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Direct transcriptomics comparison of pre-weaned bovine mammary parenchyma and fat pad

ABSTRACT: Direct transcriptomics comparison corroborated by functional and gene network analyses of pre-weaned bovine mammary parenchyma and fat pad shed light on potential cross-talk between these developing tissues. Transcriptomic characterization of mammary parenchyma and fat pad and their interaction is still incomplete. In the present experiment, the molecular epithelial-fat pad cross-talk during mammary development was assessed by transcript profiling and functional analyses. Background: Interactions between bovine mammary parenchyma (PAR) and fat pad (MFP) during neonatal tissue development are still not fully understood. It is thought that the MFP surrounding the parenchymal tissue exerts proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. Main objectives in the present study were to use bioinformatics tools to characterize differences in transcript profiles between mammary PAR and MFP from Holstein heifers at ca. 65 d age and uncover potential cross-talk between the two tissues via the analyses of signaling molecules (e.g., cytokines and growth factors) preferentially expressed in one tissue relative to the other. Results: Over 9,000 differentially expressed genes (DEG; False discovery rate ≤ 0.05) were found of which 1,478 had a ≥1.5-fold difference. Within these DEG in PAR vs. MFP (n = 736) we noted enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Few canonical pathways were among DEG and were mostly involved in cell cycle and tissue organization (p53 signaling). Enriched among DEG more highly-expressed in MFP vs. PAR (n = 742) were genes involved in lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism (fatty acid metabolism) and signaling, particularly immune- (acute phase response) and metabolism-related (cAMP signaling), were significantly enriched. Network analysis uncovered a central role of MYC, TP53, and CTNNB1 in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis revealed a central role of PPARG, KLF2, EGR2, and EPAS1 in regulating expression of highly-expressed DEG in MFP vs. PAR. Analyses revealed putative crosstalk between tissues via differential expression of cytokines and growth factors highly-expressed in PAR (angiopoietin-1, osteopontin, interleukin-1β) or in MFP (adiponectin, interleukin-13, fibroblast growth factor-2, leptin) during bovine mammary development. Conclusions: We uncovered specific transcriptomic signatures characterizing MFP and PAR tissue. Not surprisingly, the expression profile of the MFP is characteristic of adipose tissue, and the one of PAR is characteristic of an epithelial tissue undergoing expansion and remodeling. Overall, our data highlighted a large degree of interaction between the two tissues and allowed envisaging a reciprocal influence in determining the biological features (and perhaps the fate) of each of the two tissues during this stage of development. This is strongly suggested by the potential effect that the signaling molecules released preferentially by PAR have on lipid metabolism-related pathways, which from our data was what most distinguished the MFP from PAR. Similarly, the cytokines and growth factors largely expressed in MFP potentially affect the pathways related to cell cycle, development, and proliferation in PAR, which our data highlighted as the main functions represented among the genes highly expressed in PAR vs. MFP. Based on the current analysis, the number of cytokines and growth factors that potentially are secreted by each tissue and affect molecules in the other underscores the concept of crosstalk. Ultimately, these bidirectional interactions might be required to coordinate mammary tissue development under normal circumstances or in response to environmental stimuli, such as nutrition.

