The mouse mammary gland requires the actin-binding protein gelsolin for proper ductal morphogenesis

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication.     

P190-B Rho GTPase-activating protein overexpression disrupts ductal morphogenesis and induces hyperplastic lesions in the developing mammary gland

p190-B Rho GTPase activating protein is essential for mammary gland development because p190-B deficiency prevents ductal morphogenesis. To investigate the role of p190-B during distinct stages of mammary gland development, tetracycline-regulatable p190-B-overexpressing mice were generated. Short-term induction of p190-B in the developing mammary gland results in abnormal terminal end buds (TEBs) that exhibit aberrant budding off the neck, histological anomalies, and a markedly thickened stroma. Overexpression of p190-B throughout postnatal development results in increased branching, delayed ductal elongation, and disorganization of the ductal tree. Interestingly, overexpression of p190-B during pregnancy results in hyperplastic lesions. Several cellular and molecular alterations detected within the aberrant TEBs may contribute to these phenotypes. Signaling through the IGF pathway is altered, and the myoepithelial cell layer is discontinuous at sites of aberrant budding. An increase in collagen and extensive infiltration of macrophages, which have recently been implicated in branching morphogenesis, is observed in the stroma surrounding the p190-B-overexpressing TEBs. We propose that the stromal response, disruption of the myoepithelial layer, and alterations in IGF signaling in the p190-B-overexpressing mice impact the TEB architecture, leading to disorganization and increased branching of the ductal tree. Moreover, we suggest that alterations in tissue architecture and the adjacent stroma as a consequence of p190-B overexpression during pregnancy leads to loss of growth control and the formation of hyperplasia. These data demonstrate that precise control of p190-B Rho GTPase-activating protein activity is critical for normal branching morphogenesis during mammary gland development.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

Chromogranin A alters ductal morphogenesis and increases deposition of basement membrane components by mammary epithelial cells in vitro.

The extracellular function of chromogranin A (CgA), a glycoprotein widely distributed in secretory vesicles of neurons and neuroendocrine cells, has not been clearly established. To examine whether CgA might modulate the biological properties of epithelial cells, we used an in vitro model of ductal morphogenesis in which mammary epithelial (TAC-2) cells are grown in three-dimensional collagen gels. Whereas under control conditions TAC-2 cells formed thin, branched cords with pointed ends, in the presence of CgA they formed thicker cords with bulbous extremities, reminiscent of growing mammary ducts in vivo. Immunofluorescence analysis demonstrated that CgA increases the deposition of three major basement membrane components, i.e., collagen type IV, laminin, and perlecan, around the surface of the duct-like structures. Similar effects were observed with CgA partially digested with endoproteinase Lys-C, suggesting that one or more fragments of CgA are endowed with the same activity. These findings reveal a hitherto unsuspected activity for CgA, i.e., the ability to alter ductal morphogenesis and to promote basement membrane deposition in mammary epithelial cells.     

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland

Abstract. Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFα and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro – in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.     

Changes in adhesive properties of epithelial cells during early morphogenesis of the mammary gland

Previous studies have demonstrated that cell adhesion systems are downregulated in epithelial buds at the earliest stages of submandibular gland and hair follicle development, but are restored at subsequent stages. Here it is shown that epithelial cell adhesion systems are also remodeled during early mammary gland development. Immunofluorescence and electron microscopy of the mouse mammary bud demonstrated that cell-cell adhesion systems were hardly detectable, with significant downregulation of expression of desmosomal molecules, but not of E-cadherin and beta-catenin. Hemidesmosomal structures were also rarely found, although their component molecules were expressed. Differences in cell adhesivity between cells of the mammary bud and those of the overlying epidermis were shown by the finding that the mammary cells formed smaller aggregates than the epidermal cells and were not randomly mixed with the epidermal cells. At subsequent stages, the mammary epithelium restored cell-cell adhesion systems along with de novo expression of tight junction molecules. These data, together with previous findings, indicate that remodeling of epithelial cell adhesion systems is a general feature underlying the early development of several ectoderm-derived organs and support the idea that segregation and rearrangements of cells are involved in early epithelial morphogenesis.     

