To assess whether IE by the timed administration of demecolcine was reversible and whether oocytes scored as compartmentalized eventually became enucleated if allowed to develop further, oocytes were treated for 15 min with demecolcine beginning immediately after activation and fixed at 90 min pa or after overnight culture. In four replicate experiments, no significant difference was observed in the proportion of oocytes scored as IE at either time point (25% vs. 21%, respectively), indicating that for these oocytes, the enucleation was permanent (Table 3). No compartmentalized oocytes were observed after overnight culture, compared with 52% when assessed at 90 min pa. In the group that was left overnight, 66% of demecolcine-treated oocytes (n = 80) cleaved to the 2-cell stage, further confirming the reversibility of demecolcine-induced com-partmentalization of chromatin (Fig. 2i). canadian family pharmacy com
ES Cell Nuclear Transfer Using B6D2F1 Cytoplasts
The next objective was to determine the developmental competence of ethanol-activated/demecolcine-treated oocyte cytoplasts following nuclear transfer. To make best use of the 40-50% of oocytes reversibly compartmentalizing their chromatin following our optimized protocol, cyto-plasts for nuclear transfer were prepared by mechanical removal of both long flattened and closely apposed PBs. These cytoplasts were then injected with HM-1 ES cell nuclei. In each of four replicate experiments, development was compared against ethanol-activated parthenogenetic controls and embryos cloned from HM-1 ES cells by the established use of MII cytoplasts and a 2-h delay between injection and activation.
A total of 553 oocytes were ethanol-activated and treated with demecolcine for 15 min beginning immediately after activation. Of these, 307 (56%) possessing either a single long or two closely apposed PBs were selected by 90 min pa, and 192 (35%) survived mechanical removal of their PBs. Although not quantified, those oocytes with single long PBs tended to survive mechanical removal of PBs better. A total of 178 oocytes (32% of those activated and demecolcine-treated) were injected with HM-1 ES nuclei. Virtually 100% of MII oocytes survived mechanical enucleation. In total, 231 MII oocytes were enucleated, and 220 cytoplasts (95%) survived injection and, therefore, activated with SrCl2.