Development of porcine embryos produced by nuclear transfer from somatic cells treated with protein synthesis and cyclin-dependent kinase inhibitors

• Main Author: Lalonde, Annie.
• Format: Others
• Language: en
• Published: McGill University 2006
• Subjects: Swine > Cloning.; Swine > Embryos.; Cell nuclei > Transplantation.
• Online Access: http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=99190

The objective of this study was to investigate whether or not the treatment of nuclear donor cells with inhibitors of protein synthesis and cyclin-dependent kinases (CDKs) affect the development of swine embryos produced by somatic cell nuclear transfer. Host oocytes were derived from pre-pubertal females and matured in vitro for 42-46 h under standard conditions. Nuclear donor cells were fetal fibroblasts maintained in culture for 2 to 6 passages. Oocytes were reconstructed with cells treated for 22-24 hours with cycloheximide (CHX; 10mug/ml), roscovitine (ROS; 25 muM), the combination of CHX + ROS (CR), or untreated cells. Two hours after reconstruction, the oocytes were activated using ionomycin (15muM/5 min) and strontium chloride (10mM/4h), maintained for 6 h in the presence of cytochalasin B (7.5mug/ml) and CHX (10mug/ml), and then cultured in porcine zygote medium (PZM3) for 6 days. The cleavage rate, 63.7% (n=318), 55.2% (n=99), 56.7% (n=107) and 60.6% (n=347), at 48 h post-fusion were not significantly different between embryos derived from ROS, CHX, CR and control cells, respectively. Developmental rate to blastocyst stage was higher for embryos reconstructed with ROS (12.2%) and untreated cells (12.1%) when compared to CHX (5.7%) and CR (4.9%). Blastocysts produced with ROS treated cells had similar number of nuclei compared to embryos reconstructed with untreated donor cells (30.9+/-10.4 vs. 32.2+/-8.0). Phosphorylated H2A.X (gammaH2A.X) was highly expressed in donor cells treated with CR compared to non treated cells, but it was similarly expressed in most of 1-cell stage embryos reconstructed with control or treated cells. Flow cytometry analysis showed that the majority of the fibroblasts were at G 0/G1 phase of the cell cycle at the time of nuclear transfer. It was concluded that the treatment of nuclear donor cells with inhibitors of protein synthesis and CDKs did not improve the in vitro development of somatic cell nuclear transfer embryos in pigs.