Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle.
M-phase synchronized bovine blastomeres were used to study the effect of nuclear-cytoplasmic synchronization on the developmental potential after nuclear transfer (NT). The capacity of nocodazole and benomyl to reversibly synchronize blastomeres from embryos in M-phase was evaluated. Nocodazole reversibly arrested bovine embryos at the studied stages and induced high rates of M-phases in morulae and compact morulae. In contrast, benomyl was less efficient than nocodazole to synchronize in M-phase. After transfer of an M-phase blastomere, premature chromatin condensation was the prevalent finding 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equatorial plate (1-3 hpf) that acquired an organized structure over time (3-7 hpf) were subsequently observed. Anaphase-telophase structures were predominantly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 and 6-7 hpf extruded a polar body-like structure 5 hr after activation, but this was not observed in embryos activated immediately after fusion. A significantly lower activation rate was observed for oocytes activated 3-4 hpf compared to those activated 6-7 hpf. However, the ability to undergo first cleavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of activation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at a significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like structure than in those activated 3-4 hpf that extruded a polar body-like structure. Under the conditions tested M-phase donor cells cannot be properly remodeled after NT in cattle to trigger normal embryonic development. Our observations of chromatin structures together with DNA synthesis suggest that the failure in the development may be due to improper chromatin remodeling of mitotic nuclei after NT, which may result in chromosomal abnormalities incompatible with normal embryo development.
Alberio
R
R
Department of Molecular Animal Breeding and Genetics, Ludwig-Maximilian University, Oberschleissheim, Germany.
Motlik
J
J
Stojkovic
M
M
Wolf
E
E
Zakhartchenko
V
V
D000818:Animals; D001542:Benomyl / Q000494:pharmacology; D001757:Blastomeres / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D002970:Cleavage Stage, Ovum; D004247:DNA / Q000096:biosynthesis; D005260:Female; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology
D004247:DNA; D015739:Nocodazole; D001542:Benomyl