Preparación de citoplastos receptores para transferencia nuclear mediante enucleación química de ovocitos

Oocyte enucleation is a key step in mammalian cloning by nuclear transfer. Traditional mechanical enucleation, although efficient, is technically demanding and clearly damaging to the oocyte. Alternatively, oocytes can be chemically enucleated. Treatment of metaphase II oocytes with antimitotic agents induces the formation of a cortical protrusion which facilitates the mechanical removal of the chromosomes (chemically assisted enucleation). When the antimitotic treatment is applied to pre-activated oocytes, all the chromatin of the oocyte is extruded inside the second polar body, producing an enucleated oocyte (chemically induced enucleation). The aim of the present study was to optimize the efficiency of chemically assisted and induced enucleation methods in mouse oocytes, using different antimitotic drugs. In the chemically assisted enucleation experiments, CD-1 and B6CBAF1 oocytes were treated with demecolcine (0,4 μg/ml), nocodazole (0,3 y 1μg/ml) or vinblastine (0,1μg/ml) for 15, 30, 60 or 120 min, in order to determine the best treatment for the formation of cortical protrusions. In the chemically induced enucleation experiments, CD-1 oocytes were first activated with ethanol, then treated for 15, 30 or 60 min with demecolcine (0,4 μg/ml), nocodazole (0,3 y 1μg/ml), vinblastine (0,1 μg/ml) or taxol (1μM), and finally cultured in a medium with strontium chloride until second polar body extrusion. Our results show that the antimitotic drugs demecolcine, vinblastine and nocodazole can be successfully employed in both chemically assisted and chemically induced enucleation of mouse oocytes. The high rates of cortical protrusion formation (>80%) and of enucleated oocytes (∼40%) obtained in the chemically assisted and chemically induced enucleation experiments, respectively, demonstrate that these protocols represent a simpler alternative to the traditional method of mechanical enucleation.