scholarly journals  Gene expression of six major milk proteins in primary bovine mammary epithelial cells isolated from milk during the first twenty weeks of lactation

2012 ◽  
Vol 57 (No. 10) ◽  
pp. 469-480 ◽  
Author(s):  
T. Sigl ◽  
H.H.D. Meyer ◽  
S. Wiedemann
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Milk Protein ◽  
Protein Gene ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Milk Protein Gene ◽  
Bovine Mammary Epithelial Cells ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression

&nbsp;The objective of the present study was to refine a previously developed method to isolate primary bovine mammary epithelial cells (pBMEC) from fresh milk. Using this method, it was tested whether the number of pBMEC and the relation of recovered pBMEC to total somatic cell count vary within the individual lactation stages. Furthermore, the expression levels of the milk protein genes during the first twenty weeks of lactation were determined by quantitative PCR method. A total number of 152 morning milk samples were obtained from twenty-four Holstein-Friesian cows during the first 20 weeks of lactation (day 8, 15, 26, 43, 57, 113, and 141 postpartum). Numbers of extracted pBMEC were consistent at all time-points (1.1 &plusmn; 0.06 to 1.4 &plusmn; 0.03 &times;10<sup>3</sup>/ml) and an average value of RNA integrity number (RIN) was 6.3 &plusmn; 0.3. Percentage of pBMEC in relation to total milk cells (2.0 &plusmn; 0.2 to 6.7 &plusmn; 1.0%) correlated with milk yield. Expression patterns of the casein genes alpha (&alpha;)<sub>S1</sub>, (&alpha;)<sub>S2</sub>, beta (&beta;), and kappa (&kappa;) (CSN1S1, CSN1S2, CSN2, CSN3, respectively) and the whey protein genes &alpha;-lactalbumin (LALBA) and progestagen-associated endometrial protein (PAEP; known as &beta;-lactoglobulin) were shown to be comparable, i.e. transcripts of all six milk protein genes were found to peak during the first two weeks of lactation and to decline continuously towards mid lactation. However, mRNA levels were different among genes with CSN3 showing the highest and LALBA the lowest abundance. We hypothesized that milk protein gene expression has a pivotal effect on milk protein composition with no influence on milk protein concentration. This paper is the first to describe milk protein gene expression during lactation in pBMEC collected in milk. Future studies will be needed to understand molecular mechanisms in pBMEC including regulation of expression and translation throughout lactation. &nbsp;

scholarly journals Uncoupling the mechanisms that facilitate cell survival in hormone-deprived bovine mammary explants

2008 ◽  
Vol 41 (3) ◽  
pp. 103-116 ◽  
Author(s):  
Amelia J Brennan ◽  
Julie A Sharp ◽  
Christophe M Lefèvre ◽  
Kevin R Nicholas
Keyword(s):  
Gene Expression ◽  
Cell Survival ◽  
Milk Protein ◽  
Protein Gene ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Milk Protein Gene ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression ◽  
Lactogenic Hormones

Mammary explants can be hormonally stimulated to mimic the biochemical changes that occur during lactogenesis. Previous studies using mammary explants concluded that the addition of exogenous macromolecules were required for mammary epithelial cells to remain viable in culture. The present study examines the survival of mammary explants from the dairy cow using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants cultured from late pregnant cows mimicked lactogenesis and showed significantly elevated milk protein gene expression after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. During this time, the mammary explants remained hormone responsive, the alveolar architecture was maintained and the expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. We report that a population of bovine mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. In addition, we found mammary explant viability was dependent on de novo protein and RNA synthesis. Global functional microarray analysis showed that differential expression of genes involved in energy production, immune responses, oxidative stress and apoptosis signalling might contribute to cell survival. As the decline in milk production in dairy cattle after peak lactation results in considerable economic loss, the identification of novel survival genes may be used as genetic markers for breeding programmes to improve lactational persistency in dairy cows.

scholarly journals A population of mammary epithelial cells do not require hormones or growth factors to survive

