prolonged when they wcrc transplanted into a blastomere of enucleated (C57BLI6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88,67,76,71,64,45, 32, and 20% following storage for 0,48,72,96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4°C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4°C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm
The preservation of mammalian oocytes and embryos for varying periods of time has practical applications in many areas of biology, e.g., in animal husbandry, cryobiology, genetics, toxicology, and mutagenesis. The technique of frozen storage (longterm storage) for mammalian embryos has become established since a successful freezing method was reported for the mouse (23,24). Today live young have been obtained from frozen-thawed embryos of many mammalian species, including human beings. However, few researchers have conducted their studies on embryos at temperatures of 0 to 10°C (short-term storage). Limited development of preimplantation embryos from mice (5, 21), rabbits (2-4, 6), sheep (15), and cattle (8, 19, 25) has been reported following short-term storage. The
Received 1992
September 23, 1991; accepted January 13,
resistance of embryos to low temperature varies according to their developmental stages. Generally embryos at earlier developmental stages are less resistant to cold storage. A successful and reliable procedure for pronuclear transplantation has been developed for mouse zygotes (12). With this technique, it has been possible to examine the genetic and cytoplasmic factors contributing to embryonic development (11, 13, 17). Short-term low-temperature storage without freezing could be a helpful approach to the various manipulations currently in use in livestock breeding, especially in cattle, sheep, and goats. In a previous study, we investigated the resistance of mouse pronuclei to cold storage (16). In order to determine whether it is the nucleus or the cytoplasm that is more damaged during cold storage, we examined the viability of the nuclei of two-cell mouse embryos stored at 4°C.
493 001 l-2240192 Copyright
$5.00
0 1992 hy Academic
Press, Inc.
494
NAKAMURA MATERIALS
Experimental
AND METHODS
Design
Experiment 1. The first part of the study was designed to examine the effects of various sucrose concentrations and the duration of storage on the survival of freshly collected two-cell mouse embryos. Two-cell embryos were stored at 4°C for up to 96 h and then cultured at 37°C. Survival was assessed by determining their ability to develop into blastocysts after 3 days cultivation. Experiment 2. In the second part of the study, we examined the viability of nuclei from two-cell embryos after cold storage. CD-I two-cell embryos were stored at 4°C for up to 192 h in PBI containing 0.5 M sucrose. Individual nuclei were then transplanted to one enucleated blastomere of a fresh Fl (C57BL16JXCBA) two-cell embryos. The viability of reconstituted twocell embryos was examined by both in vitro culture and embryo transfer.
Source of Embryos CD-I female mice (5 weeks of age) and Fl female mice (5-8 weeks of age) were superovulated by intraperitoneal injections of 5 IU pregnant mare’s serum gonadotropin (PMSG, Teikoku Zoki Co. Ltd., Japan) and 5 IU human chorionic gonadotropin (hCG, Teikoku Zoki Co. Ltd., Japan) given 48 h apart. The mice were mated with males of same strain. Forty-two to 44 h after the hCG injection, two-cell embryos were obtained by flushing the oviducts with M2 medium (I) supplemented with 0.4% bovine serum albumin (BSA, Sigma A-4378). These two-cell embryos were then washed three times with M2 medium supplemented with 0.4% BSA. The coat color phenotypes used in this study were white (CD-l) and agouti (Fl).
Cold storage Two-cell CD-I embryos were pipetted into I ml of PBI medium (21) containing 0,0.25,0.5,0.75, or I .OM sucrose, in sterile polystyrene test tubes (Coming). The caps of the tubes were sealed with vinyl tape and the tubes were then placed in a glass beaker filled with water (20°C). The beaker was placed in a refrigerator at 4°C for periods varying from 24 to 96 h in Exp. 1 and 48 to 192 hr in Exp. 2.
Nuclear
Transplantation
Nuclear transplantation was carried out as described previously (18, 20). An area of approximately lO-20% of the zona pellucida in the two-cell embryos was cut with a tine glass needle to make manipulation procedure easy (20). Manipulations were carried out in 0.2 ml of M2 medium overlaid with parafIin oil. The two-cell embryos were then transferred to a micromanipulation glass slide in a drop of M2 medium contain-
AND TSUNODA ing cytochalasin B (5 &mg, Sigma) and Colcemid (0.1 kg/ml, Aldrich) overlaid with paraffin oil. After I5 min. a single nucleus was removed from recipient FI two-cell embryos, and a karyoplast containing the nucleus of a CD-l two-cell embryo which had been stored at 4°C for between 48 and 192 h was inserted, together with inactivated Sendai virus (HVJ, 2,500 hemagglutinating activity units/ml), into the perivitelline space next to the enucleated blastomere of the recipient Fl two-cell embryos.
