Our results demonstrate, for the first time, the unique characteristics of the transcriptomes of epithelial cells and intralobular stromal fibroblasts in mammary tissue of dry cows at three weeks prepartum. Differential gene expression between the compartments was characterized by an enrichment of genes that primarily had intracellular functions in the epithelial compartment and extracellular functions in the stromal compartment. Further, expression analysis enabled the dissecting of the tissue specific contributions for preparing the mammary gland for milk synthesis.
The epithelial transcriptome was enriched with genes that clustered in ontologies and pathways characteristic of cellular turnover and development of protein and lipid synthetic machinery. Specifically, the enrichment of genes in molecular and cellular functions: cellular growth and proliferation, cell death and cell cycle indicate that at this point in mammary development the epithelial compartment is replacing old senescent cells with new cells, and cells are preparing to synthesize proteins and lipids that are necessary for lactation. Enrichment of genes in cellular assembly and organization, RNA trafficking, protein trafficking and energy metabolism molecular and cellular functions is indicative of the development of cellular infrastructure necessary to support the increased rates of protein synthesis and secretory activity needed for lactation 
. Canonical pathways enriched with genes expressed at a greater level in epithelium reflected its metabolic and synthetic roles (oxidative phosphorylation, citrate cycle, protein ubiquitination pathway) and its role in initiating angiogenesis (angiopoetin signaling) and neurogenesis (neuregulin signaling).
IPA network analysis enabled the visualization of cellular distribution and queries into interactions among molecules encoded by genes that enriched epithelial or stromal transcriptomes. The top five IPA epithelial networks were composed of genes that encoded for molecules with intracellular functions. Functions of these molecules included regulators of transcription, translation and intracellular trafficking, reflective of the role of epithelial cells in the mammary and the building of cellular machinery necessary for milk synthesis 
Genes in stromal transcriptome enriched cellular ontologies specific for ECM and secreted molecules. Enriched cellular and molecular functions indicated that genes expressed at a greater level in the intralobular stroma encoded proteins that regulate cell-cell signaling, cellular movement, and cellular development. Stromal molecular signatures also reflected that cell turnover, proliferation and death occurred in mammary stroma of three week prepartum dry cows. Specifically, molecular and cellular functions: cellular growth and proliferation, cell death and cell cycle were enriched with genes expressed at a greater level in stromal fibroblasts.
Translation of proliferation signatures in both tissues was evident in the presence of approximately 0.34% and 0.37% BrdU labeled epithelial and stromal cells, respectively. Relative rate of stromal cell proliferation was similar to rates reported for stromal fibroblasts in dry cows three weeks prepartum 
. However the relative rate of epithelial cell proliferation was 10- to 20-fold less than rates previously reported. Norgaard et al. reported that 6% and 12% of bovine mammary epithelial cells expressed the proliferation marker Ki67 at 4 weeks and 2 weeks prepartum 
. Using a protocol similar to ours, Capuco et al. reported an epithelial labeling index of 2.8% in 3 week prepartum dry cows 
. Further, in the Norgaard study, tissue was immediately fixed after biopsy, while for both Capuco's study and our study tissue was incubated for 2 hrs in culture with a uridine label to attain labeling index prior to fixation. Thus differences in relative rates of epithelial cell proliferation, may be due to differences in approaches used to attain indexes and/or stages sampled. The lower rate in our study and Capuco's study relative to the Norgaard study also suggest that, although tissue integrity was maintained in short term culture (see ), epithelial cells in tissue culture have a lower rate of proliferation. In this regard, it is important that we also acknowledge that the transcriptional repertoire may be affected by the 2 hrs of explant culture. Our intent was to maintain state of differentiation by incubating tissue with Ins and Hyd 
. However transition into an in vitro
environment also removes the progesterone block, which may lead to secretory activation (for review 
), and thus may have induced elements of the epithelial signature indicative of lipid synthesis and secretion.
The top five stromal networks had a large proportion of genes that encoded extracellular molecules including laminins, known to regulate milk protein synthesis 
, multiple proteases including MMP and ADAM metallopeptidases, which release signaling factors from the ECM, and several growth factors including IL6, IGF1, and NOV. Cytosolic proteins encoded by genes within the stromal network were primarily related to clathrin-mediated endocytosis. Endocytosis is required for a vast number of functions that are essential for the well being of the cell, including regulation of nutrient uptake, cell adhesion and migration, and receptor signaling. Thus enrichment of these genes likely reflected the very active state of stromal tissue during this period of mammary development.
Mammary stroma is a heterogeneous environment consisting of multiple cell types including fibroblasts, immune, endothelial and nerve cells. Several of the canonical pathways enriched with genes expressed at a greater level in the intralobular stromal compartment reflected this heterogeneous environment. These pathways included Dendritic Cell Maturation, Leukocyte Extravasation Signaling, Complement System, VEGF Signaling and Acute Phase Response Signaling. Enrichment of these pathways suggests that stromal compartment regulates these processes. It is unclear whether these signatures were derived from enriched fibroblasts or other stromal cell types that may have been captured during LCM, as genes that enrich these pathways may be transcribed by stromal fibroblasts and act on corresponding tissues in a paracrine manner or may be indicative of the presence of nervous tissue, endothelial cells and/or immune cells in the stromal isolates.
