A reliable technique of nuclear transplantation for immature mammalian oocytes

Transplanting a germinal vesicle (GV) to another enucleated oocyte provides a possible way to avoid age-related aneuploidy in metaphase II (Mn) oocyte s from older women. This study was conducted to examine the efficiency of e ach step of nuclear transplantation as reflected in the survival and matura tion capacity of immature mouse oocytes subjected to this procedure. GV sta ge oocytes were retrieved from unstimulated ovaries. A GV removed with a sm all amount of cytoplasm (karyoplast) was transferred subzonally into a prev iously enucleated oocyte, which was then exposed to direct current to promo te fusion. Such reconstituted oocytes were placed in culture to allow matur ation, and some that had extruded a first polar body were fixed and process ed for chromosome analysis. Each step of nuclear transplantation - survival , enucleation, grafting, and reconstitution - was successful in >90%, with the overall efficiency of reconstitution being 80%. The observation of norm al karyotypes confirmed that the procedure did not increase chromosomal ane uploidy. An electrolytic medium, revealed to be superior for the reconstitu tion procedure, also allowed haploidization of the transplanted nucleus. Th ese findings suggest that this technique can be applied to study the effect s of a 'younger' woman's ooplasm on the disjunction of an 'older' woman's c hromosomes during meiosis I.