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Proteomic analysis of the extraembryonic tissues from cloned porcine fetus at day 35 of pregnancy.

Ko YG, Hwang S, Kim SW, Kim H, Seong HH, Kim JH, Song Y, Yang BS, Song YM, Cho JH - BMC Res Notes (2014)

Bottom Line: 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.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.These 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.

View Article: PubMed Central - PubMed

Affiliation: Animal Genetic Resources Station, National Institute of Animal Science, RDA, Namwon 590-832, Korea. kog4556@korea.kr.

ABSTRACT

Background: Somatic 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.

Results: In 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.

Conclusion: These 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.

No MeSH data available.


Up-regulated apoptotic marker proteins in the SCNT extraembryonic tissue. (A) The expression of Bax and Hsp27 were up-regulated and the Bcl2 expression was down-regulated in the SCNT extraembryonic tissue. Actin was used as a loading control. (B) Dnmt1 was highly expressed in the control extraembryonic tissue while the expression of Dnmt3a and 3b Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue. (C) TUNEL assays in the control and SCNT extraembryonic tissue. Dewaxed paraffin sections from the control and SCNT extraembryonic tissue were subjected to the TUNEL assay. Fragmented DNAs were detected by a TUNEL assay using a fluorescence microscope with either propidium iodide (PI) (left) or FITC (FITC-dUTP) (right). In normal extraembryonic tissue, apoptotic cells were rarely identified while apoptotic cells significantly increased in SCNT extraembryonic tissue. Magnification: control and SCNT extraembryonic tissue (x200).
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Fig4: Up-regulated apoptotic marker proteins in the SCNT extraembryonic tissue. (A) The expression of Bax and Hsp27 were up-regulated and the Bcl2 expression was down-regulated in the SCNT extraembryonic tissue. Actin was used as a loading control. (B) Dnmt1 was highly expressed in the control extraembryonic tissue while the expression of Dnmt3a and 3b Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue. (C) TUNEL assays in the control and SCNT extraembryonic tissue. Dewaxed paraffin sections from the control and SCNT extraembryonic tissue were subjected to the TUNEL assay. Fragmented DNAs were detected by a TUNEL assay using a fluorescence microscope with either propidium iodide (PI) (left) or FITC (FITC-dUTP) (right). In normal extraembryonic tissue, apoptotic cells were rarely identified while apoptotic cells significantly increased in SCNT extraembryonic tissue. Magnification: control and SCNT extraembryonic tissue (x200).

Mentions: To further investigate whether the developmental defects in the extraembryonic tissue of SCNT fetus affect the expression of antioxidation-related proteins or apoptotic marker proteins, such as GPX, catalase, Bax, Bcl2, and Hsp27, we performed western blot analysis using control and SCNT extraembryonic tissue. The expression levels of catalase or GPX related to oxidative stress were decreased in SCNT extraembryonic tissues (Figure 3C). The expression of Hsp27 was up regulated more than two-fold in SCNT extraembryonic tissue compared with control tissue, and these data are consistent with 2-D analysis. The expression of Bax, an apoptotic marker protein, was up-regulated, and the expression of the anti-apoptotic Bcl-2 gene decreased in the SCNT extraembryonic tissue (Figure 4A). Next, we investigated the expression of DNA methyltransferases 1, 3a and 3b (Dnmt 1, 3a and 3b) in control and SCNT extraembryonic tissue. We found that that 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). In contrast, the expression levels of Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue, while they were low in control fetus (Figure 4B). These results suggest that a relatively low level of Dnmt1 activity exists during the development of SCNT fetus.Figure 4


Proteomic analysis of the extraembryonic tissues from cloned porcine fetus at day 35 of pregnancy.

Ko YG, Hwang S, Kim SW, Kim H, Seong HH, Kim JH, Song Y, Yang BS, Song YM, Cho JH - BMC Res Notes (2014)

Up-regulated apoptotic marker proteins in the SCNT extraembryonic tissue. (A) The expression of Bax and Hsp27 were up-regulated and the Bcl2 expression was down-regulated in the SCNT extraembryonic tissue. Actin was used as a loading control. (B) Dnmt1 was highly expressed in the control extraembryonic tissue while the expression of Dnmt3a and 3b Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue. (C) TUNEL assays in the control and SCNT extraembryonic tissue. Dewaxed paraffin sections from the control and SCNT extraembryonic tissue were subjected to the TUNEL assay. Fragmented DNAs were detected by a TUNEL assay using a fluorescence microscope with either propidium iodide (PI) (left) or FITC (FITC-dUTP) (right). In normal extraembryonic tissue, apoptotic cells were rarely identified while apoptotic cells significantly increased in SCNT extraembryonic tissue. Magnification: control and SCNT extraembryonic tissue (x200).
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Fig4: Up-regulated apoptotic marker proteins in the SCNT extraembryonic tissue. (A) The expression of Bax and Hsp27 were up-regulated and the Bcl2 expression was down-regulated in the SCNT extraembryonic tissue. Actin was used as a loading control. (B) Dnmt1 was highly expressed in the control extraembryonic tissue while the expression of Dnmt3a and 3b Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue. (C) TUNEL assays in the control and SCNT extraembryonic tissue. Dewaxed paraffin sections from the control and SCNT extraembryonic tissue were subjected to the TUNEL assay. Fragmented DNAs were detected by a TUNEL assay using a fluorescence microscope with either propidium iodide (PI) (left) or FITC (FITC-dUTP) (right). In normal extraembryonic tissue, apoptotic cells were rarely identified while apoptotic cells significantly increased in SCNT extraembryonic tissue. Magnification: control and SCNT extraembryonic tissue (x200).
Mentions: To further investigate whether the developmental defects in the extraembryonic tissue of SCNT fetus affect the expression of antioxidation-related proteins or apoptotic marker proteins, such as GPX, catalase, Bax, Bcl2, and Hsp27, we performed western blot analysis using control and SCNT extraembryonic tissue. The expression levels of catalase or GPX related to oxidative stress were decreased in SCNT extraembryonic tissues (Figure 3C). The expression of Hsp27 was up regulated more than two-fold in SCNT extraembryonic tissue compared with control tissue, and these data are consistent with 2-D analysis. The expression of Bax, an apoptotic marker protein, was up-regulated, and the expression of the anti-apoptotic Bcl-2 gene decreased in the SCNT extraembryonic tissue (Figure 4A). Next, we investigated the expression of DNA methyltransferases 1, 3a and 3b (Dnmt 1, 3a and 3b) in control and SCNT extraembryonic tissue. We found that that 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). In contrast, the expression levels of Dnmt3a and 3b were highly detectable in the SCNT extraembryonic tissue, while they were low in control fetus (Figure 4B). These results suggest that a relatively low level of Dnmt1 activity exists during the development of SCNT fetus.Figure 4

Bottom Line: 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.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.These 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.

View Article: PubMed Central - PubMed

Affiliation: Animal Genetic Resources Station, National Institute of Animal Science, RDA, Namwon 590-832, Korea. kog4556@korea.kr.

ABSTRACT

Background: Somatic 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.

Results: In 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.

Conclusion: These 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.

No MeSH data available.