Culture and Transfer of Embryos After storage, embryos were allowed to equilibrate for 10 min at room temperature. After recovery or nuclear transplantation, embryos were then washed three times with M2 and Ml6 medium (22) and cultured at 37°C for 3 days in microdrops of Ml6 medium supplemented with 0.4% BSA under paraffin oil, in atmosphere of 5% CO, in air. The viability of embryos was defined as the ability to develop to blastocysts in culture at 37°C for 3 days. After 3 days cultivation, developed embryos were classified as normal and incomplete blastocysts. A normal blastocyst was defined as one in which both blastomeres had divided synchronously to form a blastocyst. An incomplete blastocyst was one in which only a single blastomere had formed a blastocyst, and in which the other blastomere had not divided. In Exp. 2, the reconstituted embryos which developed to blastocysts after 3 days in culture were transferred into the uterine horns of Day 3 pseudopregnant CD-l recipients.
Evaluation of the Viability Cold-Stored Nuclei
of
The recipient mice were allowed to litter and the young were sexed and examined for their eye and coat color after they had grown to weaning. The viability of stored nuclei was evaluated by the coat color of the young. Thus, albino (white) and chimeric young were considered to express the phenotype of the stored nuclei. Chimeric progeny were raised to sexual maturity and then mated with albino mice (CD-l) in order to determine whether the nuclei from the cold stored two-cell embryos were able to contribute to the germ cell population. The pregnant mice were sacrificed on Day 18of gestation and live fetuses were examined for eye color. The germ cell population type was classified as albino, chimeric (mixed type of albino and colored), and colored types according to the eye color of the fetuses. The albino and the chimeric types in their germ cell population were considered to express the phenotype of the stored nuclei.
Statistical
Analysis
The results were examined by x2 test with Yates correction.
VIABILITY
OF NUCLEl
495
OF MOUSE EMBRYOS
creased compared with the proportion containing the nuclei of fresh embryos.
RESULTS
Experiment 1: In Vitro Viability of Two-Cell Embryos after Cold Storage As shown in Table 1, the viability of twocell embryos developed to blastocysts was quite low after 24 and 48 h of storage in sucrose-free PBl medium (14 and 5%, respectively). In the same sample containing 0.5 and 0.75 M sucrose, however, survival times were longer; the highest survival rates were obtained with 0.5 M sucrose when embryos were examined after 24, 48, 72, and 96 h of storage (51, 42, 9, and 3%, respectively). Experiment 2: In Vitro Viability of Reconstituted Chimeric Two-Ceil Embryos As shown in Table 2, degeneration of embryos increased after 120 h of storage. The proportions of chimeric embryos that developed to normal blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage of two-cell embryos for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. After more than 120 h of storage, the proportions of chimeric embryos that developed to incomplete blastocysts were significantly in-
Evaluation of Viability of Cold-Stored Nuclei after Embryo Transfer The results in Table 3 show that the proportions of transferred blastocysts that developed into live young were 43, 60,29, 37, 37, 39, 25, and 38% following storage for 0, 48,72,96, 120, 144, 168, and 192 h, respectively. The numbers of young that died before weaning were 2, 4, and 1 following storage for 0, 120, and 168 h, respectively. The rates at which stored nuclei contributed to the coat color of the young (white and chimera) were 85, 73, 100, 50, 67, 50, 67, and 67% following storage for 0,48, 72, 96, 120, 144, 168, 192 h, respectively. It is known that the sex of chimeras tends to be male (14), since XX/XY chimera exhibit a male phenotype. In the present study, 20 of 27 (74%) of chimeric young were male. As shown in Table 4, contributions to the germ cell population of the chimeric young from stored nuclei were 33, 67, 67, 60, 67, 0, 0 and 100%following storage for 0,48, 72,96, 120, 144, 168, and 192 h, respectively. One male chimera derived from nuclei stored for
TABLE 1 In Vitro Development of Two-Cell Mouse Embryos to the Blastocyst Stage after Storage for up to 96 h at
4°C in PB 1 Medium Containing Various Concentrations of Sucrose No. blastocysts developed/No. embryos cultured (%) Duration of storage (h)
Sucrose cont.
CM) 0
0
24
48
72
96
77190
6143 (14) 6147 (13) 23/45* (51) 30/46* (65) 4131
2137 (5) 6136 (17) 15/36* 9136* (25) o/12
o/34 (0) 0134 (0) 3133 (9) 2128 (7) O/II
0129 (0) O/28 (0) l/29 (3) 0129 (0) -
(13)
(0)
(0)
(86) 0.25
-
0.5 0.75
-
1.0
-
(42)
Note. Numbers in parentheses are percentages. *Significantly different from sucrose-free PBI (x2 test) (P < 0.001).
496
NAKAMUKA
AND TSUNODA
TABLE 2 In Vitro Development of Chimeric Two-Cell Embryos Produced by Allogeneic Nuclear Transfer of Embryos Preserved at 4°C in PBl Containing 0.5 M Sucrose into One Blastomere of a Fresh Embryo Duration of preservation of donor nuclei at 4°C (h) 0 48 72 96 120
No. of embryos degenerated/ No. of embryos preserved m O/55 (0) o/50 (0) o/59 (0) 4153
(8) 144 168 192
7187 (9) lo/78 (13) 27174 (37)
No. of embryos whose karyoplast fused/manipulated (%I 56162 (90) 52155 (95) 48/50
No. of embryos developed to No. of embryos” cultured 51 52
35*
(67) 46
35
(76) 55
(98) 47149 (96) 76/80 (95) 65168 (96) 43147 (92)
45
(88)
(96) 58159
Blastocyst (%I’
44 65 60 41
39* (71) 28** (64) 29*** (45) 19*** (32)
8WW (20)
Incompleteb blastocyst (%)’
Other (%I 2 (4)
A (1:) (1G 4 (9) 10
(I:,
(18)
(1;)
,:t; 17*
(1:) 19
(26)
(2%
33** (55) 28***
8 (13) 5
(68)
(12)
0 The embryos which fused both blastomeres were not cultured. b Single blastomere developed to blastocyst. ’ Based on number of embryos cultured. *,****** Significantly different from fresh embryos (x2 test) (*P < 0.05, **P < 0.01, ***P < 0.001).
