Response Of Mammary Gland Research Articles

Acute iodine deficiency induces a transient VEGF-dependent microvascular response in mammary glands involving HIF-1, ROS, and mTOR.

Iodine deficiency (ID), which affects almost two billion people worldwide, is associated with breast pathologies such as fibrosis in human and induces breast atypia in animal models. Because ID induces vascular activation in the thyroid, another iodide-uptaking organ, and as breast is also sensitive to ID, we aimed to characterize ID-induced effects on the breast microvasculature in vivo and in two different breast cell lines in vitro. Virgin and lactating NMRI mice received an iodide-deficient diet and a Na+/I- symporter inhibitor for 1 to 20 days. Some virgin mice were treated with vascular endothelial growth factor A (VEGF) or VEGF receptor inhibitors. In vitro, ID was induced in MCF7 and MCF12A cells by replacing the iodide-containing medium by an iodide-deficient medium. In vivo, VEGF expression was increased following ID in mammary tissues. Consequently, ID induced a transient increase in mammary gland blood flow, measured after anesthesia, in virgin and lactating mice, which was repressed by VEGF or VEGF receptor inhibitors. In MCF7 cells, ID induced a transient increase in reactive oxygen species, followed by an increase in hypoxia-inducible factor-1α (HIF-1α) protein and VEGF mRNA expression. Antioxidant N-acetylcysteine and mammalian target of rapamycin (mTOR) inhibitor blocked ID-induced HIF-1α protein increase and VEGF transcription. However, mTOR activity was not inhibited by N-acetylcysteine. Similar responses were observed in MCF12A cells. These data indicate that ID activates the canonical VEGF pathway and mTOR in breast tissues, which provides new insights to better understand the correlation between ID, vascular activation, and breast pathologies.

Immunomodulation of Host Chitinase 3-Like 1 During a Mammary Pathogenic Escherichia coli Infection.

Chitin is a N-acetyl-d-glucosamine biopolymer that can be recognized by chitin-binding proteins. Although mammals lack chitin synthase, they induce proteins responsible for detecting chitin in response to bacterial infections. Our aim was to investigate whether chitinase 3-like 1 (CHI3L1) has a potential role in the innate immunity of the Escherichia coli (E. coli) infected mammary gland. CHI3L1 protein was found to be secreted in whey of naturally coliform-affected quarters compared to whey samples isolated from healthy udders. In addition, gene expression of CHI3L1 was confirmed in udder tissue of cows experimentally infected with a mammary pathogenic E. coli (MPEC) strain. Despite the known anatomical differences, the bovine udders’ innate immune response was mimicked by applying an experimental mouse model using MPEC or non-MPEC isolates. The effect of CHI3L1 expression in the murine mammary gland in response to coliform bacteria was investigated through the use of CHI3L1−/− mice as well as through treatment with either a pan-caspase inhibitor or chitin particles in wild-type mice. The local induction of CHI3L1 postinfection with different E. coli strains was demonstrated to be independent of both bacterial growth and mammary interleukin (IL)-8 levels. Indeed, CHI3L1 emerged as a regulator impacting on the transcytosis of Ly6G-positive cells from the interstitial space into the alveolar lumen of the mammary tissue. Furthermore, CHI3L1 was found to be upstream regulated by caspase activity and had a major downstream effect on the local pro-inflammatory cytokine profile, including IL-1beta, IL-6, and RANTES/CCL5. In conclusion, CHI3L1 was demonstrated to play a key role in the cytokine and caspase signaling during E. coli triggered inflammation of the mammary gland.

