Mammary Transcriptome Research Articles

Dynamic Profile of the Yak Mammary Transcriptome during the Lactation Cycle.

The objective of this study was to assess the transcriptome of the mammary tissue of four yaks during the whole lactation cycle. For this purpose, biopsies of the mammary gland were performed at -30, -15, 1, 15, 30, 60, 120, 180, and 240 days relative to parturition (d). The transcriptome analysis was performed using a commercial bovine microarray platform and the results were analyzed using several bioinformatic tools. The statistical analysis using an overall false discovery rate ≤ 0.05 for the effect of whole lactation and p < 0.05 for each comparison identified >6000 differentially expressed genes (DEGs) throughout lactation, with a large number of DEGs observed at the onset (1 d vs. -15 d) and at the end of lactation (240 d vs. 180 d). Bioinformatics analysis revealed a major role of genes associated with BTA3, BTA4, BTA6, BTA9, BTA14, and BTA28 in lactation. Functional analysis of DEG underlined an overall induction of lipid metabolism, suggesting an increase in triglycerides synthesis, likely regulated by PPAR signaling. The same analysis revealed an induction of amino acid metabolism and secretion of protein, with a concomitant decrease in proteasome, indicating a major role of amino acid handling and reduced protein degradation in the synthesis and secretion of milk proteins. Glycan biosynthesis was induced for both N-glycan and O-glycan, suggesting increased glycan content in the milk. The cell cycle and immune response, especially antigen processing and presentation, were strongly inhibited during lactation, suggesting that morphological changes are minimized during lactation, while the mammary gland prevents immune hyper-response. Transcripts associated with response to radiation and low oxygen were enriched in the down-regulated DEG affected by the stage of lactation. Except for this last finding, the functions affected by the transcriptomic adaptation to lactation in mammary tissue of yak are very similar to those observed in dairy cows.

Mammary epithelial cell transcriptome reveals potential roles of lncRNAs in regulating milk synthesis pathways in Jersey and Kashmiri cattle

BackgroundLong noncoding RNAs (lncRNAs) are now proven as essential regulatory elements, playing diverse roles in many biological processes including mammary gland development. However, little is known about their roles in the bovine lactation process.ResultsTo identify and characterize the roles of lncRNAs in bovine lactation, high throughput RNA sequencing data from Jersey (high milk yield producer), and Kashmiri cattle (low milk yield producer) were utilized. Transcriptome data from three Kashmiri and three Jersey cattle throughout their lactation stages were utilized for differential expression analysis. At each stage (early, mid and late) three samples were taken from each breed. A total of 45 differentially expressed lncRNAs were identified between the three stages of lactation. The differentially expressed lncRNAs were found co-expressed with genes involved in the milk synthesis processes such as GPAM, LPL, and ABCG2 indicating their potential regulatory effects on milk quality genes. KEGG pathways analysis of potential cis and trans target genes of differentially expressed lncRNAs indicated that 27 and 48 pathways were significantly enriched between the three stages of lactation in Kashmiri and Jersey respectively, including mTOR signaling, PI3K-Akt signaling, and RAP1 signaling pathways. These pathways are known to play key roles in lactation biology and mammary gland development.ConclusionsExpression profiles of lncRNAs across different lactation stages in Jersey and Kashmiri cattle provide a valuable resource for the study of the regulatory mechanisms involved in the lactation process as well as facilitate understanding of the role of lncRNAs in bovine lactation biology.

Bta-miR-125a Regulates Milk-Fat Synthesis by Targeting SAA1 mRNA in Bovine Mammary Epithelial Cells

The nutritional value of cow milk mainly depends on its fatty acid content and protein composition. The identification of genes controlling milk production traits and their regulatory mechanisms is particularly important for accelerating genetic progress in the breeding of dairy cows. On the basis of mammary gland transcriptome analyses, in this study we identified an miRNA, bta-miR-125a, that could control bovine milk-fat production by targeting the 3′ untranslated region (UTR) of the serum amyloid A-1 (SAA1) mRNA. The presence of synthetic bta-miR-125a (i.e., an miR-125a mimic) significantly down-regulated the expression of luciferase from mRNAs containing the binding sequence for bta-miR-125a in the 3′-UTR in a dual-luciferase reporter assay. Furthermore, the presence of the miR-125a mimic decreased the steady-state level of the SAA1 protein, but increased the accumulation of triglycerides and cholesterol content in bovine mammary epithelial cells (MAC-Ts). Blocking the function of bta-miR-125a using a specific inhibitor decreased the level of triglycerides and cholesterol content in the cells. These results indicate that bta-miR-125a can serve as a positive regulator of lipid synthesis in mammary epithelial cells, which acts by targeting SAA1 gene expression.