cysts after 96 h of storage, whereas the viability of the nuclei of two-cell embryos was significantly extended when they were transplanted into a blastomere of enucleDISCUSSION ated fresh Fl two-cell embryos. SeventyFew attempts have been made to pre- one percent of the reconstituted two-cell serve mammalian embryos at low tempera- embryos containing nuclei stored at 4°C for tures above 0°C. Kasai et al. (7) have re- 96 h developed into blastocysts (Table 2). ported that the protective effects of sucrose These findings suggested that the viability on the storage of mouse and rat morulae at of nuclei was maintained longer than that of 0°C for extended times. Furthermore, in embryos during cold storage. From the reKasai’s later work (8), he reported that sults in Table 3, it can be seen that the rates mouse morulae could be stored for as long of stored nuclei contributions to the coat as 120 h at 0°C in various sugar solutions. In color of the young were comparable to the Exp. 1, similar protective effects of sucrose contribution rates of fresh nuclei. Furtherwere obtained in the cold storage of two- more, the viability of nuclei stored at 4°C cell mouse embryos. That is, the viability of for 192 h was confirmed in the germ cell two-cell embryos stored at 4°C was much population of chimeric young (Table 4). improved when 0.5 M sucrose was added to Why the viability of embryos decreases PBl medium (Table 1). However, only 3% during cold storage is not known. Herr and of these embryos developed into blasto- Wright (5) suggested that early embryos
192 h showed the albino type in its germ cell population.
VIABILITY
497
OF NUCLEI OF MOUSE EMBRYOS
TABLE 3 Development of Reconstituted Embryos Transferred to Recipient Mice and Coat Color of the Young at Weaning Young at weaning Duration of preservation of donor nuclei at 4°C (h) 0
No. pregnant/ No. recipient (%)
No. live young/ No. blastocysts transferred (%)
414(100)
15/35(43)
Coat color (%)b Sex
No. young
AL
CH
Male Female
8 5
2 5
4 0
2. 0
13
7
4
2 (15) 2 2
(85) 48
3/3(100)
15/25(60)
Male Female
9 6
2 2
I5
4
5 2 7
(2;)
(73) 72
3/3(100)
l/24(29)
Male Female
3 4
1 2
7
3
2 2
0 0
4
0 (0) 4 1
(1W 96
3/3(100)
10/27(37)
Male Female
7 3
0 0
10
0
3 2 5 (50)
120
3/3(100)
10/27(37)
Male Female
2 4
0 1
6
I
2 1 3 (67)
144
2/3(67)
10/26(39)
Male Female
8 2
2 I
10
3
2 0 2 (50)
168
2/2(100)
4/16(25)
Male Female
2 1
0 1
3
I
1 0
l/2(50)
3/8(38)
Male Female
I 2
0 1
3
1
5 (50) 0 2 2 (33) 4 I 5 (50) I 0
1 (67)
192
co
1 0
(3:) 0 I
I (67)
(3:)
Note. Numbers in parentheses are percentages. AL, albino; CH, chimera; CO, colored a Based on number of embryos transferred to recipients. b Based on number of live young at weaning.
may be more sensitive to undesirable components such as heavy metal contamination or, alternatively, that an absence of critical substances in synthetic media may be a fac-
tor involved in the reduced survival observed during cold storage. Recently, Kono and Tsunoda (9) reported that the pronuclei of mouse zygotes were apparently damaged
498
NAKAMUKA
AND TSUNODA
TABLE 4 Contributions of Nuclei Preserved at 4°C to Germ Cell Population of Chimeras Duration of preservation of donor nuclei of 4°C (h) 0
No. of chimeras
No. of fertile chimeras
4
3
Sex of chimeras Male Female
Germ cell population type of chimeras (%)” AL
AL/CO
1 0
0 0
2 0
0
2 (67) 2 0
I (33) 48
7
6
Male Female
3 0
0 1
3
1 (67)
72
3
Male Female
1 0
0 1
1 5
Male Female
2 1
0 0
3 3
Male Female
1 0
0 1
1
1
(67) 144
2
Male Female
0 0
0 0
0
0
(0) 168
1
Male Female
(3:) I I
0 (W
120
(3:) 0 1
1 (67)
96
co
0 0
0 0
0
(4:) 1 0 1 (33) 2 0 2 (low 1 0
0 (0)
192
1
Male Female
1 0 1 (100)
0 0
0 0
0
0 (0)
Note. Numbers in parentheses are percentages. AL: albino type, AL/CO: chimeric type, CO: colored type. D Based on number of fertile chimeras.
more than the cytoplasm by the vitrification-warming cycle and by the toxicity of VSl solution. However, the present study indicated that the nuclei in the two-cell mouse embryos were more resistant than the cytoplasm to the low temperature;
these results being similar to previously reported results for mouse zygotes (16). This technique may be useful for short-term storage for embryo manipulation procedures where all combinations cannot be performed on the same day.
