Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

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 of 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 mu M 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 Mil-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. a 1997 Wiley-Liss, Inc.