Simulating properties of in vitro epithelial cell morphogenesis

How do individual epithelial cells (ECs) organize into multicellular structures? ECs are studied in vitro to help answer that question. Characteristic growth features include stable cyst formation in embedded culture, inverted cyst formation in suspension culture, and lumen formation in overlay culture. Formation of these characteristic structures is believed to be a consequence of an intrinsic program of differentiation and de-differentiation. To help discover how such a program may function, we developed an in silico analogue in which space, events, and time are discretized. Software agents and objects represent cells and components of the environment. "Cells" act independently. The "program" governing their behavior is embedded within each in the form of axioms and an inflexible decisional process. Relationships between the axioms and recognized cell functions are specified. Interactions between "cells" and environment components during simulation give rise to a complex in silico phenotype characterized by context-dependent structures that mimic counterparts observed in four different in vitro culture conditions: a targeted set of in vitro phenotypic attributes was matched by in silico attributes. However, for a particular growth condition, the analogue failed to exhibit behaviors characteristic of functionally polarized ECs. We solved this problem by following an iterative refinement method that improved the first analogue and led to a second: it exhibited characteristic differentiation and growth properties in all simulated growth conditions. It is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. The second analogue also uses an inflexible axiomatic program. When specific axioms are relaxed, growths strikingly characteristic of cancerous and precancerous lesions are observed. In one case, the simulated cause is aberrant matrix production. Analogue design facilitates gaining deeper insight into such phenomena by making it easy to replace low-resolution components with increasingly detailed and realistic components.     

Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis

Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly, suggesting common mechanisms of epithelial growth.     

Delivering the message: epimorphin and mammary epithelial morphogenesis

The mammary gland consists of a highly branched tubular epithelium surrounded by a complex mesenchymal stroma. Epimorphin is an extracellular protein that is expressed by mammary mesenchymal cells that directs epithelial morphogenesis. Depending upon the context of presentation--polar versus apolar--epimorphin can selectively direct two key processes of tubulogenesis: branching morphogenesis (processes involved in tubule initiation and extension) and luminal morphogenesis (required for enlargement of tubule caliber). Here, we outline the fundamentals of mammary gland development and describe the function of epimorphin in these processes. We conclude with a review of recent studies that suggest similar morphogenic roles for epimorphin in other glandular organs.     

Bovine mammary epithelial cell cultures for the study of mammary gland functions

In the present study, the analysis of epithelial cells derived from various sources was undertaken, beginning from the mammary gland tissue through the primary cultures and their subsequent passages. The objective of the study was the comparative analysis of the stage in which the epithelial cells obtained from individuals in different lactation cycles and disparate phases of cell culture growth are the most suitable for morphological research and analysis of gene expression activity. The cultures of primary bovine mammary epithelial cells and passages were identified morphologically using immunocytochemical methods. After positive identification, real-time PCRs were performed for the analysis of the expression level of casein genes, whey protein genes, and butyrophilin gene. The most stable reference genes in real-time PCRs for the mammary gland tissue and cell cultures were also determined. Of the reference genes, the UXT and GAPDH genes appeared to be the most stable ones for the mammary gland tissue samples and epithelial cell cultures. The results obtained allowed concluding that the mammary gland samples collected from heifers constituted the most effective material for the initiation of primary cultures. The primary cultures formed characteristic for the mammary gland tissue dome structures, which images were obtained using confocal microscopy. The highest levels of expression of the CSN1S1, CSN1S2, CSN2, and CSN3 genes were detected in primary cultures. The levels of expression of whey protein genes (LALBA and BGL) were highest in the second passage. The most abundant expression of the BTN1A1 gene was observed in primary cultures and the third passage. On the basis of the whole experiment, it can be concluded that primary cultures and cells of the second passage derived from heifer individuals appeared to be the best materials for the analysis of mammary gland function and gene expression activity.     

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.     

Milk ceruloplasmin and its expression by mammary gland and liver in pigs

Concentrations of ceruloplasmin and copper in milk and blood plasma, the nature of milk ceruloplasmin, and the effects of lactation and gestation on these parameters, as well as the expression of ceruloplasmin mRNA by the mammary gland, were examined in pigs. As seen previously in humans, ceruloplasmin and copper concentrations in sow milk were much higher a few days after birth than 1 month later, averaging 26.5 and 6.6 mg ceruloplasmin/L (by immunoassay) and 1.67 and 0.34 mg total Cu/L, on days 3 and 33 postpartum, respectively. Values for ceruloplasmin oxidase activity (measured with p-phenylene diamine) were 7.8 and 1.3 nmol/min/L, respectively. Daily milk ceruloplasmin production went from 61 to 22 mg/day and daily copper output from 38 to 12 mg/day. In contrast, there was little or no variation in serum ceruloplasmin concentration during lactation or gestation, although total plasma copper was high at the end of gestation. Milk ceruloplasmin was of the same apparent size as serum ceruloplasmin, as determined by SDS-PAGE and immunoblotting, and ceruloplasmin mRNAs of liver and mammary gland were indistinguishable by Northern analysis and RT-PCR of the various exons. Expression of total RNA and ceruloplasmin mRNA, as detected in biopsies of mammary gland, increased markedly upon onset of lactation and then declined during the next month in conjunction with a drop in milk ceruloplasmin production. The results indicate that milk ceruloplasmin, while being the same protein as in plasma, is not derived from the plasma but is produced by the mammary gland.     

