Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle

M-phase synchronized bovine blastomeres were used to study the effect of nu clear-cytoplasmic synchronization on the developmental potential after nucl ear transfer (NT). The capacity of nocodazole and benomyl to reversibly syn chronize blastomeres from embryos in M-phase was evaluated. Nocodazole reve rsibly 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 findi ng 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equato rial plate (1-3 hpf) that acquired an organized structure over time (3-7 hp f) were subsequently observed. Anaphase;telophase structures were predomina ntly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 an d 6-7 hpf extruded a polar body-like structure 5 hr after activation, but t his was not observed in embryos activated immediately after fusion. A signi ficantly lower activation rate was observed for oocytes activated 3-4 hpf c ompared to those activated 6-7 hpf. However, the ability to undergo first c leavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of acti vation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like struc ture than in those activated 3-4 hpf that extruded a polar body-like struct ure. Under the conditions tested M-phase donor cells cannot be properly rem odeled after NT in cattle to trigger normal embryonic development. Our obse rvations of chromatin structures together with DNA synthesis suggest that t he failure in the development may be due to improper chromatin remodeling o f mitotic nuclei after NT, which may result in chromosomal abnormalities in compatible with normal embryo development. (C) 2000 Wiley-Liss, Inc.