Histamine activates inflammatory response and depresses casein synthesis in mammary gland of dairy cows during SARA

BackgroundMounting evidences observed that subacute ruminal acidosis (SARA) induced by high concentration (HC) diet increases the translocation of histamine from digestive tract into circulation causing a diverse of diseases in dairy cows. However, it is largely unknown how it does affect the function of mammary gland and milk quality. Hence, this study aims to observe the effects of histamine derived from the digestive tract on the inflammatory response and casein synthesis in the mammary glands during SARA. Twelve cows fitted rumen fistula were randomly divided into either control group administrated low concentration (LC) diet (60% forage, n = 6) or treatment group administrated HC diet (40% forage, n = 6) for 18 weeks.ResultsOur data showed that HC diet resulted in significant declines in rumen pH value, milk yield and milk quality, as well as longer duration of averaged pH value below 5.6 per day (more than 180 min) compared to LC diet, these findings confirmed SARA occurence. Our study also observed that SARA increased the content of histamine in rumen fluid, plasma, liver and mammary gland, and enhanced the mRNA expression of histamine specific receptor in the mammary gland. Additionally, we found that the mRNA expression of inflammatory response genes in mammary glands was increased, which was consistent with the protein expression results, showing that the protein kinase C(PKC) / nuclear factor kappa B (NF-κB) or protein kinase A (PKA) / NF-κB signalling pathways of the inflammatory response were activated. The mRNA expression of mTOR, P70S6K and αS1 in mammary glands were significantly decreased with the protein expression of mTOR, P70S6K and αS1-casein, and the phosphorylation levels of the mTOR and P70S6K proteins were also decreased.ConclusionsOur study showed that the milk protein of lactating cows is depressed after long-term feeding of HC at the individual level, which was paralleled at the gene and protein levels. The inflammatory response in mammary gland caused by histamine derived from the digestive tract is related to the decline of casein synthesis. Our findings point to a new link between the inflammatory response and casein synthesis, but the understanding of the molecular mechanisms involved in this process will require further research.

Biological features of cows with different levels of stress resistance

The results of studies of biological features of 66 cows of Holstein breed with different reaction levels to stress have been quoted. The stress factor was the planned blood taking from them. In an hour after this procedure, blood was taken again from the jugular vein with the aim to determine the concentration of cortisol in experimental animals. Based on the different cortisol concentration in blood, the cows have been differentiated into three gro ups: I – with high stress resistance, II – with medium stress resistance and III – with low stress resistance. The aim of the studies was to establish the relationship between the different stress resistance in animals and the development and function of the udder, the milk productivity and the heat resistance of the organism under summer temperature loading in the conditions of Ukraine. It has been established that the animals of group I were milked in double-quick time (P <0.001), and their milk-ejection rate was 1.4 kg/min (P <0.001) compared to the third group. They also had a higher reaction rate at the beginning of milking (P <0.001). The parts of the udder in the cows of this group are more evenly developed, which serves as the best precondition for the implementation of the milk flow reflex by machine milking. Cows with low stress resistance have a well-defined prolonged latent period at the beginning of machine milking. This is explained by the delay in the reaction of their mammary gland in response to the beginning of milking. It has been suggested that this disadvantage can be mitigated by using a more improved milking equipment, with an udder massage regime, which is capable to compensate the disturbance of the milk yield reflex in cows, which are more sensitive to stress. The influence of different stress resistance on fat and protein content in the milk of Holstein cows has not been properly defined. But in comparison with low stress-resistant animals, milk yields for 305 days of the first lactation were higher in highly stress-resistant animals – by 639 kg (P <0.001), in cows of the middle group – by 216 kg (P <0.05). The lower resistance of cows to stress makes them vulnerable in conditions of temperature load during the hottest months (July, August) of the summer period in Ukraine. Their body reacts to such a load with sharper fluctuations in body temperature, pulse, respiration rate and thermal sensitivity index as compared to their peers with high level of stress resistance (P <0.01...0.001). To solve this problem with trans-humance grazing it is possible to use mobile canopies.

