Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.     

Heterogeneity of fibroblast response in host-tumor cell-cell interactions in metastatic tumors

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor-associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor-conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor- or mast-cell-mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum-deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density-fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor-cell-conditioned media. The stimulatory activity of tumor-cell-conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.     

Membranes of mammary gland : XV. 5-Thio- -glucose decreases lactose content and inhibits secretory vesicle maturation in lactating rat mammary gland

Short-term administration of the glucose analog 5-thio- -glucose to primiparous lactating rats reduced mammary tissue lactose concentrations to half of control levels. Treatment with colchicine alone caused slight reductions in mammary tissue lactose content. These treatments did not alter the morphology or degree of development of rough endoplasmic reticulum or Golgi apparatus, but did cause alterations in secretory vesicles. In mammary tissue from untreated lactating animals, large, swollen secretory vesicles were abundant in apical regions of epithelial cells. After thioglucose administration secretory vesicles in the apical cytoplasm were smaller and were more densely packed with contents. While administration of colchicine alone caused accumulation of large numbers of nearly fully swollen vesicles, treatment with both colchicine and thioglucose induced accumulation of smaller, less fully developed secretory vesicles which contained morphologically recognizable casein micelles. Mammary tissue from late gestation rats was low in lactose; vesicles in this tissue resembled secretory vesicles in tissue from rats treated with thioglucose in that they were small and densely packed. These observations suggest that lactose, an osmoregulator in mammary gland, is transferred from Golgi apparatus to the apical cell surface within secretory vesicles. Lactose appears to be important for secretory vesicle maturation in mammary epithelial cells.     

Characterization of transferrin receptors on plasma membranes of lactating rat mammary tissue

Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion.     

Zip3 plays a major role in zinc uptake into mammary epithelial cells and is regulated by prolactin

During lactation, a substantial amount of Zn²⁺ is transferred by the mammary gland from the maternal circulation into milk; thus secretory mammary epithelial cells must tightly regulate Zn²⁺ transport to ensure optimal Zn²⁺ transfer to the suckling neonate. To date, six Zn²⁺ import proteins (Zip1–6) have been identified; however, Zip3 expression is restricted to tissues with unique requirements for Zn²⁺, such as the mammary gland, which suggests that it may play a specialized role in this tissue. In the present study, we have used a unique mammary epithelial cell model (HC11) to characterize the role of Zip3 in mammary epithelial cell Zn²⁺ transport. Confocal microscopy demonstrated that Zip3 is localized to the cell surface in mammary epithelial cells and transiently relocalized to an intracellular compartment in cells with a secretory phenotype. Total ⁶⁵Zn transport was higher in secreting cells, while gene silencing of Zip3 decreased ⁶⁵Zn uptake into mammary epithelial cells, particularly in those with a secretory phenotype. Finally, reduced expression of Zip3 ultimately resulted in cell death, indicating that mammary epithelial cells have a unique requirement for Zip3-mediated Zn²⁺ import, which may reflect the unique requirement for Zn²⁺ of this highly specialized cell type and thus provides a physiological explanation for the restricted tissue distribution of this Zn²⁺ importer. zinc transport; mammary gland; lactation     

Adaptive shifts in the activity of amino acid transport into mammary secretory cells in changed nutritional conditions

On the basis of analysis of published data, direct (using 13C) and indirect methods of estimating the amino acid transfer into mammary secretory cells in vivo were compared and the modified indirect method was used to determine quantitatively the shifts in transport activity in lactating cows and goats in trials with amino acid deficit or excess and in investigations that used a hyperinsulemic-euglycemic clamp. The analysis suggests that inadequacy of traditional use of extraction efficiency as a measure of tissue affinity to substrate is associated with two shortcomings: 1) if the changes in mammary blood flow are more expressed compared to transport activity, the arteriovenous difference and extraction efficiency may change in opposite direction to the shifts in transport activity; 2) due to the effect of nonlinearity, in situations characterized by small ratio of blood flow: the transport activity extraction efficiency is insensitive to shifts on activity of transport. The re-analysis of published data using the modified inbdirect method indicated that the deficit of individual amino acid caused a rise in activity of their transport and the excess decreased a net transfer into cell. The insulineuglycemic clamp treatments increased the activity of amino acid transport into the mammary cell and milk protein yield. The results obtained suggest that net transmembrane transfer of amino acid into mammary secretory cell can be controlled by the cell itself according to metabolic demand.     

Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells

The mammary gland epithelium comprises two major cell types: basal and luminal. Basal cells interact directly with the extracellular matrix (ECM) and express higher levels of the ECM receptors, integrins, than luminal cells. We show that deletion of beta1 integrin from basal cells abolishes the regenerative potential of the mammary epithelium and affects mammary gland development. The mutant epithelium was characterized by an abnormal ductal branching pattern and aberrant morphogenesis in pregnancy, although at the end of gestation, the secretory alveoli developed from beta1 integrin-positive progenitors. Lack of beta1 integrin altered the orientation of the basal-cell division axis and in mutant epithelium, in contrast to control tissue, the progeny of beta1 integrin-null basal cells, identified by a genetic marker, was found in the luminal compartment. These results reveal, for the first time, the essential role of the basal mammary epithelial cell-ECM interactions mediated by beta1 integrins in the maintenance of a functional stem cell population, mammary morphogenesis and segregation of the two major mammary cell lineages.     