Impact of Fluoxetine Treatment and Folic Acid Supplementation on the Mammary Gland Transcriptome During Peak Lactation.

Serotonin is a key regulator of mammary gland homeostasis during lactation. Selective serotonin reuptake inhibitors (SSRIs) are commonly used to treat peripartum depression, but also modulates mammary gland serotonin concentrations and signaling in part through DNA methylation. The objective of this study was to determine mouse mammary transcriptome changes in response to the SSRI fluoxetine and how methyl donor supplementation, achieved by folic acid supplementation, affected the transcriptome. Female C57BL/6J mice were fed either breeder diet (containing 4 mg/kg folic acid) or supplemented diet (containing 24 mg/kg folic acid) beginning 2 weeks prior to mating, then on embryonic day 13 mice were injected daily with either saline or 20 mg/kg fluoxetine. Mammary glands were harvested at peak lactation, lactation day 10, for transcriptomic analysis. Fluoxetine but not folic acid altered circulating serotonin and calcium concentrations, and folic acid reduced mammary serotonin concentrations, however only fluoxetine altered genes in the mammary transcriptome. Fluoxetine treatment altered fifty-six genes. Elovl6 was the most significantly altered gene by fluoxetine treatment along with gene pathways involving fatty acid homeostasis, PPARγ, and adipogenesis, which are critical for milk fat synthesis. Enriched pathways in the mammary gland by fluoxetine revealed pathways including calcium signaling, serotonin receptors, milk proteins, and cellular response to cytokine stimulus which are important for lactation. Although folic acid did not impact specific genes, a less stringent pathway analysis revealed more diffuse effects where folic acid enriched pathways involving negative regulation of gene expression as expected, but additionally enriched pathways involving serotonin, glycolysis, and lactalbumin which are critical for lactation. In conclusion, peripartal SSRI use and folic acid supplementation altered critical genes related to milk synthesis and mammary gland function that are important to a successful lactation. However, folic acid supplementation did not reverse changes in the mammary gland transcriptome altered by peripartal SSRI treatment.

Hypogalactia in mammary quarters adjacent to lipopolysaccharide-infused quarters is associated with transcriptional changes in immune genes

Infusion of lipopolysaccharides (LPS) into a mammary gland can provoke inflammatory responses and impair lactation in both the infused gland and neighboring glands. To gain insight into the mechanisms controlling the spatiotemporal response to localized mastitis in lactating dairy cows, we performed RNA sequencing on mammary tissue from quarters infused with LPS, neighboring quarters in the same animals, and control quarters from untreated animals at 3 and 12 h postinfusion. Differences in gene expression were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Comparing mammary transcriptomes from all 3 treatments revealed 3,088 and 1,644 differentially expressed (DE) genes at 3 and 12 h, respectively. Of these genes, >95% were DE only in LPS-infused quarters and represented classical responses to LPS: inflammation, apoptosis, tissue remodeling, and altered cell signaling and metabolism. Although relatively few genes were DE in neighboring quarters (56 at 3 h; 74 at 12 h), these represented several common pathways. At 3 h, tumor necrosis factor (TNF), nuclear factor-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified by the upregulation of anti-inflammatory (NFKBIA, TNFAIP3) and cell adhesion molecule (VCAM1, ICAM1) genes in neighboring glands. Additionally, at 12 h, several genes linked to 1-carbon and serine metabolism were upregulated. Some responses were also regulated over time. The proinflammatory response in LPS-infused glands diminished between 3 and 12 h, indicating tight control over transcription to re-establish homeostasis. In contrast, 2 glucocorticoid-responsive genes, FKBP5 and ZBTB16, were among the top DE genes upregulated in neighboring quarters at both time points, indicating potential regulation by glucocorticoids. We conclude that a transient, systemic immune response was sufficient to disrupt lactation in neighboring glands. This response may be mediated directly by proinflammatory factors from the LPS-infused gland or indirectly by secondary factors released in response to systemic inflammatory signals.

Abundance of solute carrier family 27 member 6 (SLC27A6) in the bovine mammary gland alters fatty acid metabolism.

