title: Antimitotic treatments for chemically assisted oocyte enucleation in nuclear transfer procedures.
abstract: Chemically assisted enucleation has been successfully applied to porcine and bovine oocytes to prepare recipient cytoplasts for nuclear transfer procedures. In this study, the antimitotic drugs demecolcine, nocodazole, and vinblastine were first assessed for their ability to induce the formation of cortical membrane protrusions in mouse, goat, and human oocytes. While only 2% of the treated human oocytes were able to form a protrusion, high rates of protrusion formation were obtained both in mouse (84%) and goat oocytes (92%), once the treatment was optimized for each species. None of the antimitotics applied was superior to the others in terms of protrusion formation, but mouse oocytes treated with vinblastine were unable to restore normal spindle morphology after drug removal and their in vitro development after parthenogenetic activation was severely compromised, rendering this antimitotic useless for chemically assisted enucleation approaches. Aspiration of the protrusions in mouse oocytes treated with demecolcine or nocodazole yielded 90% of successfully enucleated oocytes and allowed the extraction of a smaller amount of cytoplasm than with mechanical enucleation, but both enucleation methods resulted in the depletion of spindle-associated gamma-tubulin from the prepared cytoplasts. Treatment of mouse oocytes with demecolcine or nocodazole had no effect on their in vitro development after parthenogenetic activation, or on their ability to repolymerize a new spindle after the removal of the drug or the reconstruction of the treated cytoplasts with a somatic nucleus. Therefore, demecolcine- and nocodazole-assisted enucleation appears as an efficient alternative to mechanical enucleation, which can simplify nuclear transfer procedures.
authors.affiliation: Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Spain.
authors.forename: Nuno
authors.initials: N
authors.lastname: Costa-Borges
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Paramio
authors.forename: Gloria
authors.initials: G
authors.lastname: Calderón
authors.forename: Josep
authors.initials: J
authors.lastname: Santaló
authors.forename: Elena
authors.initials: E
authors.lastname: Ibáñez
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D019976:Cloning, Organism / Q000379:methods*; D003703:Demecolcine / Q000494:pharmacology; D006041:Goats; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000187:drug effects*; D015739:Nocodazole / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D008941:Spindle Apparatus / Q000187:drug effects* / Q000502:physiology; D014404:Tubulin / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D014404:Tubulin; D050257:Tubulin Modulators; D014747:Vinblastine; D015739:Nocodazole; D003703:Demecolcine