Effect of the concentration of circulating prolactin on dairy cows' responsiveness to domperidone injection

The objective of this study was to determine whether the responsiveness of the mammary gland to prolactin (PRL) is affected by the concentration of the hormone. After 1 pre-experimental week (d -7 to -1), 18 Holstein cows in mid to late lactation were injected intramuscularly twice daily with either 0.5 mg of quinagolide (QN) or 2 mL of water (control) for 2 wk (d 1 to 14; treatment period). After the treatment period, all cows received daily subcutaneous injections of 300 mg of domperidone (DOMP) for 3 wk (d 15 to 35; DOMP period). The cows were monitored for an additional 2 wk as a posttreatment period (d 36 to 49). Blood and milk samples were collected 3 times per week. Additionally, blood samples were collected during the a.m. milking on d -4, 14, and 35. Milk production was not affected by QN during the treatment period but was increased during the DOMP and posttreatment periods in the QN cows. With respect to milk composition, the treatments affected only the protein content, which was greater in the QN cows during the treatment period. Blood PRL concentration declined during QN injections and was lower in the QN cows than in the control cows between d 5 and 14. The basal concentration of PRL was increased by DOMP injections during the DOMP and posttreatment periods but was not affected by previous QN injections. Prolactin concentration in milk was not affected by the QN treatments but was increased by DOMP injections during the DOMP and posttreatment periods. Milking-induced PRL release was decreased by QN on d 14. On d 35, milking did not induce a significant release of PRL above the baseline for both treatments. In conclusion, the results of this experiment support the contention that the mammary gland's responsiveness to PRL is modulated by the previous level of the hormone.

Sunflower oil supplementation affects the expression of miR-20a-5p and miR-142-5p in the lactating bovine mammary gland.

Oil supplementation in dairy cattle diets is used to modulate milk fat composition, as well as the expression of mammary lipogenic genes, whose regulation remains unclear. MiRNAs are small non-coding RNA considered as crucial regulators of gene expression, offering clues to explain the mechanism underlying gene nutriregulation. The present study was designed to identify miRNAs whose expression in the cow mammary gland is modulated by sunflower oil supplementation. MiRNomes were obtained using RNAseq technology from the mammary gland of lactating cows receiving a low forage diet, supplemented or not with 4% sunflower oil. Among the 272 miRNAs characterized, eight were selected for RT-qPCR validations, showing the significant down-regulation of miR-142-5p and miR-20a-5p by sunflower supplementation. These two miRNAs are predicted to target genes whose expression was reported as differentially expressed by sunflower supplementation. Among their putative targets, ELOVL6 gene involved in lipid metabolism has been studied. However, a first analysis did not show its significant down-regulation, in response to the over-expression of miR-142-5p, of miR-20a-5p, or both, in a bovine mammary epithelial cell line. However, a clearer understanding of the miRNA expression by lipid supplementation would help to decipher the regulation of lactating cow mammary gland in response to nutrition.

Effect of 17β-estradiol on milk production, hormone secretion, and mammary gland gene expression in dairy cows

Estradiol inhibits milk production in dairy cows. The present study evaluated the effect of 17β-estradiol (E2) injections on prolactin (PRL) secretion and the mammary gland response to this hormone. Eight mid-lactation cows were used in a crossover design. During each experimental period, the cows were injected daily with either E2 (2.5 mg) or soy oil (2.5 mL; control) for 7 d. For each period, blood and milk samples were collected from d -4 to 14 (relative to the first injection) to measure PRL, insulin-like growth factor-1, and cortisol concentrations. In addition, blood samples were collected during morning milking on d -4, 2, and 7 to determine the milking-induced PRL release. Mammary gland biopsies were collected on the last day of injections. Milk fat samples were collected from d 1 to 7 and on d 14. The mRNA levels of genes encoding proteins related to mammary activity (α-lactalbumin, β-casein, and acetyl-coenzyme A carboxylase), apoptosis (Bax, Bcl2, and caspase-3), PRL receptors (PRLR; long and short forms), signal transducer and activator of transcription 5 (STAT5A and STAT5B), and suppressors of cytokine signaling (SOCS2 and SOCS3) were evaluated by real-time reverse transcription PCR using RNA extracted from milk fat and mammary biopsies. Milk production was decreased moderately (about 9%) by E2 injections during the treatment period. Estradiol injections increased basal PRL levels in serum and milk but did not affect milking-induced PRL release. Estradiol injections increased the plasma concentration of insulin-like growth factor-1 but did not affect cortisol concentration during the treatment period. In mammary tissue, the expression of Bcl2 was downregulated, whereas that of STAT5A and B and the Bax:Bcl2 mRNA ratio was higher during E2 injections. The total STAT5 protein content in mammary tissue was elevated by E2 injections. We found no significant difference observed for the other genes in mammary tissue or milk fat. The present data do not support the contention that E2 injections inhibit milk production by interfering with PRL signaling, but enhanced basal PRL concentration and STAT5 gene expression in mammary tissue.

