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.
Highlights
Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications
Extraembryonic tissue is a multifunctional organ developed from both fetal and maternal origins. It provides nutrition, gas exchange, waste removal, and endocrine and immune support for the developing fetus [6]. This abnormally small extraembryonic tissue from SCNT embryos indicates that one of the main reasons for the low birth rate of cloned animals is due to the defective development of extraembryonic tissue
In the proteomic analysis using the abnormally small extraembryonic tissue on the 26th day of pregnancy from the SCNT embryos, 39 proteins were identified as differentially regulated proteins
Summary
Extraembryonic Tissue—In vitro maturation, nuclear transfer, and embryo culture were performed according to previous reports [22, 23]. Somatic cells were the fetal fibroblasts isolated from the F1 fetus produced by the crossing of inbred Duroc male and Landrace female pigs. 200 SCNT embryos were transferred to the oviducts of each synchronized recipient. Recipients were prepared as described previously [24]. Three extraembryonic tissues from SCNT embryos and three control tissues at the 26th day of pregnancy were prepared.
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