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Transplant Rev ; ruses. Nature ; Chem Immunol ; man T cells stimulated with the su- endogenous mammary tumour vi- Nature ; J Exp Med ; Science ; receptors are consistent with an im- staphylococcal enterotoxins to T J Exp Med ; Nature ; ing VI35 T cell antigen receptor. J receptor binding sites for the micro- Immuno; Immunology ; USA ; Cell ; MAM, a model superantigen. Im- receptor Va expression on Mlsa su- Annu Rev Im- chain influences reactivity to Mls-1 specificity. J Exp Med ; munol ; J Immu- J ture of the T cell receptor. Nature jor histocompatibility complex-spe- Exp Med ; Nature ; New York, Aca- Nature ; 24 Sprent J: T lymphocytes and the thy- lococci.

Lancet ;ii: Cell ; Science ; with S. Nature ; I MHC specificity. J Immunol ; Induction of disease and expression prevents viral infection. In- Cell ; Immunol Today ; Immu- mouse spleen. J Exp Med ; the Mycoplasma arthritidis superan- nol Rev ; Se- a critical role in autoimmunity? J Exp potential role in human disease. J Biol Med ; Adv Immunol ; Chem ; J Exp Med no; Cellular and Molecular Biology of ; A : litters were weighed daily and divided by the no.

Growth curves did not change when weights of pups in largest and smallest litters were excluded. The inset shows the rate of weight gain between days 5 and B : photographs of pups on day To examine the effect of deletion of IR in the pregnant mouse, we examined the histology of mammary glands from In addition, the growth of cytoplasmic lipid droplets in the epithelium, a reliable marker of differentiation, is substantial The views of whole mounts Fig. Differentiation of MECs is diminished in pregnant mice expressing Cre recombinase.

A and B : whole mounts fixed as described and stained with carmine red. E and F : immunocytochemistry for casein green , adipophilin perilipin 2; red , and nuclei DAPI, blue. Scale bars as shown. See Table 1 for quantitation of the properties of these images. In summary, these data provide strong evidence that IR is important for normal alveolar growth and differentiation during pregnancy.

Because the effects of deleting IR were more pronounced with respect to differentiation than proliferation, we will focus on differentiation in the remainder of this paper. The formation of acinar cultures from primary MECs placed on a base of Matrigel was first reported by Barcellos-Hoff et al. Both laboratories were able to demonstrate hollowed acini-like structures that were positive for luminal casein. Similar cultures, variously called mammospheres, three-dimensional 3D acini, or acinar cultures have found wide utility in many laboratories where they have been used to elucidate the interaction of the mammary epithelium with the extracellular matrix reviewed in Ref.

Recently, similar cultures using matrixes that do not promote attachment of isolated epithelial cells have been used to characterize mammary stem cells 17 ; the term mammospheres is now restricted to these cultures. To elucidate the ligand responsible for differentiation of MECs in 3D acinar cultures, we started with single cells isolated from the day Importantly, when these cells are mixed with full-strength Matrigel, they associate to form spheres with a single layer of epithelium.

These spheres gradually increase in size mainly because of expansion of the lumen. Unlike cultures where cells are placed on top of Matrigel, very little apoptosis is seen data not shown.

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Figure 5 shows several acini incubated in the presence of increasing doses of insulin. Differentiated acini contain numerous lipid droplets identified as hollow-appearing cytoplasmic structures surrounded by red stain for ADPH. Effect of insulin on properties of acinar cultures. A : representative three-dimensional 3D acini cultured with designated insulin doses. B , C , and D : Slidebook analysis of acini cultured for 7 days with growth medium followed by culture in differentiation medium see materials and methods for composition at concentrations of insulin from 0 to 50 nM.

Each point represents the mean parameters from three to five acini. Each parameter was fit to the Michaelis-Menton equation using the curve fit function of SlideWrite. The best-fitting parameters Table 2 were then used to plot the lines shown.

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B : acinar and luminal diameters and no. Diameters were measured using the ruler function of Slidebook. Nuclei were counted by hand from the DAPI-stained structures. C : cell, cytoplasmic, and casein areas. Cytoplasmic area was defined as the total area of green stain that did not overlap with DAPI-stained nuclei, and the casein area was defined as the area of high-intensity green stain.

Luminal stain was not always present, and, when it was, it was not included in these calculations. D : ADPH area or no. To determine whether insulin was interacting with the IR in the cultures from CD1 mice, we performed dose-response curves using concentrations of insulin ranging from 0 to 50 nM, well within the physiological range in vivo, measuring the size of the structures and their lumens, the number of nuclei per structure, and the intensity and area occupied by stain for ADPH and casein.

