48 (3) : , 2002

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1 48 (3) : , 2002 A cta Zoologica S inica 3 33 (, ) ( Wiss, ) G1 S G2, M,, G % ( 322/ 369) 8419 % ( 299/ 352) 7115 % ( 193/ 270) 5810 % (76/ 131) 3511 % (46/ 131) , S [ 7916 % ( 109/ 137) 7414 % ( 93/ 125) 3013 % (33/ 109) 1913 % (17/ 88) 618 % (6/ 88) ] G2 [6316 % (70/ 110) 60 % (60/ 100) 1619 % (11/ 65) 1613 % (7/ 43) 417 % (2/ 43) ] [ 7811 % (185/ 237) 7312 % (158/ 216) 4914 % (43/ 87) 3212 % (28/ 87) 1216 % (11/ 87) ] ( P < 0105) G , 6 19, 1312 %, S (518 %, 7/ 120) G2 (0, 0/ 126) (513 %, 8/ 150) ( P < 0105) 80,,, PCC DNA, ( Willadsen, 1986),, ( Tsunoda et al., 1987 ) ( Prather et al., 1987) ( Stice et al., 1988) ( Yong et al., 1991) ( Prather et al., 1989), MPF, (Meng et al., 1997) ; ( Wilmut et al., 1997) ( Cibelli et al., 1998) ( Wakayama et al., 1998) DNA ( G1 ), (Baguist et al., 1999) ( Onishi et al., 2000), ) G2 (DNA ), M,,, M M ( MPF ), MPF (Collas et al., 1992) PCC,, ( G0 ),, G1 S (DNA 1 (N EBD),, 111 M ( PCC) N EBD PCC kg , Wiss ,,

2 3 : 369, 98,, 6 FSH (,, ), 12 h,,, mg, 019 mg m, FSH 12 h,, hcg ( ) 100 IU hcg h,, 013 % BSA (Sigma) DPBS ( GIBCO), - ( COCs) COCs G1 G2 011 % ( Sigma) Ca 2 + Mg 2 + PBS, min, HM199, 38 10, 30 min,, DPBS , mm (2001) hcg (1998), 0129, hcg 48 h, mol/ L - 16 RM min, [ M199 ( GIBCO) + 10 %FBS (, (10 V, 1 MHz) 5 10 s ) mmol/ L ( GIBCO) mmol/ L ED TA (Sigma) g/ ml, 3 ( ) 0150 g/ ml ( 210 kv/ cm, 40 s ) ] 12 h 32, 5 min G g/ ml RM199 RM h, 012 g/ ml RM199 8 h, S g/ ml h,, RM h, 24 h RM199 6 h G g/ ml 10 g/ ml Hoechst33342 RM h, RM199 8 h, 1 g/ ml RM199 8 h, 015 % (Boehringer) mpbs min, Ca 2 + Mg 2 + PBS, DPBS g/ ml 6 8 B ( Sigma) HM199 ( M mmol/ L Hepes), min, ( M ),, - ( CE2450, Kefa, China) -,, 7 8 h (Sigma) DPBS, min, DPBS ( 10 l) ( Olympus), 2 4, 1 d,

