Gene Expression In Mammary Gland Research Articles

Transcriptomic Analysis of Pubertal and Adult Virgin Mouse Mammary Epithelial and Stromal Cell Populations

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

Preliminary monosodium glutamate-induced changes in mammary gland receptors and gene expression, water channel, oxidative stress, and some lactogenic biomarkers in lactating rats

BackgroundChanges induced by monosodium glutamate (MSG) can negatively impact milk production and secretion, among other adverse effects. This study aimed to investigate the effects of MSG consumption on receptor gene expression and quantification of hormones and receptors, as well as oxidative stress biomarkers and other lactogenic parameters in lactating animals. Twenty-four female Wistar rats, nine weeks of age, were randomly assigned to four groups, each containing six rats, at parturition. The rats in groups II, III, and IV were given varying doses of monosodium glutamate (MSG); while, group I was given distilled water and served as the control. The experimental period lasted two (2) weeks.ResultsThe groups administered with MSG showed a significant decrease in mammary PRLR gene expression (p < 0.05), as well as a marked reduction (p < 0.05) in mammary PRLR, OXT receptor, AQP-3, brain antioxidant enzymes (SOD, GPx, and CAT), and pituitary SOD compared to the control group (p < 0.05). Furthermore, there was a significant increase (p < 0.05) in reactive oxygen species levels in the serum and mammary gland homogenates, erythrocyte osmotic fragility, and elevated (p < 0.05) brain and pituitary MDA levels in the MSG-administered groups compared to the control group. Daily milk yields were significantly decreased (p < 0.05) in the MSG-administered groups between days 10 and 14 of lactation.ConclusionThe findings of this study suggest that prolonged consumption of MSG could interfere with lactation-associated functions via increased ROS production, reduced antioxidants, decreased AQP-3, mammary prolactin and oxytocin receptors, and prolactin receptor mRNA in lactating Wistar rats.Graphical

Abstract B001: The effect of chronic psychological stress during puberty and early adulthood on breast cancer risk and development

Abstract Breast cancer is the second most common cancer among women in the United States. Puberty is a well-established sensitive period in women when breast cancer (BC) risk is increased by the physical environment for example exposure to chemical carcinogens, synthetic estrogens, endocrine disrupters, radiation, etc. However, relatively little is known about effects of exposure to chronic psychological stress from the social environment during this time period (puberty and early adulthood). In previous studies using the Sprague-Dawley rat model of spontaneous mammary cancer, chronically heightened glucocorticoid stress reactivity arising from imposed social isolation after weaning increased duration of glucocorticoid release in response to everyday stressors, impeded mammary gland ductal development, and caused earlier and more aggressive mammary gland cancer incidence during adulthood. However due to the period of isolation lasting from weaning to death, a critical time period in which these changes occurred was not established. Here we found that chronic social isolation only during puberty and early adulthood results in alterations in mammary gland gene expression patterns and preservation of mammary stem/progenitor cells. We have observed that social isolation beginning at five weeks and ending at 21 weeks leads to a 4-fold increase in the key metabolic lipogenic enzymes ATP citrate lyase (ACLY; p-value = 0.008), 2-fold increase in acetyl-CoA carboxylase (Acaca; p-value=0.01), and 1.88-fold change in stearoyl-CoA desaturase 1 (Scd1; p-value = 0.50). Interestingly, we have also observed that social isolation during this time period, leads to a slight decrease (0.70-fold change) in hexokinase 2 (HK2; p-value= 0.007). Regarding mammary stem/progenitor cell preservation, we found that social isolation beginning at five weeks and ending at 17 weeks leads to a significant increase in the number of mammary gland stem/progenitor cells (isolated by FACS sorting). This correlation was only observed in response to corticosterone reactivity, and null to exposure to estrogen or progesterone. In summary, our findings suggest that social isolation during puberty and early adulthood correlates to increased glucocorticoid exposure resulting in metabolic reprogramming in the mammary gland and inappropriate preservation of mammary stem/progenitor cells. Altogether these factors may increase the risk of mammary cancer occurrence later in adulthood. Citation Format: Briana Banks, Matthew Brady. The effect of chronic psychological stress during puberty and early adulthood on breast cancer risk and development [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B001.