Enrichment of genes expressed at a greater level in intralobular stroma in the Hepatic Fibrosis/Hepatic Stellate Cell Activation canonical pathway, stromal network 3 (collagens, TGFBR2, SPARC, ENG), ECM gene ontology and VEGF signaling pathway suggest that stromal activation is being induced during this period of mammary gland development. Stromal activation is associated with remodeling during wound healing and pathologically with cancer and fibrosis 
and is likely a critical component of normal mammary remodeling during the prepartum period. Stromal activation is characterized by fibroblast activation to the myofibroblast phenotype, stimulation of collagen type I deposition and induction of angiogenesis 
. Myofibroblasts express smooth muscle α-actin, produce proteases such as uPA and stromelysin-3 as well as ECM molecules including collagen I, II and III, fibronectin, and tenascin 
. It is possible that stromal activation is initiated by transforming growth factor beta 2 (TGFB2) and vascular endothelial growth factor (VEGF) secreted by epithelial cells. TGFB activates mammary fibroblasts into myofibroblasts in vitro 
and in vivo 
. We have also shown that TGFB1 increases the number of myofibroblasts in mammary fibroblasts from dry cows, which supports this signature (unpublished data).
There was also transcriptome evidence for paracrine interactions between the tissues with the enrichment of the IGF1 canonical pathway and expression of IGF binding proteins. The paracrine effects of stromally derived IGF1 on epithelial cell proliferation in the bovine mammary gland is well established 
. Our stromal and epithelial transcriptomes were consistent with these studies and the molecular signatures suggest that the IGF1 signaling pathway leading to proliferation of epithelial cells is stimulated via IGF1 that was transcribed in the stromal compartment.
Examination of genes that encode proteins with plasma membrane and extracellular distributions gave insight into the interaction between the tissues and pathways that may be active during this period of mammogenesis (). Epithelial and stromal contributions to angiogenesis, neurogenesis, immune response and tissue remodeling were unique and were reflected in differential expression of several VEGF molecules, cytokines, and protease molecules. The neurogenesis signatures suggest that both stromal and epithelial tissue participate in increasing nervous innervation of the mammary gland prior to lactation. Increased innerveration of the mammary gland during pregnancy likely reflects preparation for the neuroendocrine response to suckling/milking that is important to milk let down and maintaining lactation. Greater expression of GHR and PRLR transcripts in the epithelial compartment suggests the mammary gland was able to respond to the systemic effects of respective hormones during this period of development, although this would need to be confirmed with protein expression. Differences in expression of growth factors and receptors suggested that local signals for cellular proliferation and differentiation were mediated by paracrine interactions between the compartments at this time. For example higher expression of the growth factors that stimulate fibroblast proliferation/activation (TGFB2 and PDGF) in the epithelial compartment was mirrored by higher expression of their receptors (TGFBR2 and PDGFR) in the stromal compartment.
Interestingly although both epithelial and stromal transcriptomes showed differential contributions to the expression of proteases that remodel the ECM, only the stromal compartment showed evidence of expression of ECM molecules. These signatures were validated using immunohistochemistry. Staining showed the differential distribution of proteins that make up the extracellular matrix, fibronectin, and remodel the ECM, MMP3, as well as TGFB1 and its type II receptor (TGFBR2) ( and 
Both compartments showed unique expression of integrin receptors that serve as cell-ECM receptors and are responsible for initiating signals important to cell proliferation and differentiation. These findings suggest that stromal fibroblasts play an important role in regulating mammary function and differentiation through the generation of developmentally specific ECM molecules. The greater expression of SFRP (secreted fizzled-related proteins) in the stromal cell compartment also suggested a role for Wnts and related molecules in mediating mammogenesis during this stage of development. SFRPs have been shown to block Canonical Wnt Signaling pathways and alternatively to behave in a paracrine manner to stimulate epithelial branching 
Mammary epithelial and intralobular stromal cells contribute distinct molecular signatures to the mammary transcriptome in dry cows at 3 weeks prepartum. The epithelial signature was characterized by enrichment of molecules that regulate protein synthesis, metabolism and secretion. In contrast the stromal signature was primarily characterized as being enriched with genes that encode extracellular molecules important to signaling, ECM synthesis and ECM remodeling. Tissue specific signatures also provided evidence of paracrine stimulation of the IGF1 signaling pathway in the epithelial compartment from stromally produced IGF1 and stimulation of the stromal reaction from the epithelial compartment. Genes representing angiogenesis and neurogenesis were transcribed in both the epithelium and stroma. Molecular signatures point to the dynamic role the stroma plays in synthesis of signaling molecules that stimulate epithelial morphogenesis and proliferation, as well as in the synthesis of ECM conducive to milk protein synthesis. These signatures also highlight the importance of examining the roles of cells within the mammary gland when targeting therapies and studying mechanisms that impact milk production.