3 Table 1 Effects of cell cycle phases of rabbit donor blastomeres on the cell fusion of oocyte2blastomere couplets, the activation of reconstituted embryos and the development of rabbit nuclear transfer embryos in vitro Donor nuclei ( n) 1) Replicates Fused Activated Cleaved Morulae Blastocysts Hatched Number of B. cell ( %) ( %) ( %) ( %) ( %) ( %) (Means + SD) 2) 2) 2) G1 ( G1 blastomere) a (352/ 369) 8713 a (322/ 369) 8419 a (299/ 352) 7115 a (193/ 270) 5810 a (76/ 131) 3511 a (46/ 131) a (15) S (S blastomere) a (125/ 137) 7916 b (109/ 137) 7414 b (93/ 125) 3013 b (33/ 109) 1913 b (17/ 88) 618 b (6/ 88) b (13) G2 ( G2 blastomere) a (100/ 110) 6316 ab (70/ 110) 6010 ab (60/ 100) 1619 c (11/ 65) 1613 b (7/ 43) 417 b (2/ 43) ab (13) (Not synchronized blastomere) a (216/ 237) 7811 b (185/ 237) 7312 b (158/ 216) 4914 ab (43/ 87) 3212 b (28/ 87) 1216 b (11/ 87) b (14), ( P < 0105) [ One2Way ANOVA, different superscripts in the same column represent significant difference ( P < 0105) ] 1) (Based on the fused embryos) 2) (Based on the activated embryos) SAS ( SAS Instit ute Inc1, 6112 ) ( P < 0105),, ( P > 0105),, (DUNCAN), S ( P > 0105) G1 K2S, ( P < 0105), S,, ( P > 0105),, G G1 S G M, 2, 2 G1 1 1 ( P < 0105), ( G1 S G2) S, G1 ( P < 0105), G2 ( P < 0105), G2 ( P < 0105) G1 S ( P < 0105) 212 G1 S G2 3 ( G1 S G2) ( P < 0105) G2 ( G1 S G2)

4 3 : Table 2 In vivo development of nuclear transfer rabbit embryos obtained by using blastomeres at different cell cycle phases as donors Donor nuclei Replicates Number of embryos Number of recipients Number of pregnancy ( %) Number of born ( %) G1 ( G1 blastomere) (1312 a ) S (S blastomere) (518 b ) G2 ( G2 blastomere) (0 c ) (Not2synchronized blastomere) (513 b ), ( P < 0105) [ One2Way ANOVA, different superscripts in the same column represent significant difference ( P < 0105) ],, G1 M, (Collas et al.,, G1 1992),, G2, S DNA G2 MPF 4,,, ( Campbell et al., 1993),,, G2 S G1,, DNA S G2, MPF, ( Otaegui et al., DNA,, 1994 ) ( Campbell et al., 1993 ) DNA, ( Rao et al,. ( Takano et al., 1996) ( Campbell et al., 1977), MPF, 1994) DNA, M, (Blow et al., 1988) MPF, DNA, (Collas et al., 1992), G1 S G1 DNA,, (Stumpf et al., 1992), MPF,,,