Immunohistological localization of IgG1, IgA and secretory component in the bovine mammary gland during involution

Summary Immunoperoxidase methods were used to localize secretory component, immunoglobulin A and immunoglobulin G1 in mammary tissue from dairy cows. In lactating tissue, immunostaining for immunoglobulin A and secretory component was observed primarily in the luminal contents of alveoli. By day 2 of involution, alveolar epithelial cells stained for both immunoglobulin A and secretory component. Staining of alveolar epithelial cells for immunoglobulin A and secretory component continued throughout the period of mammary involution. No staining for secretory component was observed in the interalveolar stromal area. Immunoglobulin G1 immunostaining was localized primarily in the interalveolar areas in lactating tissue, but was localized at the apical and basolateral surface of alveolar cells on day 2 of involution. In contrast to immunoglobulin A, immunoglobulin G1 staining of epithelial cells did not persist and was primarily in the interalveolar areas by day 4. These results suggest that an increased localization of immunoglobulin G1 in bovine mammary epithelial cells may occur transiently in early involution, while an increase in immunoglobulin A and secretory component localization in epithelial cells persists throughout involution.     

Effect of prepartum blockade of microtubule formation on ultrastructural differentiation on the mammary epithelium in holstein heifers

1. Prepartum infusion of colchicine into mammary quarters of heifers inhibited normal development of bovine mammary secretory tissue. 2. In comparison with control, tissue from colchicine-infused quarters exhibited 22.6% less alveolar luminal area and 17.1% more interalveolar connective tissue area indicating a reduction in secretory activity. 3. Cytological analysis demonstrated that undifferentiated cells predominated in treated quarters whereas the majority of cells in control quarters were fully differentiated. 4. Although control quarters reached normal histological development by the third week postpartum, tissue from colchicine-infused quarters remained relatively immature. 5. Results suggest that (1) an intact microtubule system is necessary during the prepartum period if the mammary gland is to respond normally to lactogenic stimuli and (2) transient disruption of microtubule integrity during the prepartum period irreversibly suppresses differentiation of mammary epithelia during the subsequent lactation.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).     

Prolactin receptor expression in monolayer cultures of rabbit mammary epithelial cells: pre- and postpartum [125I]-prolactin binding activity

Expression of specific [125I]-prolactin-binding sites under culture conditions has been investigated for isolated mammary epithelial cells from virgin, pregnant, and lactating rabbits. Primary monolayer cultures were obtained by sequential enzymatic dispersion of mammary tissue followed by 48 hr incubation in a medium selective for epithelial cells. Scatchard analyses of binding data obtained from these cultures indicated a single class of receptor sites, the affinity constant of which (2.5 X 10(9) M-1) did not vary significantly during mammary development. The number of prolactin receptors, however, expressed by virgin and early pregnant epithelial cells was significantly increased over those from late pregnancy or lactation. Less differentiated cells also respond to growth in pregnant rabbit serum with an increase in specific [125I]-prolactin binding. The diminished receptor expression by cells obtained after 17 days of pregnancy coincides with the attainment of secretory capacity in the animal, and may reflect the influence of the low serum prolactin or high progesterone levels circulating during the last trimester in the rabbit, or be the cultural expression of secretory differentiation.     

Role of microtubules in milk secretion — Action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells

Summary Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.     

Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling

Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.     

IRF6 in development and disease: A mediator of quiescence and differentiation

Post utero development of the mammary gland is a complex developmental process characterized by states of rapid cell proliferation (branching morphogenesis) followed by functional differentiation (lactation) and the consequent apoptosis (involution) of the secretory mammary epithelial cell. This process is cyclical, such that involution returns the mammary gland to a near-virgin-like state capable of responding to morphogenic cues with each consecutive pregnancy. Importantly, many of the regulatory processes which oversee mammary gland development are corrupted or otherwise compromised during the development of breast cancer. For example, Interferon Regulatory Factor 6 (IRF6) is a novel protein with growth inhibitory properties that was initially identified in mammary epithelial cells through its interaction with maspin, a known tumor suppressor in normal breast tissue. Recent findings from our laboratory suggest that IRF6 functions synergistically with maspin to regulate mammary epithelial cell differentiation by acting on the cell cycle. This perspective focuses on the possible involvement of IRF6 in promoting differentiation by regulating exit from the cell cycle and entry into the G(0) phase of cellular quiescence, and how these new findings shed light on normal mammary gland development and the initiation and progression of breast cancer.