Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

Expression, location, and interactions of ErbB2 and its intramembrane ligand Muc4 (sialomucin complex) in rat mammary gland during pregnancy

Muc4 (also called Sialomucin complex) is a heterodimeric glycoprotein complex consisting of a peripheral O-glycosylated subunit ASGP-1 (ascites sialoglycoprotein-1) tightly but non-covalently bound to an N-glycosylated transmembrane subunit ASGP-2. Muc4/SMC can act as an intramembrane ligand for ErbB2 via an EGF-like domain present in the transmembrane subunit. The complex is developmentally regulated in normal rat mammary gland and overexpressed in a number of mammary tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance, promote metastasis, and protect from apoptosis. We have investigated whether Muc4/SMC and ErbB2 are co-expressed and co-localized in normal rat mammary gland and whether Muc4/SMC-ErbB2 complex formation is developmentally regulated in this tissue. Muc4/SMC and ErbB2 have different expression patterns and regulatory mechanisms in the developing rat mammary gland, but both are maximally expressed during late pregnancy and lactation. The two proteins form a complex in lactating mammary gland which is not detected in the virgin gland. Moreover, this complex does not contain ErbB3. ErbB2 is co-localized with Muc4/SMC at the apical surfaces of ductal and alveolar cells in lactating gland; however, another form of ErbB2, recognized by a different antibody, localizes to the basolateral surfaces of these cells. ErbB2 phosphorylated on Tyr 1248 co-localized with Muc4/SMC at the apical surface but not at the basolateral surfaces of these cells. To investigate the function of Muc4 in the mammary gland, transgenic mice were derived using an MMTV-Muc4 construct. Interestingly, mammary gland development in the transgenic mice was aberrant, exhibiting a bifurcated pattern, including invasion down the blood vessel, similar to that exhibited by transgenic mice inappropriately expressing activated ErbB2 in the mammary gland. These data provide further evidence of the ability of Muc4/SMC to interact with ErbB2 and influence its behavior in normal epithelia.     

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Progesterone receptors in normal mammary gland: receptor modulations in relation to differentiation

The biological basis for the observed modulation in cytoplasmic progesterone receptors (PgR) of normal mammary gland occurring during mammary development was investigated. Specifically, the relative roles of hormones vs. differentiation on (a) the decrease in PgR concentration during pregnancy and lactation and (b) the loss of mammary responsiveness to estrogen during lactation were examined. PgR were measured using the synthetic progestin, R5020, as the ligand. The hormones estrogen and progesterone were tested in vivo for their effect of PgR concentration. Mammary gland differentiation was assessed morphologically and by measuring enzymatically active alpha- lactalbumin. These studies show that there is a stepwise decrease in PgR that occurs in two stages. The first decrease is completed by day 12 of pregnancy and the second decrease occurs only after parturition. There appears to be a hormonal basis for the first decrease and it appears to be caused by the negative effect of progesterone on estrogen- mediated increase in PgR. In direct contrast, the absence of PgR during lactation and the mammary tissue insensitivity to estrogenic stimulation of PgR were not related to the hormonal milieu of lactation but were directly related to the secretory state of the mammary gland and lactation per se.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the rat mammary gland

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP) family of peptides, is present in the brain and in neuronal elements of a number of peripheral organs. Since no information on PACAP in the mammary gland exists, we have investigated, by radioimmunoassay and immunohistochemistry, the occurrence and distribution of PACAP immunoreactivity in the mammary gland of lactating and non-lactating rats. A specific monoclonal mouse anti-PACAP antibody'has been used to show that the peptide is located in nerve fibres associated with bundles of circular and longitudinal smooth muscle surrounding the lactiferous duct of the nipple. PACAP-immunoreactive nerve fibres and nerve bundles are present in the subepidermal connective tissue of the nipple and in the mammary parenchyma, some of the fibres being in close contact with blood vessels. Occasionally, a few delicate varicose fibres are associated with secretory alveoli and lactiferous ducts. The majority of PACAP-positive nerve fibres are, however, located in the glabrous skin of the nipple and the hairy skin adjacent to the nipple forming a subepithelial plexus from which delicate varicose nerve fibres enter the overlying epithelium. Double immunostaining for PACAP and a marker for sensory neurons, calcitonin gene-related peptide, has disclosed that the two peptides are almost completely co-localized. A minor population of the PACAP-immunoreactive nerve fibres shows co-existence with VIP. Although no obvious changes at the immunohistochemical level could be observed during pregnancy or lactation, elevated concentrations of immunoreactive PACAP-38 in mammary extracts have been found during lactation. Our data suggest that PACAP is involved in the nervous control of mammary gland function, probably in the transmission of suckling stimuli.     

