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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.


Immunohistochemical analysis of antioxidant proteins in the control and SCNT extraembryonic tissue. (A) Catalase expression in the control extraembryonic tissue was mainly localized on the luminal epithelium (LE) and endometrial glandular (EG) cells. Magnification: control and SCNT extraembryonic tissue (x200). Scale bars represent 100 μm. (B) Immunohistochemical analysis of GPX in the control and SCNT extraembryonic tissue. GPX was strongly expressed in the luminal epithelium (LE) of the control extraembryonic tissue. The insect in (a, x200) and (c, x200) showed (b, x400) and (d, x400), respectively. Scale bars represent 100 μm. (C) Western blot analysis of antioxidant enzyme proteins in the control and SCNT extraembryonic tissue. The expression of GPX and Catalase was down-regulated in the SCNT extraembryonic tissue.
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Fig3: Immunohistochemical analysis of antioxidant proteins in the control and SCNT extraembryonic tissue. (A) Catalase expression in the control extraembryonic tissue was mainly localized on the luminal epithelium (LE) and endometrial glandular (EG) cells. Magnification: control and SCNT extraembryonic tissue (x200). Scale bars represent 100 μm. (B) Immunohistochemical analysis of GPX in the control and SCNT extraembryonic tissue. GPX was strongly expressed in the luminal epithelium (LE) of the control extraembryonic tissue. The insect in (a, x200) and (c, x200) showed (b, x400) and (d, x400), respectively. Scale bars represent 100 μm. (C) Western blot analysis of antioxidant enzyme proteins in the control and SCNT extraembryonic tissue. The expression of GPX and Catalase was down-regulated in the SCNT extraembryonic tissue.

Mentions: To determine the effect of the abnormal development of extraembryonic tissue in SCNT fetus, we performed immunohistochemical analysis of control and SCNT extraembryonic tissue. As shown in Figure 3, histological analysis of SCNT extraembryonic tisssues showed clusters of terminal villi with apparent syncytial bridging and sprouting. Adhesion occurred between the maternal and fetal epithelium with the formation of microvilli resulting in an interdigitation of both tissues. The expression of antioxidant proteins GPX and catalase revealed extensive positive staining in the extraembryonic tissues of control fetus; however, SCNT-derived extraembryonic tissue exhibited significantly lower expression levels of GPX and catalase (Figure 3A and B).Figure 3


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)

Immunohistochemical analysis of antioxidant proteins in the control and SCNT extraembryonic tissue. (A) Catalase expression in the control extraembryonic tissue was mainly localized on the luminal epithelium (LE) and endometrial glandular (EG) cells. Magnification: control and SCNT extraembryonic tissue (x200). Scale bars represent 100 μm. (B) Immunohistochemical analysis of GPX in the control and SCNT extraembryonic tissue. GPX was strongly expressed in the luminal epithelium (LE) of the control extraembryonic tissue. The insect in (a, x200) and (c, x200) showed (b, x400) and (d, x400), respectively. Scale bars represent 100 μm. (C) Western blot analysis of antioxidant enzyme proteins in the control and SCNT extraembryonic tissue. The expression of GPX and Catalase was down-regulated in the SCNT extraembryonic tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4289280&req=5

Fig3: Immunohistochemical analysis of antioxidant proteins in the control and SCNT extraembryonic tissue. (A) Catalase expression in the control extraembryonic tissue was mainly localized on the luminal epithelium (LE) and endometrial glandular (EG) cells. Magnification: control and SCNT extraembryonic tissue (x200). Scale bars represent 100 μm. (B) Immunohistochemical analysis of GPX in the control and SCNT extraembryonic tissue. GPX was strongly expressed in the luminal epithelium (LE) of the control extraembryonic tissue. The insect in (a, x200) and (c, x200) showed (b, x400) and (d, x400), respectively. Scale bars represent 100 μm. (C) Western blot analysis of antioxidant enzyme proteins in the control and SCNT extraembryonic tissue. The expression of GPX and Catalase was down-regulated in the SCNT extraembryonic tissue.
Mentions: To determine the effect of the abnormal development of extraembryonic tissue in SCNT fetus, we performed immunohistochemical analysis of control and SCNT extraembryonic tissue. As shown in Figure 3, histological analysis of SCNT extraembryonic tisssues showed clusters of terminal villi with apparent syncytial bridging and sprouting. Adhesion occurred between the maternal and fetal epithelium with the formation of microvilli resulting in an interdigitation of both tissues. The expression of antioxidant proteins GPX and catalase revealed extensive positive staining in the extraembryonic tissues of control fetus; however, SCNT-derived extraembryonic tissue exhibited significantly lower expression levels of GPX and catalase (Figure 3A and B).Figure 3

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.