VIABILITY
OF NUCLEI OF MOUSE EMBRYOS
ACKNOWLEDGMENT We thank Dr. Y. Okada, Cellular Biology, and Osaka University for the gift of HVJ for cell fusion and Dr. D. G. Whittingham, M. R. C. Experimental Embryology and Teratology Unit, and Dr. H. Imai, National Institute of Animal Industry, for critical and helpful evaluation of the manuscript. REFERENCES I. Fulton, B. P., and Whittingham, D. G. Activation of mammalian oocyte by intracellular injection of calcium. Nature (London) 273, 149-151 (1978). 2. Hafez, E. S. E. Storage of fertilized ova. In?. J. Fertil. 8, 459-466 (1963). 3. Hafez, E. S. E. Storage media for rabbit ova. J. Appl. Physiol. 20, 731-736 (1965). 4. Hafez, E. S. E. Egg storage. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., Ed.), pp. 117-132. Freeman, San Francisco, 1971. 5. Herr, C. M., and Wright, R. W., Jr. Cold storage of mouse embryos of different stages of development. Theriogenology 29, 765-770 (1988). 6. Hughes, M. A., and Anderson, G. B. Short-term storage of rabbit embryos at 4°C. Theriogenology 18, 275-282 (1982). 7. Kasai, M., Niwa, K., and Iritani, A. Protective effect of sucrose on the survival of mouse and rat embryos stored at 0°C. J. Reprod. Ferril. 68, 377-380 (1983). 8. Kasai, M. Nonfreezing technique for short-term storage of mouse embryos. J. In Vitro Fertil. Embryo Transf. 3, l&14 (1986). 9. Kono, T.. and Tsunoda, Y. Ovicidal effects of vitrification solution and vitrification-warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse zygotes. Cryobiology 25, 197-202 (1988). 10. Linder, G. M., Anderson, G. B., Bon Durant, R. H., and Cupps, P. T. Survival of bovine embryos stored at 4°C. Theriogenology 20, 311320 (1983). 11. Mann, J. R. DDK egg-foreign sperm incompatibility in mice is not between the pronuclei. J. Reprod. Ferril. 76, 779-781 (1986).
12. McGrath, J., and Solter, D. Nuclear transplanta-
499
tion in the mouse embryo by microsurgery and ceilfusion. Science 220, 1300-1303 (1983). 13. McGrath, J., and Solter, D. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 226, 1317-1319(1984). 14. McLaren, A. “Mammalian Chimeras.” Cambridge Univ. Press, London, 1976. 15. Moore, N. W., and Bilton, R. J. The storage of fertilized sheep ova at 5°C. Amt. J. Biol. Sci. 26, 1421-1427 (1973). 16. Nakamura, K., and Tsunoda, Y. Investigation on the low temperature resistance of mouse pronuclei by using nuclear transplantation technique. Jpn. J. Anim. Reprod. 32, 134-137 (1986). 17. Nakamura, K., and Tsunoda, Y. An analysis of in vitro 2-cellblock by using pronuclear transplantation technique. Jpn. J. Anim. Reprod. 33, 8287 (1987). 18. Nakamura, K., and Tsunoda, Y. Chimeras obtained by the nuclear transplantation technique in the mouse. Jpn. J. Anim. Reprod. 33, 15-18 (1987). 19. Trounson, A. 0.. Willadsen, S. M., and Rowson, L. E. A. The influence of in vitro culture and cooling on the survival and development of cow embryos. J. Reprod. Fertil. 47, 367-370 (1976). 20. Tsunoda, Y., Yasui, T., Nakamura, K., Uchida, T., and Sugie, T. Effects of cutting zona pellucida on the pronuclear transplantation in the mouse. J. Exp. Zoo/. 240, 119-125 (1986). 21. Whittingham, D. G., and Wales, R. G. Storage of two-cell mouse embryos in vitro. Aust. J. Biol. Sci. 22, 1065-1068 (1969). 22. Whittingham, D. G. Culture of mouse ova. J. Reprod. Ferril. 14, 7-21 (1971). 23. Whittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to - 196°C and -269°C. Science 187, 411414 (1972). 24. Wilmut, I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11, 1071-1079 (1972). 25. Wilmut, I., Polge, C., and Rowson, L. E. A. The effect on cow embryos of cooling to 20, 0, and - 196°C.J. Reprod. Fertil. 45, 409-114 (1975).