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions.

Epithelial cell layers are interconnected by a meshwork of tight junction (TJ) protein strands, which are localized within apicolateral membranes. The proteins that form TJs are regarded to provide a static barrier, determining epithelial properties. However, recent findings in the field of barriology suggest that TJs contribute to more physiological aspects than indicated by the sum of the qualities of the single TJ proteins. Generally, TJs exhibit four major functions: (i) a "gate function," defining transepithelial permeability (i.e., barrier) properties, (ii) a "fence function" determining epithelial cell polarity, (iii) a "signaling function," affecting regulatory pathways, and (iv) a "stabilizing function," maintaining the integrity of the epithelium. This review presents a critical view on how the efficacy of physiological processes in epithelia and thus organ function might be improved by changes in the expression of claudins, the latter representing the largest and most variable family of TJ proteins. Major focus is set on (i) the coordinated regulation of transport and barrier in the intestine, (ii) the role of TJs in defining the route for antigen uptake and presentation in intestinal Peyer's patches, and (iii) the TJ function in mammary glands in response to milk accumulation, which represent impressive examples to highlight the amplification of epithelial functions by TJ proteins. © 2017 IUBMB Life, 69(5):290-296, 2017.

Genome-Wide Transcriptional and Post-transcriptional Regulation of Innate Immune and Defense Responses of Bovine Mammary Gland to Staphylococcus aureus

Staphylococcus aureus (S. aureus) is problematic for lactating mammals and public health. Understanding of mechanisms by which the hosts respond to severe invasion of S. aureus remains elusive. In this study, the genome-wide expression of mRNAs and miRNAs in bovine mammary gland cells were interrogated at 24 h after intra-mammary infection (IMI) with high or low concentrations of S. aureus. Compared to the negative control quarters, 194 highly-confident responsive genes were identified in the quarters with high concentration (109 cfu/mL) of S. aureus, which were predominantly implicated in pathways and biological processes pertaining to innate immune system, such as cytokine-cytokine receptor interaction and inflammatory response. In contrast, only 21 highly-confident genes were significantly differentially expressed in face of low concentration (106 cfu/mL) of S. aureus, which slightly perturbed the cell signaling and invoked corresponding responses like vasoconstriction, indicating limited perturbations and immunological evading. Additionally, the significant up-regulations of bta-mir-223 and bta-mir-21-3p were observed in the quarters infected by high concentration of S. aureus. Network analysis suggested that the two miRNAs' pivotal roles in defending hosts against bacterial infection probably through inhibiting CXCL14 and KIT. The significant down-regulation of CXCL14 was also observed in bovine mammary epithelial cells at 24 h post-infection of S. aureus (108 cfu/mL) in vitro. Integrated analysis with QTL database further suggested 28 genes (e.g., CXCL14, KIT, and SLC4A11) as candidates of bovine mastitis. This study first systematically revealed transcriptional and post-transcriptional responses of bovine mammary gland cells to invading S. aureus in a dosage-dependent pattern, and highlighted a complicated responsive mechanism in a network of miRNA-gene-pathway interplay.

Effects of Pubertal Exposure to Dietary Soy on Estrogen Receptor Activity in the Breast of Cynomolgus Macaques.

