Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield

Abstract

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Figure 1. MECs isolation from milk using immunomagnetic beads.

The purified MECs which were separated from other somatic cells using immunomagnetic beads coated with anti-cytokeratin antibodies specific to mammary epithelial cells and observed under a microscope at 200×. White arrows indicate MECs and black arrows indicate immunomagnetic beads.

Figure 2. RT-PCR analysis of Bovine Mammary Epithelial cells.

MECs specific gene's primers (I); M: 100 bp ladder; A: SMA; B: Cytokeratin 8; C: β-casien and D: α-lactalbumin; (II): Loading control represents the house keeping gene β-actin (A). Other cell types using for CD19 (I), CD4 (II), CD45 (III) and IL-8 (IV); M: 100 bp ladder; A: Skin fibroblast (Negative control); B: Mammary Epithelial cells and C: Somatic cells (Positive control); Loading control represents the house keeping gene β-actin (B).

Figure 3. Schematic representation of 2D-DIGE experimental design.

Comparative proteome analysis of bovine mammary epithelial cells at Early, Peak and Late stages of lactation (A) and CyDye labeling and dye swapping for early, peak and late stage samples (B). Schematic representation of 2D-DIGE experimental plan for comparative proteome analysis of bovine MEC isolated from high and low milk yielding animals (C) and CyDye labeling for corresponding samples (D).

Figure 4. 2D-DIGE gel images.

All 12 gels of both experiments (gels 1–6 represents early vs peak vs late lactation; gels 7–12 represents low yielding Sahiwal cows vs high yielding Sahiwal cows vs high yielding Karan Fries (KF) cross bred cows) represented above. The gels were scanned using all the three lasers corresponding to Cy2, Cy3 and Cy5 wave lengths. The images were taken at 200 µ resolution. The green color spots are down regulating and red color spots are up regulating proteins.

Figure 5. Principal Component Analysis (PCA) of spot maps.

The different stages of lactation (A) and high and low-yielding samples (B) to represent high reproducibility among biological samples within each group.

Figure 6. Unsupervised Hierarchical Clustering (HC).

The expression maps and spot maps with relative expression values for each are displayed as heat map using a relative scale from −1 (green) to +1 (red). A. HC of total differentially regulated proteins identified at different stages of lactation and B. HC of total differentially regulated proteins identified from high and low milk yielding samples.

Figure 7. Categorization of identified proteins based on molecular function and biological process.

Differentially expressed proteins identified were categorized based on Molecular function (A) and Biological process (B) using panther classification system .

Figure 8. Network analysis of differentially expressed proteins.

The differentially expressed proteins were mapped onto existing mammalian pathways and networks of protein-protein interactions and other biochemical pathways reported previously in the literature. In this network 1, Red color features are the up-regulated proteins during peak stage and green color features are down-regulated during early stage of lactation. The full names of these proteins are shown in supporting information table S4.

Figure 9. Pathway analysis of differentially expressed proteins at early and late stages of lactation.

The differentially expressed proteins during early and late stages were mapped onto existing mammalian pathways and networks and in this network 2, the red color proteins represent up-regulated in late and down-regulated in early stages of lactation.

Figure 10. Pathway analysis of differentially expressed proteins in high and low milk yielding cows.

The differentially regulated proteins of high and low milk yielding samples were mapped onto existing mammalian pathways and networks. In this network 3, the red color proteins are up-regulated in high-yielding (Hy) and down-regulating in low-yielding (Ly) samples. The full names of these proteins are shown in supporting information table S5.

Figure 11. Validation of 2D-DIGE data by western blot.

Western blot analysis of annexin A1, ARP3, PGAM1 and Lamin-B1 proteins were differentially regulated during lactation stages and annexin A1 and vimentin were differentially regulated in high and low-milk yielding samples.

Figure 12. Validation of 2D-DIGE data by quantitative PCR and compared with the 2D-DIGE data.

Quantitative PCR analysis was performed for the genes shown in the figure and compared its fold expression change in relation to the 2D-DIGE data. A. The expression profile of proteins at peak and late stage of lactation were represented in comparison to early stage that was normalized to 1. B. The expression profile of proteins of Hy and KF samples were represented in comparison to Ly samples that was normalized to 1.