Limits to sustained energy intake. XXXIII. Thyroid hormones play important roles in milk production but do not define the heat dissipation limit in Swiss mice.

The limits to sustained energy intake set physiological upper boundaries that affect many aspects of human and animal performance. The mechanisms underlying these limits however remain unclear. We exposed Swiss mice to either supplementary thyroid hormones (THs) or methimazole during lactation at 21 °C or 32.5 °C, and measured food intake, resting metabolic rate (RMR), milk energy output (MEO), serum THs and mammary gland gene expression of females, and litter size and mass of their offspring. Lactating females developed hyperthyroidism following exposure to supplementary THs at 21 °C, but they did not significantly change body temperature, asymptotic food intake, RMR or MEO, and litter and mass were unaffected. Hypothyroidism, induced by either methimazole or 32.5 °C exposure, significantly decreased asymptotic food intake, RMR and MEO, resulting in significantly decreased litter size and litter mass. Furthermore, gene expression of key genes in the mammary gland were significantly decreased by either methimazole or hot exposure, including gene expression of THs and prolactin receptors, and Stat5a and Stat5b. This suggests that endogenous THs are necessary to maintain sustained energy intake and milk energy output. Suppression of the thyroid axis seems to be an essential aspect of the mechanism by which mice at 32.5 °C reduce their lactation performance to avoid overheating. However, THs do not define the upper limit to sustained energy intake and milk energy output at peak lactation at 21 °C. Another, as yet unknown, factor, prevents supplementary thyroxine exerting any stimulatory metabolic impacts on lactating mice at 21 °C.

Malic Enzyme 1 (ME1) Promotes Adiposity and Hepatic Steatosis and Induces Circulating Insulin and Leptin in Obese Female Mice.

Malic Enzyme 1 (ME1) supports lipogenesis, cholesterol synthesis, and cellular redox potential by catalyzing the decarboxylation of L-malate to pyruvate, and the concomitant reduction of NADP to NADPH. We examined the contribution of ME1 to the development of obesity by provision of an obesogenic diet to C57BL/6 wild type (WT) and MOD-1 (lack ME1 protein) female mice. Adiposity, serum hormone levels, and adipose, mammary gland, liver, and small intestine gene expression patterns were compared between experimental groups after 10 weeks on a diet. Relative to WT female mice, MOD-1 female mice exhibited lower body weights and less adiposity; decreased concentrations of insulin, leptin, and estrogen; higher concentrations of adiponectin and progesterone; smaller-sized mammary gland adipocytes; and reduced hepatosteatosis. MOD-1 mice had diminished expression of Lep gene in abdominal fat; Lep, Pparg, Klf9, and Acaca genes in mammary glands; Pparg and Cdkn1a genes in liver; and Tlr9 and Ffar3 genes in the small intestine. By contrast, liver expression of Cdkn2a and Lepr genes was augmented in MOD-1, relative to WT mice. Results document an integrative role for ME1 in development of female obesity, suggest novel linkages with specific pathways/genes, and further support the therapeutic targeting of ME1 for obesity, diabetes, and fatty liver disease.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Abstract 24: The effect of chronic psychological stress during puberty and early adulthood on mammary gland development and mammary cancer risk in adulthood