Milk fatty acid (FA) composition is associated with the nutritional value of milk and is known to vary with the stage of lactation. Although biochemical aspects controlling FA metabolism in the bovine mammary gland are well-established, less is known about the underlying molecular mechanisms. Thus, to address some of these shortcomings, the present study sought to evaluate milk FA composition and mammary transcriptome profiles at different stages of lactation. Compared with 90 d of lactation, at 315 d of lactation, there was an increase in the concentrations of C18:2, polyunsaturated fatty acids (PUFA), and short-chain fatty acids (SCFA), and a decrease in C16:0 and long-chain fatty acids (LCFA) in milk. To further identify candidate genes and pathways responsible for these phenotypic differences, the transcriptome of bovine mammary tissue at 90 d (peak) and 315 d (late) of lactation was profiled using RNA-seq. A total of 827 differentially expressed genes were identified. Bioinformatic analysis revealed that the major differentially modulated lipid metabolic pathways were the PPAR signaling pathway, alpha-linolenic acid metabolism and linoleic acid metabolism. Compared with peak lactation, the mammary tissue at late lactation had lower abundance of genes related to FA transport and activation (CD36, SLC27A6, ACSM1, FABP3 and FABP4). Thus, to further explore the role of FA transport into mammary cells, we knocked down fatty acid transport protein 6 (solute carrier family 27 member 6, SLC27A6) in the bovine mammary epithelial cells (BMECs) using siRNA. The knockdown of SLC27A6 dramatically downregulated the mRNA abundance of genes associated with FA activation (ACSL4), oxidation (CPT1A) and transport (CD36), while the abundance of genes associated with transcription regulation (PPARG), diacylglycerol acyltransferase 1 (DGAT1), FA binding (FABP3), and desaturation (FADS2) was upregulated. In addition, SLC27A6 silenced the intracellular content of triglyceride (TG) and the percentage of C18:1cis9 and C20:4cis5,8,11,14 was greater, whereas that of C16:0 and C18:0 was lower. Overall, in vivo results indicated that LCFA transport into mammary cells during late lactation partly explains the difference in the FA profiles. In vitro analyses underscored how FA transport via SLC27A6 could dictate in part the intracellular utilization of FA for TG synthesis versus oxidation. The data provide strong support for a central role of SLC27A6 in the regulation of FA metabolism in BMECs.

155 Effects of sustained hyperprolactinemia in late gestation on the mammary parenchymal transcriptome of gilts

Abstract The study objective was to determine the effects of hyperprolactinemia on the mammary parenchymal transcriptome in late-pregnant gilts. Gilts were divided into 3 groups on day 90 of gestation to receive IM injections of 1) canola oil (CTL, n = 18) until day 109 of gestation, 2) domperidone (dopamine antagonist) until day 96 (T7, n = 17) or, 3) domperidone until day 109 (T20, n = 17). Mammary glands were collected on day 110 and parenchymal tissue was sampled for transcriptomic analyses. Total RNA was isolated from 6 CTL and 6 T20 gilts for microarray analysis. The GeneChip® Porcine Gene 1.0 ST Array (19,202 genes) was used for hybridization. Array quality control, data normalization and expression level analyses were performed with the Affymetrix Expression Console and Transcriptome Analysis Console (TAC) software. Using a threshold cut-off of 1.5 fold (P &amp;lt; 0.05), a total of 313 upregulated and 480 downregulated gene transcripts were identified in T20 vs CTL gilts. A qPCR validation analysis of selected upregulated (n = 13) and downregulated (n = 13) genes was conducted on all animals (CTL, T7, T20). The MIXED procedure of SAS was used for statistical analyses. All selected genes were validated for the CTL vs T20 comparison (P &amp;lt; 0.01). Only 4 selected genes (CAMK1G, COL9A1, P2RX7, TDRD1) were downregulated in the T7 treatment (vs CTL, P &amp;lt; 0.05). Functional analyses of differentially expressed genes were performed using the PANTHER classification system. The top upregulated Biological Process enriched GO terms were Inflammatory Response (GO:0006954) and Response to Lipid (GO:0033993). The Positive Regulation of Cell Population Proliferation (GO:0008284) and Regulation of Cellular Catabolic Process (GO:0031329) GO terms were identified for downregulated genes. Results suggest that a sustained hyperprolactinemia during late-pregnancy (T20 treatment) may increase mammary inflammatory response and reduce cell proliferation.

Folic Acid Induces Intake-Related Changes in the Mammary Tissue Transcriptome of C57BL/6 Mice.

Folic acid (FA) intake has been associated with increased breast cancer risk in some studies. Although underlying mechanisms are unknown, epigenetic modifications that persistently alter transcription have been suggested. We tested the hypothesis that high FA (HFA) intake alters the adult mammary transcriptome in a manner consistent with increased potential for carcinogenesis, detectable beyond the period of intake. C57BL/6 mice were fed control FA (CFA) (1 mg/kg diet) or HFA (5 mg/kg diet) diets for 4 weeks, followed by AIN93M maintenance diet for 4 weeks. Plasma 5-methyltetrahydrofolate, p-aminobenzoylglutamate and unmetabolised FA concentrations were greater (1.62, 1.56, 5.80-fold, respectively) in HFA compared to CFA mice. RNA sequencing of the mammary transcriptome (~20 million reads) showed 222 transcripts (191 upregulated) differentially expressed between groups. Gene Set Enrichment showed upregulated genes significantly enriched in Epithelial Mesenchymal Transition, Myogenesis and Apical Junction and downregulated genes in E2F targets, MYC targets and G2M checkpoint. Cancer was the most altered Disease and Disorder pathway, with Metastasis, Mammary Tumour and Growth of Tumour the most upregulated pathways. ChIP-seq enrichment analysis showed that targets of histone methyltransferase EZH2 were enriched in HFA mice. This study demonstrates HFA intake during adulthood induces mammary transcriptome changes, consistent with greater tumorigenic potential.

Mammary transcriptome reveals cell maintenance and protein turnover support milk synthesis in early-lactation cows.

A more complete understanding of the molecular mechanisms that support milk synthesis is needed to develop strategies to efficiently and sustainably meet the growing global demand for dairy products. With the postulate that coding gene transcript abundance reflects relative importance in supporting milk synthesis, we analyzed the global transcriptome of early lactation cows across magnitudes of normalized RNA-Seq read counts. Total RNA was isolated from milk samples collected from early-lactation cows (n = 6) following two treatment periods of postruminal lysine infusion of 0 or 63 g/day. Twelve libraries were prepared and sequenced on an Illumina NovaSeq6000 platform using paired end reads. Normalized read counts were averaged across both treatments, because EBseq analysis found no significant effect of lysine infusion. Approximately 10% of the total reads corresponded to 12,730 protein coding transcripts with a normalized read count mean ≥5. For functional annotation analysis, the protein coding transcripts were divided into nine categories by magnitude of reads. The 13 most abundant transcripts (≥50K reads) accounted for 67% of the 23M coding reads and included casein and whey proteins, regulators of fat synthesis and secretion, a ubiquitinating protein, and a tRNA transporter. Mammalian target of rapamycin, JAK/STAT, peroxisome proliferator-activated receptor alpha, and ubiquitin proteasome pathways were enriched with normalized reads ≥100 counts. Genes with ≤100 reads regulated tissue homeostasis and immune response. Enrichment in ontologies that reflect maintenance of translation, protein turnover, and amino acid recycling indicated that proteostatic mechanisms are central to supporting mammary function and primary milk component synthesis.

Diet Composition Affects Liver and Mammary Tissue Transcriptome in Primiparous Holstein Dairy Cows.

Simple SummaryCorn stover (CS) diets are still adopted by small dairy farms in China. Compared with mixed forage (MF) diets, feeding CS diet affects milk composition, digestibility, feed intake, ruminal fermentation, and lactation performance. Mammary gland and liver are two of the most important organs for lactation in cows. However, research related to the effect of CS diet on the metabolism of liver and mammary tissues of dairy cows is limited. Overall evaluation of the biological response of liver and mammary tissues of dairy cows to changes in CS diet compared with MF diet is essential. Thus, the objective of the present study was to evaluate the overall adaptation of liver and mammary tissue to a CS diet in mid-lactation primiparous dairy cows. Modest effect on the transcriptome of the liver and mammary tissue by the CS diet was observed. The analysis of the genes affected by CS indicated mammary gland responding to lower level of linoleate from the diet (lower in CS vs. MF) by activating the associated biosynthesis metabolism while the liver adaptively activated potassium transport to compensate for a lower K ingestion. The results of this study may facilitate the development of better feeding and management strategies and the increase of profitability of dairy farms.The objective of the present study was to evaluate the overall adaptations of liver and mammary tissue to a corn stover (CS) compared to a mixed forage (MF) diet in mid-lactation primiparous dairy cows. Twenty-four primiparous lactating Holstein cows were randomly allocated to 2 groups receiving either an alfalfa forage diet (MF, F:C = 60:40) with Chinese wildrye, alfalfa hay and corn silage as forage source or a corn stover forage diet (CS, F:C = 40:60). A subgroup of cows (n = 5/diet) was used for analysis of liver and mammary transcriptome using a 4 × 44K Bovine Agilent microarray chip. The results of functional annotation analysis showed that in liver CS vs. MF inhibited pathways related to lipid metabolism while induced the activity of the potassium channel. In mammary tissue, fatty acid metabolism was activated in CS vs. MF. In conclusion, the analysis of genes affected by CS vs. MF indicated mammary gland responding to lower level of linoleate from the diet (lower in CS vs. MF) by activating the associated biosynthesis metabolic pathway while the liver adaptively activated potassium transport to compensate for a lower K ingestion.

Investigation of the adolescent female breast transcriptome and the impact of obesity

BackgroundEarly life environmental exposures affect breast development and breast cancer risk in adulthood. The breast is particularly vulnerable during puberty when mammary epithelial cells proliferate exponentially. In overweight/obese (OB) women, inflammation increases breast aromatase expression and estrogen synthesis and promotes estrogen-receptor (ER)-positive breast cancer. In contrast, recent epidemiological studies suggest that obesity during childhood decreases future breast cancer risk. Studies on environmental exposures and breast cancer risk have thus far been limited to animal models. Here, we present the first interrogation of the human adolescent breast at the molecular level and investigate how obesity affects the immature breast.MethodsWe performed RNA-seq in 62 breast tissue samples from adolescent girls/young women (ADOL; mean age 17.8 years) who underwent reduction mammoplasty. Thirty-one subjects were non-overweight/obese (NOB; mean BMI 23.4 kg/m2) and 31 were overweight/obese (OB; BMI 32.1 kg/m2). We also compared our data to published mammary transcriptome datasets from women (mean age 39 years) and young adult mice, rats, and macaques.ResultsThe ADOL breast transcriptome showed limited (30%) overlap with other species, but 88% overlap with adult women for the 500 most highly expressed genes in each dataset; only 43 genes were shared by all groups. In ADOL, there were 120 differentially expressed genes (DEG) in OB compared with NOB samples (padj < 0.05). Based on these DEG, Ingenuity Pathway Analysis (IPA) identified the cytokines CSF1 and IL-10 and the chemokine receptor CCR2 as among the most highly activated upstream regulators, suggesting increased inflammation in the OB breast. Classical ER targets (e.g., PR, AREG) were not differentially expressed, yet IPA identified the ER and PR and growth factors/receptors (VEGF, HGF, HER3) and kinases (AKT1) involved in hormone-independent ER activation as activated upstream regulators in OB breast tissue.ConclusionsThese studies represent the first investigation of the human breast transcriptome during late puberty/young adulthood and demonstrate that obesity is associated with a transcriptional signature of inflammation which may augment estrogen action in the immature breast microenvironment. We anticipate that these studies will prompt more comprehensive cellular and molecular investigations of obesity and its effect on the breast during this critical developmental window.

Gene expression profiles for low-dose exposure to diethyl phthalate in rodents and humans: a translational study with implications for breast carcinogenesis

Phthalates are commonly included as ingredients in personal care products such as cosmetics, shampoos and perfumes. Diethyl phthalate (DEP) has been found to be anti-androgenic and linked with adverse reproductive effects on males, but effects on females are poorly understood. We designed an integrative and translational study to experimentally examine the effects of DEP exposure at a human-equivalent dose on the mammary transcriptome in rats and to subsequently examine the DEP gene signature in breast tissues (both pre-malignant and tumor) from a population study. In Sprague-Dawley rats treated orally with DEP from birth to adulthood, we identified a signature panel of 107 genes predominantly down-regulated by DEP exposure. Univariate analysis of this 107 DEP gene signature in pre-malignant breast tissues revealed that six genes (P4HA1, MPZL3, TMC4, PLEKHA6, CA8, AREG) were inversely associated with monoethyl phthalate (MEP; the urinary metabolite of DEP) concentration (p < 0.05) among postmenopausal women; all six genes loaded on to one of seven factors identified by factor analysis. Transcription factor enrichment analysis revealed that genes in this factor were enriched for androgen receptor binding sites. These six genes were also significantly down-regulated in pre-malignant adjacent tissues compared to the corresponding tumor tissues in pair-wise analyses (p < 0.05). Results from our translational study indicate that low level exposure to diethyl phthalate results in measurable genomic changes in breast tissue with implications in breast carcinogenesis.

