Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling.
Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.
Choi
Young-Ho
YH
Departments of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station, Texas 77843-4466, USA.
Love
Linda B
LB
Westhusin
Mark E
ME
Hinrichs
Katrin
K
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002457:Cell Extracts; D002467:Cell Nucleus / Q000502:physiology*; D002843:Chromatin / Q000502:physiology*; D019976:Cloning, Organism / Q000379:methods*; D003571:Cytochalasin B / Q000494:pharmacology; D003600:Cytosol / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005306:Fertilization / Q000187:drug effects / Q000502:physiology; D006736:Horses; D009865:Oocytes / Q000502:physiology*; D010312:Parthenogenesis / Q000187:drug effects / Q000502:physiology*
D002457:Cell Extracts; D002843:Chromatin; D004791:Enzyme Inhibitors; D003571:Cytochalasin B; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine