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

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.     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

Mammary adenocarcinoma in a young female hypercholesterolemic rat.

Pathological examination of a 12-week-old female rat with hypercholesterolemia revealed mammary adenocarcinoma. Grossly, one grayish-white nodule was found in the hypoderm near the right posterior mammary gland. Histologically, the nodule was composed of variable-sized epithelial cells which lined the tubules and ducts in one to several layers, accompanied by proliferation of connective tissue in the stroma. Mitotic figures, necrosis and hemorrhage appeared frequently throughout the lesion. The tumor cells had invaded the surrounding muscular tissue.     

Localization of mammary-derived growth inhibitor in capillary endothelial cells of the bovine mammary gland

Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parencyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto-and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.     

Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.     

Immunohistochemical studies on various aspects of glandular immunoglobulin transport in man

Summary Gland-associated immunocyte populations have been characterized in human tissue specimens from which extracellular immunoglobulins have been removed by saline extraction. There is a striking preponderance of IgA-producing immunocytes adjacent to glands of the gastro-intestinal and respiratory tracts, in minor and major salivary glands, and in lactating mammary glands. Immunohistochemically, these cells have been found to contain dimeric IgA with incorporated J chain. Despite this local IgA production, immunohistochemical tests on alcohol-fixed specimens demonstrate that the glandular stroma is normally permeated predominantly by IgG, most of which is obviously serum-derived. However, the serous glandular cells selectively transmit dimeric IgA, which appears along their lateral borders and apically in the cytoplasm, whereas the epithelial occurrence of IgG is less conspicuous and is restricted to the interstices.The same epithelial cells produce a glycoprotein called the secretory component (SC) which exhibits specific affinity for J chain-containing dimeric IgA and pentameric IgM. In saline-extracted tissue, IgA, but IgG, is retained regularly along the borders of SC-producing cells; this probably reflects complexing between locally formed IgA and SC in the epithelial cell membranes. SC apparently functions as a glandular receptor for dimeric IgA which thus most likely enters the epithelial cells by adsorptive pinocytosis. After covalent stabilization, the IgA-SC complexes are extruded to the gland lumen. Immunohistochemically the Golgi zone has been found to contain free SC but no IgA, whereas SC occurring more apically in the epithelial cell exhibits characteristics of being IgA-associated. Pentameric IgM is handled by the glands in a way similar to dimeric IgA, but local synthesis of IgM is normally negligible, except in the gut.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Reprogramming non-mammary and cancer cells in the developing mouse mammary gland

The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the "niche" and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary cells, including mouse and human cancer cells, may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells.     

Colocalization of α-lactalbumin and a major casein in secretory vesicles of rat mammary epithelial cells

Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles.     

Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors

Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

Ultrastructural localization of intracellular immunoglobulins in Epon-embedded human lymph nodes. An immunoelectron microscopic investigation using the immunogold staining (IGS) and the avidin-biotin-peroxidase complex (ABC) methods

The ultrastructural localization of intracellular immunoglobulins on ultrathin sections of glutaraldehyde-fixed, postosmicated, and Epon-embedded human lymph nodes has been achieved using such highly sensitive immunocytochemical techniques as immunogold staining and avidin-biotin-peroxidase complex. These immunoelectron microscopic techniques allow the identification of intracellular immunoglobulins without affecting the ultrastructural morphology of the tissue, since they do not require any pretreatment of the sections with proteolytic enzymes or deresinating agents. Therefore, immunoglobulins can be precisely localized in the cell organelles; structures whose morphology is well preserved. The availability of a reliable postembedding staining procedure for the ultrastructural localization of immunoglobulins is of definite value for investigations on human lymphoid tissue, both normal and pathological.     

