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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

Morphology of fetus and extraembryonic tissues in the control and SCNT-derived fetus from 35 day of pregnancy. (A) Control fetus and extraembryonic tissue from 35 day of pregnancy. (B) The SCNT fetus and extraembryonic tissue from 35 day of pregnancy. a and a’, embryo; b and b’, amnionic sac; c and c’ extraembryonic tissue. The weight of fetus was measured after removal of all extraembryonic tissues including chorioallantois, amniotic and allantoic fluid, and umbilical cord. Bar, 10 mm.
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Fig1: Morphology of fetus and extraembryonic tissues in the control and SCNT-derived fetus from 35 day of pregnancy. (A) Control fetus and extraembryonic tissue from 35 day of pregnancy. (B) The SCNT fetus and extraembryonic tissue from 35 day of pregnancy. a and a’, embryo; b and b’, amnionic sac; c and c’ extraembryonic tissue. The weight of fetus was measured after removal of all extraembryonic tissues including chorioallantois, amniotic and allantoic fluid, and umbilical cord. Bar, 10 mm.

Mentions: A total of 1500 nuclear transferred embryos at the 1- to 2-cell stage were surgically transferred to the oviducts of 18 recipients. Eight recipients (8 pregnancy/18 recipient) were confirmed to be pregnant at day 35 after the transfer of the embryo. As shown in Table 1, the number of recovered SCNT-derived fetus (44.4% pregnancy) at gestational day 35 is significantly lower than the number of fetus recovered from the control (100% pregnancy) (Table 1). The control conceptuses (normal) were obtained from recipients that were impregnated by natural mating. The size and shape of fetus and extraembryonic tisssues were observed in the microscopic analysis. 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). Abnormal blood vessel development was observed in the extraembryonic tissue of SCNT fetus. Interestingly, the sizes of the allantoic and the amniotic sac of both SCNT fetus and control fetus were similar. The fetal weight of the SCNT groups (2.71 ± 0.4) was significantly smaller than the weight of the control groups (3.34 ± 0.26) (Table 1). The number of recovered fetuses from the control group (11.33 ± 1.33) was higher than the number recovered from the SCNT group (4.67 ± 2.33) (Table 1). The number of abnormal fetus with SCNT group (2.33 ± 0.33) was significantly higher than the control group (1 ± 0) (Table 1). Together, these results demonstrated that the extraembryonic tissue in the SCNT fetus showed an abnormally small size and shape, and displayed abnormal morphology of the extraembryonic tissue compared with control (natural mating).Table 1


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)

Morphology of fetus and extraembryonic tissues in the control and SCNT-derived fetus from 35 day of pregnancy. (A) Control fetus and extraembryonic tissue from 35 day of pregnancy. (B) The SCNT fetus and extraembryonic tissue from 35 day of pregnancy. a and a’, embryo; b and b’, amnionic sac; c and c’ extraembryonic tissue. The weight of fetus was measured after removal of all extraembryonic tissues including chorioallantois, amniotic and allantoic fluid, and umbilical cord. Bar, 10 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Morphology of fetus and extraembryonic tissues in the control and SCNT-derived fetus from 35 day of pregnancy. (A) Control fetus and extraembryonic tissue from 35 day of pregnancy. (B) The SCNT fetus and extraembryonic tissue from 35 day of pregnancy. a and a’, embryo; b and b’, amnionic sac; c and c’ extraembryonic tissue. The weight of fetus was measured after removal of all extraembryonic tissues including chorioallantois, amniotic and allantoic fluid, and umbilical cord. Bar, 10 mm.
Mentions: A total of 1500 nuclear transferred embryos at the 1- to 2-cell stage were surgically transferred to the oviducts of 18 recipients. Eight recipients (8 pregnancy/18 recipient) were confirmed to be pregnant at day 35 after the transfer of the embryo. As shown in Table 1, the number of recovered SCNT-derived fetus (44.4% pregnancy) at gestational day 35 is significantly lower than the number of fetus recovered from the control (100% pregnancy) (Table 1). The control conceptuses (normal) were obtained from recipients that were impregnated by natural mating. The size and shape of fetus and extraembryonic tisssues were observed in the microscopic analysis. 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). Abnormal blood vessel development was observed in the extraembryonic tissue of SCNT fetus. Interestingly, the sizes of the allantoic and the amniotic sac of both SCNT fetus and control fetus were similar. The fetal weight of the SCNT groups (2.71 ± 0.4) was significantly smaller than the weight of the control groups (3.34 ± 0.26) (Table 1). The number of recovered fetuses from the control group (11.33 ± 1.33) was higher than the number recovered from the SCNT group (4.67 ± 2.33) (Table 1). The number of abnormal fetus with SCNT group (2.33 ± 0.33) was significantly higher than the control group (1 ± 0) (Table 1). Together, these results demonstrated that the extraembryonic tissue in the SCNT fetus showed an abnormally small size and shape, and displayed abnormal morphology of the extraembryonic tissue compared with control (natural mating).Table 1

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