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Developmental kinetics of cloned, parthenogenetic and in vitro fertilized bovine embryos — Preliminary observations
Theriogenology
223
DEVELOPMENTAL KINETICS OF CLONED, PARTHENOGENETIC AND IN VITRO FERTILIZED BOVINE EMBRYOS - PRELIMINARY OBSERVATIONS P. Holm, P.J. Booth & H. Callesen Embryo Technology Center, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark In the present study, the kinetics of the first cell cycles of bovine embryos produced in vitro by nuclear transfer (NTE), parthenogenetic activation (PA) and conventional in vitro fertilization (IVF) were compared. Immature oocytes with tight cumulus and even ooplasm were aspirated from abattoir ovaries and matured for 24 h in TCM199 with calf serum and hormones. Mature oocytes were randomly allocated into 3 treatment groups: (1; IVF) in vitro fertilization in TALP with heparin (5 W/ml) and PHE, (2; PA) parthenogenetic activation in TCM199 with calcium ionophore A23 187 (10 pM, 5 min) followed by cycloheximide (10 l&ml, 6 h) or (3; NTE) nuclear transfer by enucleation in TCM199 with cytochalasin B (5 pg/ml) and Hoechst 33342 (5 pg/ml) followed by activation as described for treatment 2 and fusion of activated cytoplasts to blastomeres of day 5 in vitro produced embryos by electropulse (0.8 kV/cm, 30 ps) in a mamritol-MgSO,-CaCl,solution. At 20 h after initiation of treatments, presumptive zygotes were transferred to modified SOFaa medium (Holm et al. 1997, Proc. 13th AETE-meeting, 158) and cultured for 7 days in a time-lapse culture system on a microscopic stage (Holm et al. 1997, Theriogenology 47, 324). Images of each treatment group were recorded at 30 min intervals throughout the culture period. The developmental kinetics of the individual embryos identified within the focal plane of the images were analyzed retrospectively. In treatment groups IVF, PA and NTE, 13/27, 8/31 and 12/29 cleaved ova, respectively, developed to the compact morula or blastocyst stages within the 7 days of culture. The first appearance (mean + sem) of a blastocoel cavity were observed 136 rt:2 h (n=12), 136 + 3 h (n=8) and 13 1 + 3 h (n=l l), respectively, after fertilization/activation. The cell cycle lengths within the respective treatment groups are shown in the table. Cell cycle lengths (mean f sem in h) of in vitro produced bovine IVF, NTE and PA embryos. Treatment Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 M-CM CM-EB IVF 28.3kO.6 8.8kO.7 6.9kO.7” 36.3+1.7” 16.6k1.8” 22.Ok4.9 17.4f2.7 PA 25.9+2.6ab 11.2k1.8 8.2+0.5ab 35.8k3.5” 19.5*1.5” 12.Ok2.0 24.5?2.6 NTE 24.9+ l.Ob 9.150.8 10.7*1.6” 17.0f2.7b 39.2+12.4b 16.4f5.0 23.8k3.0 “,bValues with different superscripts are significantly different, ~~0.05. GLM-procedure, SAS@. These preliminary data indicate that the developmental kinetics of NTE embryos and IVF embryos differ in respect to the length of the lst, 3rd, 4th and 5th cell cycles, while the kinetics of PA embryos only vary significantly from IVF embryos in respect to the 1st cell cycle. The longer 1st cell cycle of IVF embryos corresponds to the expected time interval from sperm addition until oocyte penetration and thus activation. The later cell cycles in which differences were observed, accommodate major genomic events, such as transition from maternal to embryonic control at 4th cell cycle. Thus, the respective shortened and prolonged 4th and 5th cell cycles of NTE embryos suggest insufficient reprogramming and altered function of the transferred nuclei (Smith et al. 1996, Mol. Reprod. Dev., 45,444-450).
223
DEVELOPMENTAL KINETICS OF CLONED, PARTHENOGENETIC AND IN VITRO FERTILIZED BOVINE EMBRYOS - PRELIMINARY OBSERVATIONS P. Holm, P.J. Booth & H. Callesen Embryo Technology Center, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark In the present study, the kinetics of the first cell cycles of bovine embryos produced in vitro by nuclear transfer (NTE), parthenogenetic activation (PA) and conventional in vitro fertilization (IVF) were compared. Immature oocytes with tight cumulus and even ooplasm were aspirated from abattoir ovaries and matured for 24 h in TCM199 with calf serum and hormones. Mature oocytes were randomly allocated into 3 treatment groups: (1; IVF) in vitro fertilization in TALP with heparin (5 W/ml) and PHE, (2; PA) parthenogenetic activation in TCM199 with calcium ionophore A23 187 (10 pM, 5 min) followed by cycloheximide (10 l&ml, 6 h) or (3; NTE) nuclear transfer by enucleation in TCM199 with cytochalasin B (5 pg/ml) and Hoechst 33342 (5 pg/ml) followed by activation as described for treatment 2 and fusion of activated cytoplasts to blastomeres of day 5 in vitro produced embryos by electropulse (0.8 kV/cm, 30 ps) in a mamritol-MgSO,-CaCl,solution. At 20 h after initiation of treatments, presumptive zygotes were transferred to modified SOFaa medium (Holm et al. 1997, Proc. 13th AETE-meeting, 158) and cultured for 7 days in a time-lapse culture system on a microscopic stage (Holm et al. 1997, Theriogenology 47, 324). Images of each treatment group were recorded at 30 min intervals throughout the culture period. The developmental kinetics of the individual embryos identified within the focal plane of the images were analyzed retrospectively. In treatment groups IVF, PA and NTE, 13/27, 8/31 and 12/29 cleaved ova, respectively, developed to the compact morula or blastocyst stages within the 7 days of culture. The first appearance (mean + sem) of a blastocoel cavity were observed 136 rt:2 h (n=12), 136 + 3 h (n=8) and 13 1 + 3 h (n=l l), respectively, after fertilization/activation. The cell cycle lengths within the respective treatment groups are shown in the table. Cell cycle lengths (mean f sem in h) of in vitro produced bovine IVF, NTE and PA embryos. Treatment Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 M-CM CM-EB IVF 28.3kO.6 8.8kO.7 6.9kO.7” 36.3+1.7” 16.6k1.8” 22.Ok4.9 17.4f2.7 PA 25.9+2.6ab 11.2k1.8 8.2+0.5ab 35.8k3.5” 19.5*1.5” 12.Ok2.0 24.5?2.6 NTE 24.9+ l.Ob 9.150.8 10.7*1.6” 17.0f2.7b 39.2+12.4b 16.4f5.0 23.8k3.0 “,bValues with different superscripts are significantly different, ~~0.05. GLM-procedure, SAS@. These preliminary data indicate that the developmental kinetics of NTE embryos and IVF embryos differ in respect to the length of the lst, 3rd, 4th and 5th cell cycles, while the kinetics of PA embryos only vary significantly from IVF embryos in respect to the 1st cell cycle. The longer 1st cell cycle of IVF embryos corresponds to the expected time interval from sperm addition until oocyte penetration and thus activation. The later cell cycles in which differences were observed, accommodate major genomic events, such as transition from maternal to embryonic control at 4th cell cycle. Thus, the respective shortened and prolonged 4th and 5th cell cycles of NTE embryos suggest insufficient reprogramming and altered function of the transferred nuclei (Smith et al. 1996, Mol. Reprod. Dev., 45,444-450).