CRYOBIOLOGY
Viability
(1992)
of Nuclei of Two-Cell Fused with Blastomeres
Mouse Embryos Stored at 4°C and of Fresh Two-Cell Embryos
K. NAKAMURA”
AND
Y. TSUNODAt
*Sufety Evaluation Laboratory of Kaken Pharmaceutical Co., Ltd., 301 Gensuke Fujieda, Shizuoka 426 Japan: and fFaculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631 Japan This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0°C. Two-cell embryos were stored at 4’C for 24-96 h in PBl containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4°C fresh Fl was significantly prolonged when they wcrc transplanted into a blastomere of enucleated (C57BLI6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88,67,76,71,64,45, 32, and 20% following storage for 0,48,72,96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4°C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4°C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm
The preservation of mammalian oocytes and embryos for varying periods of time has practical applications in many areas of biology, e.g., in animal husbandry, cryobiology, genetics, toxicology, and mutagenesis. The technique of frozen storage (longterm storage) for mammalian embryos has become established since a successful freezing method was reported for the mouse (23,24). Today live young have been obtained from frozen-thawed embryos of many mammalian species, including human beings. However, few researchers have conducted their studies on embryos at temperatures of 0 to 10°C (short-term storage). Limited development of preimplantation embryos from mice (5, 21), rabbits (2-4, 6), sheep (15), and cattle (8, 19, 25) has been reported following short-term storage. The
Received 1992
September 23, 1991; accepted January 13,
resistance of embryos to low temperature varies according to their developmental stages. Generally embryos at earlier developmental stages are less resistant to cold storage. A successful and reliable procedure for pronuclear transplantation has been developed for mouse zygotes (12). With this technique, it has been possible to examine the genetic and cytoplasmic factors contributing to embryonic development (11, 13, 17). Short-term low-temperature storage without freezing could be a helpful approach to the various manipulations currently in use in livestock breeding, especially in cattle, sheep, and goats. In a previous study, we investigated the resistance of mouse pronuclei to cold storage (16). In order to determine whether it is the nucleus or the cytoplasm that is more damaged during cold storage, we examined the viability of the nuclei of two-cell mouse embryos stored at 4°C.
493 001 l-2240192 Copyright
$5.00
0 1992 hy Academic
Press, Inc.
494
NAKAMURA MATERIALS
Experimental
AND METHODS
Design
Experiment 1. The first part of the study was designed to examine the effects of various sucrose concentrations and the duration of storage on the survival of freshly collected two-cell mouse embryos. Two-cell embryos were stored at 4°C for up to 96 h and then cultured at 37°C. Survival was assessed by determining their ability to develop into blastocysts after 3 days cultivation. Experiment 2. In the second part of the study, we examined the viability of nuclei from two-cell embryos after cold storage. CD-I two-cell embryos were stored at 4°C for up to 192 h in PBI containing 0.5 M sucrose. Individual nuclei were then transplanted to one enucleated blastomere of a fresh Fl (C57BL16JXCBA) two-cell embryos. The viability of reconstituted twocell embryos was examined by both in vitro culture and embryo transfer.
Source of Embryos CD-I female mice (5 weeks of age) and Fl female mice (5-8 weeks of age) were superovulated by intraperitoneal injections of 5 IU pregnant mare’s serum gonadotropin (PMSG, Teikoku Zoki Co. Ltd., Japan) and 5 IU human chorionic gonadotropin (hCG, Teikoku Zoki Co. Ltd., Japan) given 48 h apart. The mice were mated with males of same strain. Forty-two to 44 h after the hCG injection, two-cell embryos were obtained by flushing the oviducts with M2 medium (I) supplemented with 0.4% bovine serum albumin (BSA, Sigma A-4378). These two-cell embryos were then washed three times with M2 medium supplemented with 0.4% BSA. The coat color phenotypes used in this study were white (CD-l) and agouti (Fl).
Cold storage Two-cell CD-I embryos were pipetted into I ml of PBI medium (21) containing 0,0.25,0.5,0.75, or I .OM sucrose, in sterile polystyrene test tubes (Coming). The caps of the tubes were sealed with vinyl tape and the tubes were then placed in a glass beaker filled with water (20°C). The beaker was placed in a refrigerator at 4°C for periods varying from 24 to 96 h in Exp. 1 and 48 to 192 hr in Exp. 2.
Nuclear
Transplantation
Nuclear transplantation was carried out as described previously (18, 20). An area of approximately lO-20% of the zona pellucida in the two-cell embryos was cut with a tine glass needle to make manipulation procedure easy (20). Manipulations were carried out in 0.2 ml of M2 medium overlaid with parafIin oil. The two-cell embryos were then transferred to a micromanipulation glass slide in a drop of M2 medium contain-
AND TSUNODA ing cytochalasin B (5 &mg, Sigma) and Colcemid (0.1 kg/ml, Aldrich) overlaid with paraffin oil. After I5 min. a single nucleus was removed from recipient FI two-cell embryos, and a karyoplast containing the nucleus of a CD-l two-cell embryo which had been stored at 4°C for between 48 and 192 h was inserted, together with inactivated Sendai virus (HVJ, 2,500 hemagglutinating activity units/ml), into the perivitelline space next to the enucleated blastomere of the recipient Fl two-cell embryos.