Hydrolase histochemistry of the thymus: development and species variations

In the present investigation the localization and activity of alkaline, neutral, and acid hydrolases of the thymus were studied during development of rats and mice and of various adult species using histochemical methods. If different procedures of tissue pretreatment were employed, several inhibition effects and morphological as well as enzyme histochemical artifacts occurred dependent on the mode of tissue pretreatment. After embedding in glycol methacrylate, sections of the thymus showed a better structural preservation than cryostat sections but were accompanied by a drastic decrease of activity and low localization quality of the final reaction products especially in the case of protease studies with 4-methoxy-2-naphthylamine peptides as substrates. Smears of thymic cells facilitated the allocation of enzymes to mobile or fixed cells in the stroma of the thymus. The perivascular localization of aminopeptidase M could only be shown with combined techniques. In comparison, primarily the proteases yielded information on the thymic stroma and in this context especially on the epithelial reticular cells and the stroma proper but also on thymocytes (lymphocytes) and enabled a species-dependent subdivision of the thymic reticulum already in the light microscope. Enzyme histochemically the development of the rat and mouse thymus could be subdivided into an early period and perinatal (pre- and postnatal) period of functional differentiation. Morphological (proliferation of cortical lymphocytes) and enzyme histochemical changes (disappearance of dipeptidylpeptidase IV, significant loss of alkaline phosphatase activity and beginning activity increase of aminopeptidase M) occurred primarily at the transition from the early to the prenatal period. During the postnatal phase, a significant activation of lysosomal enzymes in the thymic medulla and general enzymatic differentiation of the cortical epithelial reticular cells were found. Species differences and species similarities for the respective enzymes and their localization as well as for the thymic cells were noticed for adult rats, mice, guinea-pigs, hamsters, and marmoset monkeys. Differences were true especially for the thymocytes; less species differences were seen for the epithelial reticular cells; capsular and perivascular connective tissue and the macrophages behaved rather similarly. Species-independently certain medullary epithelial reticular cells showed high and typically localized alkaline phosphatase activities and species-dependently also high activities of neutral hydrolases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of the basement membrane during regeneration of the corneal epithelium

Summary The epithelium regenerating after a surface lesion of the cornea forms a new basement membrane. This process begins 6–8 days after the lesion when the wound is completely covered, and epithelial cells have ceased to migrate. Only that part of the epithelial cells facing the stroma is involved. First, tufts of fine filamentous structures (about 30 Å thick) appear on the internal side of the plasma membrane, and apparently penetrate it reaching the extracellular space where they form a loose network. This then differentiates into two discrete layers, a very thin discontinuous one, restricted to areas with tufts, very close to the plasma membrane (juxtamembranous layer), and a thick continuous layer, the basement membrane proper, parallel to and much further away from the plasma membrane. The basement membrane appears to be the product of cytoplasmic secretion by epithelial cells, and there is no evidence for connective tissue cells taking part in this process.Supported by the Deutsche Forschungsgemeinschaft.     

Immunofluorescence location using monoclonal antibodies of prolactin receptors in mammary epithelial cells of lactating rabbits

Immunofluorescent staining of PRL receptors on frozen sections of lactating rabbit mammary gland with a monoclonal antibody (IgG1 M110) shows that receptors are localized in the cytoplasm of epithelial cells and on short portions of plasma membrane. In vivo treatment by bromocriptine or in vitro treatment of mammary tissue slices by monensin modifies localization of receptors.     

The stroma reaction myofibroblast: a key player in the control of tumor cell behavior

The cooperation between epithelial and mesenchymal cells is essential for embryonic development and probably plays an important role in pathological phenomena such as wound healing and tumor progression. It is well known that many epithelial tumors are characterized by the local accumulation of connective tissue cells and extracellular material; this phenomenon has been called the stroma reaction. One of the cellular components of the stroma reaction is the myofibroblast, a modulated fibroblast which has acquired the capacity to neoexpress alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells, and to synthesize important amounts of collagen and other extracellular matrix components. It is now well accepted that the myofibroblast is a key cell for the connective tissue remodeling which takes place during wound healing and fibrosis development. Myofibroblasts are capable of remodeling connective tissue but also interact with epithelial cells and other connective tissue cells and may thus control such phenomena as tumor invasion and angiogenesis. In this review we discuss the mechanisms of myofibroblast evolution during fibrotic and malignant conditions and the interaction of myofibroblasts with other cells in order to control tumor progression. On this basis we suggest that the myofibroblast may represent a new important target of antitumor therapy.     