Endogenous estrogens influence mammary gland development during puberty and breast cancer risk during adulthood. Early-life exposure to dietary or environmental estrogens may alter estrogen-mediated processes. Soy foods contain phytoestrogenic isoflavones (IF), which have mixed estrogen agonist/antagonist properties. Here, we evaluated mammary gland responses over time in pubertal female cynomolgus macaques fed diets containing either casein/lactalbumin (n = 12) or soy protein containing a human-equivalent dose of 120 mg IF/day (n = 17) for approximately 4.5 years spanning menarche. We assessed estrogen receptor (ER) expression and activity, promoter methylation of ERs and their downstream targets, and markers of estrogen metabolism. Expression of ERα and classical ERα response genes (TFF1, PGR, and GREB1) decreased with maturity, independent of diet. A significant inverse correlation was observed between TFF1 mRNA and methylation of CpG sites within the TFF1 promoter. Soy effects included lower ERβ expression before menarche and lower mRNA for ERα and GREB1 after menarche. Expression of GATA-3, an epithelial differentiation marker that regulates ERα-mediated transcription, was elevated before menarche and decreased after menarche in soy-fed animals. Soy did not significantly alter expression of other ER activity markers, estrogen-metabolizing enzymes, or promoter methylation for ERs or ER-regulated genes. Our results demonstrate greater ER expression and activity during the pubertal transition, supporting the idea that this life stage is a critical window for phenotypic modulation by estrogenic compounds. Pubertal soy exposure decreases mammary ERα expression after menarche and exerts subtle effects on receptor activity and mammary gland differentiation. Cancer Prev Res; 9(5); 385-95. ©2016 AACR.

The Peptide Oxytocin Antagonist F-792, When Given Systemically, Does Not Act Centrally in Lactating Rats.

Oxytocin secreted by nerve terminals in the posterior pituitary has important actions for ensuring a successful outcome of pregnancy: it stimulates uterine contractions that lead to birth and it is essential in the milk‐ejection reflex, enabling milk to be expelled from the mammary glands into the mouths of suckling young. Oxytocin also has important actions in the brain: released from dendrites of neurones that innervate the posterior pituitary, oxytocin auto‐excites the neurones to fire action potentials in co‐ordinated bursts, causing secretion of pulses of oxytocin. Central oxytocin actions are blocked by an oxytocin antagonist given into the brain and, consequently, milk transfer stops. Systemic peptide oxytocin antagonist (atosiban) treatment is used clinically in management of pre‐term labour, a major obstetric problem. Hence, it is important to know whether an oxytocin antagonist given peripherally can enter the brain and interfere with central oxytocin actions. In the present study, we tested F792, a peptide oxytocin antagonist. In urethane‐anaesthetised suckled rats, we show that the mammary gland responsiveness to oxytocin is blocked by i.v. injections of 7 μg/kg of F792, and the milk‐ejection reflex is blocked when F792 is given directly into the brain at a dose of 0.2 μg. To critically test whether F792 given systemically can enter the brain, we recorded the suckling‐ and oxytocin‐induced burst‐firing of individual antidromically identified oxytocin neurones in the paraventricular nucleus. Given systemically at 100 μg/kg i.v., F792 acted only peripherally, blocking the milk‐ejecting actions of oxytocin, but not the burst‐firing of oxytocin neurones during suckling (n = 5 neurones in five rats). Hence, this peptide oxytocin antagonist does not enter the brain from the circulation to interfere with an essential oxytocin function in the brain. Furthermore, the functions of oxytocin in the brain evidently cannot be explored with a systemic peptide antagonist.

Lactoferrin and Immunoglobulin G Concentration in Bovine Milk from Cows with Subclinical Mastitis during the Late Lactation Period

