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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-?B stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

SUBMITTER: Janjanam J

PROVIDER: S-EPMC4128602 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Janjanam Jagadeesh J Singh Surender S Jena Manoj K MK Varshney Nishant N Kola Srujana S Kumar Sudarshan S Kaushik Jai K JK Grover Sunita S Dang Ajay K AK Mukesh Manishi M Prakash B S BS Mohanty Ashok K AK

PloS one 20140811 8

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 diff ...[more]

PMID: 25111801

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Project description:Arginine, a semi-essential functional amino acid, has been found to promote the synthesis of casein in mammary epithelial cells to some extent. Data from mouse indicated that microRNA (miRNA) are important in regulating the development of mammary gland and milk protein synthesis. Whether there are potential links among arginine, miRNA and casein synthesis in bovine mammary gland is uncertain. The objective of the present work was to detect the effects of arginine supplementation on the expression of miRNA associated with casein synthesis in mammary tissue and mammary epithelial cells (BMEC). The first study with bovine mammary epithelial cells (BMEC) focused on screening for miRNA candidates associated with the regulation of casein production by arginine. The BMEC were cultured with three different media, containing 0, 1.6 and 3.2 mM arginine, for 24 h. The expression of candidate miRNA was evaluated. Subsequently, in an in vivo study, 6 Chinese Holstein dairy cows with similar BW (mean ± SE) (512.0 ± 19.6 kg), parity (3), BCS (4.0) and DIM (190 ± 10.3 d) were randomly assigned to three experimental groups. The experimental cows received an infusion of casein, arginine (casein plus double the concentration of arginine in casein), and alanine (casein plus alanine, i.e., iso-nitrogenous to the arginine group) in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). Mammary gland biopsies were obtained from each cow at the end of each infusion period. Results of the in vitro study showed differences between experimental groups and the control group for the expression of nine miRNA: miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, miR-712, miR-574-5p, miR-468 and miR-875-3p. The in vivo study showed that arginine infusion promoted milk protein content, casein yield and the expression of CSN1S1 and CSN1S2. Furthermore, the expression of miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712 was also greater in response to arginine injection compared with the control or alanine group. Overall, results both in vivo and in vitro revealed that arginine might partly influence casein yield by altering the expression of 6 miRNAs (miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712).

Project description:BACKGROUND: The lactating mammary gland responds to changes in milking frequency by modulating milk production. This response is locally regulated and, in dairy cows, the udder is particularly sensitive during early lactation. Relative to cows milked twice-daily throughout lactation, those milked four-times-daily for just the first 3 weeks of lactation produce more milk throughout that lactation. We hypothesized that the milk yield response would be associated with increased mammary cell turnover and changes in gene expression during frequent milking and persisting thereafter. Cows were assigned to unilateral frequent milking (UFM; left udder halves milked twice-daily; right udder halves milked four-times daily) on days 1 to 21 of lactation, followed by twice-daily milking for the remainder of lactation. Relative to udder halves milked twice-daily, those milked four-times produced more milk during UFM; the difference in milk yield declined acutely upon cessation of UFM after day 21, but remained significantly elevated thereafter. We obtained mammary biopsies from both udder halves on days 21, 23, and 40 of lactation. RESULTS: Mammary cell proliferation and apoptosis were not affected by milking frequency. We identified 75 genes that were differentially expressed between paired udder halves on day 21 but exhibited a reversal of differential expression on day 23. Among those genes, we identified four clusters characterized by similar temporal patterns of differential expression. Two clusters (11 genes) were positively correlated with changes in milk yield and were differentially expressed on day 21 of lactation only, indicating involvement in the initial milk yield response. Two other clusters (64 genes) were negatively correlated with changes in milk yield. Twenty-nine of the 75 genes were also differentially expressed on day 40 of lactation. CONCLUSIONS: Changes in milking frequency during early lactation did not alter mammary cell population dynamics, but were associated with coordinated changes in mammary expression of at least 75 genes. Twenty-nine of those genes were differentially expressed 19 days after cessation of treatment, implicating them in the persistent milk yield response. We conclude that we have identified a novel transcriptional signature that may mediate the adaptive response to changes in milking frequency.

Dataset Information

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

Publications

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

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