title: Nuclear transfer in sheep embryos: the effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes.
abstract: The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation if IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 microM nocodazole for 14-17 hr. Three types of transfers were compared directly: "S-->S," early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; "M-->MII," nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S-->MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M-->MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S-->S and M-->MII, and between S-->S and control, though developmental rate was also increased for S-->S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M-->MII or S-->S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M-->MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos.
authors.affiliation: College of Animal Science and Technology, Beijing Agricultural University, Beijing, People's Republic of China.
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Dai
authors.forename: R M
authors.initials: RM
authors.lastname: Moor
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002453:Cell Cycle; D002467:Cell Nucleus / Q000502:physiology*; D018920:Coculture Techniques; D003593:Cytoplasm / Q000502:physiology*; D004261:DNA Replication; D004558:Electric Stimulation; D018274:Electroporation; D004624:Embryo Transfer / Q000379:methods*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect; D008940:Mitotic Index; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D011247:Pregnancy; D012756:Sheep / Q000196:embryology*
chemical_list: D000970:Antineoplastic Agents; D015739:Nocodazole