Background: Lactoferrin and immunoglobulin G in milk have an important role in udder resistance to infection in the involution period. Both proteins express antimicrobial activity- lactoferrin by the binding and sequestration of iron ion; and immunoglobulin G by complement activation, bacterial opsonization and agglutination. Many factors affect lactoferrin and immunoglobulin G concentrations in bovine milk, such as the stage of lactation, milk production, and intramammary infections. The aim of this study was to determine concentrations of lactoferrin and immunoglobulin G in milk from healthy cows and subclinical mastitic cows during the late lactation period, and to evaluate the relationship between them.Materials, Methods &amp; Results: A total of 150 quarter milk samples from 41 cows (Holstein-Friesian breed) in late lactation period were reviewed in this study. Milk samples were collected during morning milking, using aseptic techniques in sterile test tubes. From each sample, 0.1 mL of milk was plated on Columbia blood agar base with 5% defibrinated ovine blood, MacConkey agar and Sabouraud dextrose agar and incubated for 24 h - 48 h (bacteria) and 5 days (yeasts, mould) at 37oC. Milk samples for detection lactoferrin and immunoglobulin G concentration were skimmed at 1,400 g for 45 min and stored at -20°C until analysis. Lactoferrin concentration in bovine milk was determined using the Bovine Lactoferrin ELISA Quantitation Set. Milk samples were diluted at a ratio of 1:10,000. Plates were read at 450 nm absorbence values. Immunoglobulin G concentration was determined by the immunodiffusion method using radial immunodiffusion (RID) plates. Milk samples were diluted in a ratio of 1:30. Reading of results was done after incubation for 48 h by measuring the diameter of the precipitation ring. The highest mean lactoferrin concentration was observed in udder quarters infected with contagious pathogens (Streptococcus agalactiae and Staphylococcus aureus), while the highest mean immunoglobulin G concentration was detected in milk samples where minor mastitis pathogens (coagulase-negative staphylococci and Corynebacterium spp.) were isolated. Milk samples where Staphylococcus aureus was isolated had the lowest immunoglobulin G concentration, and the lowest lactoferrin concentration was observed in samples infected with enviromental pathogens (Streptococcus dysgalactiae).Discussion: This study showed that lactoferrin and immunoglobulin G concentrations are higher in milk samples from subclinical mastitic cows than in milk from normal lactating cows. Lactoferrin concentrations in milk samples from udder quarters infected with major mastitis pathogens were significantly higher than in milk infected with minor mastitis pathogens. The lowest concentration of immunoglobulin G was detected in milk samples where Staphylococcus aureus was isolated, while the highest immunoglobulin G concentration was observed in milk samples from quarters infected with minor mastitis pathogens. Lactoferrin and immunoglobulin G concentrations were significantly and positively correlated in all milk samples. This means that cows with high lactoferrin concentrations have high immunoglobulin G concentrations. In quarter milk samples infected with Staphylococcus aureus, lactoferrin and immunoglobulin G concentrations were negatively correlated. The cause of these findings could be the suppression of local immune response of mammary gland.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 3. Untargeted metabolomics.

Intramammary infection leading to bovine mastitis is the leading disease problem affecting dairy cows and has marked effects on the milk produced by infected udder quarters. An experimental model of Streptococcus uberis mastitis has previously been investigated for clinical, immunological and pathophysiological alteration in milk, and has been the subject of peptidomic and quantitative proteomic investigation. The same sample set has now been investigated with a metabolomics approach using liquid chromatography and mass spectrometry. The analysis revealed over 3000 chromatographic peaks, of which 690 were putatively annotated with a metabolite. Hierarchical clustering analysis and principal component analysis demonstrated that metabolite changes due to S. uberis infection were maximal at 81 hours post challenge with metabolites in the milk from the resolution phase at 312 hours post challenge being closest to the pre-challenge samples. Metabolic pathway analysis revealed that the majority of the metabolites mapped to carbohydrate and nucleotide metabolism show a decreasing trend in concentration up to 81 hours post-challenge whereas an increasing trend was found in lipid metabolites and di-, tri- and tetra-peptides up to the same time point. The increase in these peptides coincides with an increase in larger peptides found in the previous peptidomic analysis and is likely to be due to protease degradation of milk proteins. Components of bile acid metabolism, linked to the FXR pathway regulating inflammation, were also increased. Metabolomic analysis of the response in milk during mastitis provides an essential component to the full understanding of the mammary gland's response to infection.

