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Discovery of porcine maternal factors related to nuclear reprogramming and early embryo development by proteomic analysis.

Zhao Q, Guo Z, Piao S, Wang C, An T - Proteome Sci (2015)

Bottom Line: In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05).Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05).These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Northeast Forestry University, 26 Hexing Road, Xiangfang Dist., Harbin, Helongjiang 150040 China.

ABSTRACT

Background: Differentiated cell nuclei can be reprogrammed to a pluripotent state in several ways, including incubation with oocyte extracts, transfer into enucleated oocytes, and induced pluripotent stem cell technology. Nuclear transfer-mediated reprogramming has been proven to be the most efficient method. Maternal factors stored in oocytes have critical roles on nuclear reprogramming and early embryo development, but remain elusive.

Results: In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05). Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05). But 33O could sustain IVF embryo development with higher cleavage and blastocyst rates comparing to 42O (p < 0.05). To clarify the development potential difference between 33O and 42O, 18 differentially expressed proteins were identified by proteomic analysis, and randomly selected proteins were confirmed by Western blot. Bioinformatic analysis of these proteins revealed that 33O highly synthesized proteins related to fertilization, and 42O was rich in nuclear reprogramming factors.

Conclusions: These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.

No MeSH data available.


Related in: MedlinePlus

2D DIGE data validation. Though PCR results of 13 genes were inconsistent with the 2D DIGE results (a), the protein expression of 3 randomly selected genes (PDIA3, SOD1 and VIM) checked by Western blot (c) were in agreement with the gray values of these spots in the 2D DIGE (b). Histone H2B served as loading control
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Fig4: 2D DIGE data validation. Though PCR results of 13 genes were inconsistent with the 2D DIGE results (a), the protein expression of 3 randomly selected genes (PDIA3, SOD1 and VIM) checked by Western blot (c) were in agreement with the gray values of these spots in the 2D DIGE (b). Histone H2B served as loading control

Mentions: To uncover the basis for nuclear reprogramming and embryo development, global protein changes between 33O and 42O were examined by proteomic analysis. After oocytes collection and treatment, the total proteins were separated by 2D DIGE (Fig. 2). Analysis of the gel images showed 994 paired protein spots, and we used independent t-test to calculate differentially expressed proteins. We considered only spots with fold-changes greater than 1.5 to exclude the possibility of false positives by multiple comparisons. In total, 25 protein spots were found to be differentially expressed with fold-changes greater than 1.5 (p < 0.05; Fig. 3a), and finally 18 proteins were identified by MALDI-TOF/MS. Based on 2D DIGE analysis, 7 proteins were down-regulated and 11 proteins were up-regulated in comparison of 42O to 33O (Fig. 3b and Table 3). The 7 over-expressed proteins in 33O were indentified as protein-arginine deiminase type-6 (PADI6), major vault protein isoform 1(MVP), inositol polyphosphate-1-phosphatase (INPP1), glucose regulated protein 58 (PDIA3), glial fibrillary acidic protein (GFAP), 90-kDa heat shock protein (HSP90B1), and beta-actin(ACTG1). The 11 high expressed proteins in 42O were trypsinogen precursor (PRSS1), ADP-sugar pyrophosphatase-like isoform 1 (NUDT5), vimentin-like (VIM), heat shock 70 kDa protein 5 (HSPA5), heat shock 90kD protein 1 (HSP90AB1), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), similar to GLUD1 protein (GLUD1), glutathione S-transferasemu2 (GSTM2), superoxide dismutase [Cu-Zn] (SOD1), DJ-1 protein (PARK7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To comfirm the results, 2D DIGE data were examined by Quantitative realtime PCR and Western blot (Fig. 4). Though PCR results of 13 genes were inconsistent with the 2D DIGE results (Fig. 4a), the gray values of 3 randomly selected spots (PDIA3, SOD1 and VIM) in the 2D DIGE (Fig. 4b) were in agreement with the protein expressions of these genes checked by Western blot (Fig. 4c).Fig. 2


Discovery of porcine maternal factors related to nuclear reprogramming and early embryo development by proteomic analysis.

