Reproductive Technology

Demecolcine-Induced Oocyte Enucleation for Somatic Cell Cloning: Coordination Between Cell Cycle Egress, Kinetics of Cortical Cytoskeletal Interactions, and Second Polar Body Extrusion

Abstract

Studies were designed to further explore the use of pharmacological agents to create developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase-II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times post-activation. Chromosome segregation, spindle dynamics and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA, microtubule and microfilament selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain and treatment specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly due to a combination of factors such as the time course of meiotic cell cycle progression after ethanol-activation, the degree of spindle rotation and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.