Transcriptome Analysis of Epithelial and Stromal Contributions to Mammogenesis in Three Week Prepartum Cows

Abstract

Transcriptome analysis of bovine mammary development has provided insight into regulation of mammogenesis. However, previous studies primarily examined expression of epithelial and stromal tissues combined, and consequently did not account for tissue specific contribution to mammary development. Our objective was to identify differences in gene expression in epithelial and intralobular stromal compartments. Tissue was biopsied from non-lactating dairy cows 3 weeks prepartum, cut into explants and incubated for 2 hr with insulin and hydrocortisone. Epithelial and intralobular stromal tissues were isolated with laser capture microdissection. Global gene expression was measured with Bovine Affymetrix GeneChips, and data were preprocessed using RMA method. Moderated t-tests from gene-specific linear model analysis with cell type as a fixed effect showed more than 3,000 genes were differentially expressed between tissues (P<0.05; FDR<0.17). Analysis of epithelial and stromal transcriptomes using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathways Analysis (IPA) showed that epithelial and stromal cells contributed distinct molecular signatures. Epithelial signatures were enriched with gene sets for protein synthesis, metabolism and secretion. Stromal signatures were enriched with genes that encoded molecules important to signaling, extracellular matrix composition and remodeling. Transcriptome differences also showed evidence for paracrine interactions between tissues in stimulation of IGF1 signaling pathway, stromal reaction, angiogenesis, neurogenesis, and immune response. Molecular signatures point to the dynamic role the stroma plays in prepartum mammogenesis and highlight the importance of examining the roles of cell types within the mammary gland when targeting therapies and studying mechanisms that affect milk production.

Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVβ3-integrin receptor

“…Thrombin and cathepsin D have been shown to cleave hOPN in vivo and could potentially also cleave bOPN in bovine milk. Thrombin generally has the same specificity as plasmin, although it preferentially cleaves after arginine residues (Weinstein and Doolittle, 1972); however, to our knowledge, thrombin has never been identified in bovine milk, although its expression has been detected by microarray analysis in lactating epithelial cells (Casey et al, 2011). The lysosomal aspartic proteinase cathepsin D is another well characterized milk protease and its concentration in bovine skim milk has been estimated to be approximately 0.4 μg/mL (Larsen et al, 1996).…”

Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes

“…Fibroblasts play crucial roles in the development and involution of murine, human, and bovine mammary glands, as well as in lactation678. Accumulating evidence suggests that the fibroblast functions as a structural element and as a vital immunoregulatory cell9.…”

“…The mammary gland is a complex organ of various tissue and cell types, and epithelia cells and fibroblasts are predominant components of mammary parenchyma and stroma, respectively 4 5 . Fibroblasts play crucial roles in the development and involution of murine, human, and bovine mammary glands, as well as in lactation 6 7 8 . Accumulating evidence suggests that the fibroblast functions as a structural element and as a vital immunoregulatory cell 9 .…”

Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation

“…Other transcriptome analyses were separately conducted on epithelial and intralobular stromal compartments of non-lactating dairy cows 3 weeks prepartum. Besides the differences between epithelial and stromal signatures, genes representing angiogenesis and neurogenesis were transcribed in both the epithelium and stroma (Casey et al 2011).…”