Demecolcine-assisted enucleation of goat oocytes: protocol optimization, mechanism investigation, and application to improve the developmental potential of cloned embryos.

Abstract

Although demecolcine-assisted enucleation has been performed successfully in porcine and cattle, the mechanism and protocol optimization of chemically assisted enucleation need further investigation. The present study optimized the protocol for goat oocyte enucleation and demonstrated that a 30-min treatment with 0.8 ng/mL demecolcine-induced cytoplasmic protrusions in over 90% of the oocytes. Rates of enucleation, cell fusion, and blastocyst formation were significantly higher after demecolcine-assisted than after blind aspiration enucleation, although differences in rates of live births remain to be unequivocally determined between the two treatments. The ability to form protrusions decreased significantly as spindles became less organized in aged oocytes and the oocytes with a poor cumulus expansion. More than 93% of the demecolcine-induced protrusions persisted for 2 h in the absence of cytochalasin B (CB) but most disappeared within 30 min of CB treatment. The spindle disintegrated, an actin-rich ring formed around the chromosome mass and the MAP kinase activity increased significantly after demecolcine treatment. When oocytes with induced protrusions were treated with CB, however, the contractile ring disappeared, the spindle reintegrated, and both MPF and MAP kinase activities decreased significantly. It is concluded that (1) cytoplasmic protrusions can be induced in goat oocytes with a very low concentration of demecolcine; (2) oocyte selection and enucleation can be achieved simultaneously with demecolcine treatment; and (3) an interactive effect between the MAP kinase, MPF, microfilaments and microtubules might be implicated in the control of cytoplasmic protrusion formation after demecolcine treatment.