Abstract Breast cancer is the second most common cancer among women in the United States. Puberty is a well-established sensitive period in women when breast cancer (BC) risk is increased by the physical environment for example exposure to chemical carcinogens, synthetic estrogens, endocrine disrupters, radiation, etc. However, relatively little is known about effects of exposure to chronic psychological stress from the social environment during this time period (puberty and early adulthood). In previous studies using the Sprague-Dawley rat model of spontaneous mammary cancer, chronically heightened glucocorticoid stress reactivity arising from imposed social isolation after weaning increased duration of glucocorticoid release in response to everyday stressors, impeded mammary gland ductal development, and caused earlier and more aggressive mammary gland cancer incidence during adulthood. However due to the period of isolation lasting from weaning to death, a critical time period in which these changes occurred was not established. Here we found that chronic social isolation only during puberty and early adulthood results in alterations in mammary gland gene expression patterns and preservation of mammary stem/progenitor cells. We have observed that social isolation beginning at five weeks and ending at 21 weeks leads to a 4-fold increase in the key metabolic lipogenic enzymes ATP citrate lyase (ACLY; p-value = 0.008), 2-fold increase in acetyl-CoA carboxylase (Acaca; p-value=0.01), and 1.88-fold change in stearoyl-CoA desaturase 1 (Scd1; p-value = 0.50). Interestingly, we have also observed that social isolation during this time period, leads to a slight decrease (0.70-fold change) in hexokinase 2 (HK2; p-value= 0.007). Regarding mammary stem/progenitor cell preservation, we found that social isolation beginning at five weeks and ending at 17 weeks leads to a significant increase in the number of mammary gland stem/progenitor cells (isolated by FACS sorting). This correlation was only observed in response to corticosterone reactivity, and null to exposure to estrogen or progesterone. In summary, our findings suggest that social isolation during puberty and early adulthood correlates to increased glucocorticoid exposure resulting in metabolic reprogramming in the mammary gland and inappropriate preservation of mammary stem/progenitor cells. Altogether these factors may increase the risk of mammary cancer occurrence later in adulthood. Citation Format: Briana Banks, Marianna Johnson, Joscelyn Hoffmann, Hannah You, Matt Piron, Suzanne Conzen, Martha McClintock, Matthew Brady. The effect of chronic psychological stress during puberty and early adulthood on mammary gland development and mammary cancer risk in adulthood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 24.

Effects of Water Extract from Artemisia argyi Leaves on LPS-Induced Mastitis in Mice.

Simple SummaryMastitis is a common disease in dairy cows. On the one hand, it will reduce milk yield and milk quality of dairy cows, thus increasing the cost of animal husbandry, and, on the other hand, it will influence the health of infected animals and even human beings. Generally speaking, because mastitis is caused by pathogenic microorganisms, antibiotic treatment is commonly used. However, antibiotic resistance of microorganisms caused by wrongful use of antibiotics and antibiotic residues after treatment has become an urgent problem to be solved. Chinese herbal medicines are pure natural substances, and many of them have antibacterial, anti-inflammatory, or immune-enhancing effects. In this experiment, Artemisia argyi (A. argyi) was selected as the research object to construct the cell model of cow mastitis. Studies have found that A. argyi extract can play a positive role in the regulation of inflammation, which is rich in organic acids and flavonoids. Therefore, A. argyi extract may be a potential treatment for mastitis.In the context of the unsatisfactory therapeutic effect of antibiotics, the natural products of plants have become a research hotspot. Artemisia argyi (A. argyi) is known as a traditional medicine in China, and its extracts have been reported to have a variety of active functions, including anti-inflammatory. Therefore, after establishing the mouse mastitis model by lipopolysaccharide (LPS), the effects of A. argyi leaves extract (ALE) were evaluated by pathological morphology of the mammary gland tissue, gene expression, and serum oxidation index. Studies have shown that ALE has a restorative effect on LPS-induced mammary gland lesions and significantly down-regulated the rise of myeloperoxidase (MPO) induced by LPS stimulation. In addition, ALE played a positive role in LPS-induced oxidative imbalance by restoring the activities of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) and preventing the increase in nitric oxide (NO) concentration caused by the over-activation of total nitric oxide synthase (T-NOS). Further analysis of gene expression in the mammary gland showed that ALE significantly down-regulated LPS-induced up-regulation of inflammatory factors IL6, TNFα, and IL1β. ALE also regulated the expression of MyD88, a key gene for toll-like receptors (TLRs) signaling, which, in turn, regulated TLR2 and TLR4. The effect of ALE on iNOS expression was similar to the effect of T-NOS activity and NO content, which also played a positive role. The IκB gene is closely related to the NF-κB signaling pathway, and ALE was found to significantly alleviate the LPS-induced increase in IκB. All of these results indicated that ALE may be considered a potential active substance for mastitis.