New insights into the importance of prolactin in dairy ruminants

In most mammals, prolactin (PRL) is essential for maintaining lactation, and the suppression of PRL inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear, because inconsistent effects on milk yield have been observed with the short-term suppression of PRL by bromocriptine. Therefore, several experiments have been conducted to assess the galactopoietic role of PRL. In an initial experiment, cows in early lactation received daily injections of the dopamine agonist quinagolide for 9wk. Quinagolide reduced milking-induced PRL release and caused a faster decline in milk production. Quinagolide also reduced mammary epithelial cell activity, survival, and proliferation. In goats, cabergoline, another dopamine agonist, caused a 28% decrease in milk yield the day after injection. In another experiment, cows were injected for 5d with quinagolide, with quinagolide plus bovine PRL injected at milking time, or with vehicles only. Again, quinagolide reduced milk, protein, and lactose yields. Although PRL injections were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of mammary epithelial cells purified from milk. Recently, our team stimulated PRL secretion with daily injections of the dopamine antagonist domperidone for 5wk. Milk production increased gradually and was greater in domperidone-treated cows during the last 4wk of the treatment period. In most experiments where PRL secretion was manipulated, feed intake paralleled the changes of PRL concentration, supporting the idea that PRL increases feed intake to provide the nutrients necessary to support lactation in dairy ruminants. In late-lactation cows, quinagolide and cabergoline decreased milk production within the first day of treatment and induced more rapid changes in several markers of mammary gland involution after drying-off. In addition, quinagolide improved the resistance to intramammary infection, suggesting that PRL inhibition could be an alternative strategy for facilitating drying-off. Prolactin appears to directly affect mammary gland functions, but mammary gland responsiveness to PRL appears to be modulated by local and systemic factors. Therefore, the modulation of the number and isoforms of the PRL receptors as well as the expression of intracellular modulators of cell signaling in the mammary gland require further investigation. In conclusion, these data, combined with those from other studies, provide a good body of evidence that PRL is galactopoietic in dairy ruminants.

Food Deprivation Affects the miRNome in the Lactating Goat Mammary Gland

BackgroundNutrition affects milk composition thus influencing its nutritional properties. Nutrition also modifies the expression of mammary genes, whose regulation is not fully understood. MicroRNAs (miRNA) are small non coding RNA which are important post-transcriptional regulators of gene expression by targeting messenger RNAs. Our goal was to characterize miRNA whose expression is regulated by nutrition in the lactating goat mammary gland, which may provide clues to deciphering regulations of the biosynthesis and secretion of milk components.Methodology/principal findingsUsing high-throughput sequencing technology, miRNomes of the lactating mammary gland were established from lactating goats fed ad libitum or deprived of food for 48h affecting milk production and composition. High throughput miRNA sequencing revealed 30 miRNA with an expression potentially modulated by food deprivation; 16 were down-regulated and 14 were up-regulated. Diana-microT predictive tools suggested a potential role for several nutriregulated miRNA in lipid metabolism. Among the putative targets, 19 were previously identified as differently expressed genes (DEG). The functions of these 19 DEG revealed, notably, their involvement in tissue remodelling.Conclusion/significanceIn conclusion, this study offers the first evidence of nutriregulated miRNA in the ruminant mammary gland. Characterization of these 30 miRNA could contribute to a clearer understanding of gene regulation in the mammary gland in response to nutrition.

Expression patterns of β-defensin and cathelicidin genes in parenchyma of bovine mammary gland infected with coagulase-positive or coagulase-negative Staphylococci.