2008 ◽  
Vol 196 (3) ◽  
pp. 483-496 ◽  
Author(s):  
Amelia J Brennan ◽  
Julie A Sharp ◽  
Elie Khalil ◽  
Matthew R Digby ◽  
Sonia L Mailer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Milk Protein ◽  
Protein Gene ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Milk Protein Gene ◽  
Culture Model ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression ◽  
Lactogenic Hormones

Hormonal stimulation of mammary explants mimics many of the biochemical changes observed during lactogenesis. Previous studies using eutherian species conclude that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. The present study examines the survival of mammary explants from the wallaby and mouse using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants from pregnant tammars and mice showed that milk protein gene expression was significantly elevated after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. Surprisingly, mammary explants remained hormone responsive and expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. Furthermore, the alveolar architecture was maintained. Global functional microarray analysis showed that classic involution markers were not differentially expressed, although two stress-induced survival genes were significantly up-regulated. We report that a population of mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. We propose that the mammary explant culture model uncouples the first phase of involution, as milk accumulation that normally provides involution stimuli is absent in this culture model allowing a population of cells to survive.

scholarly journals Vinculins interaction with talin is essential for mammary epithelial differentiation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pengbo Wang ◽  
Jian Wu ◽  
Amber Wood ◽  
Matthew Jones ◽  
Robert Pedley ◽  
...  
Keyword(s):  
Cellular Differentiation ◽  
Milk Protein ◽  
Protein Gene ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Milk Protein Gene ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression ◽  
Extracellular Microenvironment

AbstractVinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.

scholarly journals Cell proliferation and milk protein gene expression in rabbit mammary cell cultures

1983 ◽  
Vol 96 (5) ◽  
pp. 1435-1442 ◽  
Author(s):  
YML Suard ◽  
M Haeuptle ◽  
E Farinon ◽  
J Kraehenbuhl
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Protein Synthesis ◽  
Milk Protein ◽  
Protein Gene ◽  
Milk Proteins ◽  
Collagen Gels ◽  
Milk Protein Gene ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression

We analyzed the synthesis of DNA, the rate of cell proliferation, and the expression of milk protein genes in mammary cells grown as primary cultures on or in collagen gels in chemically defined media. We assessed DNA synthesis and cell growth, measured by [(3) H]- thymidine incorporation into acid-insoluble material, DNA content, and cell counts, in a progesterone- and prolactin-containing medium. In some experiments, cultures were pulsed for 1 h with [(3)H]thymidine and dissociated into individual cells which were cytocentrifuged and processed for immunocytochemistry and autoradiography. We analyzed expression of milk protein genes at the transcriptional, translation and posttranslational levels in progesterone-depleted medium in the presence or absence of prolactin. We measured protein secretion by radioimmunoassays with antisera directed against caseins, α-lactalbumin and milk transferrin1. We determined protein synthesis by incorporating radio-labeled amino acids into acid-precipitable material and by immunoprecipitating biosynthetically labeled milk proteins. We assessed the accumulation of casein mRNA by hybridizing total cellular RNA extracted from cultured cells with (32)P-labeled casein cDNA probes. On attached collagen gels, the cells synthesized DNA and replicated until they became confluent. The overall protein synthetic activity was low, and no milk proteins were synthesized or secreted even in the presence of prolactin. The block in milk protein gene expression was not restricted to translational or posttranslational events but also included transcription, since no casein mRNA accumulated in these cells. On floating gels, protein synthesis was threefold higher than in cells from attached gels. Overall protein synthesis as well as casein and α-lactalbumin synthesis and secretion were prolactin-dependent with maximal stimulation at around 10(-9) M. A marked inhibition occurred at higher hormone concentrations. Casein mRNA accumulated in these cells, provided prolactin was present in the medium. In contrast, these cells did not synthesize DNA, nor did they replicate. In embedding gels, the rate of cell proliferation was exponential over 25 d with a doubling time of approximately 70 h. The overall protein synthesis increase was parallel in time with the increase in cell number. Caseins and α-lactalbumin (in contrast to transferrin) were synthesized only in the presence of prolactin. We observed the same hormone dependency as with cells growing on floating gels. The number of casein- and transferring-positive cells was measured after dissociating the cell cultures. At day 12, 60 percent of the total cells stored transferring in small cytoplasmic vesicles, whereas only 25 percent of the cells accumulated casein. Differences in the organization and in the shape of mammary cells depending on cell surface conditions suggest that the geometry of the cells, their interaction with extracellular matrix constituents, and cell-to-cell interactions play a role in the expression of two mammary functions: DNA synthesis and growth, as well as milk protein gene expression.

Examination of methionine stimulation of gene expression in dairy cow mammary epithelial cells using RNA-sequencing

2020 ◽  
Vol 87 (2) ◽  
pp. 226-231
Author(s):  
Xiaoming Hou ◽  
Minghui Jiang ◽  
Jinyu Zhou ◽  
Shuyuan Song ◽  
Feng Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Epithelial Cells ◽  
Rna Sequencing ◽  
Milk Protein ◽  
Mammary Epithelial Cells ◽  
Cell Model ◽  
Expression Profiles ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells

AbstractIn this research communication, a cell model with elevated β-CASEIN synthesis was established by stimulating bovine mammary epithelial cells with 0.6 mM methionine, and the genome-wide gene expression profiles of methionine-stimulated cells and untreated cells were investigated by RNA sequencing. A total of 458 differentially expressed genes (DEGs; 219 upregulated and 239 downregulated) were identified between the two groups. Gene Ontology (GO) analysis showed that the two highest-ranked GO terms in ‘molecular function’ category were ‘binding’ and ‘catalytic activity’, suggesting that milk protein synthesis in methionine-stimulated cells requires induction of gene expression to increase metabolic activity. Kyoto Encyclopedia of Genes and Genomes analysis revealed that within the ‘environmental information processing’ category, the subcategory that is most highly enriched for DEGs was ‘signal transduction’. cGMP-PKG, Rap1, calcium, cAMP, PI3K-AKT, MAPK, and JAK-STAT are the pathways with the highest number of DEGs, suggesting that these signaling pathways have potential roles in mediating methionine-induced milk protein synthesis in bovine mammary epithelial cells. This study provides valuable insights into the physiological and metabolic adaptations in cells stimulated with methionine. Understanding the regulation of this transition is essential for effective intervention in the lactation process.

Gene expression in the mammary gland of the tammar wallaby during the lactation cycle reveals conserved mechanisms regulating mammalian lactation

2016 ◽  
Vol 28 (9) ◽  
pp. 1241 ◽  
Author(s):  
C. J. Vander Jagt ◽  
J. C. Whitley ◽  
B. G. Cocks ◽  
M. E. Goddard
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Milk Protein ◽  
Mammary Epithelial Cells ◽  
Tammar Wallaby ◽  
Mammary Epithelial ◽  
Molecular Networks ◽  
Milk Protein Gene ◽  
Milk Protein Gene Expression ◽  
Lactation Cycle

The tammar wallaby (Macropus eugenii), an Australian marsupial, has evolved a different lactation strategy compared with eutherian mammals, making it a valuable comparative model for lactation studies. The tammar mammary gland was investigated for changes in gene expression during key stages of the lactation cycle using microarrays. Differentially regulated genes were identified, annotated and subsequent gene ontologies, pathways and molecular networks analysed. Major milk-protein gene expression changes during lactation were in accord with changes in milk-protein secretion. However, other gene expression changes included changes in genes affecting mRNA stability, hormone and cytokine signalling and genes for transport and metabolism of amino acids and lipids. Some genes with large changes in expression have poorly known roles in lactation. For instance, SIM2 was upregulated at lactation initiation and may inhibit proliferation and involution of mammary epithelial cells, while FUT8 was upregulated in Phase 3 of lactation and may support the large increase in milk volume that occurs at this point in the lactation cycle. This pattern of regulation has not previously been reported and suggests that these genes may play a crucial regulatory role in marsupial milk production and are likely to play a related role in other mammals.

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