In vitro expansion of the mammary stem/progenitor cell population by xanthosine treatment

Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC) with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU) labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B) and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.https://doi.org/10.1186/1471-2121-13-1

Consumption of endophyte-infected fescue seed during the dry period does not decrease milk production in the following lactation.

Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90±4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (-60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation

Effect of consuming endophyte-infected fescue seed on transcript abundance in the mammary gland of lactating and dry cows, as assessed by RNA sequencing

Ergot alkaloids in endophyte-infected grasses inhibit prolactin secretion and reduce milk production in lactating cows. However, we previously showed that prepartum consumption of infected seed throughout the dry period did not inhibit subsequent milk production and prior exposure to bromocriptine (ergot peptide) actually increased production in the next lactation. To identify changes in the transcriptome and molecular pathways mediating the mammary gland's response to ergot alkaloids in the diet, RNA sequencing (RNA-seq) was performed on mammary tissues obtained from 22 multiparous Holstein cows exposed to 1 of 3 treatments. Starting at 90 ± 4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (BROMO; 0.1 mg/kg of BW), or endophyte-infected fescue seed (INF) as 10% of the diet. Cows were dried off 60 ± 2 d prepartum. Mammary biopsies from 4 (BROMO, INF) or 5 (CON) cows/treatment at each of the 3 phases were obtained: 7 d before dry off during the initial lactation (L1), mid-dry period (D), and 10 d postpartum (L2). Although tissue from the same cow was preferentially used at 3 phases (L1, D, L2), tissue from additional cows were used to as necessary to provide RNA of sufficient quality. Individual samples were used to generate individual RNA-seq libraries. Normalized reads of the RNA-seq data were organized into technical and biological replicates before processing with the RSEM software package. Each lactation phase was processed separately and genes that differed between any of 3 treatments were identified. A large proportion of genes differentially expressed in at least 1 treatment (n = 866) were found to be similarly expressed in BROMO and INF treatments, but differentially expressed from CON (n = 575, total for 3 phases). Of genes differentially expressed compared with CON, 104 genes were common to the L1 and L2 phases. Consistent with the production findings, networks most affected by treatments in L1 and L2 included lipid metabolism, small molecule biochemistry, and molecular transport, whereas networks related more to developmental and cellular functions and maintenance were evident during D phase. Similar patterns of expression in BROMO and INF during late and early lactation suggest involvement of similar cell signaling pathways or mechanisms of action for BROMO and INF and the importance of prolactin messaging pathways

Bacterial Endotoxin Induces Oxidative Stress and Reduces Milk Protein Expression and Hypoxia in the Mouse Mammary Gland

The aim of this study was to investigate the mechanisms underlying the reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces hypoxia, oxidative stress, and cell apoptosis while inhibiting milk gene expression in the mammary gland. To test this hypothesis, the left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, E. coli 055: B5, 100 μL of 0.2 mg/mL) or sterile PBS through the teat meatus 3 days postpartum. At 10.5 and 22.5 h postinjection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the 4th glands were individually collected (n=8) and analyzed. LPS treatment induced mammary inflammation at both 12 and 24 h but promoted cell apoptosis only at 12 h. Consistently, H2O2 content was increased at 12 h (P<0.01), but dropped dramatically at 24 h (P<0.01) in the LPS-treated gland. Nevertheless, the total antioxidative capacity in tissue tended to be decreased by LPS at both 12 and 24 h (P=0.07 and 0.06, respectively). In agreement with these findings, LPS increased or tended to increase the mRNA expression of antioxidative genes Nqo1 at 12 h (P=0.05) and SLC7A11 at 24 h (P=0.08). In addition, LPS inhibited mammary expression of Csn2 and Lalba across time and protein expression of Csn1s1 at 24 h (P<0.05). Furthermore, hypoxyprobe staining intensity was greater in the alveoli of the PBS-treated gland than the LPS-treated gland at both 12 and 24 h, demonstrating a rise in oxygen tension by LPS treatment. In summary, our observations indicated that while intramammary LPS challenge incurs inflammation, it induces oxidative stress, increases cell apoptosis and oxygen tension, and differentially inhibits the milk protein expression in the mammary gland