5 ( References) Baguist, A., E. Behboodi, D. Melican, J. S. Pollock, M. M. Destrempes, C. Cammuso, J. L. Williams, S. D. Nims, C. A. Porter, P. Midura, M. J. Palacios, S. L. Ayres, R. S. Denniston, M. L. Hayes, C. A. Ziomek, H. M. Meade, R. A. Godke, W. G. Gavin, E. W. Overstrom and Y. Echelard 1999 Production of goats by somatic cell nuclear transfer. N at ure Biotech. 17 : Blow, J. J. and R. A. Laskey 1988 A role for the nuclear envelope in controlling DNA replication within the cell cycle. N at ure 332 : Campbell, K. H. S., P. Loi, P. Cappai and I. Wilmut 1994 Improved development to blastocysts of ovine nuclear transfer embryos reconstructed during the presumptive S2phase of enucleated activated oocytes. Biol. Reprod. 50 : Campbell, K. H. S., W. A. Ritchie and I. Wilmut 1993 Nuclear2cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos : implication for deoxyribonucleic acid replication and development. Biol. Reprod. 49 : Cibelli, J. B., S. L. Stice, P. J. Golueke, J. J. Kane, J. Jerry, C. Blackwell, Ponce de Leon FA. and J. M. Robl 1998 Cloned transgenic calves produced from non2quiescent fetal fibroblasts. Science 280 : Collas, P., C. Pinto2Correia, Ponce de Leon F. A. and J. M. Robl 1992 Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos. Biol. Reprod. 46 : Liu, L., Z. C. Zhang and Y. S. Wang 1998 Electrical activation of i n vit ro matured ( IVM) ovine oocytes and their parthenogenetic development i n vit ro. Acta Zool. Si n.. 44 (1) : [,, (1) : ] Meng, L., J. J. Ely, R. L. Stouffer and D. P. Wolf 1997 Rhesus monkeys produced by nuclear transfer. Biol. Reprod. 57 : Onishi, A., M. Iwamoto, T. Akita, S. Mikawa, K. Takeda, T. Awata, H. Hanada and A. C. Perry 2000 Pig cloning by microinjection of fetal fibroblast nuclei. Science 289 : Otaegui, P. J., G. T. OπNeill, K. H. S. Campbell and I. Wilmut 1994 Transfer of nuclei from 82cell stage mouse embryos following use of nocodazole to control the cell cycle. Mol. Reprod. Dev. 39 : Prather, R. S., F. L. Barnes, M. M. Sims, J. M. Robl, W. H. Eyestone and N. L. First 1987 Nuclear transplantation in the bovine embryos : assessment of donor nuclei and recipient oocyte. Biol. Reprod. 37 : Prather, R. S., M. M. Sims and N. L. First 1989 Nuclear transplantation in early pig embryos. Biol. Reprod. 41 : Rao, P. N., B. A. Wilson and T. T. Puck 1977 Premature chromosome condensation for the cell cycle analysis. J. Cell. Physiol. 91 : Stice, S. L. and J. M. Robl 1988 Nuclear reprogramming in nuclear transplant rabbit embryos. Biol. Reprod. 39 : Stumpf, T. T., S. L. Terlouw, H. Funahashi and R. S. Prather 1993 Premature chromosomal condensation of nuclei transplanted into i n vit ro matured porcine oocytes. Biol. Reprod. 48 ( S upp. ) : 175. Takano, H., K. Kayama, C. Kozai, S. Shimizu, Y. Kato and Y. Tsunoda 1996 Effects of cell cycle stage of donor nuclei on the development of bovine nuclear transferred embryos. J. Reprod. Dev. 42 : Tsunoda, Y., T. Yasui, Y. Shioda, K. Nakamura, T. Uchida and T. Sugie 1987 Full2term development of mouse blastomere nuclei transplanted into enucleated two2cell embryos. J. Ex p. Zool. 242 (2) : Wakayama, T., A. C. F. Perry, M. Zuccotti, K. R. Johnson and R. Yanagimachi 1998 Full2term development of mice from enucleated oocytes injected with cumulus cell nuclei. N at ure 394 : Wang, Y. S., S. M. Zeng, S. E. Zhu, J. H. Tian, S. J. Li, W. L. Yu, Z. C. Zhang and Y. F. Chen 2001 The control of rabbit embryonic cell cycle. Acta Veteri naria et Zootechnica Si nica 32 (6) : [,,,,,,, (6) : ] Willadsen, S. M Nuclear transplantation in sheep embryos. N at ure 320 : Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind and K. H. S. Campbell 1997 Viable offspring derived from fetal and adult mammalian cells. N at ure 385 : Yong, Z., W. Jianchen, Q. J ufen and H. Zhiming 1991 Nuclear transplantation in goats. Theriogenology 35 : 299.

