Effects of transforming growth factor-β on mammary remodeling during the dry period of dairy cows

Mammary remodeling in dairy cows involves coordinated changes in stromal and epithelial tissue. Tissue remodeling is characterized by changes in cell proliferation, activation of fibroblasts into myofibroblasts, and changes in extracellular matrix content. Transforming growth factor β-1 (TGF-β1) increases differentiation of fibroblasts to myofibroblasts, regulates expression of extracellular matrix proteins and proteases, and has cell-type dependent effects on proliferation. The objective of this study was to determine whether TGF-β1 treatment of mammary tissue from cows in late lactation and the dry period affects cell proliferation, expression of matrix metalloproteinase-3 (MMP-3) and fibronectin (FN), and the differentiation of fibroblasts into myofibroblasts that express smooth muscle α actin (SMA). Tissue was biopsied from 7 Holstein cows at 4 time points: late lactation, 1 wk after dry-off, 3 wk before expected calving, and 1 wk before expected calving. Explants of biopsied tissue were incubated for 2 h in Waymouth's medium containing insulin, hydrocortisone, and 0 or 5 ng of TGF-β1/mL; a subset of cultures was also incubated with bromodeoxyuridine to measure epithelial and stromal cell proliferation. Tissues were fixed, embedded in paraffin, sectioned, and stained by immunohistochemistry. Stage at biopsy had an overall effect on rate of epithelial and stromal cell proliferation, and TGF-β1 treatment increased rate of bromodeoxyuridine incorporation more than 2-fold in both cell types at 1 wk after dry-off. The number of fibroblasts expressing SMA was 19% higher in the intralobular stroma at 1 wk after dry-off compared with that at 1 wk before expected calving, and the percentage of activated fibroblasts tended to be higher in tissue incubated with TGF-β1. Biopsy stage had an overall effect on percentage area of epithelium expressing FN and MMP-3. Incubation with TGF-β1 had no effect on percentage intralobular stroma area expressing FN or MMP-3. Effects of TGF-β1 treatment were most apparent at 1 wk after dry-off, indicating that the first week of dry period may be an ideal target for testing effects in vivo.