Examination of the xanthosine response on gene expression of mammary epithelial cells using RNA-seq technology

Abstract Background Xanthosine treatment has been previously reported to increase mammary stem cell population and milk production in cattle and goats. However, the underlying molecular mechanisms associated with the increase in stem cell population and milk production remain unclear. Methods Primiparous Beetal goats were assigned to the study. Five days post-partum, one mammary gland of each goat was infused with xanthosine (TRT) twice daily (2×) for 3 days consecutively, and the other gland served as a control (CON). Milk samples from the TRT and CON glands were collected on the 10th day after the last xanthosine infusion and the total RNA was isolated from milk fat globules (MEGs). Total RNA in MFGs was mainly derived from the milk epithelial cells (MECs) as evidenced by expression of milk synthesis genes. Significant differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) terms using PANTHER and gene networks were generated using STRING db. Results Preliminary analysis indicated that each individual goat responded to xanthosine treatment differently, with this trend being correlated with specific DEGs within the same animal’s mammary gland. Several pathways are impacted by these DEGs, including cell communication, cell proliferation and anti-microbials. Conclusions This study provides valuable insights into transcriptomic changes in milk producing epithelial cells in response to xanthosine treatment. Further characterization of DEGs identified in this study is likely to delineate the molecular mechanisms of increased milk production and stem or progenitor cell population by the xanthosine treatment

Effect of consuming endophyte-infected fescue seed on transcript abundance in the mammary gland of lactating and dry cows, as assessed by RNA sequencing

Ergot alkaloids in endophyte-infected grasses inhibit prolactin secretion and reduce milk production in lactating cows. However, we previously showed that prepartum consumption of infected seed throughout the dry period did not inhibit subsequent milk production and prior exposure to bromocriptine (ergot peptide) actually increased production in the next lactation. To identify changes in the transcriptome and molecular pathways mediating the mammary gland's response to ergot alkaloids in the diet, RNA sequencing (RNA-seq) was performed on mammary tissues obtained from 22 multiparous Holstein cows exposed to 1 of 3 treatments. Starting at 90 \ub1 4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3 7/wk subcutaneous injections of bromocriptine (BROMO; 0.1 mg/kg of BW), or endophyte-infected fescue seed (INF) as 10% of the diet. Cows were dried off 60 \ub1 2 d prepartum. Mammary biopsies from 4 (BROMO, INF) or 5 (CON) cows/treatment at each of the 3 phases were obtained: 7 d before dry off during the initial lactation (L1), mid-dry period (D), and 10 d postpartum (L2). Although tissue from the same cow was preferentially used at 3 phases (L1, D, L2), tissue from additional cows were used to as necessary to provide RNA of sufficient quality. Individual samples were used to generate individual RNA-seq libraries. Normalized reads of the RNA-seq data were organized into technical and biological replicates before processing with the RSEM software package. Each lactation phase was processed separately and genes that differed between any of 3 treatments were identified. A large proportion of genes differentially expressed in at least 1 treatment (n = 866) were found to be similarly expressed in BROMO and INF treatments, but differentially expressed from CON (n = 575, total for 3 phases). Of genes differentially expressed compared with CON, 104 genes were common to the L1 and L2 phases. Consistent with the production findings, networks most affected by treatments in L1 and L2 included lipid metabolism, small molecule biochemistry, and molecular transport, whereas networks related more to developmental and cellular functions and maintenance were evident during D phase. Similar patterns of expression in BROMO and INF during late and early lactation suggest involvement of similar cell signaling pathways or mechanisms of action for BROMO and INF and the importance of prolactin messaging pathways

Consumption of endophyte-infected fescue seed during the dry period does not decrease milk production in the following lactation

Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90\ub14 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3 7/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (-60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation