Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.     

Dispersed mammary gland epithelial cells. I. Isolation and separation procedures

The mammary gland from midpregnant rabbits has been dissociated into individual cells by enzymatic digestion, divalent cation chelation, and gentle shearing. A heterogeneous cell population is obtained, comprising approximately 60% parenchymal cells, approximately 10% myoepithelial cells, and approximately 30% connective tissue cells, including fibroblasts, plasma cells, and microphages. The epithelial cells are characterized by the presence of fat droplets, which in 65% of the cells form large supranuclear vacuoles. Their buoyant density is less than 1.045, allowing their separation from myoepithelial cells and connective tissue cells by isopycnic centrifugation in a density gradient. The homogeneity of the epithelial cell fraction has been assessed by light and electron microscopy. The cells are viable and functionally active as indicated by their ability to exclude vital dyes, incorporate labeled precursors, consume oxygen, maintain intracellular Na+ and K+ concentrations, and retain their structural integrity. In addition, when cultured in Petri dishes, the cells grow as a monolayer, reestablish junctional complexes and retain cell polarity.     

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.     

v-myc alters the response of a cloned mouse mammary epithelial cell line to lactogenic hormones

Several oncogenes have now been implicated in mammary carcinogenesis. We investigated the phenotypic effects of expressing three representative oncogenes in mammary epithelial cells. v-myc (coding for a nuclear protein), v-Ha-ras (a G-protein homologue) and v-fgr (a tyrosine kinase) genes were introduced into the nontumorigenic clone 14 of the mouse mammary epithelial cell line COMMA-1D. Their effects upon growth and differentiation were determined. Anchorage-independent growth was induced by all three oncogenes with low efficiency. v-Ha-ras and v-fgr induced tumorigenicity in nude mice. The effect of oncogenes upon parameters unique to mammary epithelial cells in vitro was assayed. Both v-myc and v-fgr abolished the ability of clone 14 to grow as three-dimensional branching structures in hydrated collagen gel. v-fgr completely and v-myc partially inhibited the expression of the epithelium specific cytokeratins. Clone 14 can be induced to produce the beta-casein milk protein by the combination of the lactogenic hormones, dexamethasone, insulin, and PRL. Introduction of v-myc into clone 14 cells resulted in an estimated 50-fold increased induction of beta-casein protein and at least a 60-fold increase in beta-casein mRNA. The number of cells stained with anti-beta casein antibodies also showed a 10-fold increase after v-myc introduction. This still required the synergistic action of all three lactogenic hormones. Thus v-myc can alter the normal response of mammary epithelial cells to lactogenic hormones.     

Biosynthesis of medium-chain fatty acids by mammary epithelial cells from virgin rats.

Epithelial cells were isolated from the undifferentiated mammary glands of mature virgin female rats, and their lipogenic characteristics were studied. These cells synthesized predominantly medium-chain fatty acids, albeit at a low rate. In contrast, whole tissue from mammary glands of virgin rats synthesized predominantly long-chain fatty acids at a relatively higher rate, indicating that the lipogenic activity is dominated by the adipocyte component of the gland. Enzyme assays revealed that thioesterase II, the enzyme which regulates production of medium-chain fatty acids by the fatty acid synthetase, was present at a high activity in the undifferentiated mammary epithelial cells of virgin rats. Immunohistochemical studies confirmed this observation and showed that the regulatory enzyme was present exclusively in the epithelial cells lining the alveolar and ductal elements of the undifferentiated gland. This study demonstrates that the potential to elaborate tissue-specific medium-chain fatty acids is already expressed in the undifferentiated tissue of virgin rats and is not acquired as a result of the differentiation associated with the lactogenic phase of development. In this species mammary epithelial cells apparently synthesize predominantly medium-chain fatty acids at all stages of development, and only the overall rate of synthesis is increased on induction of the fatty acid synthetase during lactogenesis.     

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.     

Mechanical stimulation of mammary gland development in virgin and pregnant rats

The mammary glands of pregnant Sprague-Dawley rats prevented from self-licking by collars around the neck (n = 9) were only about 50% as developed as those of pregnant rats without collars (n = 9). However, when the ventral body surface of pregnant collared rats was stimulated mechanically (n = 9), mammary glands increased in size, underwent lobuloalveolar growth, and secreted milk in a manner similar to normal pregnant rats.When virgin rats were stimulated mechanically for 22 days (n = 9), their mammary glands had significantly greater secretory tissue and lobuloalveolar development than did the glands of nonstimulated virgin rats (n = 9).Stimulated rats were tied to the stimulating device under ether anesthesia. Because ether and restraint have been shown under some conditions to be Stressors that increase development of the mammary glands, two additional pregnant collared control groups were added. One was restrained on the device without stimulation (n = 9). The other was lightly anesthetized with ether (n = 10). Mammary underdevelopment occurred in both groups. This suggested that stress of ether and restraint were unlikely causes of the mammary development in the stimulated group.     