Detection of circulating mammary mucin (Muc1) and MUC1 immune complexes (Muc1-CIC) in healthy women

There is convincing epidemiological evidence that multiparity provides protection against the development of breast cancer. In the present study we evaluated the levels of MUC1 and MUC1 circulating immune complexes (MUC1-CIC) in 135 serum samples obtained from healthy women. The study population included 13 women who had never been pregnant, 31 primiparous pregnant women, 36 multiparous pregnant women who had lactated, 5 multiparous pregnant women who had never lactated, 24 multiparous non-pregnant women who were lactating at the time of the study, 24 multiparous non-pregnant women who had lactated, and 2 multiparous non-pregnant women who had never lactated. The purpose of this work was to detect MUC1 variations during pregnancy and lactation as well as to study the possible induction of a humoral immune response against MUC1 in these conditions. We employed ELISA techniques to measure MUC1 (CASA test) and MUC1-CIC (IgM and IgG) using two anti-MUC1 monoclonal antibodies (MAbs): C595 and SM3. Statistical analysis was performed using the ANOVA test. The pooled results pertaining to pregnant versus non-pregnant women were compared and significant differences were observed in MUC1 and MUC1-CIC-lgM levels detected with both MAbs; the MUC1-CIC-lgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-lgG levels detected with SM3 did not show any significant differences. When the results were compared between lactating and non-lactating women, no significant differences were found. In conclusion, MUC1 and MUC1-CIC-lgM, detected with both MAbs, and MUC1-CIC-4gG levels detected with the MAb C595 are apparently induced by pregnancy.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

Glycerogenic pathway in the rat mammary gland

The conversions of the isotope from [1-14C]acetate, [1-14C]glucose and [6-14C]glucose to CO2 and fatty acids in acini isolated from the mammary gland at the peak of lactation were studied. The incorporation of [9,10-3H]oleate into triacylglycerol synthesis as single substrate or in combination with substrates that potentially may supply trioses-phosphate was also determined. The rate of fatty acid synthesis paralleled the activity of the hexose monophosphate shunt and the data obtained reveal that little carbon from triose stage enters the phosphohexose pool via reversal of glycolytic pathway. The results are interpreted in terms of the NADPH producing systems and phosphoenolpyruvate carboxykinase activities as well as the possible implications in lipogenic and glyceroneogenic pathways.     

Alterations in estrogen receptor isoforms in the mammary gland and uterus of the rat during differentiation

1. Estrogen receptors in lactating mammary glands and uteri of rats which were 10 and 19 days postpartum exhibited molecular heterogeneity based on their surface charge properties. 2. The polymorphism of estrogen receptors detected by high-performance ion exchange chromatography may be monitored in-line with a radioisotope detector. 3. Estrogen receptors from the mammary gland and uterus of rats at 10 days of lactation exhibited primarily two receptor isoforms eluting at 200-250 mM and 250-300 mM phosphate, whereas three ionic isoforms (eluting at 50-150, 200-250 and 325-375 mM phosphate) were found in the mammary glands of rats at 19 days of lactation. Similar changes in the profiles of estrogen receptor isoforms were observed in uterine cytosol preparations at each stage of postpartum differentiation. 4. The elution pattern of receptor-associated radioactivity was not altered by the addition of diisopropylphosphate, a potent inhibitor of trypsin-like proteases, either before, during or immediately after homogenization. This indicates that the differences observed in the receptor elution profile of 10 and 19 day postpartum lactating mammary glands were not due to artifactual proteolysis. 5. In summary, our data indicate that the differentiation stage of lactating mammary glands may dictate the final profile of receptor isoforms detected.     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

Properties of the lectin from the hog peanut (Amphicarpaea bracteata)

An N-acetyl-D-galactosamine-specific lectin has been isolated from the two seed forms of the hog peanut (Amphicarpaea bracteata) using an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1,3)-[alpha-L-Fuc-(1,2)]-beta-D-Gal (Synsorb A). The affinity-purified lectin appears to be identical in both seed types. Gel filtration on Sephadex G-200 gives a single symmetrical peak corresponding to Mr 135,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows four subunit forms, each of which contains carbohydrate. Limited amino terminal sequencing indicates heterogeneity in two of the first 10 residues. The lectin contains no cysteine. There are four equivalent, noninteracting GalNAc binding sites per 135,000-Da molecule, having an association constant for methyl N-acetyl-alpha-D-galactosaminide of 4.0 X 10(4) M-1. Precipitin and hapten inhibition studies show the lectin to be specific for terminal, nonreducing D-GalNAc units, with a preference for the alpha-anomer and enhanced specificity for the disaccharide, GalNAc alpha 1,3GalNAc. There is also a single adenine binding site per Mr 135,000 lectin molecule with an association constant of 1.3 X 10(6) M-1.     

The interrelationships among poly(ADP-ribosyl)ation, DNA synthesis and mammary gland differentiation

Because mammary epithelium from virgin mice must undergo DNA synthesis prior to differentiation and because poly(ADP-ribosyl)ation has been linked to the cell cycle, it was hypothesized that this requirement for DNA synthesis might be related to the poly(ADP-ribosyl)ation of nuclear proteins. However, 3-methoxybenzamide, an inhibitor of poly(ADP-ribosyl)ation, stimulates alpha-lactalbumin accumulation even when added after DNA replication is completed. Furthermore, in parous mice this compound is still effective when DNA synthesis is blocked by cytosine arabinoside-beta-D-arabinofuranoside. Therefore, poly(ADP-ribosyl)ation appears to be associated, not with DNA synthesis, but with some other event in mammary gland differentiation.     

黄牛泌乳系统正常菌群的检测

采用稀释滴种法对30头健康黄牛泌乳系统的10种主要正常菌群分四个部位(乳头、乳池、输乳管和腺泡)进行定性、定量检测。结果有6种菌群被检测出,按数量由多至少依次为棒状杆菌、乳杆菌、葡萄球菌、芽胞杆菌、双歧杆菌和肠杆菌。乳头的优势菌群为棒状杆菌、芽胞杆菌和葡萄球菌;乳池、输乳管和腺泡的优势菌群均为棒状杆菌和乳杆菌。黄牛泌乳系统各正常菌群的数量均较少,远低于其他孔道系统。     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.