Cloned Mice: DISCUSSION(4)
Although the efficacy of IE and the resulting cytoplast developmental competence in different strains of mouse oocytes were not compared directly, a slight improvement in cleavage and blastocyst development and a live offspring were obtained using B6CBAF1 oocytes. In this strain, ethanol-induced meiotic progression was initially delayed, so that by 15 min pa, 97% of eggs sampled were still in MII, compared with 85% of B6D2F1 observed in AII. The significance of this is unclear given the requirement for an intact MII spindle for ethanol-induced calcium oscillations. However, an oocyte actively in the process of chromosome segregation would possibly be less able to recover from microtubule spindle disruption, thus yielding an inferior cytoplast for nuclear transfer development. Interestingly, impaired development of cloned mice has recently been described using, as nuclear donors, fibroblasts and ES cells arrested in M phase by microtubule destabilization with nocodazole. Using fibroblasts as nuclear donors in experiments where activation was delayed, serial nuclear transfer of pronuclei from cloned embryos was required to produce healthy offspring. natural asthma treatment
It must be noted that both demecolcine and nocodazole are likely to affect other microtubule-mediated processes, such as zygote polarity, pronucleus formation, cell cleavage, and mitosis, the extent of which may relate to their respective reversibility. The potential asynchrony in cytoplasmic events created by transient microtubule disruption during M phase may also generally diminish the developmental potential of cloned embryos.
In summary, our study exemplifies the cloning of mice from ES cells using a new method involving ethanol-activated cytoplasts induced to enucleate and compartmentalize endogenous oogenetic chromatin. Although cloned embryo development was impaired relative to the traditional method involving delayed activation, our results demonstrate that no de facto requirement exists in the mouse to preexpose donated nuclei to unactivated oocyte cytoplasm to attain development to term. Future improvements in the efficacy of IE and cytoplast competence may involve methods of synchronizing meiotic progression after activation, activation strategies capable of eliciting multiple transients in intracellular calcium, and enhancement of nuclear readiness for reprogramming before nuclear transfer.