We observed that transient treatment with the microtubule-destabilizing agent demecolcine during meiotic resumption resulted in the irreversible enucleation and reversible compartmentalization of chromatin in activated mouse oocytes. This treatment was effective in different mouse strains, with its efficacy being dependent on both the timing and the duration of its administration. Activated cytoplasts produced following mechanical removal of PBs associated with demecoline-treated oocytes were competent to develop after receipt of ES cell nuclei, although at lower rates than with established methods using MII cytoplasts. However, such cytoplasts still supported development of live offspring. canadian health & care mall
We observed that resumption of meiosis following ethanol activation was both asynchronous and dependent on mouse strain. This alone was likely to account for the reduced efficacy of inducing complete enucleation to the rate of approximately 20% observed by our standardized protocol. Theoretically, the rate of IE could be improved by combining demecolcine with agents synchronizing meiotic progression. One possibility would be the protein synthesis-inhibitor CHX, which when applied to activated eggs can hasten the decline of maturation-promoting factor activity.
Induced enucleation and compartmentalization of chromatin was likely achieved by the transient disruption of cytoplasmic and spindle microtubule dynamics following oocyte activation. One consequence of this could have been the inhibition or delay of spindle rotation during the resumption of meiosis and its subsequent uncoupling from PB emission. This was suggested by the parallel orientation of spindles with respect to the plasma membrane in de-mecolcine-induced PBs. In mouse oocytes, the meiotic spindle and cytoplasmic microtubules are organized by cen-triole-less microtubule organizing centers nucleated by 7-tubulin. In activated mammalian oocytes, the mechanism of spindle positioning and rotation is poorly understood, but it likely involves interactions between microtubule spindles and cortical microfilaments. During mouse meiotic maturation, treatment with the microfilament-inhib-itor cytochalasin D disrupts microtubule spindle position within the oocyte and centrosome organization.. Using the same inhibitor, cortical microfilaments have been shown to be required for spindle anchoring and rotation during maturation and activation of amphibian oocytes. Although in our study the microfilament-inhibitor CB was used to suppress PB emission in embryos cloned from MII cytoplasts, CB was not used in the creation of activated/ demecolcine-treated cytoplasts.