Lactation Biology 1

We hypothesized that accumulation of milk would influence gene expression in the mammary gland of lactating dairy cows. To test this hypothesis, we enrolled 4 multiparous Holstein cows (150 ± 10 DIM) in a half-udder milk stasis experiment. On d 1 of the experiment, right udder halves were milked at 0430 h and 1430 h. On d 2 of the experiment, right udder halves were milked at 0430 h and mammary biopsies were obtained from both udder halves immediately thereafter. At the time of biopsy, it had been 24 h since left udder halves had last been milked. Using Affymetrix GeneChip Bovine Genome Arrays, we identified 32 genes that were differentially expressed between left (full) and right (empty) udder halves (fold change >1.5; P < 0.05). Four of the genes were downregulated in response to milk stasis, whereas 28 were upregulated. Differentially expressed genes were associated with extracellular matrix remodeling, tight junction formation, regulation of blood flow, and apoptosis. In addition, 4 of the differentially expressed genes had been previously identified as candidates for local regulation of milk production in dairy cows. Expression of 2 of these candidates, early growth response-1 (EGR-1) and thrombospondin-1 (THBS-1), was validated using real-time quantitative RT-PCR. Consistent with microarray results, both genes were upregulated in response to 24-h of milk stasis (P < 0.03). Immunofluorescence was used to localize expression of EGR-1 protein, which was restricted to epithelia and was uniformly distributed. We conclude that accumulation of milk alters gene expression in the bovine mammary gland. In particular, EGR-1 and THBS-1 have emerged as strong candidates for local regulation of milk production in dairy cows.