Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos

This study determines the efficiency of sequential calcium treatments (elec troporation or ionomycin) combined with protein synthesis (cycloheximide) o r phosphorylation inhibitors (6-dimethylaminopurine) or the specific matura tion promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfe r embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cyclohe ximide and cytochalasin B induced pronuclear formation and development to t he blastocyst stage at a rate similar to control embryos produced by intrac ytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In con trast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that o bserved in controls. Following reconstruction by nuclear transfer, activati on with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activ ated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective met hods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethyla minopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/ros covitine, which are applicable to parthenote or nuclear transfer embryo pro duction.