6 3 : 373 ( Abstract) EFFECTS OF THE DONOR BLASTOMERE CELL CYCL E STAGES ON THE D EVELOPMENT OF RABBIT NUCL EAR TRANSPLANTATION EMBRYOS 3 WAN G Yong2Sheng ZEN G Shen2Ming 33 ZHU Shi2En TIAN Jian2Hui YU Wen2Li ZHAN G Zhong2Cheng CHEN Yong2Fu SHAN G Jia2Ji WEI Jian2Sheng ( College of A ni mal Science and Technology, Chi na A gricult ural U niversity, Beiji ng , Chi na) ( Hongkong Wiss Biotechnology Center, Chi na) We studied the effects of donor cell cycle stages on the development of rabbit nuclear transfer embryos. The donor nuclei were f rom early rabbit embryos t hat were t reated wit h chemicals to synchronize blastomeres at different cell cycle stages. 162cell rabbit embryos were recovered f rom t he oviduct s of New Zealand white rabbit does administ rated wit h FSH and hc G for superovulation. The embryos were cult ured to 322cell stage in TCM199 with Earleπs salt supplemented with 10 % FBS, 1125 mmol/ L Na pyruvate, 011 mmol/ L ED TA, 0175 g/ L penicillin and 0150 g/ L streptomycin (RM199). The embryos were treated with 015 % pronase to separate blastomeres. The blastomeres wit hout chemical t reat ment were used as non2synchronized donor cells. The cells at G1 stage were isolated from the 162cell embryos that were cultured first in RM199 containing 1100 g/ ml nocodazole for 12 h, and then in RM199 containing 0120 g/ ml aphidicolin for 8 h. The blastomeres at S stage were isolated from the 162cell embryos that were cultured first in RM199 containing 1100 g/ ml nocodazole for 12 h, and then in RM199 for 6 h. The blastomeres at G2 phase were isolated from the 162cell embryos that were cultured first in RM199 containing 1100 g/ ml nocodazole for 12 h, then in RM199 for 8 h, and finally in 1100 g/ ml cycloheximide for 8 h. The M oocytes were recovered from superovulated New Zealand white rabbit does at h after hc G injection and mechanically enucleated as recipient cytoplasm. The G1, S, G2 stage or non2synchronized blastomeres were t ransferred into t he enucleated oocytes, respectively. The oocyte2blastomere couplet s were elect rically f used and activated using t hree DC pulses of 210 kv/ cm at 5 min intervals after AC (10V, 1MHz) for 5210 s in 0129 mol/ L mannitol solution containing 011 mmol/ L CaCl 2, 011 mmol/ L MgSO 4 and 10 mmol/ L histidine (p H = 710). The reconstituted embryos were transferred into RM199 and cultured in humidified air with 5 % CO 2 at 38 to observe the in vit ro develop mental potentials of nuclear t ransplantation embryos. The f usion rates were 9514 %, 9112 %, 9019 % and 9111 % in G1, S, G2 and non2synchronized group, respectively. There was no statistical difference among different groups ( P > 0105). The result implied t hat cell f usion did not have relationship to t he cell cycle stage of t he two cells. However, t he cell cycle stages of donor nuclei significantly affected t he develop ment of nuclear t ransplantation embryos. The activation rate in G1 group ( 8713 %) was significantly higher t han t hat in S (7916 %), G2 (6316 %) or non2synchronized group (7811 %). The rates of cleavage, morula, blastocyst, hatched blastocyst and blastocyst cell number of cloning embryos f rom G1 group (8419 %, 7115 %, 5810 %, 3511 % and ) were significantly higher than in S (7414 %, 3013 %, 1913 %, 3 This work was supported by Research on Highly Efficient Techniques in Procine Somatic Cell Nuclear Transfer funded by Hongkong Wiss Biotechnology Center 33 Corresponding author E2mail : edu. cn

7 % and ), G2 ( 6010 %, 1619 %, 1613 %, 417 % and ) or non2synchronized group (7312 %, 4914 %, 3212 %, 1216 % and ) ( P < 0105). The embryos f rom G2 nuclei had poor develop mental potential i n vit ro. The rates of cleavage, morula and blastocyst cell number were significantly lower t han ot her groups ( P < 0105). The i n vivo develop ment of cloning embryos f rom different cell cycle nuclei was observed in this experiment. A total of 144 nuclear transplantation embryos from G1 stage were t ransferred into 12 recipient does. Six of t hem became pregnant and gave birt h 19 live pups. The pupping rate was 1312 %. It was significantly higher t han S ( 518 %, 7/ 120 ), G2 group ( 0, 0/ 126 ) or non2 synchronized donor nuclei ( 513 %, 8/ 150) ( P < 0105). The cloning embryos f rom G2 nuclei could not develop to normal off spring alt hough a few cloning embryos developed to blastocyst s. Nevert heless, t here was no statistical difference in develop mental potential of cloning embryos between S and non2synchronized group, indicating that the cell cycle stage of early rabbit embryonic cells is in S stage. The results in this experiment show that the cycle stages of donor cells significantly affect the developmental potential of rabbit cloning embryos. i n vit ro and i n vivo. significantly decreases. The G1 nuclei can successf ully support t he develop ment of cloning embryos Alt hough t he embryos f rom S nuclei can develop to live off spring t he pupping rate The embryos f rom G2 nuclei have poor develop mental rate i n vit ro and i n vivo. Key words Rabbit, Cell cycle, Nuclear transplantation, Development, Embryo