Figure 5 shows the effect of increasing insulin doses on acinar morphology Fig. Each point represents the mean and SE from analysis of three to five acini. The lines are theoretically plotted using the parameters calculated from the best fit to the Michaelis-Menton equation, as shown in Table 2.

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It is possible that this problem reflects the difficulties Bl6C57 mice the parent strain have with lactation in general The conclusion from these experiments is that insulin has a strong effect on alveolar differentiation at concentrations in the physiological range. IGF-I had a small but significant effect on acinar diameter and number of nuclei only at a concentration of 50 nM. It had no effect on luminal diameter. IGF-II had no effects at the concentrations shown on any of these parameters.

At nM, it began to promote differentiation as shown by increases in staining for casein and ADPH data not shown. Thus neither of these ligands promotes acinar differentiation at concentrations in physiologically relevant ranges. However, it had no effect on insulin action, as shown by the effect of 2 nM insulin on acinar diameter in the presence and absence of antibody Fig. Similar results were obtained for other differentiation parameters. We conclude that insulin acting through IR is essential for alveolar differentiation in the latter part of pregnancy in the mouse.

The images in Figs. To further test this hypothesis and to begin to identify genes whose expression is affected by insulin signaling in midpregnancy, we prepared MECs from the mammary glands of There was a distinct separation between gene expression in the two strains as shown by the PCA analysis in Fig.

The signal intensities obtained from processing these arrays, which allow evaluation of the expression of 28, genes, were put into GeneSpring X and analyzed as described in materials and methods to give a list of genes of which 93 were upregulated and were downregulated 1. Pathway analysis of these genes using standard programs proved unsatisfactory because the literature on pathway analysis in the differentiating mammary gland is not yet reflected in most standard pathway programs 5.

For this reason, we used a combination of our own knowledge of synthesis pathways for the production of milk, Gene Ontology analysis from Genespring X, and pathway analysis using Metacore. This combinatorial approach yielded 11 categories of genes that were predominantly downregulated when IR expression is abrogated Table 3.

Affected genes include those for the major milk proteins and milk fat globule proteins, genes involved in both lipid synthesis and lipid degradation, and genes for lactose synthesis as well as for ion transport, immune regulation, and ubiquination. More detailed data on the genes in each of these categories are given in Supplemental Tables S1 and S2 Supplemental data for this article may be found on the American Journal of Physiology: Endocrinology and Metabolism website.

The same approach yielded five categories of genes that were predominantly upregulated, including cell cycle genes, extracellular matrix and cytoskeleton genes, as well as genes for mRNA transport and processing, and a few signal transduction genes. Again the detailed gene lists can be found in Supplemental Tables S1 and S2. Finally, five categories, the largest of which was small molecule metabolic processes, had more or less equal numbers of up- and downregulated genes, suggesting that they belong to more complex metabolic pathways.

Table 3. See Supplemental Table 2 for gene identities by category. Physiologically, what do these findings mean? Because many categories of mammary differentiation specific genes were downregulated with diminished insulin signaling, our findings suggest that secretory differentiation in the mammary gland of the late pregnant mouse is dependent on insulin. Finally, changes in both the cytoskeleton and interactions with the extracellular matrix that prepare the gland for secretory activity may also have a similar dependency on insulin.

If these conclusions are correct, it seems likely that the expression trajectories for the downregulated, differentiation-related genes would be upward in the latter half of pregnancy, and, conversely, the expression trajectories for the upregulated genes would be downward in the same period. We are able to use previously published data on the time course of gene expression in the developing mammary gland 37 to test these hypotheses. Again the detailed time course data are consistent with this interpretation Fig. Not all genes fit these predictions.

The three genes in Fig.


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These genes are Pold2 , which encodes the kDa catalytic subunit of DNA polymerase delta; Reln , which encodes a large secreted extracellular matrix protein REELIN involved in cell-cell interactions; and Srm , which encodes spermidine synthase, a src-related kinase lacking COOH-terminal regulatory tyrosine and NH 2 -terminal myristylation sites. Two other categories of genes also do not fit the overall prediction; both are found in Fig. These genes fall into a number of categories; their major characteristic in the time course is that their expression is mostly level in pregnancy, decreasing only with the onset of secretory activation around pregnancy day Time course of expression of genes changed 1.

A : expression intensity ratios pregnancy day Genes in the upper two quadrants increased 1.

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Genes in the right hand quadrants are down 1. Data for each gene plotted is given in Supplemental Table 2 organized into the categories shown in the legend. For this reason, the analysis of correlation with the time course of gene expression includes only 42 down-going and 33 up-going genes. In the remaining panels, gene names are shown to the right of the graph, and categories are indicated by color in the list within the graph.

