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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.


Related in: MedlinePlus

Two-dimensional protein separation of the control and SCNT extraembyonic tissue as visualized by the silver staining. Proteins were isolated from the control and SCNT extraembryonic tissue, and 1000 μg of total protein were loaded to the 2-D gel. The first dimension was 24-cm pH 4–10 nonlinear IPG, and the second dimension was 10 ~ 16% gradient gels (Control I and II, control extraembryonic tissues; SCNT I and II, SCNT extraembryonic tissues).
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Fig2: Two-dimensional protein separation of the control and SCNT extraembyonic tissue as visualized by the silver staining. Proteins were isolated from the control and SCNT extraembryonic tissue, and 1000 μg of total protein were loaded to the 2-D gel. The first dimension was 24-cm pH 4–10 nonlinear IPG, and the second dimension was 10 ~ 16% gradient gels (Control I and II, control extraembryonic tissues; SCNT I and II, SCNT extraembryonic tissues).

Mentions: To examine protein expression differences between control and SCNT fetus, we performed proteomics analysis using the extraembryonic tissue of SCNT and control fetus on day 35 of pregnancy. Figure 2 shows the profile of extraembryonic proteins from SCNT and control fetus using 2-D with a nonlinear IPG gradient of pH 4–10 and 10 ~ 16% gradient gels in the second dimension. A representative 2-D image of proteins in the control and SCNT extraembryonic tissue is shown in Figure 2, and 33 spots were identified as shown in Tables 2 and 3. After protein identification with mass spectrophotometry and protein database search, 3 proteins were up-regulated (2.0- to 4.5-fold increases) and 30 proteins were down-regulated (2.0- to 5.5-fold decreases) in the SCNT extraembryonic tissue (Tables 2 and 3). Tables 2 and 3 summarizes the primary properties of the 33 proteins that were differentially regulated in SCNT and control fetus. The proteins are classified into six groups based on function: (1) lipid binding proteins (2/33, 6%), (2) antioxidant proteins (3/33, 9%), (3) translation elongation factors (3/33, 9%), (4) ATP binding proteins (6/33, 18%), (5) transcription factors (2/36, 6%), and (6) others (15/36, 51%). The majority of the ATP binding proteins, antioxidant proteins, and metabolic-related proteins were down-regulated, whereas the expression levels of peroxisome proliferative activated receptor gamma (PPARγ), Hsp27, and elongation factor 1-gamma (EF1-γ) were up-regulated in SCNT extraembryonic tissue.Figure 2


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)

Two-dimensional protein separation of the control and SCNT extraembyonic tissue as visualized by the silver staining. Proteins were isolated from the control and SCNT extraembryonic tissue, and 1000 μg of total protein were loaded to the 2-D gel. The first dimension was 24-cm pH 4–10 nonlinear IPG, and the second dimension was 10 ~ 16% gradient gels (Control I and II, control extraembryonic tissues; SCNT I and II, SCNT extraembryonic tissues).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Two-dimensional protein separation of the control and SCNT extraembyonic tissue as visualized by the silver staining. Proteins were isolated from the control and SCNT extraembryonic tissue, and 1000 μg of total protein were loaded to the 2-D gel. The first dimension was 24-cm pH 4–10 nonlinear IPG, and the second dimension was 10 ~ 16% gradient gels (Control I and II, control extraembryonic tissues; SCNT I and II, SCNT extraembryonic tissues).
Mentions: To examine protein expression differences between control and SCNT fetus, we performed proteomics analysis using the extraembryonic tissue of SCNT and control fetus on day 35 of pregnancy. Figure 2 shows the profile of extraembryonic proteins from SCNT and control fetus using 2-D with a nonlinear IPG gradient of pH 4–10 and 10 ~ 16% gradient gels in the second dimension. A representative 2-D image of proteins in the control and SCNT extraembryonic tissue is shown in Figure 2, and 33 spots were identified as shown in Tables 2 and 3. After protein identification with mass spectrophotometry and protein database search, 3 proteins were up-regulated (2.0- to 4.5-fold increases) and 30 proteins were down-regulated (2.0- to 5.5-fold decreases) in the SCNT extraembryonic tissue (Tables 2 and 3). Tables 2 and 3 summarizes the primary properties of the 33 proteins that were differentially regulated in SCNT and control fetus. The proteins are classified into six groups based on function: (1) lipid binding proteins (2/33, 6%), (2) antioxidant proteins (3/33, 9%), (3) translation elongation factors (3/33, 9%), (4) ATP binding proteins (6/33, 18%), (5) transcription factors (2/36, 6%), and (6) others (15/36, 51%). The majority of the ATP binding proteins, antioxidant proteins, and metabolic-related proteins were down-regulated, whereas the expression levels of peroxisome proliferative activated receptor gamma (PPARγ), Hsp27, and elongation factor 1-gamma (EF1-γ) were up-regulated in SCNT extraembryonic tissue.Figure 2

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.


Related in: MedlinePlus