Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer

Abstract

The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4 degrees C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

Effect of Oocyte Aging on Parthenogenetic Activation with Cycloheximide and A Iteration of the Activity of Maturation Promoting Factor during Aging of Bovine Oocytes.

“…Similar results were reported by Nagai [13] who used another activation agent (ethanol) for parthenogenetic activation. The promotion of activation in oocytes by aging has also been shown with other activation agents including Ca 2+ ionophore A23187 and electrical stimulation in cattle [14][15][16], and with a variety of mammals including mice [17,18], rabbits [19], and pigs [20]. Taken together, these results suggest that the agedoocytes are more sensitive to various activation stimuli than newly matured oocytes in all mammalian species.…”

Mouse zygotes as recipients in embryo cloning

“…Full-term development was obtained after the transfer of embryonic nuclei into telophase I and anaphase II oocytes (Kono et al 1992) and into two-cell blastomeres (mouse, Tsunoda et al 1987, Kono & Tsunoda 1989, Kono et al 1991, rat, Roh et al 2003, as well as after the transfer of somatic nuclei into telophase II oocytes (cow, Bordignon & Smith 1998, Liu et al 2000goat, Baguishi et al 1999) and two-cell embryos (rabbit, Skrzyszowska et al 2006). However, in almost all cloning procedures performed during the last 20 years, metaphase II oocytes were the cytoplasts of choice.…”

Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

“…When the time between activation (by electric stimulation or ionomycin followed by 4-6 h incubation in 6-DMAP or cycloheximide respectively) and NT increased to 4-6 h, development past the eightcell stage was almost completely abolished both with M-or G 0 /G 1 -phase donors (Tani et al 2001(Tani et al , 2003. However, there are cases where artificially pre-activated TII cytoplasts have supported somatic chromatin remodeling after NT in cattle and mice (Bordignon et al 1999(Bordignon et al , 2001, and even resulted in viable cloned goats (Baguisi et al 1999) and cattle (Bordignon & Smith 1998, Kurosaka et al 2002, Bordignon et al 2003. Some of these studies differ from ours in using different species, different activation stimuli (e.g., electrical current, ionomycin, and ethanol/cycloheximide) and activation times, and oocytes of different age post-maturation at the time of telophase enucleation.…”