Culture and Transfer of Embryos After storage, embryos were allowed to equilibrate for 10 min at room temperature. After recovery or nuclear transplantation, embryos were then washed three times with M2 and Ml6 medium (22) and cultured at 37°C for 3 days in microdrops of Ml6 medium supplemented with 0.4% BSA under paraffin oil, in atmosphere of 5% CO, in air. The viability of embryos was defined as the ability to develop to blastocysts in culture at 37°C for 3 days. After 3 days cultivation, developed embryos were classified as normal and incomplete blastocysts. A normal blastocyst was defined as one in which both blastomeres had divided synchronously to form a blastocyst. An incomplete blastocyst was one in which only a single blastomere had formed a blastocyst, and in which the other blastomere had not divided. In Exp. 2, the reconstituted embryos which developed to blastocysts after 3 days in culture were transferred into the uterine horns of Day 3 pseudopregnant CD-l recipients.
Evaluation of the Viability Cold-Stored Nuclei
of
The recipient mice were allowed to litter and the young were sexed and examined for their eye and coat color after they had grown to weaning. The viability of stored nuclei was evaluated by the coat color of the young. Thus, albino (white) and chimeric young were considered to express the phenotype of the stored nuclei. Chimeric progeny were raised to sexual maturity and then mated with albino mice (CD-l) in order to determine whether the nuclei from the cold stored two-cell embryos were able to contribute to the germ cell population. The pregnant mice were sacrificed on Day 18of gestation and live fetuses were examined for eye color. The germ cell population type was classified as albino, chimeric (mixed type of albino and colored), and colored types according to the eye color of the fetuses. The albino and the chimeric types in their germ cell population were considered to express the phenotype of the stored nuclei.
Statistical
Analysis
The results were examined by x2 test with Yates correction.
VIABILITY
OF NUCLEl
495
OF MOUSE EMBRYOS
creased compared with the proportion containing the nuclei of fresh embryos.
RESULTS
Experiment 1: In Vitro Viability of Two-Cell Embryos after Cold Storage As shown in Table 1, the viability of twocell embryos developed to blastocysts was quite low after 24 and 48 h of storage in sucrose-free PBl medium (14 and 5%, respectively). In the same sample containing 0.5 and 0.75 M sucrose, however, survival times were longer; the highest survival rates were obtained with 0.5 M sucrose when embryos were examined after 24, 48, 72, and 96 h of storage (51, 42, 9, and 3%, respectively). Experiment 2: In Vitro Viability of Reconstituted Chimeric Two-Ceil Embryos As shown in Table 2, degeneration of embryos increased after 120 h of storage. The proportions of chimeric embryos that developed to normal blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage of two-cell embryos for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. After more than 120 h of storage, the proportions of chimeric embryos that developed to incomplete blastocysts were significantly in-
Evaluation of Viability of Cold-Stored Nuclei after Embryo Transfer The results in Table 3 show that the proportions of transferred blastocysts that developed into live young were 43, 60,29, 37, 37, 39, 25, and 38% following storage for 0, 48,72,96, 120, 144, 168, and 192 h, respectively. The numbers of young that died before weaning were 2, 4, and 1 following storage for 0, 120, and 168 h, respectively. The rates at which stored nuclei contributed to the coat color of the young (white and chimera) were 85, 73, 100, 50, 67, 50, 67, and 67% following storage for 0,48, 72, 96, 120, 144, 168, 192 h, respectively. It is known that the sex of chimeras tends to be male (14), since XX/XY chimera exhibit a male phenotype. In the present study, 20 of 27 (74%) of chimeric young were male. As shown in Table 4, contributions to the germ cell population of the chimeric young from stored nuclei were 33, 67, 67, 60, 67, 0, 0 and 100%following storage for 0,48, 72,96, 120, 144, 168, and 192 h, respectively. One male chimera derived from nuclei stored for
TABLE 1 In Vitro Development of Two-Cell Mouse Embryos to the Blastocyst Stage after Storage for up to 96 h at
4°C in PB 1 Medium Containing Various Concentrations of Sucrose No. blastocysts developed/No. embryos cultured (%) Duration of storage (h)
Sucrose cont.
CM) 0
0
24
48
72
96
77190
6143 (14) 6147 (13) 23/45* (51) 30/46* (65) 4131
2137 (5) 6136 (17) 15/36* 9136* (25) o/12
o/34 (0) 0134 (0) 3133 (9) 2128 (7) O/II
0129 (0) O/28 (0) l/29 (3) 0129 (0) -
(13)
(0)
(0)
(86) 0.25
-
0.5 0.75
-
1.0
-
(42)
Note. Numbers in parentheses are percentages. *Significantly different from sucrose-free PBI (x2 test) (P < 0.001).
