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Abstract
Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, α-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin β subunit, and potassium channel tetramerisation domain-containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, α-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and α-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and α-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.
References
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Fig. 1.
Two-dimensional gel electrophoresis of bovine endometrium (A, B) Representative two-dimensional gel electrophoresis profiles of endometrium obtained from Korean cattle with and without endometritis. The soluble protein (100 μg) of each isolate was loaded on the 2-DE gel. The 2-DE gels were focused on 17-cm IPG strips with immobilized pH ranging 3 to 10 (A) or 5 to 8 (B). Proteins were then separated on 12.5% polyacrylamide gel. Molecular size markers are shown on the left-hand side of the gels. The proteins were visualized by silver staining. Number (1 to 14) represents DEP identified by PDQuest.
Fig. 2.
Differentially expressed proteins in bovine endometrium with endometritis (A) Details of 2-DE gel images showing selected DEPs. The enlarged images of DEPs contain some additional protein spots. (B) Summary of intensities of DEP spots. Each bar represents mean±SD of four repeated experiments. The asterisks indicate the significant difference from the corresponding control value obtained for normal endometrium (p<0.05).
Fig. 3.
Validation of 2-DE analysis by semi-quantitative PCR and western blot analyses in bovine endometrium, with and without endometritis. (A) RT-PCR products for desmin (453-bp), transferring (488-bp), α-actin (329-bp), HSP27 (300-bp), PRSIN-6 (402-bp), KCTD11 (344-bp), and MHC-I (428-bp) derived from bovine endometrium. The first lanes show the 1-kb DNA ladder. Bar graph shows normalized mRNA levels of desmin, transferrin, α-actin, HSP27, PRSIN-6, KCTD11, and MHC-I in the endometria with and without endometritis. The expression levels were normalized to GAPDH. Each bar represents mean±SD of five repeated experiments. The asterisks indicate the significant difference from the corresponding control value obtained for normal endometrium (p<0.05). N and E represent normal endometrium and endometrium with endometritis, respectively. (B, C) Western blot analyses of desmin, α-actin, HSP27 and HSP70 proteins. Ponceau S staining of total proteins served as a loading control. The bar graph shows the up-regulation of desmin and α-actin in endometrium with endometritis. Each bar represents the mean±SD of three repeated experiments. Asterisks indicate the significant difference from the corresponding control value (normal), p<0.05.
Table 1.
Primer sequences used for RT-PCR
Table 2.
List of differentially expressed proteins identified by MALDI-TOF-MS in the endometrium with endometritis
| Spot No. | Annotation | MW /pI | Swiss-Prot. Acc. Nos. | CV (%) | Fold change | MOWSE score | % Cov |
|---|---|---|---|---|---|---|---|
| 1 | Desmin | 53.4/5.2 | O62654 | 24.8/33.9 | 3.7 (Δ) | 558,976 | 43.7 |
| 2 | Serotransferrin precursor (Transferrin) | 77.8/6.8 | Q29443 | 58.9/37.9 | 0.37 (∇) | 387 | 21.7 |
| 3 | Actin, aortic smooth muscle (α-actin-2) | 42.0/5.2 | P62739 | 0/47.9 | 1.53 (Δ) | 10,758 | 42.2 |
| 4 | Interleukin-2 precursor (IL-2) | 17.6/6.1 | P05016 | – (∇)∗ | 228 | 18.1 | |
| 5 | Heat-shock protein β-1 (HSP27) | 22.4/6.0 | Q3T149 | 26.7/31.4 | 1.68 (Δ) | 123,678 | 45.9 |
| 6 | Peroxiredoxin-6 | 24.9/6.0 | O77834 | 43.4/23.9 | 1.82 (Δ) | 269 | 26.9 |
| 7 | Heat-shock protein beta-1 (HSP27) | 22.4/6.0 | Q3T149 | 10.8/8.2 | 3.93 (Δ) | 35,712 | 44.8 |
| 8 | Luteinizing hormone receptor isoform 1 (Fragment) | 24.7/5.2 | Q28026 | 43.2/22.6 | 2.27 (Δ) | 931 | 36.8 |
| 9 | Collectin-43 precursor (CL-43) | 33.6/5.0 | P42916 | 28.9/12.2 | 2.3 (Δ) | 194 | 28.4 |
| 10 | Hemoglobin β subunit | 16.0/7.0 | P02070 | 10.4/0 | - (∇)∗ | 226 | 51.7 |
| 11 | Hemoglobin β subunit | 16.0/7.0 | P02070 | 0.73 (∇) | 159 | 44.1 | |
| 12 | Potassium channel tetramerisation domain containing 11 (KCTD11) | 20.5/6.4 | Q58DF7 | 0.71 (∇) | 822 | 36.3 | |
| 13 | Deoxyribonuclease-1 | 31.3/5.3 | P00639 | + (Δ)† | 222 | 21.7 | |
| 14 | MHC class I heavy chain (Fragment) | 40.4/5.4 | O78186 | 0/56.4 | + (Δ)† | 23,348 | 42.5 |
Table 3.
List of hypothetical proteins identified by MALDI-TOF-MS in the endometrium with endometritis
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