Cultivation of epithelia from the secretory coil of the ovine apocrine gland: Evidence of secretory cell function and ductal morphogenesis In vitro

Summary The secretory coil of the ovine apocrine gland is composed predominantly of two cell types, secretory cells lining the lumen and myoepithelial cells adjacent to the basement membrane. The glands synthesize a number of hormones and growth factors, but analysis of the functions of these molecules may be hampered by the mixing of apocrine and sebaceous secretions in the pilary canal. The purpose of this study was to isolate the glands and devise simple culture procedures to facilitate investigations of secretory cell function. The most successful approach involved microdissection of the secretory coils individually from skin biopsies and culture in Dulbecco’s modified Eagle’s medium. After 1–2 wk in medium, cell outgrowths were seen from explants. These consisted predominantly of populations of epithelial cells, many containing granules. Smaller granules were usually concentrated around the cell nuclei and accumulated lipophilic dyes. Large granules were unreactive. Western analysis showed that cells in culture synthesized nerve growth factor-like peptides, a feature consistent with one of the functions of the gland in vivo. When isolated secretory coils were explanted to culture dishes coated with matrigel, highly compact, multilayered masses of cells grew out. Subsequently, tubular structures formed. The observations suggest that some differentiated functions of gland cells were retained in vitro and that the procedures described provide a system for the study of apocrine secretions in isolation from those of other skin glands.     

Milk lipid and protein traffic in mammary epithelial cells: joint and independent pathways

In mammary epithelial cells, milk lipids and proteins are synthesised in the same compartment, the endoplasmic reticulum. Lipids, carried through the cytoplasm, associate with the apical membrane which then pinches off and releases the lipid globule. Proteins, carried through membrane compartments are released in the lumen after fusion of secretory vesicles with the apical membrane. These processes assure a relatively constant composition of milk but it is not known whether lipid and protein secretion are linked. The protein composition of the milk fat globule membrane and the stimulatory effects of prolactin and oxytocin on lipid and protein secretion suggest that these processes are coupled and co-regulated. However, it is possible to observe a dissociation between the formation and the secretion of the two constituents, during differentiation and in various experimental conditions, and this suggests that coupling is not strictly required.     

Mammary gland morphogenesis in vitro: Formation of branched tubules in collagen gels by a cloned rat mammary cell line

The three-dimensional growth in vitro of cloned rat mammary cell lines on floating type I collagen gels has been investigated. Multicellular outgrowths formed by the various cell types show morphological differences on serial histological sectioning and electron microscopy. One cell line, Rama 25, an epithelial cell line derived from a dimethylbenz(a)anthracene (DMBA)-induced mammary adenocarcinoma can form branched tubules within the matrix. The amount of collagen in the matrix modified the structure of the predominant outgrowths formed by this cell line. High-concentration (0.6% w/v) collagen gels support the growth of tubules up to 0.5 mm in length which have an extensive lumen surrounded by rings of up to 26 cells. Absence of differentiated myoepithelial elements around the ring suggests a resemblence to primitive ducts found in the mammary glands of neonatal rats. The spectrum of cellular polarity toward the lumen seen throughout the tubules and the occasional irregular arrangement of epithelial cells are features of adenocarcinoma. Lumen formation occurs by central cell necrosis and separation of the external layers of initially solid cords. The tubules branch either dichotomously, by bifurcation at the distal ends or monopodially, by budding at the sides of the outgrowths. Rama 25 grown on gels containing lower concentrations of collagen (0.1 or 0.3% w/v) produce narrow branching structures with incomplete lumina and spikes of elongated cells. Tubular structures are not formed by Rama 25 grown on nonfloating gels. At the light microscopic level the layer of spindle cells formed beneath the surface monolayer on nonfloated gels resembles the sarcomatous regions of tumors, however at the ultrastructural level the spindle cells show some evidence of being myoepithelial-like rather than fibroblast-like. Sandwiching the epithelial cell sheet between two layers of collagen gel results in loss of contact with the media and the formation of spindle cells. The myoepithelial-like cell lines Rama 29 and Rama 401 form spiked branches of elongated cells and solid branching cords of cells, respectively. However, no lumen formation is observed. The fibroblast-like cell line Rama 27 shows extensive migration of either single cells or chains of cells into the gel. Thus only one cell type (Rama 25) is necessary to form branched tubules in vitro and the structure of the tubules can be modified by collagen, a component of the extracellular matrix.     

Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions

This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.