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Bovine mammary epithelial cell cultures for the study of mammary gland functions.

ABSTRACT: In the present study, the analysis of epithelial cells derived from various sources was undertaken, beginning from the mammary gland tissue through the primary cultures and their subsequent passages. The objective of the study was the comparative analysis of the stage in which the epithelial cells obtained from individuals in different lactation cycles and disparate phases of cell culture growth are the most suitable for morphological research and analysis of gene expression activity. The cultures of primary bovine mammary epithelial cells and passages were identified morphologically using immunocytochemical methods. After positive identification, real-time PCRs were performed for the analysis of the expression level of casein genes, whey protein genes, and butyrophilin gene. The most stable reference genes in real-time PCRs for the mammary gland tissue and cell cultures were also determined. Of the reference genes, the UXT and GAPDH genes appeared to be the most stable ones for the mammary gland tissue samples and epithelial cell cultures. The results obtained allowed concluding that the mammary gland samples collected from heifers constituted the most effective material for the initiation of primary cultures. The primary cultures formed characteristic for the mammary gland tissue dome structures, which images were obtained using confocal microscopy. The highest levels of expression of the CSN1S1, CSN1S2, CSN2, and CSN3 genes were detected in primary cultures. The levels of expression of whey protein genes (LALBA and BGL) were highest in the second passage. The most abundant expression of the BTN1A1 gene was observed in primary cultures and the third passage. On the basis of the whole experiment, it can be concluded that primary cultures and cells of the second passage derived from heifer individuals appeared to be the best materials for the analysis of mammary gland function and gene expression activity.

SUBMITTER: Jedrzejczak M

PROVIDER: S-EPMC4047483 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fibroblast growth factor (FGF) signaling plays essential roles in tissue development and homeostasis. Accumulating evidence reveals that fibroblast growth factor 2 (FGF2) regulates ductal elongation, which requires cell proliferation and epithelial expansion in the mammary gland. However, the function and mechanisms by which FGF2 controls functionality of epithelial cells is less well defined. Here, we demonstrate the functional effects of FGF2 on bovine mammary epithelial (MAC-T) cells and the intracellular signaling mechanisms for these FGF2-induced actions. The current results show that treatment of MAC-T cells with a recombinant FGF2 induced cell proliferation and cell-cycle progression with increased expression of proliferating cell nuclear antigen and cyclin D1. Moreover, FGF2 increased phosphorylation of serine/threonine protein kinase (protein kinase B [AKT]), extracellular signal-regulated kinases 1 and 2 (ERK1/2), Jun N-terminal kinase (JNK), 70 kDa ribosomal S6 kinase (P70S6K), 90 kDa ribosomal S6 kinase (P90S6K), ribosomal protein S6 (S6), and cyclin D1 proteins. These FGF2-induced activations of signaling pathway proteins were inhibited by blocking AKT, ERK1/2, or JNK phosphorylation. The effect of FGF2 to stimulate MAC-T cell proliferation was mediated by activation of FGF receptors (FGFR) and AKT, ERK1/2, and JNK mitogen-activated protein kinase pathways in response to FGF2 stimulation. Furthermore, expression and activation of endoplasmic reticulum (ER) stress-related factors and ER stress-induced MAC-T cell death was reduced by FGF2. Together, these results suggest that the FGF2-FGFR-intracellular signaling cascades may contribute to maintaining and/or increasing numbers of mammary epithelial cells by inducing proliferation of mammary epithelial cells and by protecting cells from ER stress responses. Therefore, this study provides evidence that FGF2 signaling is a positive factor for mammary gland remodeling and for increasing persistency of milk production.

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Bovine mammary epithelial cell cultures for the study of mammary gland functions.

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