BackgroundMastitis is still considered to be the most economically important infectious disease in dairy cattle breeding. The immune response in mammary gland tissues could help in developing support strategies to combat this disease. The role of neutrophils and macrophages in the innate response of mammary gland is well known. However, the immune response in mammary gland tissues, including levels of antimicrobial peptide transcripts, has not been well recognized. Moreover, most studies are conducted in vitro, on cell cultures, or on artificially infected animals, with analysis being done within a several dozen hours after infection.The aim of the study was to examine the in vivo transcript levels of beta-defensin and cathelicidins genes in cow mammary gland secretory tissue (parenchyma) with the chronic, recurrent and incurable mammary gland inflammation induced by coagulase-positive or coagulase-negative Staphyloccoci vs. bacteria-free tissue.ResultsThe mRNA of DEFB1, BNBD4, BNBD5, BNBD10 and LAP genes, but not of TAP gene, were detected in all investigated samples regardless of the animals’ age and microbiological status of the mammary gland, but at different levels. The expression of most of the beta-defensin genes was shown to be much higher in tissues derived from udders infected with bacteria (CoPS or CoNS) than from bacteria-free udders, regardless of parity. Cathelicidins (CATH4, CATH5 and CATH6) showed expression patterns contrasting those of β-defensins, with the highest expression in tissues derived from bacteria-free udders.ConclusionIncreased expression of genes encoding β-defensins in the infected udder confirms their crucial role in the defense of the cow mammary gland against mastitis. On the other hand, the elevated cathelicidin transcripts in non-infected tissues indicate their role in the maintenance of healthy mammary tissues. The expression levels of investigated genes are likely to depend on the duration of the infection and type of bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-014-0246-z) contains supplementary material, which is available to authorized users.

Aspectos de la respuesta inmune innata en las infecciones intramamarias causadas por Staphylococcus aureus en bovinos

Staphylococcus aureus is the pathogen most frequently isolated from bovine mastitis worldwide, causing chronic intramammary infections that limit profitable dairying. The objective of this article is to characterize the mechanisms involved in S. aureus mammary gland infections considering two different aspects of the infectious process; on the one hand, the aspects involved in the host innate immune response and on the other hand, the capacity of this organism to evade the immune system and interact with different cell types. The exploration of S. aureus interactions with the immune response of bovine mammary gland will help identify targets to outline new preventive or curative alternatives for intramammary infections caused by this organism.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide.

To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. 5 lactating Red Holsteins. Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 μg of lipopolysaccharide [LPS], 100 μg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1β in milk somatic cells were analyzed. Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1β, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1β in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1β during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Life‐time genistein consumption alters unfolded protein response in mammary glands and tumors, and prevents antiestrogen resistance in rats (121.5)

We have previously observed that life‐time dietary intake of genistein (GEN) prevents development of tamoxifen (TAM) resistance in rat mammary tumor. This study investigated the mechanisms explaining this effect.Female Sprague‐Dawley rats were supplemented with 0 or 500 ppm GEN before puberty. Mammary carcinogen DMBA was administered at 48 days of age, and four dietary groups were established: lifelong GEN, prepubertal GEN, adult GEN and controls. Rats were then treated with 337 ppm TAM citrate via diet, and those fed GEN lifelong or during adult life were kept on GEN during TAM treatment. GEN was also given again to the rats consuming it at prepuberty and to some control rats. The remaining TAM treated control rats were never fed GEN. Tumors of rats fed GEN during prepuberty or lifelong exhibited reduced resistance to TAM, compared to no GEN controls, whilst adult only GEN intake caused TAM resistance (p&lt;0.001). Mammary cancer recurrence rate was highest in the rats which started consuming GEN during TAM treatment (p&lt;0.001).Chronic activation of the unfolded protein response (UPR) pathway has been linked to TAM resistance. We found that the active form of ATF6, ATF6 p50, was higher in normal mammary glands of lifelong GEN fed rats than in the controls (p&lt;0.05). mRNA expression of its target genes, Xbp1 and GRP78, were also higher in the lifelong GEN group (p&lt;0.05). However, the levels of GRP78, IRE1α, ATF4 and Beclin were significantly lower in the tumors of lifelong GEN exposed rats compared with adulthood only GEN fed rats (p&lt;0.05).Thus, lifelong GEN intake may stimulate responsiveness of mammary tumors to TAM by affecting the UPR pathway:members of this pathway were activated in the normal mammary gland and suppressed in the tumors of lifelong GEN consuming rats. Mammary tumor response to TAM treatment no GEN control post‐diagnosis GEN adult GEN prepubertal GEN lifelong GEN Complete response (%) 54 33 38 56 52 Partial response (%) 8 16 21 21 24 Resistance (%) 38 51 41 23 24 Recurrence (% of complete response) 23 33 7 11 18 Grant Funding Source: Supported by NCI U54CA149147