Dynamic imaging of the metaphase II spindle and maternal chromosomes in bovine oocytes: Implications for enucleation efficiency verification, avoidance of parthenogenesis, and successful embryogenesis

Manipulations of DNA and cellular structures are essential for the propagat ion of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that impro ves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindl e microtubules with microinjected polymerization-competent X-rhodamine-tubu lin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so th at the maternal chromosomes can be verifiably removed to make enucleated eg gs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enuc leation removed the smallest amount of cytoplasm (4-7%) and was 100% effici ent only when performed under continuous fluorescence, i.e., longer fluores cence exposure. This was in part due to the finding that the second metapha se spindle is frequently displaced (60.7 +/- 10%) from its previously assum ed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fl uorochromes, often resulted in enucleation failure (36 +/- 6%). When labele d oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3 %), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oo cytes (37%). This suggests that longer wavelength fluorochromes can be empl oyed for live visualization of metaphase spindle components, verification o f their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without comp romising the oocyte's developmental potential after activation.