Korean J Physiol Pharmacol.  2010 Aug;14(4):205-212. 10.4196/kjpp.2010.14.4.205.

Proteomic Analysis of Differentially Expressed Proteins in Bovine Endometrium with Endometritis

• Choe C
• Park JW
• Kim ES
• Lee SG
• Park SY
• Lee JS
• Cho MJ
• Kang KR
• Han J
• Kang D

• KMID: 2285404
• DOI: http://doi.org/10.4196/kjpp.2010.14.4.205

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, alpha-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 beta 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, alpha-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 alpha-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and alpha-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

Keyword

Actin; Desmin; Endometritis; Proteomics

MeSH Terms

Actins
Collectins
Desmin
Endometritis
Endometrium
Female
Heat-Shock Proteins
Hemoglobins
Interleukin-2
Potassium Channels
Proteins
Proteomics
Receptors, LH
RNA, Messenger
Transferrin
Actins
Collectins
Desmin
Heat-Shock Proteins
Hemoglobins
Interleukin-2
Potassium Channels
Proteins
RNA, Messenger
Receptors, LH
Transferrin

Figure

• 

  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.

Reference

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