Proteomic Analysis of the Extraembryonic Tissue from Cloned Porcine Embryos

Abstract

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy. Molecular & Cellular Proteomics 5:1559 -1566, 2006.

Serial cloning of pigs by somatic cell nuclear transfer: Restoration of phenotypic normality during serial cloning

“…Histological analyses of 28 of the 38 scNT clones that died after birth revealed 11 had distinct histological anomalies, the most common of which was meningitis, followed by poor development of the elbow joint bone, flexor tendons, congestion, and Leydig cell hypoplasia (Park et al, 2004(Park et al, , 2005. As shown in our very recent report, the abnormal histopathologic findings of the deceased cloned pigs are closely associated with aberrant protein expression patterns (Park et al, 2005;Chae et al, 2006;Lee et al, 2007).…”

“…ROS levels increase gradually during early pregnancy, resulting in the apoptotic cell death of extraembryonic tissues (Chae et al, 2006). Very recently, we also reported that term placenta Fig.…”

“…This finding is in part because of a high incidence of fetal death that is due to a variety of afflictions during pregnancy, including fetal overgrowth and placental malformations (Hiendleder et al, 2004;Arnold et al, 2006;Chae et al, 2006;Lee et al, 2007). These studies have revealed that some of the subtle abnormalities of adult scNT-derived animals are species-specific.…”

“…NT was carried out as described previously (Yin et al, 2003;Park et al, 2004, Chae et al, 2006Lee et al, 2007). Briefly, the matured eggs with the first polar body were cultured in NCSU-23 medium supplemented with 0.4 mg/ml demecolcine (Sigma) and 0.05 mol/L sucrose for 1 hr.…”

“…Estrus synchronization of the recipients was established as reported previously (Yin et al, 2003;Park et al, 2004Park et al, , 2005Chae et al, 2006;Lee et al, 2007). One or two cells of scNT embryos were then surgically transferred into the oviducts of the synchronized recipients.…”