scholarly journals Transgenic mice carrying the guinea-pig α-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation

1991 ◽  
Vol 275 (2) ◽  
pp. 459-467 ◽  
Author(s):  
A Maschio ◽  
P M Brickell ◽  
D Kioussis ◽  
A L Mellor ◽  
D Katz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Guinea Pig ◽  
Milk Protein ◽  
Mouse Skin ◽  
Sebaceous Glands ◽  
Milk Protein Gene ◽  
Milk Protein Genes ◽  
Milk Protein Gene Expression ◽  
Alpha Lactalbumin

We have generated transgenic mice carrying the entire guinea-pig alpha-lactalbumin gene. Lactating transgenic mice expressed high levels of correctly initiated and processed guinea-pig alpha-lactalbumin mRNA in the secretory epithelium of their mammary glands, and secreted guinea-pig alpha-lactalbumin in their milk. Transcripts were detectable after 7 days of pregnancy, indicating that the transgene was under correct hormonal control. Whereas no or negligible transcription was detectable in all other tissues tested, high levels of transcripts were found in the skin of lactating transgenic mice. Guinea-pig alpha-lactalbumin protein was undetectable in the skin, however. In situ hybridization analysis showed that expression was localized to the undifferentiated cells in the basal layer of the sebaceous glands. Further studies revealed high levels of endogenous beta-casein mRNA in normal lactating mouse skin, demonstrating that the transcription of milk protein genes in lactating mouse skin is a normal event, and is not peculiar to the transgene. This surprising finding highlights the developmental relationship of the mammary gland to other specialized structures of the skin, supports a role for epithelial-extracellular matrix interactions in the regulation of milk protein gene expression in vivo, and identifies the skin as a particularly accessible model system in which to study the regulation of milk protein gene expression. In addition, the guinea-pig alpha-lactalbumin gene will be a source of regulatory sequences with which to direct heterologous gene expression to the sebaceous glands of transgenic mice.

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 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.

Hormone-dependent milk protein gene expression in bovine mammary explants from biopsies at different stages of pregnancy

2004 ◽  
Vol 71 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Paul A Sheehy ◽  
James J Della-Vedova ◽  
Kevin R Nicholas ◽  
Peter C Wynn
Keyword(s):  
Gene Expression ◽  
Milk Protein ◽  
Protein Gene ◽  
Bovine Milk ◽  
Mammary Tissue ◽  
Endocrine Regulation ◽  
Surgical Biopsy ◽  
Milk Protein Gene ◽  
Milk Protein Gene Expression

A method for the collection of mammary biopsies developed previously was refined and used to study the endocrine regulation of bovine milk protein gene expression. Our surgical biopsy method used real-time ultrasound imaging and epidural analgesia to enable recovery of a sufficient quantity of mammary tissue from late-pregnant dairy cows for explant culture in vitro. The time of biopsy was critical for prolactin-dependent induction of milk protein gene expression in mammary explants, as only mammary tissue from cows nearing 30 d prepartum was hormone-responsive. This suggests that during the later stages of pregnancy a change in the responsiveness of milk protein gene expression to endocrine stimuli occurred in preparation for lactation. This may relate to the diminution of a putative population of undifferentiated cells that were still responsive to prolactin. Alternatively, the metabolic activity of the tissue had increased to the level whereby the response of the tissue was no longer assessable using this model in vitro.

Effects of compensatory growth on milk protein gene expression and mammary differentiation

1988 ◽  
Vol 2 (10) ◽  
pp. 2619-2624 ◽  
Author(s):  
Chung S. Park ◽  
Yun J. Choi ◽  
Wanda L. Keller ◽  
Robert L. Harrold
Keyword(s):  
Gene Expression ◽  
Compensatory Growth ◽  
Milk Protein ◽  
Protein Gene ◽  
Milk Protein Gene ◽  
Milk Protein Gene Expression

281. The role of insulin in milk protein synthesis

2005 ◽  
Vol 17 (9) ◽  
pp. 117
Author(s):  
K. K. Menzies ◽  
K. L. Macmillan ◽  
K. R. Nicholas ◽  
C. Lefevre ◽  
C. Ormandy
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Dairy Cows ◽  
Milk Protein ◽  
Explant Culture ◽  
Milk Protein Gene ◽  
Culture Model ◽  
Milk Protein Genes ◽  
Milk Volume

The mammary explant culture model has been frequently used to mimic lactation and to examine the endocrine control of milk protein gene expression. Studies in the mouse show the expression of the milk protein genes in explants requires insulin in the presence of prolactin and cortisol. The role of insulin in milk protein synthesis in the dairy cow is not as clear. The bovine mammary explant culture model has been utilised to show that insulin is essential for alpha-s1-casein gene expression and the synthesis of the casein proteins. In addition, mouse culture experiments were undertaken to provide an insight into the underlying molecular mechanisms of insulin action in hte mammary gland. A global analysis of the genes induced in the cultured explants was done using Affymetrix microarray and showed 132 genes, including the major milk protein genes, required the complement of insulin, cortisol and prolactin for maximal expression. Twenty-seven genes showed a 3-fold change in gene expression in response to insulin. The function of these genes can be largely categorised into maintenance of cell integrity, signal transduction, transport mechanisms, cellular metabolism and a direct effect on gene transcription in the nucleus. The requirement for insulin in milk protein synthesis is highlighted by its role in inducing the STAT5 gene, known to be a key transcription factor for the milk protein genes. Interestingly, dairy cows of high genetic merit have unusually low serum concentrations of insulin. This has occured in association with a high selection pressure for milk volume that has altered the regulation of blood glucose homeostasis. Our study indicates that this intensity of selection for high milk volume could be compromising the dairy cow’s potential for milk protein production: Has selecting for milk volume in many populations of dairy cows been achieved by lowering circulating insulin levels with consequent effects on the efficiency for milk protein yield as well as compromised reproductive performance.

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