Mammary Transcriptome Profile during Peak and Late Lactation Reveals Differentially Expression Genes Related to Inflammation and Immunity in Chinese Holstein.

Somatic cell count (SCC) in milk is widely used in the dairy industry, as an indicator of the health of mammary gland. While the SCC of dairy cattle was higher in late lactation than in peak lactation, its association with gene expressions of mammary gland were largely unknown. In this study, a transcriptomic sequencing approach and bioinformatics analysis were used to investigate the differential expressed genes (DEGs) associated with inflammation and immunity between peak and late periods of lactation in Chinese Holstein. A total of 446 DEGs (padj < 0.05 and fold change >2) were identified, 50 of which belonged to seven pathways and five terms related to inflammation and immunity. Our data suggested that the activation of nuclear transcription factor-κB (NF-κB) pathway and Toll-like receptor signaling pathway caused inflammatory response, and the activation of chemokine signaling pathway and cytokine-cytokine receptor interaction signaling pathway caused a protective immune response to ensure dairy cows health during late lactation. Our findings deepen the understanding of the molecular mechanism and physiological functions of mammary inflammation in Chinese Holstein during late lactation.

Genetic and genomic analyses underpin the feasibility of concomitant genetic improvement of milk yield and mastitis resistance in dairy sheep.

Milk yield is the most important dairy sheep trait and constitutes the key genetic improvement goal via selective breeding. Mastitis is one of the most prevalent diseases, significantly impacting on animal welfare, milk yield and quality, while incurring substantial costs. Our objectives were to determine the feasibility of a concomitant genetic improvement programme for enhanced milk production and resistance to mastitis. Individual records for milk yield, and four mastitis-related traits (milk somatic cell count, California Mastitis Test score, total viable bacterial count in milk and clinical mastitis presence) were collected monthly throughout lactation for 609 ewes of the Chios breed. All ewes were genotyped with a mastitis specific custom-made 960 single nucleotide polymorphism (SNP) array. We performed targeted genomic association studies, (co)variance component estimation and pathway enrichment analysis, and characterised gene expression levels and the extent of allelic expression imbalance. Presence of heritable variation for milk yield was confirmed. There was no significant genetic correlation between milk yield and mastitis traits. Environmental factors appeared to favour both milk production and udder health. There were no overlapping of SNPs associated with mastitis resistance and milk yield in Chios sheep. Furthermore, four distinct Quantitative Trait Loci (QTLs) affecting milk yield were detected on chromosomes 2, 12, 16 and 19, in locations other than those previously identified to affect mastitis resistance. Five genes (DNAJA1, GHR, LYPLA1, NUP35 and OXCT1) located within the QTL regions were highly expressed in both the mammary gland and milk transcriptome, suggesting involvement in milk synthesis and production. Furthermore, the expression of two of these genes (NUP35 and OXCT1) was enriched in immune tissues implying a potentially pleiotropic effect or likely role in milk production during udder infection, which needs to be further elucidated in future studies. In conclusion, the absence of genetic antagonism between milk yield and mastitis resistance suggests that simultaneous genetic improvement of both traits be achievable.

Conjugated linoleic acid (CLA)-induced milk fat depression: application of RNA-Seq technology to elucidate mammary gene regulation in dairy ewes

Milk fat depression (MFD) is characterized by a reduction in the content of milk fat, presumably caused by the anti-lipogenic effects of rumen biohydrogenation intermediates, such as trans-10 cis-12 conjugated linoleic acid (CLA). In this study, RNA-Seq technology was used to help elucidate the mammary responses involved in CLA-induced MFD in lactating ewes. To this end, we compared the milk somatic cell transcriptome of ewes suffering from CLA-induced MFD with control ewes (i.e., those without MFD), as well as with ewes fed a diet supplemented with fish oil (FO-MFD) that we previously reported affects the mammary transcriptome. In the differential expression analysis between CLA-MFD and controls, we identified 1,524 differentially expressed genes (DEGs), whereas 653 were detected between CLA- and FO-MFD groups. Although this article focuses on lipid metabolism, CLA affected the expression of many genes related to other biological processes, especially immunity. Among the 55 genes shared by both MFD conditions, some genes linked to fatty acid synthesis, such as ACACA, AACS, ACSS2, or ACSS3, were downregulated. In addition, this study provides a list of candidate genes that are not usually considered in the nutrigenomics of MFD but that may act as key regulators of this syndrome in dairy ewes.