496
NAKAMUKA
AND TSUNODA
TABLE 2 In Vitro Development of Chimeric Two-Cell Embryos Produced by Allogeneic Nuclear Transfer of Embryos Preserved at 4°C in PBl Containing 0.5 M Sucrose into One Blastomere of a Fresh Embryo Duration of preservation of donor nuclei at 4°C (h) 0 48 72 96 120
No. of embryos degenerated/ No. of embryos preserved m O/55 (0) o/50 (0) o/59 (0) 4153
(8) 144 168 192
7187 (9) lo/78 (13) 27174 (37)
No. of embryos whose karyoplast fused/manipulated (%I 56162 (90) 52155 (95) 48/50
No. of embryos developed to No. of embryos” cultured 51 52
35*
(67) 46
35
(76) 55
(98) 47149 (96) 76/80 (95) 65168 (96) 43147 (92)
45
(88)
(96) 58159
Blastocyst (%I’
44 65 60 41
39* (71) 28** (64) 29*** (45) 19*** (32)
8WW (20)
Incompleteb blastocyst (%)’
Other (%I 2 (4)
A (1:) (1G 4 (9) 10
(I:,
(18)
(1;)
,:t; 17*
(1:) 19
(26)
(2%
33** (55) 28***
8 (13) 5
(68)
(12)
0 The embryos which fused both blastomeres were not cultured. b Single blastomere developed to blastocyst. ’ Based on number of embryos cultured. *,****** Significantly different from fresh embryos (x2 test) (*P < 0.05, **P < 0.01, ***P < 0.001).
cysts after 96 h of storage, whereas the viability of the nuclei of two-cell embryos was significantly extended when they were transplanted into a blastomere of enucleDISCUSSION ated fresh Fl two-cell embryos. SeventyFew attempts have been made to pre- one percent of the reconstituted two-cell serve mammalian embryos at low tempera- embryos containing nuclei stored at 4°C for tures above 0°C. Kasai et al. (7) have re- 96 h developed into blastocysts (Table 2). ported that the protective effects of sucrose These findings suggested that the viability on the storage of mouse and rat morulae at of nuclei was maintained longer than that of 0°C for extended times. Furthermore, in embryos during cold storage. From the reKasai’s later work (8), he reported that sults in Table 3, it can be seen that the rates mouse morulae could be stored for as long of stored nuclei contributions to the coat as 120 h at 0°C in various sugar solutions. In color of the young were comparable to the Exp. 1, similar protective effects of sucrose contribution rates of fresh nuclei. Furtherwere obtained in the cold storage of two- more, the viability of nuclei stored at 4°C cell mouse embryos. That is, the viability of for 192 h was confirmed in the germ cell two-cell embryos stored at 4°C was much population of chimeric young (Table 4). improved when 0.5 M sucrose was added to Why the viability of embryos decreases PBl medium (Table 1). However, only 3% during cold storage is not known. Herr and of these embryos developed into blasto- Wright (5) suggested that early embryos
192 h showed the albino type in its germ cell population.
VIABILITY
497
OF NUCLEI OF MOUSE EMBRYOS
TABLE 3 Development of Reconstituted Embryos Transferred to Recipient Mice and Coat Color of the Young at Weaning Young at weaning Duration of preservation of donor nuclei at 4°C (h) 0
No. pregnant/ No. recipient (%)
No. live young/ No. blastocysts transferred (%)
414(100)
15/35(43)
Coat color (%)b Sex
No. young
AL
CH
Male Female
8 5
2 5
4 0
2. 0
13
7
4
2 (15) 2 2
(85) 48
3/3(100)
15/25(60)
Male Female
9 6
2 2
I5
4
5 2 7
(2;)
(73) 72
3/3(100)
l/24(29)
Male Female
3 4
1 2
7
3
2 2
0 0
4
0 (0) 4 1
(1W 96
3/3(100)
10/27(37)
Male Female
7 3
0 0
10
0
3 2 5 (50)
120
3/3(100)
10/27(37)
Male Female
2 4
0 1
6
I
2 1 3 (67)
144
2/3(67)
10/26(39)
Male Female
8 2
2 I
10
3
2 0 2 (50)
168
2/2(100)
4/16(25)
Male Female
2 1
0 1
3
I
1 0
l/2(50)
3/8(38)
Male Female
I 2
0 1
3
1
5 (50) 0 2 2 (33) 4 I 5 (50) I 0
1 (67)
192
co
1 0
(3:) 0 I
I (67)
(3:)
Note. Numbers in parentheses are percentages. AL, albino; CH, chimera; CO, colored a Based on number of embryos transferred to recipients. b Based on number of live young at weaning.
may be more sensitive to undesirable components such as heavy metal contamination or, alternatively, that an absence of critical substances in synthetic media may be a fac-
tor involved in the reduced survival observed during cold storage. Recently, Kono and Tsunoda (9) reported that the pronuclei of mouse zygotes were apparently damaged
498
NAKAMUKA
AND TSUNODA
TABLE 4 Contributions of Nuclei Preserved at 4°C to Germ Cell Population of Chimeras Duration of preservation of donor nuclei of 4°C (h) 0
No. of chimeras
No. of fertile chimeras
4
3
Sex of chimeras Male Female
Germ cell population type of chimeras (%)” AL
AL/CO
1 0
0 0
2 0
0
2 (67) 2 0
I (33) 48
7
6
Male Female
3 0
0 1
3
1 (67)
72
3
Male Female
1 0
0 1
1 5
Male Female
2 1
0 0
3 3
Male Female
1 0
0 1
1
1
(67) 144
2
Male Female
0 0
0 0
0
0
(0) 168
1
Male Female
(3:) I I
0 (W
120
(3:) 0 1
1 (67)
96
co
0 0
0 0
0
(4:) 1 0 1 (33) 2 0 2 (low 1 0
0 (0)
192
1
Male Female
1 0 1 (100)
0 0
0 0
0
0 (0)
Note. Numbers in parentheses are percentages. AL: albino type, AL/CO: chimeric type, CO: colored type. D Based on number of fertile chimeras.
more than the cytoplasm by the vitrification-warming cycle and by the toxicity of VSl solution. However, the present study indicated that the nuclei in the two-cell mouse embryos were more resistant than the cytoplasm to the low temperature;
these results being similar to previously reported results for mouse zygotes (16). This technique may be useful for short-term storage for embryo manipulation procedures where all combinations cannot be performed on the same day.
