101 DEVELOPMENTAL ARREST AND CYTOSKELETAL ANOMALIES OF RAT EMBRYOS RECONSTRUCTED BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Many factors influence success rates in animal cloning by somatic cell nuclear transfer (SCNT), including cell cycle stage of the donor cells and recipient oocyte, the procedure of micromanipulation, and the activation protocol. It has been reported that the development of rat embryos is inhibited at the 2-cell stage during in vitro culture and that microtubule (MT) distribution is involved in the developmental arrest. This study was conducted to determine the effects of oocyte activation methods and the cell cycle coordination for cloning rats from fetal fibroblasts (FF). Recipient oocytes were activated with cycloheximide, 6-dimethylaminopurine, and roscovitine and used either before (metaphase-II, MII) or after (telophase-II, TII) activation for fusion with FF at the G0/G1-phase, metaphase, and S/G2-phase. Moreover, enucleated zygotic and parthenogenetic ooplasts were used for serial cloning with pronuclear and 2-cell-stage blastomeres derived from SCNT. There was no significant difference in the first cleavage rate in the 3 activation groups after SCNT. Metaphase donor cells (85.2%) had a significantly higher cleavage rate than G0/G1-phase FF with MII oocytes (70.4%) and G2-phase FF with TII oocytes (72.2%). However, reconstructed embryos were unable to develop beyond the 2-cell stage either in vitro or in vivo. Moreover, serial cloning with zygotic and parthenogenetic recipients was also unable to overcome the developmental arrest at the 2-cell-stage. To assess the cytoskeleton after SCNT, reconstructed 2-cell-stage embryos were harvested at different times after cleavage for immunostaining (anti alpha-tubulin) and mRNA abundance (beta-actin and alpha-tubulin). Reconstructed 2-cell embryos showed much thicker fibrous or disconnected MT distribution concomitant with a reduction in cytoskeletal transcripts at the late 2-cell stage. In summary, our results indicate that the developmental arrest of rat SCNT embryos was a result of abnormal MT distribution caused by improper transcription of cytoskeleton genes; however, further studies are needed to understand the early developmental block of rat SCNT-derived embryos.