ORGANISM(S): Bos taurus

PROVIDER: GSE20363 | GEO | 2010/06/01

SECONDARY ACCESSION(S): PRJNA125457

REPOSITORIES: GEO

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Project description:Background: To reduce costs of rearing replacement heifers, researchers have focused on decreasing age at breeding and first calving, and to increase returns upon initiation of lactation the focus has been on increasing mammary development prior to onset of first lactation. Enhanced plane of nutrition pre-weaning may benefit the entire replacement heifer operation by promoting mammary gland development and greater future production. Methods: Twelve Holstein heifer calves (<1 wk old) were reared on 1 of 2 dietary treatments (n = 6/group) for 8 wk: a control group fed a restricted milk replacer at 0.45 kg/d (R, 20% crude protein, 20% fat), or an accelerated group fed an enhanced milk replacer at 1.13 kg/d (EH, 28% crude protein, 25% fat). At weaning (8 wk), calves were euthanized and sub-samples of mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested upon removal from the body. Total RNA from both tissues was extracted and sequenced using the Illumina HiSeq2500 platform. The Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis (IPA) were used for pathway analysis and functions, gene networks, and cross-talk analyses of the two tissues. Results: When comparing EH vs R 1,561 genes (↑895, and ↓666) and 970 genes (↑506, and ↓464) were found differentially expressed in PAR and MFP, respectively. DIA and IPA results highlight a greater proliferation and differentiation activity in both PAR and MFP, supported by an increased metabolic activity. When calves were fed EH, the PAR displayed transcriptional signs of greater overall organ development, with higher ductal growth and branching, together with a supportive blood vessel and nerve network. These activities were mediated by intracellular cascades, such as AKT, SHH, MAPKs, and Wnt, probably activated by hormones, growth factors, and endogenous molecules. The analysis also revealed strong communication between MFP and PAR, with the first enhancing the development of PAR through secreted growth factors and the recruitment of immune cells. Conclusion: The transcriptomics and bioinformatics approach highlighted the mechanisms that mediate the mammary gland response to a higher plane of nutrition in the pre-weaning period.

Project description:Background Our objective was to compare mammary tissue gene expression profiles during a Streptococcus uberis (S. uberis) mastitis challenge between lactating cows subjected to dietary-induced negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5). The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 d, and cows assigned to PEB were fed the same diet for ad libitum intake. Five days after feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of S. uberis (O140J). At 20 h post-inoculation, both the S. uberis-infected mammary quarters (i.e., YES) and non-infected rear quarters (i.e., NO) from all cows were biopsied for RNA extraction. Effect of S. uberis Intramammary Infection (IMI; YES vs NO) S. uberis IMI resulted in 2,102 oligos (1,939 annotated genes) differentially expressed genes (DEG; YES versus NO). Of these, 1,082 genes were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA1. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Pathway analysis of all DEG indicated that IL-10 Signaling, IL-6 Signaling, and Glucocorticoid Receptor Signaling were among the top canonical pathways affected by infection. Interestingly, LXR/RXR Signaling was also a primary affected pathway, indicating a relationship between immune system response and metabolism during an IMI. Network analysis indicated that TNF had positive relationships (i.e. up-regulation) with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships (i.e., down-regulation) with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1). Results provided information into the early response factors associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to an overall up-regulation of genes involved in the innate immune response and a down-regulation of genes involved with lipid metabolism. Effect of NEB Within Infected Quarters (YES) Energy balance (NEB vs. PEB) resulted in 285 DEG (FDR ≤ 0.05), with 85 DEG up-regulated and 200 DEG down-regulated. Canonical pathways most affected by NEB were IL-8 Signaling (10 genes), Glucocorticoid Receptor Signaling (13), and NRF2-mediated Oxidative Stress Response (10). Among genes differentially expressed by NEB, Cell Growth and Proliferation (48) and Cellular Development (36) were the most enriched functions. Up-regulated genes included ANXA1, HMOX1, and HLAA; down-regulated genes included NOTCH1 and CCND1. Regarding immune response, HLAA was up-regulated due to NEB, whereas the majority of genes involved in immune response were down-regulated (e.g., AKT1, IRAK1, MAPK9, and TRAF6). Results helped identify mechanisms affected by dietary-induced NEB that may be associated with the impairment of immune function and increased risk of mastitis in cows experiencing postpartal NEB. Effect of NEB Within Non-Infected Quarters (NO) Energy balance (NEB vs. PEB) resulted in 278 DEG within non-infected rear mammary quarters. Up-regulated DEG most affected by NEB (genes = 180) included genes involved in lipid metabolism and amino acid metabolism. The down-regulated DEG most affected by NEB (n = 98) included genes involved in carbohydrate metabolism, anti-inflammatory response, and apoptosis. Among genes up-regulated, Ingenuity Pathway Analysis® identified Lipid Metabolism (8), Molecular Transport (14), and Small Molecule Biochemistry (15) as some of the most enriched molecular functions. Genes down-regulated by NEB were associated with Cell Growth and Proliferation (21) and Cell Death (18). Results indicate that dietary induced-NEB alters genes primarily associated with cellular metabolism, proliferation, and tumorigenesis but no candidate genes were identified as being associated with risk of disease.

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Direct transcriptomics comparison of pre-weaned bovine mammary parenchyma and fat pad

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