Investigating the effect of positional variation on mid-lactation mammary gland transcriptomics in mice fed either a low-fat or high-fat diet.

Little attention has been given to the effect of positional variation of gene expression in the mammary gland. However, more research is shedding light regarding the physiological differences that mammary gland location can have on the murine mammary gland. Here we examined the differentially expressed genes between mammary gland positions under either a low-fat diet (LFD) or a high-fat diet (HFD) in the mid-lactation mammary gland (lactation day 11; L11). Three-week old WT C57BL/6 mice were randomly assigned to either a low-fat diet (LFD) or high fat diet (HFD) (n = 3/group) and either the right thoracic mammary gland (TMG) or inguinal mammary gland (IMG) was collected from each dam for a total of 12 unique glands. Within each diet, differentially expressed genes (DEGs) were first filtered by adjusted p-value (cutoff ≤ 0.05) and fold-change (FC, cutoff ≥2). Genes were further filtered by mean normalized read count with a cutoff≥10. We observed that mammary gland position had a significant impact on mammary gland gene expression with either LFD or HFD diet, with 1264 DEGs in LFD dams and 777 DEGs in HFD dams. We found that genes related to snRNP binding and translation initiation were most significantly altered between the TMG and IMG. Although we were not able to discern a molecular mechanism, many small nuclear RNAs and small nucleolar RNAs were differentially expressed between the TMG and IMG responsible for cellular functions such as splicing and ribosome biogenesis, which provides and interesting avenue for future research. Our study supports the hypothesis that collection of the mammary gland from a particular location influences mammary gland gene expression, thereby highlighting the importance for researchers to be vigilant in documenting and reporting which mammary gland they are using for their studies.

Short communication: Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci

Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.

Effect of heat stress during the early and late dry period on mammary gland development of Holstein dairy cattle

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ∼45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, CSN2, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Immunophenotyping and cytokine gene expression in experimental intramammary infection with staphylococcal species in mice

Immunophenotyping and proinflammatory cytokine gene expression in mice mammary gland inoculated with Staphylococcus epidermidis, S. chromogenes, S. haemolyticus and S. aureus isolated from bovine milk were studied. Swiss albino mice were inoculated with 50 μl (2×104 CFU organisms) per mammary gland (L4, L5, R4 and R5) through intramammary route. Mice were sacrificed at 6, 12, 24, 48, 72 and 96 h, and blood and mammary gland tissues were collected for blood immunophenotyping and cytokine gene expression studies, respectively. CD4+ cells increased in S. aureus, S. epidermidis, S. haemolyticus and decreased in S. chromogenes inoculated mice. CD8 + cells increased in S. epidermidis compared to other groups. Proinflammatory cytokines showed increased expression of IL-1β (6 folds), IL-4 (3 folds), IL-6 (1 fold), IL-12 (5 folds), TNF-α (5 folds) and IFN-γ (2 folds) in S. aureus inoculated mice. S. epidermidis revealed 2 fold increase in IL-1β and IL-4, and 1 fold increase in IL-6, IL-12, TNF-α and IFN-γ. S. chromogenes showed increased expression of 2 folds in IL-1β, IFN-γ; 3 folds in IL-4, IL-6, TNF-α and 7 folds in IL-12. S. haemolyticus revealed 2 folds increase in IL-1β, IL-12, TNF-α; 3 folds in IL-6 and 5 folds in IL-4. Hence, S. aureus caused severe mastitis in mice when compared to three coagulase negative staphylococcal (CNS) species. The proinflammatory cytokines (IL-1β, IL-4 and IFN-γ) can be used as an indicator for early detection and mice can be used as mastitis model to study CNS mastitis.

