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Nuclear transfer in rabbits and cattle by electric pulse-induced fusion of blastomeres to enucleated oocytes
THERIOGENOLOGY NUCLEAR TRANSFER IN RABBITSAND CAlTLE BY ELECTRIC PULSE-INDUCED FUSION OF BLASTOMERES TO ENUCLEATED OOCYTES X.
Yang, S. Jiang and R. H. Foote Department of Animal Science Cornell University Ithaca, New York 14853-4801 USA
Experiments were conducted to evaluate the developmental potential of transferred nuclei (blastomeres) in vitro (rabbits and cattle) and in vivo (rabbits). In the rabbit trial, oocytes were collected from the oviducts of superovulated does approximately 14 h after injection of luteinizing hormone. cells were Cumulus removed by 0.2% hyaluronidase treatment and by pipetting. Cumulus-free oocytes were then placed into micromanipulation droplets of Dulbecco's phosphatebuffered saline containing 0.4% bovine serum albumin and 7.5 fig/ml cytochalasin B. Individual blastomeres isolated from 32-cell-stage embryos by the procedure of Yang et al. (Molec. Reprod. Devel. 27:118, 1990) were then added to the droplet containing the oocytes. The oocytes were enucleated blindly by aspirating the polar body and the adjacent cytoplasm expected to contain the oocyte nuclear material. The enucleation efficiency was 77% (n=lll). The same pipette was used to insert one blastomere into the perivitelline space of each oocyte through the initial puncture. Blastomere/oocyte complexes were gradually exposed to fusion medium (0.3 M mannitol) and groups of 3 to 5 were placed between the two electrodes (0.5 mm apart) of a 8TX fusion They were aligned by 5 to 7.5 V, 1.0 MHz AC current, and when chamber. alignment was observed a DC pulse of 120 V, 60 psec was applied to induce fusion. There were 331 attempted fusions, of which 14% resulted in lysis, 72% were fused and 14% were nonfused. After culture in Medium 199 (Earle's salt) with 15% fetal calf serum (FCS) at 39OC in 5% CO2:95% humidified air for 18 to 20 h, 138 (42%) developed into 2- to 4-cells and 115 of these embryos were transferred to the oviduct of 10 ovulated recipients synchronized about 12 h after the donors. Seven recipients were not pregnant and two produced two dead fetuses (Csection) and one produced four live young. Homozygosity can not be confirmed because mixed donor cells were used. In the bovine trial, 163 in vitro matured oocytes (26 h culture in Medium 199 + 7.5% FCS) with an identifiable polar body were used as nuclear recipients, and blastomeres from 32- to 64-cell embryos derived from IVF were used as nuclear donors. Enucleation efficiency for cattle was 75% (n=138). Nuclear transfer and fusion were performed as described for the rabbit excepting that a lower DC pulse (60 V, 30 psec) was used instead. This resulted in a fusion rate of 70% (n=60) and a development rate into 28-cell embryos of 24% (n=163) following culture in vitro for 2 days. Micromanipulation and electric pulses resulted in only 3% (n=60) lysis of bovine oocytes. In conclusion, electrofusion without mechanical alignment resulted in a high rate of fusion both for the rabbit and the bovine. At least some blastomeres from 32-cell embryos were capable of supporting embryo development to term in the rabbit and to early cleavage stages in cattle. Supported
298
in part
by the Cornell
Biotechnology
Program.
JANUARYlBBlVOL.35NO.l
Yang, S. Jiang and R. H. Foote Department of Animal Science Cornell University Ithaca, New York 14853-4801 USA
Experiments were conducted to evaluate the developmental potential of transferred nuclei (blastomeres) in vitro (rabbits and cattle) and in vivo (rabbits). In the rabbit trial, oocytes were collected from the oviducts of superovulated does approximately 14 h after injection of luteinizing hormone. cells were Cumulus removed by 0.2% hyaluronidase treatment and by pipetting. Cumulus-free oocytes were then placed into micromanipulation droplets of Dulbecco's phosphatebuffered saline containing 0.4% bovine serum albumin and 7.5 fig/ml cytochalasin B. Individual blastomeres isolated from 32-cell-stage embryos by the procedure of Yang et al. (Molec. Reprod. Devel. 27:118, 1990) were then added to the droplet containing the oocytes. The oocytes were enucleated blindly by aspirating the polar body and the adjacent cytoplasm expected to contain the oocyte nuclear material. The enucleation efficiency was 77% (n=lll). The same pipette was used to insert one blastomere into the perivitelline space of each oocyte through the initial puncture. Blastomere/oocyte complexes were gradually exposed to fusion medium (0.3 M mannitol) and groups of 3 to 5 were placed between the two electrodes (0.5 mm apart) of a 8TX fusion They were aligned by 5 to 7.5 V, 1.0 MHz AC current, and when chamber. alignment was observed a DC pulse of 120 V, 60 psec was applied to induce fusion. There were 331 attempted fusions, of which 14% resulted in lysis, 72% were fused and 14% were nonfused. After culture in Medium 199 (Earle's salt) with 15% fetal calf serum (FCS) at 39OC in 5% CO2:95% humidified air for 18 to 20 h, 138 (42%) developed into 2- to 4-cells and 115 of these embryos were transferred to the oviduct of 10 ovulated recipients synchronized about 12 h after the donors. Seven recipients were not pregnant and two produced two dead fetuses (Csection) and one produced four live young. Homozygosity can not be confirmed because mixed donor cells were used. In the bovine trial, 163 in vitro matured oocytes (26 h culture in Medium 199 + 7.5% FCS) with an identifiable polar body were used as nuclear recipients, and blastomeres from 32- to 64-cell embryos derived from IVF were used as nuclear donors. Enucleation efficiency for cattle was 75% (n=138). Nuclear transfer and fusion were performed as described for the rabbit excepting that a lower DC pulse (60 V, 30 psec) was used instead. This resulted in a fusion rate of 70% (n=60) and a development rate into 28-cell embryos of 24% (n=163) following culture in vitro for 2 days. Micromanipulation and electric pulses resulted in only 3% (n=60) lysis of bovine oocytes. In conclusion, electrofusion without mechanical alignment resulted in a high rate of fusion both for the rabbit and the bovine. At least some blastomeres from 32-cell embryos were capable of supporting embryo development to term in the rabbit and to early cleavage stages in cattle. Supported
298
in part
by the Cornell
Biotechnology
Program.
JANUARYlBBlVOL.35NO.l