B : most of the genes that decreased with decreased IR expression increased on time course arrays during pregnancy. F : time course of expression of genes that increase to pregnancy day 15 and then decrease before secretory activation. The expression patterns of the most interesting genes in Fig. Interestingly mammary cell expression of Gjb6 , gap junction protein 6, also known as connexin 30 has been shown to coincide with the gene expression levels shown in Fig.

It has been associated with negative regulation of cell proliferation. One could speculate that this category of molecules is involved in preventing premature secretory activation as the reproductive cycle nears the termination of pregnancy and onset of lactation. The novelty of this work is that we have demonstrated that the IR plays an important role in the development of the mammary gland during gestation. Selective deletion of the IR in the mammary epithelium reduced proliferation and markers of differentiation by midpregnancy and resulted in blunted litter growth rates. In addition, studies in cultured acini from midpregnant MECs provide strong evidence that insulin signaling through its receptor underlies differentiation of the gland in preparation for secretory activation.

These observations extend the critical role that insulin and its receptor plays in milk production during lactation to the development of the mammary gland as it prepares for lactation during gestation. These data raise important questions regarding the physiological consequences of perturbed IR signaling in the mammary gland during gestation. To address that question in future work, the role that insulin and its receptor plays must be integrated with the complex set of temporal hormonal signals that prepare the mammary gland and support its function during lactation.

The development of the mammary epithelium during pregnancy takes place in two stages. The first is a proliferative stage that depends on increases in systemic levels of progesterone from the ovary and PRL from the pituitary 27 ; in this stage the number of cells increases exponentially as alveoli are formed and multiply. By midpregnancy the rate of proliferation has decreased 49 significantly, and differentiation marked by cytoplasmic lipid droplet formation, increasing casein accumulation, and progressive expansion of the lumina takes place The hormonal regulation of this stage is not well understood: in rodents, PRL and its receptor are low, and placental lactogen, present in two forms, likely serves to foster differentiation At the same time, progesterone, secreted by the ovary, inhibits secretory activation Whether progesterone plays a role in differentiation is not known.

In rodents, withdrawal of progesterone leads to both parturition and activation of copious milk secretion. Lactation itself depends on the increase in PRL and its receptor that occurs at secretory activation 23 as well as corticosterone and insulin 4 , Although strong evidence for a role for the IGF-IR in both proliferation and differentiation during pregnancy has been presented 36 , 45 , our studies provide the first clear evidence that the IR plays a more important role in late pregnancy.

Insulin and IR are known to support milk production during lactation 4 , 16 , so our studies cannot definitively attribute the entire reduction in growth rate to impaired mammary gland development. A decline in IGF-II signaling through the IR could be responsible for the effects on proliferation, since this growth factor is expressed in early pregnancy 34 , 36 and does bind to IRA 10 , the form of IR most prevalent during the early stages of gestation.

Our studies in acinar cultures of isolated MECs, on the other hand, suggest that the critical ligand for IR-mediated differentiation is insulin. Insulin stimulated luminal expansion, increased cell size, increased high-intensity staining for casein, and increased the number of cytoplasmic lipid droplets in acini.

Much higher concentrations of both IGF-I and IGF-II had little or no effect on these parameters although our studies do not rule out effects on proliferation, particularly earlier in pregnancy The inescapable conclusion is that insulin interacting with IR is necessary for the full differentiation of the mammary epithelium in the latter half of pregnancy. We then began to address a problem that has been somewhat intractable in the mammary gland of pregnancy: what exactly are the downstream molecules involved in secretory differentiation?

Thus the physiological changes in the midpregnant gland leading to a decrease in cell proliferation and an increase in differentiation parameters appear to be dependent on insulin signaling. At midpregnancy, it is also important that mechanisms be present to inhibit secretory activation. The predominant pattern was an increase in expression beginning in midpregnancy Fig. However, another group of genes tended to be flat during most of pregnancy, decreasing markedly only around pregnancy day 18 , when secretory activation begins Fig.

A final, most interesting group increased up to fold beginning at about day 8 of pregnancy but sharply decreased after day 15 with the onset of secretory activation Fig. Surprisingly, while this MED1 mutant mouse did show developmental delays in the pubertal mammary gland, the mice were viable and fertile and there were no defects in other ER-responsive tissues like the uterus or bone, indicating a tissue-, gene-, and cell-specific role for MED1s interaction with ER.

To investigate this question further, we have crossed the above mentioned MED1 LxxLL-motif mutant mouse with a transgenic mammary tumor prone mouse whose tumorigenesis is under the control of the HER2 oncogene. You must be logged in to post a comment. Those changes are essential for the functional mammary gland. MTA3, a member of metastasis-associated protein MTA … Noni leaf, black tea and Black cohosh encouraged… High intake of black tea and certain types of flavonoids were associated with lower risk of fracture-related hospitalizations in elderly women at high risk of fracture.

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