Effects of Azgp1−/−on mammary gland, adipose tissue and liver gene expression and milk lipid composition in lactating mice

The expression of Azgp1 gene, an adipokine involved in the mobilization of body reserves, was observed in mammary gland of ruminants. Its regulation by different dietary conditions suggests a potential role in the mechanisms controlling the composition of milk fat. The aim of this study was to evaluate the role of Azgp1 during lactation. Azgp1−/− mice were compared to wild-type to determine its effects on milk fatty acid composition and offspring growth. To determine its effects on mammary gland, adipose tissue and liver gene expression, gene expression was analyzed using RT-qPCR via TLDA analyses. The body weight of Azgp1−/− mothers was slightly higher after parturition and at 10 days of lactation compared to the wild type. The milk polyunsaturated fatty acid content was increased in Azgp1−/− mice. Among the 40 genes studied, Azgp1−/− modified the expression of 9, 10 and 3 genes in mammary gland, adipose tissue and liver, respectively. These genes, involved in fatty acid synthesis, transport and triglyceride synthesis, were downregulated in Azgp1−/− mice showing a particularity during lactation. Changes in mammary gland gene expression may explain the modifications observed in milk fatty acid composition. This study supports a role of Azgp1 on lipid metabolism, in particular in mammary gland, during lactation function.

Effects of anthocyanin-rich purple corn (Zea mays L.) stover silage on nutrient utilization, rumen fermentation, plasma antioxidant capacity, and mammary gland gene expression in dairy goats1.

Eight healthy multiparous Saanen dairy goats (41.50 ± 1.84 kg) were assigned to a double 4 × 4 Latin square design. The four treatment diets were: (i) negative control, rice straw (NC); (ii) first positive control, sticky corn stover silage (PC1); (iii) second positive control, PC1 with 1 g/d commercial purple corn pigment (PC2); and (iv) anthocyanin-rich purple corn stover silage (PSS; AR). DMI did not differ (P > 0.05) among the treatments. Goats receiving the NC tended (P < 0.05) to reduce nutrient apparent digestibility, nitrogen (N) absorption, N retention, and volatile fatty acid production relative to the other groups. The levels of 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and superoxide dismutase (SOD) in plasma were greater (P < 0.05) in goats fed PC2 and AR compared with NC and PC1. The inclusion of PC2 and AR increased (P < 0.05) the abundance of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), but decreased (P < 0.05) the level of tumor necrosis factor in the mammary gland. Moreover, goats receiving AR tended to increase (P < 0.05) the levels of SOD2, GPX1, and GPX2 mRNA expression in the mammary gland. There were significant (P < 0.05) positive correlations between DPPH scavenging activity, total antioxidant capacity, SOD, catalase enzymes in plasma, and the abundance of NFE2L2 in the mammary gland. In addition, stronger (P < 0.05) positive correlations were noted between the expression of several inflammation related and antioxidant genes. Collectively, the results from the current study indicated that the consumption of anthocyanin-rich PSS by dairy goats had the potential to enhance antioxidant potential by improving antioxidant capacity in plasma and by modulating the abundance of several inflammation related and antioxidant genes in the mammary gland.

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.

Alteration of Mammary Gland Development and Gene Expression by In Utero Exposure to Cadmium.

Environmental exposure to estrogens and estrogen like contaminants during early development is thought to contribute to the risk of developing breast cancer primarily due to an early onset of puberty; however, exposure during key developing windows may also influence the risk of developing the disease. The goal of this study was to ask whether in utero exposure to the metalloestrogen cadmium alters mammary gland development due to acceleration of puberty onset or to an effect on early development of the mammary gland. The results show that, in addition to advancing the onset of puberty, in utero exposure to the metalloestrogen cadmium altered mammary gland development prior to its effect on puberty onset. In utero exposure resulted in an expansion of the number of mammosphere-forming cells in the neonatal mammary gland and an increase in branching, epithelial cells, and density in the prepubertal mammary gland. In the postpubertal mammary gland, there was a further expansion of the mammary stem/progenitor cell population and overexpression of estrogen receptor-alpha (ERα) that was due to the overexpression and altered regulation of the ERα transcripts derived from exons O and OT in response to estradiol. These results suggest that in utero exposure to cadmium increases stem/progenitor cells, cell density, and expression of estrogen receptor-alpha that may contribute to the risk of developing breast cancer.

Gestational high-fat diet and bisphenol A exposure heightens mammary cancer risk.