Functional analysis of the dairy cow mammary transcriptome between early lactation and mid-dry period.

In this research communication we used digital gene expression (DGE) analysis to identify differences in gene expression in the mammary glands of dairy cows between early lactation and the mid-dry period. A total of 741 genes were identified as being differentially expressed by DGE analysis. Compared with their expression in dry cows, 214 genes were up-regulated and 527 genes were down-regulated in lactating cow mammary glands. Gene Ontology analysis showed that lactation was supported by increased gene expression related to metabolic processes and nutrient transport and was associated with decreased gene expression related to cell proliferation. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes showed that 579 differentially expressed genes had pathway annotations related to 204 pathways. Metabolic pathway-related genes were the most significantly enriched. Genes and pathways identified by the present study provide insights into molecular events that occur in the mammary gland between early lactation and mid-dry period, which can be used to facilitate further investigation of the mechanisms underlying lactation and mammary tissue remodeling in dairy cows.

Preweaning nutrient supply alters mammary gland transcriptome expression relating to morphology, lipid accumulation, DNA synthesis, and RNA expression in Holstein heifer calves

The objective of this study was to analyze the mammary gland transcriptome to determine how preweaning nutrient supply alters the molecular mechanisms that regulate preweaning mammary development. Holstein heifers were fed via milk replacer (MR) either an elevated level of nutrient intake (ELE; on average, 5.9 ± 0.2 Mcal of ME in 8.4 L of MR/d, n = 6) or a restricted amount of nutrients (RES; 2.8 ± 0.2 Mcal of ME in 4 L of MR/d, n = 5) for 54 d after birth, at which point they were slaughtered and samples of mammary parenchyma tissue were obtained. Parenchymal mRNA was analyzed, and the fold change (FC) of 18,111 genes (ELE relative to RES) was uploaded to Ingenuity Pathway Analysis (IPA) software (Qiagen Bioinformatics, Redwood City, CA) for transcriptomic analysis. Using a threshold of P < 0.05, IPA identified that the FC of 1,931 of 18,811 differentially expressed genes (DEG) could be used for the analysis. A total of 18 molecular and cellular functions were relevant to DEG arising from the treatments; the 5 functions most associated with DEG were cell death and survival, cellular movement, cellular development, cellular growth and proliferation, and lipid metabolism. Based on the directional FC of DEG, the mammary gland of ELE heifers was predicted to have increased epithelial-mesenchymal transition (Z = 2.685) and accumulation of lipid (Z = 2.322), whereas the synthesis of DNA (Z = -2.137), transactivation of RNA (Z = -2.254), expression of RNA (Z = -2.405), transcription (Z = -2.482), and transactivation (Z = -2.611) were all predicted to be decreased. Additionally, IPA predicted the activation status of 13 upstream regulators with direct influence on DEG as affected by ELE feeding that were ligand-dependent nuclear receptors (n = 2), enzymes (n = 1), or transcription regulators (n = 10). Of these, 6 were activated (Z > 2) and 7 were inhibited (Z < -2). In summary, feeding ELE preweaning altered the mammary transcriptome of Holstein heifers, affecting cell functions involved in the morphological and physiological development of the mammary gland.

Widespread cis-regulation of RNA editing in a large mammal.

