Abstract
BackgroundSomatic cell cloning by nuclear transfer (SCNT) in pig is clearly of great benefit for basic research and biomedical applications. Even though cloned offspring have been successfully produced in pig, SCNT is struggling with the low efficiency.ResultsIn the present study, we investigated differentially expressed proteins of the extraembryonic tissue from pig SCNT fetus compared to control (normal) fetus. We obtained the extraembryonic tissue from embryos at day 35 of pregnancy and examined the protein expression profiles using two-dimensional electrophoresis (2-D) and Western blotting. The extraembryonic tissue of fetus in control pregnancy was compared to the extraembryonic tissue of SCNT fetus, which showed an abnormally small size and shape as well as exhibited abnormal placental morphology compared to control fetus. A proteomic analysis showed that the expression of 33 proteins was significantly increased or decreased in the extraembryonic tissue of SCNT fetus compared to control fetus. The differentially expressed proteins in the extraembryonic tissue of SCNT fetus included ATP or lipid binding proteins, antioxidant proteins, translation elongation factors, and transcription factors. Western blotting analysis indicated that antioxidant enzymes and anti-apoptotic proteins were down-regulated; however, the expression levels of apoptotic proteins, Bax and Hsp27, were increased in the extraembryonic tissue of SCNT fetus. Moreover, immunohistochemical analysis also showed that the expression of the catalase or GPX genes was decreased in the extraembryonic tissue with SCNT fetus compared to those with control fetus. In addition, we observed a significant decrease in DNA methytransferase1 (Dnmt1) expression in SCNT extraembryonic tissue, and the expression levels of Dnmt3a and Dnmt3b were abnormally higher in SCNT fetus compared to control fetus. Moreover, a marked increase in the frequency of TUNEL-positive cells was observed in the extraembryonic tissue in SCNT fetus.ConclusionThese results demonstrated that pig SCNT fetus showed abnormal protein expression in the extraembryonic tissue, and extensive apoptosis occurred in the extraembryonic tissue of the SCNT fetus due to an increase in apoptotic protein expression or a decrease in antioxidant protein expression.
Highlights
Somatic cell cloning by nuclear transfer (SCNT) in pig is clearly of great benefit for basic research and biomedical applications
SCNT extraembryonic tissue from day 35 of pregnancy was smaller in size and more abnormal in appearance compared to the control extraembryonic tissue (Figure 1)
We found that that DNA methytransferase1 (Dnmt1) was highly expressed in the extraembryonic tissue of control fetus, whereas the activation of the protein is restricted in the SCNT extraembryonic tissue (Figure 4B)
Summary
Somatic cell cloning by nuclear transfer (SCNT) in pig is clearly of great benefit for basic research and biomedical applications. The birth of cloned pigs is achieved by the nuclear transfer of the nuclei of somatic cells [3,4]. Early embryonic mortality in SCNT embryos pregnant pigs is high during the first 30 days of pregnancy. The high rates of embryonic death of SCNT embryos are thought to be a result of inadequate nuclear reprogramming and extraembryonic tissue formation defects in the cloned embryo. Aberrant methylation in the trophectoderm of cloned blastocysts induces global gene dysregulation in extraembryonic tissues, and this type of gene dysregulation can potentially lead to the development of a dysfunctional placenta and have detrimental effects on fetal development [10,11]
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