Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

INRA

Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzyme-linked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a function-blocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of beta-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases.

Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells.

We previously described that mouse oocytes and preimplantation embryos express the two subunits of interferon-gamma receptor. We now report that, despite the presence of STAT1 (signal transducer and activator of transcription 1) at both the mRNA and protein levels, interferon gamma (IFNgamma) as well as IFNalpha are unable to trigger massive nuclear translocation of STAT1 in these cells, even at high cytokine concentrations. Conversely, nuclear accumulation of STAT1 was readily observed in murine L929 somatic cells under the same conditions. However, in the absence of any stimulation, both tyrosine (Y701p) and serine (S727p) phosphorylated forms of STAT1 were already detected in the nuclei of oocytes and early embryos. Phosphorylated STAT1 appeared concentrated in large nuclear dots, which were identified by indirect immunofluorescence and electron microscopy as clusters of interchromatin granules (IGCs or speckles). A similar distribution was also observed for the serine (S727p) phosphorylated form of STAT3 as well as for tyrosine (Y689p) phosphorylated STAT2. Western blot analysis confirmed that STAT factors present in mouse oocytes are predominantly phosphorylated. In parallel, we showed that the transcription of two IFNgamma-target genes, namely interferon regulatory factor-1 (IRF-1) and suppressor of cytokine signaling-1 (SOCS-1) is indeed increased in two-cell embryos in response to IFNgamma. Altogether, our results suggest that, despite the lack of massive nuclear accumulation of STAT1 in response to exogenous IFNs and the permanent presence of phosphorylated STATs in the nucleus, JAK/ STAT pathways are functional during early development.

Presence of permanently activated signal transducers and activators of transcription in nuclear interchromatin granules of unstimulated mouse oocytes and preimplantation embryos.

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.

Localisation of caveolin in mammary tissue depends on cell type.

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.

Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells.

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells.

The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells.

MicroRNA in the ovine mammary gland during early pregnancy: spatial and temporal expression of miR-21, miR-205, and miR-200.

During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.

Sandrine Truchet

Jouy-en-Josas Centre

INRA

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Sandrine Truchet

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INRA

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Jouy-en-Josas Centre