42 ASSISTED ENUCLEATION IN GOLDEN HAMSTER OOCYTES TREATED WITH COLCHICINE AND DEMECOLCINE

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The golden hamster is an excellent experimental animal in many research fields. In an effort to establish experimental protocols necessary for cloning golden hamsters, optimized conditions for induced cytoplasmic protrusion and assisted enucleation of golden hamster oocytes with different concentrations of colchicine and demecolcine were examined in this study. The Golden hamsters (female, six weeks of age) were superovulated with eCG (30 IU, ip) followed by hCG (30 IU, ip) at intervals of 72 h. Hamsters were sacrificed at 13.5 h, 15 h and 18 h after hCG injection. The different ages of cumulus–oocyte complexes (COCs) were collected from oviducts and cumulus cells were removed with 0.1% hyaluronidase. (1) denuded oocytes of different ages were treated with 2.5 μg mL–1, 5 μg mL–1, and 10 μg mL–1 of colchicine for 4 h in M199TE, and they were examined each hour; (2) denuded oocytes of different ages were treated with 0.02 μg mL–1, 0.04 μg mL–1, 0.06 μg mL–1, 0.1 μg mL–1, 0.2 μg mL, 0.4 μg mL–1, and 0.6 μg mL–1 of demecolcine for 1 h in M199TE, and they were examined every 15 min; 3) according to the results of processes (1) and (2), cytoplasmic protrusions of oocytes treated with 10 μg mL–1 of colchicine (60 oocytes) or 0.4 μg mL–1 demecolcine (88 oocytes) for 1 h were removed with a micromanipulation pipette. Then the oocytes were examined by being stained with Hoechst 33342, and the percentage of assisted enucleation was compared with that of blind enucleation in which position of the chromosomes was indirectly determined by the location of the first polar body. The results showed that: (1) about 90% of oocytes at 13.5 h and 15 h post hCG injection were induced to form protrusions under the treatment of 10 μg mL–1 of colchicine for 1 h; (2) oocytes of 13.5 h post hCG were very sensive to demecolcine, even the treatment of 0.02 μg mL–1 for 1 h could also induce cytoplasmic protrusions (45.38% of 31 oocytes); (3) when treated with 0.4 μg mL–1 of demecolcine for 1 h, the cytoplasmic protrusion rate of oocytes of 13.5 h–18 h post-hCG could reach to 99–100%; (4) when the cytoplasmic protrusions induced by 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were removed, assisted enucleation rates were over 80%, significantly higher (P < 0.05) than blind enucleation (31.53% of 60 oocytes). In conclusion, the results of this study demonstrate that both colchicine and demecolcine can induce the hamster oocytes to form cytoplasmic protrusions. The treatments of 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were the best conditions to induce oocytes (13.5 h–18 h post hCG) to form protrusion. Also, the assisted enucleation rate with colchicine and demecolcine is much higher than that of blind enucleation. These results define conditions for chemical assisted enucleation and should facilitate the development of cloned golden hamsters as an animal model for human diseases.