Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

Variable duration of DNA synthesis in mammary gland cells during pregnancy and lactation of C3H/He mouse

Labeling index as well as the duration of DNA synthesis in alveolar cells of C3H mouse mammary gland at various stages of development was determined by autoradiographic methods. Labeling index of the alveolar cells is highest during pregnancy followed by a marked decrease in the lactating gland. The labeling index of the prelactating cells is significantly reduced after the same cells are transplanted into virgin females. Duration of DNA synthesis in the alveolar cells at eighth and fifteenth day of pregnancy is 14.1 and 8.2 hours respectively. During early lactogenesis, duration of DNA synthesis in the mammary alveolar cells was estimated as 8.5 hours. There is a 2–3 fold increase of the DNA replication time (21.5 hours) in the outgrowth cells of 15 day prelactating tissue after transplantation into virgin host. A possible role of the hormones of pregnancy, estrogens and progesterone for stimulation of DNA synthesis in the prelactating tissue has been discussed. It has been suggested that the marked inhibition of DNA synthesis in the lactating tissue may be due to the increased stimulation of the same tissue by endogenous adrenocorticoid hormones. Variability of the duration of DNA synthesis (8.5–21.5 hours) in alveolar cells indicates that in mouse mammary gland, DNA synthetic time is not an unadjustable process. Control of DNA synthesis in mouse mammary gland cells by exogenous 17-_β-estradiol and progesterone has been previously reported (Bresciani, '65). It is suggested that the same hormones of endogenous origin also may influence the duration of DNA synthesis and cell proliferation during mammogenesis.     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.     

Monocarboxylate transporter genes in the mammary gland of lactating cows

This study is the first to examine the expression of the 14 monocarboxylate transporter genes (MCT1–MCT14) in the mammary gland of mammals. RT-PCR, Western blot, immunohistochemistry, and immunofluorescence confocal laser microscopy were applied in a comprehensive approach to assess the expression and cellular localization of MCTs in the mammary gland of lactating cattle. RT-PCR revealed the existence of nine MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT10, MCT13, and MCT14 in cow mammary gland. The amplified cDNA segments were confirmed by sequence analysis and deposited in the GenBank. Using the commercially available antibodies against MCT1–MCT8, Western blotting verified the protein expression of MCT1, MCT2, MCT3, MCT4, MCT5, and MCT8 in the cow mammary gland. The precise cellular localization of the identified MCT proteins showed that both MCT1 and MCT2 were basolaterally localized on the cow mammary alveolar epithelial cells. In contrast, MCT4 protein signal was expressed on the apical membrane of these alveolar epithelia. MCT8, however, was predominantly localized on the basolateral membranes of the lactocytes, along with its weak labeling on the apical membrane of the same cells. No immunoreactive staining for MCT3 and MCT5 proteins could be detected histochemically in lactating bovine mammary tissue. Additionally, we proved the colocalization of CD147 with both MCT1 and MCT4 on the boundaries of the cow mammary alveolar epithelia. The existence and localization pattern of MCT genes in the mammary gland of lactating cows suggest their possible involvement in the transport of essential elements required for milk synthesis and secretion.     

Keratin-like proteins in normal and neoplastic cells of human and rat mammary gland as revealed by immunofluorescence microscopy

Normal and neoplastic human breast tissue as well as lactating and nonlactating rat mammary glands and 7,12-dimethylbenz(alpha)-anthracene-induced mammary adenocarcinomas of rat, were examined by indirect immunofluorescence microscopy using guinea pig antibodies to human and bovine epidermal prekeratin and to cytokeratin polypeptide D from mouse hepatocytes. In normal mammary glands of both species, lactating rats included, the antibodies raised against human and bovine epidermal prekeratins strongly stained ductal and myoepithelial cells, whereas antibodies to hepatic cytokeratin D revealed, in addition, fibrillar staining in cells of the alveolus-like terminal lobular units and in milk secreting cells of the rat. The presence of some finely dispersed intermediate-sized filaments of the cytokeratin type in lactating alveolar cells of rat mammary gland was also demonstrated by electron microscopy. In human intraductal mammary carcinomas the antibodies to epidermal prekeratins showed staining in myoepithelial cells and intralumenal papillary protrusions of the tumor, whereas the antibodies to hepatic cytokeratin D presented an almost complementary pattern in that they showed strongest staining in the more basally located layers of tumor cells. Intraductal adenocarcinomas of rats showed strong staining with all keratin antibodies examined. In contrast to previous studies using exclusively antisera raised against epidermal prekeratin, out results show that all types of neoplastic and non-neoplastic epithelial cells of mammary gland of both species contain-at least some-filaments of the cytokeratin type identifiable by immunologic reaction, if antibodies are used that recognize a broad range of epidermal and nonepidermal cytokeratins. Consequently, such broad range antibodies to keratin-like proteins provide adequate tools to identify and characterize neoplastic and non-neoplastic epithelial cells and to eliminate false negative immunocytochemical findings in tumor diagnosis. In addition, our observation that in the same human carcinoma two cell types can be distinguished by their reaction with two different antibodies to cytokeratins from epidermis and liver, respectively, indicates that the cells of a given carcinoma can differ in their cytoskeletal composition, thus presenting further criteria for diagnostic differentiation.     

Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland

During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.     

Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution

Involution of the mammary gland following weaning is divided into two distinct phases. Initially, milk stasis results in the induction of local factors that cause apoptosis in the alveolar epithelium. Secondly after a prolonged absence of suckling, the consequent decline in circulating lactogenic hormone concentrations initiates remodeling of the mammary gland to the virgin-like state. We have shown that immediately following weaning TGFbeta3 mRNA and protein is rapidly induced in the mammary epithelium and that this precedes the onset of apoptosis. Unilateral inhibition of suckling and hormonal reconstitution experiments showed that TGFbeta3 induction is regulated by milk stasis and not by the circulating hormonal concentration. Directed expression of TGFbeta3 in the alveolar epithelium of lactating mice using a beta-lactoglobulin promoter mobilized SMAD4 translocation to the nucleus and caused apoptosis of these cells, but not tissue remodeling. Transplantation of neonatal mammary tissue derived from TGFbeta3 null mutant mice into syngenic hosts resulted in a significant inhibition of cell death compared to wild-type mice upon milk stasis. These results provide direct evidence that TGFbeta3 is a local mammary factor induced by milk stasis that causes apoptosis in the mammary gland epithelium during involution.     

EGF receptors on plasma membranes purified from bovine mammary gland of lactating and pregnant animals

A procedure for purification of plasma membranes from bovine mammary gland has been developed. The binding capacity of EGF to the plasma membranes from mammary tissue of pregnant cows was equal to 335 fmol per mg of protein thus being twofold higher than in membranes from lactating gland. The KD values were not changed. Autoradiographs of the membrane receptor linked to [125J]-EGF revealed three labeled bands corresponding to 160 kDa, 145 kDa and 115 kDa polypeptides. The main band of 145 kDa was labeled stronger in membranes from pregnant animals. The results suggest that the EGF receptor level is enhanced in fast proliferating normal mammary tissue.     

Sodium-dependent phosphate transport across the apical membrane of alveolar epithelium in caprine mammary gland

NaPi IIb cotransporter is expressed in various tissues including mammary glands of mice. The physiological role of NaPi IIb in lactating mammary glands is still unclear. Therefore, it was the aim of the study to detect and to localize NaPi IIb protein in lactating goat mammary glands by Western analysis and immunohistochemistry. Furthermore, Na(+)-dependent P(i) uptake into apical membrane vesicles isolated from goat milk was determined using rapid filtration technique. NaPi IIb protein could specifically be detected in the apical membranes of lactating alveolar epithelial cells. Na(+)-dependent P(i) uptake into apical membrane vesicles could be measured, which was inhibited by phosphonoformic acid. The kinetic parameters were V(max) with 0.9 nmol/mg protein/10 s and K(m) with 0.22 mmol/L for P(i) affinity, K(m) value for Na(+) affinity 11 mmol/L. Stoichiometry of this mammary gland Na(+)/P(i) transport across the apical membranes seemed to be 1:1 P(i):Na(+) without cooperativity in P(i) and Na(+) binding as assessed by Scatchard and Hill plots. These features of Na(+)/P(i) transport suggest that it could be mediated by NaPi IIb. The quantitative role of this P(i) transport which is directed from the alveolar lumen into the epithelial cell of goat mammary gland will be the topic of further investigations.     

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.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Parathyroid hormone-related protein production by primary cultures of mammary epithelial cells.

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult non-neoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/10(5) cells x 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)-beta, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.     

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.     

Lactation-dependent down regulation of leptin production in mouse mammary gland

Lactation-dependent regulation of leptin expression in mouse mammary gland and parametrial adipose tissue was estimated by RT-PCR analysis for virgin, pregnant, lactating and post-lactating mice, and the serum and milk leptin levels of these mice were also determined by ELISA. Leptin gene expression in mammary gland as well as in adipose tissue was obviously detected before pregnancy, markedly decreased to 30-50% after parturition and kept at the low level during lactation period, and restored to the original level after weaning. The leptin concentration of milk collected just before weaning was about two-fold higher than that of the milk collected at mid-lactating stages. The serum leptin levels of the mid- and late-lactating mice were not significantly higher than those of non-pregnant mice. These results suggested that the lactation-induced down regulation of leptin was associated with autocrine/paracrine action of leptin in mammary and adipose tissues, and that the milk leptin, especially at the latter stages of lactation, was not only ascribed to diffusive transport from maternal blood stream, but also regional production and secretion by mammary epithelial cells. This possible production of leptin by mammary epithelial cells was further supported by the fact that leptin was expressed by cultured cells of mammary epithelial cell line, COMMA-1D, in a manner negatively dependent on the lactogenic hormones.     

Changes in rat milk quantity and quality due to variations in litter size and high ambient temperature.

The effects of variations in suckling stimulus (changes in letter size) and exposure to high ambient temperatures on the quality and quantity of rat's milk and mammary tissue structure were determined. By increasing the number of suckling pups per dam, it was found that the amount of milk produced by the animals was proportionally increased, as each pup was able to drink the same amount of milk per day. With more than 6 pups per litter, the quality of the milk was not significanly changed. Smaller letters led to changes in the amount of milk suckled per milking and to considerable changes in the content of the milk. Exposure to high ambient temperatures, although for only 8 hr per day, drastically affected the amount of milk produced by the lactating rat as well as the quality of the milk produced. Microscopic examination of mammary tissue revealed changes in the alveolar area and height of the alveolar cells as the suckling stimulus varied. After heat exposure the gland changed to one of diminished synthesizing capability. It was concluded that 10-pup litters are the optimal litter size for research concerning rat milk, and the exposure to high ambient temperatures affects the milk yield by directly affecting the alveolae.