生长抑素在大鼠乳腺组织中的分布和定位

目的研究生长抑素在大鼠乳腺组织中的分布和定位。方法本实验应用即用型快速免疫组化方法对处女期、妊娠6 d、12 d、18 d和泌乳6 d、12 d、18 d的SD大鼠的乳腺进行生长抑素检测。结果发现从处女期到泌乳期大鼠乳腺组织中均有生长抑素的表达,且主要分布于上皮细胞的胞质和腺泡的分泌物中。结论大鼠乳腺上皮细胞的胞质和腺泡的分泌物中有生长抑素的分布。     

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.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Localization of the GLUT1 glucose transporter to brefeldin A-sensitive vesicles of differentiated CIT3 mouse mammary epithelial cells

Glucose is a precursor of lactose, the major carbohydrate and osmotic constituent of human milk, which is synthesized in the Golgi. The GLUT1 glucose transporter is the only glucose transporter isoform expressed in the mammary gland. The hypothesis that lactogenic hormones induce GLUT1 and cause its localization to the Golgi of mammary epithelial cells was tested in CIT(3)mouse mammary epithelial cells. Treatment with prolactin and hydrocortisone caused a 15-fold induction of GLUT1 by Western blotting, but 2-deoxyglucose uptake decreased. Subcellular fractionation and density gradient centrifugation demonstrated enrichment of Golgi fractions with GLUT1. Lactogenic hormones enhanced GLUT1 glycosylation, but did not determine whether GLUT1 was targeted to plasma membrane or to Golgi. Confocal microscopy revealed that lactogenic hormones alter GLUT1 targeting from a plasma membrane pattern to a predominant perinuclear distribution with punctate scattering through the cytoplasm. GLUT1 is targeted to a compartment which is more sensitive to Brefeldin A than the compartments in which GM130 and beta-COP reside. Targeting of GLUT1 to endosomes was specifically excluded. We conclude that prolactin and hydrocortisone induce GLUT1, enhance GLUT1 glycosylation, and cause glycosylation-independent targeting of GLUT1 to Brefeldin A-sensitive vesicles which may represent a subcompartment of cis-Golgi. These results demonstrate a hormonally-regulated targeting mechanism for GLUT1 and are consistent with an important role for GLUT1 in the provision of substrate for lactose synthesis.     

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.     

Mouse mammary epithelial histamine system.

Histamine is suggested to play a role in mammary gland growth regulation, differentiation and functioning during pregnancy and lactation. Two pools of histamine are thought to be involved in these processes: mastocyte- and epithelial cell related histamine. In the present study we focused on epithelial cells. Immunohistochemistry has shown that the epithelial cells positive for histamine and L-histidine decarboxylase (HDC), the primary enzyme regulating histamine biosynthesis, were mainly found in cells forming alveolar structures in the mammary gland. Cultured primary mouse mammary epithelial cells (MMEC) expressed strong HDC immunoreactivity, especially dividing cells and non-differentiated ones. Histidine decarboxylase activity undergoes significant changes during pregnancy and lactation. Pregnancy associated intensive growth of the mammary gland coincided with an increase and the first days of lactation with a decrease of HDC protein expression. Binding studies with mammary tissue membranes and epithelial cell membranes revealed the presence of H1 and H3 but not H2 receptors. Summarizing, our data have shown that mammary epithelial cells are capable of synthesizing and excreting histamine and they bear histamine receptors. These findings further substantiate the role of histamine in mammary gland physiology.     

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.     

GLUT12 expression and regulation in murine small intestine and human Caco-2 cells

GLUT12 was cloned from the mammary cancer cell line MCF-7, but its physiological role still needs to be elucidated. To gain more knowledge of GLUT12 function in the intestine, we investigated GLUT12 subcellular localization in the small intestine and its regulation by sugars, hormones, and intracellular mediators in Caco-2 cells and mice. Immunohistochemical methods were used to determine GLUT12 subcellular localization in human and murine small intestine. Brush border membrane vesicles were isolated for western blot analyses. Functional studies were performed in Caco-2 cells by measuring α-methyl-d -glucose (αMG) uptake in the absence of sodium. GLUT12 is located in the apical cytoplasm, below the brush border membrane, and in the perinuclear region of murine and human enterocytes. In Caco-2 cells, GLUT12 translocation to the apical membrane and α-methyl- d -glucose uptake by the transporter are stimulated by protons, glucose, insulin, tumor necrosis factor-α (TNF-α), protein kinase C, and AMP-activated protein kinase. In contrast, hypoxia decreases GLUT12 expression in the apical membrane. Upregulation of TNF-α and hypoxia-inducible factor-1α ( HIF-1α) genes is found in the jejunal mucosa of diet-induced obese mice. In these animals, GLUT12 expression in the brush border membrane is slightly decreased compared with lean animals. Moreover, an intraperitoneal injection of insulin does not induce GLUT12 translocation to the membrane, as it occurs in lean animals. GLUT12 rapid translocation to the enterocytes’ apical membrane in response to glucose and insulin could be related to GLUT12 participation in sugar absorption during postprandial periods. In obesity, in which insulin sensitivity is reduced, the contribution of GLUT12 to sugar absorption is affected.     

Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation.

Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs alpha, delta, epsilon and zeta were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC alpha, delta, epsilon and zeta proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs alpha, delta, epsilon and zeta were stained in the luminal epithelium and myoepithelium, in varying isoform-and developmental stage-specific locations. PKC alpha was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC epsilon was present in the nucleus, and PKC zeta was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs alpha, delta, epsilon, and zeta was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs alpha, delta, epsilon and zeta. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs epsilon and zeta) or more intensely (PKCs alpha and delta), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.     

Peroxisome proliferator-activated receptor-binding protein null mutation results in defective mammary gland development

A conditional null mutation of peroxisome proliferator-activated receptor-binding protein (PBP) gene was generated to understand its role in mammary gland development. PBP-deficient mammary glands exhibited retarded ductal elongation during puberty, and decreased alveolar density during pregnancy and lactation. PBP-deficient mammary glands could not produce milk to nurse pups during lactation. Both the mammary ductal elongation in response to estrogen treatment and the mammary lobuloalveolar proliferation stimulated by estrogen plus progesterone were attenuated in PBP-deficient mammary glands. The proliferation index was decreased in PBP-deficient mammary glands. PBP-deficient mammary epithelial cells expressed abundant beta-casein, whey acidic protein, and WDNM1 mRNA, indicating a relatively intact differentiated function. PBP-deficient epithelial cells were unable to form mammospheres, which were considered to be derived from mammary progenitor/stem cells. We conclude that PBP plays a pivotal role in the normal mammary gland development.     

An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal

Mammary gland biologists have long assumed that differentiated secretory epithelial cells undergo programmed cell death at the end of lactation and that the alveolar compartment is reconstituted from undifferentiated precursor cells in subsequent pregnancies. It is generally agreed that the remodeled gland in a parous animal resembles that of a mature virgin at the morphological level. However, several physiological differences have been noted in comparing the responses of mammary epithelia from nulliparous versus parous females to hormonal stimulation and carcinogenic agents. We present genetic evidence that an involuted mammary gland is fundamentally different from a virgin gland, despite its close morphological resemblance. This difference results from the formation of a new mammary epithelial cell population that originates from differentiating cells during pregnancy. In contrast to the majority of fully committed alveolar cells, this epithelial population does not undergo cell death during involution or remodeling after lactation. We show that these cells can function as alveolar progenitors in subsequent pregnancies and that they can play an important role in functional adaptation in genetically engineered mice, which exhibit a reversion of a lactation-deficient phenotype in multiparous animals. In transplantation studies, this parity-induced epithelial population shows the capacity for self-renewal and contributes significantly to the reconstitution of the resulting mammary outgrowth (i.e. ductal morphogenesis and lobulogenesis). We propose that this parity-induced population contributes importantly to the biological differences between the mammary glands of parous and nulliparous females.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Defects in mouse mammary gland development caused by conditional haploinsufficiency of Patched-1.

In vertebrates, the hedgehog family of cell signaling proteins and associated downstream network components play an essential role in mediating tissue interactions during development and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer and other tumors. The mammary gland is a specialized skin derivative requiring epithelial-epithelial and epithelial-stromal tissue interactions similar to those required for development of other organs, where these interactions are often controlled by hedgehog signaling. We have investigated the role of the Patched-1 (Ptc1) hedgehog receptor gene in mammary development and neoplasia. Haploinsufficiency at the Ptc1 locus results in severe histological defects in ductal structure, and minor morphological changes in terminal end buds in heterozygous postpubescent virgin animals. Defects are mainly ductal hyperplasias and dysplasias characterized by multilayered ductal walls and dissociated cells impacting ductal lumens. This phenotype is 100% penetrant. Remarkably, defects are reverted during late pregnancy and lactation but return upon involution and gland remodeling. Whole mammary gland transplants into athymic mice demonstrates that the observed dysplasias reflect an intrisic developmental defect within the gland. However, Ptc1-induced epithelial dysplasias are not stable upon transplantation into a wild-type epithelium-free fat pad, suggesting stromal (or epithelial and stromal) function of Ptc1. Mammary expression of Ptc1 mRNA is both epithelial and stromal and is developmentally regulated. Phenotypic reversion correlates with developmentally regulated and enhanced expression of Indian hedgehog (Ihh) during pregnancy and lactation. Data demonstrate a critical mammary role for at least one component of the hedgehog signaling network and suggest that Ihh is the primary hedgehog gene active in the gland.