Zhao Q, Guo Z, Piao S, Wang C, An T - Proteome Sci (2015)

2D DIGE data validation. Though PCR results of 13 genes were inconsistent with the 2D DIGE results (a), the protein expression of 3 randomly selected genes (PDIA3, SOD1 and VIM) checked by Western blot (c) were in agreement with the gray values of these spots in the 2D DIGE (b). Histone H2B served as loading control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493956&req=5

Fig4: 2D DIGE data validation. Though PCR results of 13 genes were inconsistent with the 2D DIGE results (a), the protein expression of 3 randomly selected genes (PDIA3, SOD1 and VIM) checked by Western blot (c) were in agreement with the gray values of these spots in the 2D DIGE (b). Histone H2B served as loading control
Mentions: To uncover the basis for nuclear reprogramming and embryo development, global protein changes between 33O and 42O were examined by proteomic analysis. After oocytes collection and treatment, the total proteins were separated by 2D DIGE (Fig. 2). Analysis of the gel images showed 994 paired protein spots, and we used independent t-test to calculate differentially expressed proteins. We considered only spots with fold-changes greater than 1.5 to exclude the possibility of false positives by multiple comparisons. In total, 25 protein spots were found to be differentially expressed with fold-changes greater than 1.5 (p < 0.05; Fig. 3a), and finally 18 proteins were identified by MALDI-TOF/MS. Based on 2D DIGE analysis, 7 proteins were down-regulated and 11 proteins were up-regulated in comparison of 42O to 33O (Fig. 3b and Table 3). The 7 over-expressed proteins in 33O were indentified as protein-arginine deiminase type-6 (PADI6), major vault protein isoform 1(MVP), inositol polyphosphate-1-phosphatase (INPP1), glucose regulated protein 58 (PDIA3), glial fibrillary acidic protein (GFAP), 90-kDa heat shock protein (HSP90B1), and beta-actin(ACTG1). The 11 high expressed proteins in 42O were trypsinogen precursor (PRSS1), ADP-sugar pyrophosphatase-like isoform 1 (NUDT5), vimentin-like (VIM), heat shock 70 kDa protein 5 (HSPA5), heat shock 90kD protein 1 (HSP90AB1), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), similar to GLUD1 protein (GLUD1), glutathione S-transferasemu2 (GSTM2), superoxide dismutase [Cu-Zn] (SOD1), DJ-1 protein (PARK7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To comfirm the results, 2D DIGE data were examined by Quantitative realtime PCR and Western blot (Fig. 4). Though PCR results of 13 genes were inconsistent with the 2D DIGE results (Fig. 4a), the gray values of 3 randomly selected spots (PDIA3, SOD1 and VIM) in the 2D DIGE (Fig. 4b) were in agreement with the protein expressions of these genes checked by Western blot (Fig. 4c).Fig. 2

Bottom Line: In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05).Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05).These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Northeast Forestry University, 26 Hexing Road, Xiangfang Dist., Harbin, Helongjiang 150040 China.

ABSTRACT

Background: Differentiated cell nuclei can be reprogrammed to a pluripotent state in several ways, including incubation with oocyte extracts, transfer into enucleated oocytes, and induced pluripotent stem cell technology. Nuclear transfer-mediated reprogramming has been proven to be the most efficient method. Maternal factors stored in oocytes have critical roles on nuclear reprogramming and early embryo development, but remain elusive.

Results: In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05). Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05). But 33O could sustain IVF embryo development with higher cleavage and blastocyst rates comparing to 42O (p < 0.05). To clarify the development potential difference between 33O and 42O, 18 differentially expressed proteins were identified by proteomic analysis, and randomly selected proteins were confirmed by Western blot. Bioinformatic analysis of these proteins revealed that 33O highly synthesized proteins related to fertilization, and 42O was rich in nuclear reprogramming factors.

Conclusions: These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.

No MeSH data available.


Related in: MedlinePlus