Immunhistochemische Darstellung der Casein- und β-Lactoglobulinsynthese in der Rindermilchdrüse

Zusammenfassung Es wurden Milchdrüsen von gesunden, laktierenden Kühen ohne Berücksichtigung des Alters und des Laktationsstadiums untersucht. Mit der direkten Immunfluoreszenzmethode konnten Casein und -Lactoglobulin in den Epithelzellen nachgewiesen werden.Für die Präparatherstellung eigneten sich die Formalin-oder Alkoholfixation mit Paraffineinbettung am besten.Beide Proteine wurden während der ganzen zwölfstündigen Zwischenmelkzeit von allen Drüsenzellen synthetisiert. Bei einer Milchstauung (24–48 Std ohne Milchentzug) wird die Produktion von Casein und -Lactoglobulin in den Milchdrüsenzellen eingestellt.

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Immunofluorescent demonstration of casein and -lactoglobulin synthesis in the bovine mammary gland

Summary Fluorescein isothiocyanate labeled anti-casein and anti--laotoglobulin were applied in a system of direct staining to sections of bovine mammary gland. The following methods of tissue preparation were compared: cryostat sectioning, freeze substitution and fixations followed by paraffin embedding. Formalin, 95% ethanol, Bouin's, Carnoy's and solutions containing mercury salts were employed in tissue fixation. Formalin or 95% ethanol fixation with paraffin embedding proved to be the most satisfactory techniques.Both casein and -lactoglobulin were detected in all secretory epithelial cells with brightest fluorescence in the apical cytoplasm. Similar fluorescence occurred in milk retained within alveolar lumina. Both proteins could be located in alveolar epithelial cells at any time between normal milkings (12 hour intervals) but not when milking was delayed to 24–48 hours. In the latter situation epithelial cells became flattened.

     

ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.     

Heterogeneous localization of adenylate and guanylate cyclases in R3230AC rat mammary adenocarcinoma cells

Adenylate and guanylate cyclase activities were demonstrated in R3230AC rat mammary adenocarcinomas by electron microscopic cytochemistry. Adenylate (AC) and guanylate (GC) cyclases were detected on plasma membrane of tumor epithelial cells, but not on fibroblasts and endothelial cells in the perivascular space. Both AC and GC activities were enriched in tumor epithelial cells at the periphery of the tumor lobular parenchyma rather than in cells in central core of the lobular parenchyma. Furthermore, the tumor cell plasma membranes facing the connective tissue stroma were in paucity or devoid of either enzyme activity. These heterogeneous distributions of both AC and GC among tumor epithelia suggest that R3230AC epithelial cells in different parts of the tumor mass may vary significantly in their regulation of cellular physiology.     

Immunohistochemical localization of basic fibroblast growth factor within the mouse uterus.

Uterine samples were either rapidly frozen in liquid nitrogen or placed in Bouin's fixative. A commercial primary polyclonal antibody made in rabbits against human recombinant basic fibroblast growth factor (bFGF) was used. Western blot analysis indicated that the antibody was specific for bFGF and did not react with acidic FGF. The primary antibody was followed by either goat anti-rabbit immunoglobulin G (IgG) conjugated to the fluorescent phycobiliprotein tracer phycoerythrin or biotinylated goat anti-rabbit IgG and a biotin-avidin-peroxidase complex. Specificity controls using adjacent sections were carried out by (i) substituting normal rabbit sera for the primary antisera, (ii) omitting the primary antisera or (iii) extracting sections with NaCl (2 mol l-1) prior to the immunochemical procedures. No binding of the antibody was observed with any of the specificity control sections. The connective tissue stroma and the basal lamina associated with uterine glandular and surface epithelial layers were positive for bFGF. Localization was not observed within surface or glandular epithelial cells. The basal lamina and endothelial cells associated with blood vessels within the uterus and the smooth muscle cells of the myometrium were positive for bFGF. There were no differences in uterine localization patterns or intensity during the oestrous cycle or after ovariectomy and steroid hormone supplementation. These studies demonstrate the specific localization of bFGF within the mouse uterus.