Lipogenesis in interscapular brown adipose tissue of virgin, pregnant and lactating rats. The effects of intragastric feeding.

Lipogenesis in brown adipose tissue of virgin rats increased 8--10-fold after intragastric feeding with glucose or medium-chain triacylglycerol, and this increase was prevented by short-term insulin deficiency. Brown adipose tissue increased in weight during pregnancy, regressed during lactation and hypertrophied again on weaning; the rate of lipogenesis paralleled these changes. Glucose did not increase brown-adipose-tissue lipogenesis at mid-lactation.     

Secretory function of lactating mouse mammary epithelial cells cultured on collagen gels

Mammary epithelial cells dissociated from lactating mouse mammary glands form confluent monolayer cultures on collagen gel substrates. For these cultures, the substrate is more significant than the presence of lactogenic hormones in the maintenance of cell differentiation, as indicated by both morphological and biochemical criteria. Only cells cultured on floating collagen gels are able to maintain their lactose pool over several days in culture, although their ability to synthesize and secrete lactose becomes impaired. These cells are cuboidal in shape. In contrast, cells cultured on attached gels, which are constrained from changing shape and whose basolateral surfaces are inaccessible, lose their differentiation with time in culture. These flattened, dedifferentiated cells respond to the same hormonal environment by showing a mild proliferative response. Therefore, the response of cells to their hormonal milieu may be correlated with their shape: the squamous cells dedifferentiate and proliferate; the cuboidal cells maintain their differentiation and do not proliferate.     

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.     

Induction of lipid synthesis in mammary organ cultures from mature virgin and pregnant goats.

The hormones needed to induce lipogenesis in mammary organ cultures from mature virgin and pregnant goats were studied. In tissues from both mature virgin goats and goats at week 10 of pregnancy, cultivated in Waymouth medium without hormones, the rate of the incorporation of (1-(14C))-acetate into the lipids was low and decreased throughout culture. In the presence of insulin, the rate of acetate incorporation was maintained at a higher level. Cortisol acted synergistically with insulin, to produce a rate of lipid synthesis higher than that using insulin alone. The further addition of prolactin had little effect on the incorporation of acetate into the lipids of mammary explants from mature virgin goats, but markedly stimulated it in tissue from animals at weeks 9--10 of pregnancy. The maximum increase in the rate of lipid synthesis was achieved in the presence of 0.5 microgram prolactin/ml, whereas with growth hormone 50 microgram/ml was needed for the maximum effect. The initial rate of acetate incorporation into mammary explants from goats at weeks 13 and 18 of pregnancy was high. It was not stimulated by the hormones during culture, however, and decreased more rapidly in the absence of hormones than in their presence. The rate of acetate incorporation into the lipids was in agreement with the histological evaluation of the secretory response of the mammary explants after cultivation. The secretory response to prolactin and the rate of the incorporation of acetate into the lipids were highest in goats at weeks 9--10 of pregnancy while in tissues from goats at weeks 13 and 18 were not stimulated and decreased during culture.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.     

Degradation of newly-synthesised casein in mammary explants from pregnant and lactating goats

Casein degradation was measured by a pulse-chase method in explants from freshly-prepared from goat mammary tissue. An initial increase in L-[5-3H]proline radioactivity incorporated in casein during the chase period was followed in some conditions by a decrease resulting from intracellular degradation of the major casein polypeptides. Differences in the proportion of casein degraded in explants cultured with or without prolactin suggested that the process was under hormonal control. Greater degradation of casein by explants obtained in late pregnancy and early lactation suggested that the process may affect net casein production in vivo.     

Cyclic nucleotides, adenylate cyclase, and cyclic AMP phosphodiesterase in mammary glands from pregnant and lactating mice.

In the mammary glands of mice, levels of cyclic AMP increased during pregnancy and then fell precipitously following parturition. In contrast, levels of cyclic GMP fell during the gestation period and then rose rapidly during the early days of lactation. Adenylate cyclase and cyclic AMP hohsphodiesterase activities were elevated during the pregnancy and lactation periods.