Post-transcriptional RNA editing may regulate transcript expression and diversity in cells, with potential impacts on various aspects of physiology and environmental adaptation. A small number of recent genome-wide studies in Drosophila, mouse, and human have shown that RNA editing can be genetically modulated, highlighting loci that quantitatively impact editing of transcripts. The potential gene expression and physiological consequences of these RNA-editing quantitative trait loci (edQTL), however, are almost entirely unknown. Here, we present analyses of RNA editing in a large domestic mammal (Bos taurus), where we use whole-genome and high-depth RNA sequencing to discover, characterize, and conduct genetic mapping studies of novel transcript edits. Using a discovery population of nine deeply sequenced cows, we identify 2413 edit sites in the mammary transcriptome, the majority of which are adenosine to inosine edits (98.6%). Most sites are predicted to reside in double-stranded secondary structures (85.1%), and quantification of the rates of editing in an additional 355 cows reveals editing is negatively correlated with gene expression in the majority of cases. Genetic analyses of RNA editing and gene expression highlight 152 cis-regulated edQTL, of which 15 appear to cosegregate with expression QTL effects. Trait association analyses in a separate population of 9989 lactating cows also shows 12 of the cis-edQTL coincide with at least one cosegregating lactation QTL. Together, these results enhance our understanding of RNA-editing dynamics in mammals, and suggest mechanistic links by which loci may impact phenotype through RNA editing mediated processes.

343 Effect of once daily milking on mammary transcriptome and cell turnover in dairy goat

Once-daily milking is known to modify cell number and activity in the bovine mammary gland. However, in goats, the effect of once-daily milking on mammary cell death is controversial. To assess the effect of once-daily milking on mammary transcriptome and cell turnover and the effect of the duration of once-daily milking, 10 goats producing 2.5 kg of milk per day at 100 DIM were divided into 2 groups. All goats were twice daily milked during a pre-experimental period of 2 wk. Then, the goats were once daily milked for the 3 following weeks. Mammary biopsy samples were collected at d −1 and 7 after the start of once-daily milking for one group of 5 goats and at d −1 and 21 for a second group of 5 goats. Cell apoptosis and proliferation rates were analyzed in mammary tissue by immunohistological analyses after TUNEL and PCNA staining, respectively. Ribonucleic acid was extracted from mammary tissues. A transcriptomic analysis using the Agilent Bovine 4x44k microarrays has been performed to compare the effect of once-daily milking on mammary transcript profiles. Data were normalized and statistical significant raw P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. A similar reduction in milk yield (−19%) was observed in both groups of goats during once-daily milking compared with twice-daily milking. Cell apoptosis was higher during once-daily milking than during twice-daily milking for both groups of goats (P < 0.05) whereas cell proliferation did not vary (P = 0.27). The transcriptomic analysis showed a differential gene expression of 4,039 transcripts, 2,238 and 1,801 transcripts up- and downregulated, respectively, by once-daily milking compared with twice-daily milking. More than 1,000 transcripts were commonly regulated between the 2 groups of goats. IPA analysis showed that these transcripts were part of networks associated with DNA replication, cellular growth and proliferation, and cell-to-cell signaling and interaction in both groups of goats. RT-qPCR analysis of 11 genes confirmed the differential gene expression with a downregulation of genes involved in milk synthesis and an upregulation of genes involved in cellular cycle and apoptosis. These results showed that once-daily milking induce cell turnover in goat mammary tissue, with a small impact of time.

Abstract 1401: Deterring breast cancer progression. The critical role of a lipid-sphingolipid epigenetic mechanism of cell fate

Abstract Genetic and environmental factors cooperate to assure precise genome-wide epigenetic regulation of the mammary epithelia cell transcriptome. Our interest in regulatory epigenetic mechanisms that, by determining mammary epithelial cell decisions, are pivotal to deter the onset and progression of breast cancer, let us identify a regulatory epigenetic mechanism of mammary morphogenesis that links a physiological regulatory lipid of environmental origin (all-trans retinoic acid, RA) with sphingolipids capable of determining either cell death (ceramide, CER) or cell life (sphingosine 1 phosphate, S1P). Whenever RA, for different reasons, fails to perform the epigenetic transcriptional control of neutral sphingomyelinase 2 (nSMase2/SMDP3), involved in the synthesis of pro-apoptotic CER, S1P fosters both pro-proliferative and pro-invasive activity. Apparently, in the absence of epigenetic control of CER production by RA, S1P contributes to determine RA tumorigenic action. This study provides the rationale for combination therapeutic approaches with epigenetic drugs and inhibitors of either sphingosine kinase, the enzyme involved in S1P synthesis, or S1P receptors. This study was supported by the NCI R01 CA127614 grant (NS). Citation Format: Stefano Rossetti, Vincenzo Gagliostro, Nicoletta Sacchi. Deterring breast cancer progression. The critical role of a lipid-sphingolipid epigenetic mechanism of cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1401. doi:10.1158/1538-7445.AM2017-1401