In utero exposure to bisphenol A (BPA) increases mammary cancer susceptibility in offspring. High-fat diet is widely believed to be a risk factor of breast cancer. The objective of this study was to determine whether maternal exposure to BPA in addition to high-butterfat (HBF) intake during pregnancy further influences carcinogen-induced mammary cancer risk in offspring, and its dose–response curve. In this study, we found that gestational HBF intake in addition to a low-dose BPA (25 µg/kg BW/day) exposure increased mammary tumor incidence in a 50-day-of-age chemical carcinogen administration model and altered mammary gland morphology in offspring in a non-monotonic manner, while shortening tumor-free survival time compared with the HBF-alone group. In utero HBF and BPA exposure elicited differential effects at the gene level in PND21 mammary glands through DNA methylation, compared with HBF intake in the absence of BPA. Top HBF + BPA-dysregulated genes (ALDH1B1, ASTL, CA7, CPLX4, KCNV2, MAGEE2 and TUBA3E) are associated with poor overall survival in The Cancer Genomic Atlas (TCGA) human breast cancer cohort (n = 1082). Furthermore, the prognostic power of the identified genes was further enhanced in the survival analysis of Caucasian patients with estrogen receptor-positive tumors. In conclusion, concurrent HBF dietary and a low-dose BPA exposure during pregnancy increases mammary tumor incidence in offspring, accompanied by alterations in mammary gland development and gene expression, and possibly through epigenetic reprogramming.

Aryl Hydrocarbon Receptor Activation via Beta Naphthoflavone Disrupts Mouse Mammary Gland Development during Lactogenesis

Aryl hydrocarbon receptor (AHR) ligands including 2,3,7,8-tetrachlorodibenzodioxin (TCDD) have been shown to have deleterious effects on lactation. In the current study, the effects of dietary beta naphthoflavone (5,6- benzoflavone, BNF), a potent AHR agonist, were characterized in lactating dams. Using morphologic and molecular techniques we observed disruption of mammary gland differentiation, gene expression, and metabolism in lactating Ahrb (high affinity) mice. Similar effects were absent in lactating Ahrd (low affinity) mice. Quantitative PCR analysis revealed ~50% reduction in the expression of key lactogenesis mammary genes including whey acid protein (Wap), α-lactalbumin (Lalba), and β-casein that is consistent with previous reports. All together results including those from Ahrd (low affinity) mice support that the AHR is an possible important regulator of lactation and BNF in the mammary gland cause serve disruption.

Physical activity induced protection against breast cancer risk associated with delayed parity

Epidemiological evidence indicates that physical activity between menarche and first pregnancy is associated with a lower risk of breast cancer among women with at least 20years between these reproductive events. The mechanism by which physical activity during this interval confers protection is unknown. This study used a novel animal model to assess potentially protective effects of physical activity on tumor development in delayed parity. Thirty-six female Sprague Dawley rats received an i.p. injection of 50mg/kg N-methyl-N-nitrosourea (MNU) at 5weeks of age. Estrogen and progesterone pellets were implanted subcutaneously 1week (early parity, EP, n=8) or 4weeks (delayed parity, DP, n=11) following MNU injection. An additional group of DP rats were progressively exercise trained (Ex+DP, n=9) on a treadmill following MNU injection for 7weeks (up to 20m/min at 15% incline for 30min). We observed the greatest tumor latency and smallest tumor burden in Ex+DP animals. Ductal hyperplasia and inflammation of non-tumor bearing mammary glands were only found in DP, and we detected a significant increase in collagen for DP and Ex+DP compared to EP. Exercise induced differential gene expression of cyclin-dependent kinase-inhibitor 1C (Cdkn1c) and urokinase-plasminogen activator (Plau) in mammary tissue of Ex+DP animals compared to DP alone. While there are delayed parity-induced changes in mammary gland collagen and gene expression levels, Ex+DP animals had longer tumor latency, smaller tumor burden, and glandular tissue resistant to ductal hyperplasia. Exercise may induce protection through beneficial regulation of gene expression profiles.

0845 Effect of 17β-estradiol on milk production, hormone secretion, and mammary gland gene expression of dairy cows

0845 Effect of 17β-estradiol on milk production, hormone secretion, and mammary gland gene expression of dairy cows