VIABILITY
OF NUCLEI OF MOUSE EMBRYOS
ACKNOWLEDGMENT We thank Dr. Y. Okada, Cellular Biology, and Osaka University for the gift of HVJ for cell fusion and Dr. D. G. Whittingham, M. R. C. Experimental Embryology and Teratology Unit, and Dr. H. Imai, National Institute of Animal Industry, for critical and helpful evaluation of the manuscript. REFERENCES I. Fulton, B. P., and Whittingham, D. G. Activation of mammalian oocyte by intracellular injection of calcium. Nature (London) 273, 149-151 (1978). 2. Hafez, E. S. E. Storage of fertilized ova. In?. J. Fertil. 8, 459-466 (1963). 3. Hafez, E. S. E. Storage media for rabbit ova. J. Appl. Physiol. 20, 731-736 (1965). 4. Hafez, E. S. E. Egg storage. In “Methods in Mammalian Embryology” (J. C. Daniel, Jr., Ed.), pp. 117-132. Freeman, San Francisco, 1971. 5. Herr, C. M., and Wright, R. W., Jr. Cold storage of mouse embryos of different stages of development. Theriogenology 29, 765-770 (1988). 6. Hughes, M. A., and Anderson, G. B. Short-term storage of rabbit embryos at 4°C. Theriogenology 18, 275-282 (1982). 7. Kasai, M., Niwa, K., and Iritani, A. Protective effect of sucrose on the survival of mouse and rat embryos stored at 0°C. J. Reprod. Ferril. 68, 377-380 (1983). 8. Kasai, M. Nonfreezing technique for short-term storage of mouse embryos. J. In Vitro Fertil. Embryo Transf. 3, l&14 (1986). 9. Kono, T.. and Tsunoda, Y. Ovicidal effects of vitrification solution and vitrification-warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse zygotes. Cryobiology 25, 197-202 (1988). 10. Linder, G. M., Anderson, G. B., Bon Durant, R. H., and Cupps, P. T. Survival of bovine embryos stored at 4°C. Theriogenology 20, 311320 (1983). 11. Mann, J. R. DDK egg-foreign sperm incompatibility in mice is not between the pronuclei. J. Reprod. Ferril. 76, 779-781 (1986).
12. McGrath, J., and Solter, D. Nuclear transplanta-
499
tion in the mouse embryo by microsurgery and ceilfusion. Science 220, 1300-1303 (1983). 13. McGrath, J., and Solter, D. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 226, 1317-1319(1984). 14. McLaren, A. “Mammalian Chimeras.” Cambridge Univ. Press, London, 1976. 15. Moore, N. W., and Bilton, R. J. The storage of fertilized sheep ova at 5°C. Amt. J. Biol. Sci. 26, 1421-1427 (1973). 16. Nakamura, K., and Tsunoda, Y. Investigation on the low temperature resistance of mouse pronuclei by using nuclear transplantation technique. Jpn. J. Anim. Reprod. 32, 134-137 (1986). 17. Nakamura, K., and Tsunoda, Y. An analysis of in vitro 2-cellblock by using pronuclear transplantation technique. Jpn. J. Anim. Reprod. 33, 8287 (1987). 18. Nakamura, K., and Tsunoda, Y. Chimeras obtained by the nuclear transplantation technique in the mouse. Jpn. J. Anim. Reprod. 33, 15-18 (1987). 19. Trounson, A. 0.. Willadsen, S. M., and Rowson, L. E. A. The influence of in vitro culture and cooling on the survival and development of cow embryos. J. Reprod. Fertil. 47, 367-370 (1976). 20. Tsunoda, Y., Yasui, T., Nakamura, K., Uchida, T., and Sugie, T. Effects of cutting zona pellucida on the pronuclear transplantation in the mouse. J. Exp. Zoo/. 240, 119-125 (1986). 21. Whittingham, D. G., and Wales, R. G. Storage of two-cell mouse embryos in vitro. Aust. J. Biol. Sci. 22, 1065-1068 (1969). 22. Whittingham, D. G. Culture of mouse ova. J. Reprod. Ferril. 14, 7-21 (1971). 23. Whittingham, D. G., Leibo, S. P., and Mazur, P. Survival of mouse embryos frozen to - 196°C and -269°C. Science 187, 411414 (1972). 24. Wilmut, I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 11, 1071-1079 (1972). 25. Wilmut, I., Polge, C., and Rowson, L. E. A. The effect on cow embryos of cooling to 20, 0, and - 196°C.J. Reprod. Fertil. 45, 409-114 (1975).
