Efficient production of nuclear transferred rat embryos by modified methods of reconstruction.
In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.
Max-Delbrück-Center for Molecular Medicine (MDC), Berlin-Buch, Germany.
Elena
E
Popova
Michael
M
Bader
Alexander
A
Krivokharchenko
D000818:Animals; D019976:Cloning, Organism / Q000379:methods*; D003571:Cytochalasin B; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D011256:Pregnancy Outcome; D051381:Rats; D016019:Survival Analysis
D003571:Cytochalasin B