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 donor cells and recipient oocytes, the procedure of micromanipulation, and the activation protocol. This study was conducted to determine the effects of cell cycle coordination for cloning rats from fetal fibroblasts (FFs). Moreover, enucleated zygotic and parthenogenetic ooplasts were used for serial cloning with pronuclear and two-cell stage blastomeres derived from SCNT. Metaphase donor cells had a significantly higher cleavage rate than G0/G1-phase FFs with MII oocytes and G2-phase FFs with TII oocytes. However, reconstructed embryos were unable to develop beyond the two-cell stage, neither in vitro nor in vivo. Moreover, the developmental arrest at the two-cell stage was not overcome, even when using serial cloning with zygotic and parthenogenetic recipients. To assess the cytoskeleton after SCNT, reconstructed two-cell stage embryos were harvested at different times after cleavage for immunostaining (anti-alpha-tubulin) and mRNA abundance (beta-actin, alpha-tubulin, alpha-actinin). Reconstructed two-cell embryos showed abnormal microtubule distribution and down-regulated expression of several cytoskeletal transcripts. Therefore, it seems that the developmental arrest of rat SCNT embryos is associated with improper transcription of cytoskeleton genes, presumably resulting in abnormal microtubule distribution.