Mammary glucose uptake in the lactating ewe and the use of methionine arterio-venous difference for the calculation of mammary blood flow

(1) The validity of using the arterio-venous concentration difference of methionine to calculate mammary blood flow in the ewe, on the basis of the Fick principle, is discussed. (2) Calculation of mammary blood flow in the lactating Merino ewe indicated that blood flow per unit weight of tissue and the ratio of blood flow : milk yield were approximately twice that found in the lactating cow and goat. (3) Calculated mammary blood flow in Merino ewes was used in conjunction with glucose arterio-venous difference to determine mammary glucose uptake. Glucose uptake per unit weight of tissue in the ewe was almost double that found in the cow and goat. The ratio of mammary glucose uptake to lactose output was also higher in the ewe than that found in the cow and goat. The utilization of glucose by the mammary gland of the ewe is discussed in relation to the possible greater requirement for NADPH and glycerol for milk fat synthesis in this species.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Plasma membrane fragments in bovine and caprine skim milks

By either differential or linear gradient ultracentrifugation of bovine or caprine skim milks it was possible to obtain fractions which contained 45–75% of the lipid phosphorus and unesterified cholesterol of the skim milk. Electron microscopy of these fractions revealed the presence of numerous membrane-bound vesicles, microvilli and membrane fragments. Assay of the fractions for certain membrane-bound enzymes; i.e. 5′-nucleotide pyrophosphatase, alkaline phosphatase and ATPases, established the presence of all but the latter in the membrane-rich fractions. The distributions of the enzymes in the various fractions were correlated with their lipid phosphorus and cholesterol contents.Compositions of the phospholipids from skim milk membranes, milk fat globule membranes and the plasma membrane of the lactating mammary cell were observed to be similar and unique for having a relatively high level (20–25%) of sphingomyelin. By virtue of secretory processes, all of these membranes appear to be interrelated with each other and with Golgi vesicle membranes. It is concluded that the membrane material in the skim milk originates primarily from plasma membrane of the lactating cell. The possibiltiy that Golgi vesicle membranes form a substantial part of this material is not precluded by the results of this study.Separation of bovine skim milk on a Sepharose 4B gel column demonstrated that virtually all of the 5′-nucleotidase and lipid phosphorus are recovered together in the void volume of the column. Considering the particle size discriminating characteristics of this gel, the skim milk membrane material appears to be constituted of relatively large structures rather than of discrete subunits.     

Milk prolactin is biologically active

Fat-free milk from cow and goat was chromatographed on Sephadex G-100 and the prolactin (PRL) activity of the fractions determined by radioimmunoassay (RIA). A single prolactin component was observed in 3 cow and 3 goat milk samples with a Vf/Vt ratio of approximately 0.5. Fractions in which PRL was detected by RIA and fractions on either side of the PRL peak were combined, dialyzed and freeze dried. The fractions were assayed for biological activity using the pseudopregnant rabbit mammary gland in organ culture; the degree of secretory response was evaluated histologically. Milk prolactin was biologically active. In the RIA cow milk PRL and one of 2 samples of goat milk PRL gave dose response curves parallel with the bovine PRL standard. In the bioassay the dose response curves for cow milk PRL and ovine PRL were parallel while goat milk PRL was parallel when the results were compared on a weight basis but not on the basis of prolactin content of the preparations assayed by RIA.     

Selenium content of milk and milk products of Turkey. II

Selenium content of 1028 milk and milk products of Turkey are presented in this study. The selenium content of human milk (colostrum, transitional, and mature milk), various kinds of milk [cow, sheep, goat, buffalo, paper boxes (3%, 1.5%, 0.012% fat), bottled milk, condensed milk (10% fat), mineral added milk (1.6%), and banana, strawberry, and chocolate milk] and milk products (kefir, yogurt, Ayran, various cheese, coffee cream, ice cream, butter, margarine, milk powder, and fruit yogurt) in Turkey were determined by a spectrofluorometric method. The selenium levels of cow milks collected from 57 cities in Turkey were also determined. Selenium levels in cow milk varied with geographical location in Turkey and were found to be lowest for Van and highest for Aksaray. The results [milk (cow, sheep, goat, buffalo and human) and milks products] were compared with literature data from different countries.     

Relationship of milk iron and the changing concentration of mammary tissue transferrin receptors during the course of lactation

The concentration of iron in all mammalian milks falls during lactation while the infant's iron requirement increases. Little is known, however, about the entry of iron into milk. Recently, transferrin receptors have been identified on lactating rat mammary plasma membranes, which may regulate iron entry into mammary tissue, potentiating its availability for subsequent transport into milk. This study was conducted to determine what relationship exists between the declining concentration of milk iron and the transferrin receptor concentration during various stages of lactation. Minimal transferrin receptors were detected in nulliparous rats. Total mammary transferrin receptor content increased during early and mid-lactation while milk iron concentration decreased. The continued appearance of high levels of transferrin receptors throughout lactation, without a concomitant increase in milk iron concentration, suggests a need for iron for functions other than cellular growth or secretion into milk to meet infant needs.     

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.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue.     

Isolation and characteristics of poly(A+) RNA in the lactating bovine udder

Total cellular poly(A+)-RNA was isolated from a lactating cow mammary gland. The poly(A+)-RNA molecules exhibit a heterogeneous distribution from 500 to 5000 nucleotides (average size--1600 nucleotides) and are made up of three main fractions (1550, 950 and 600 nucleotides) possessing a high template activity during translation in vitro. Optimal conditions for poly(A+)-RNA translation in a cell-free protein-synthesizing system from wheat embryos were elaborated. Immunochemical analysis of translation products revealed that 30% of the synthesized polypeptides are precipitated by immunoglobulins against cow milk proteins. Using hybridization with homologous cDNA, the kinetic complexity and heterogeneity of total cellular poly(A+)-RNA were investigated. This population was shown to consist of four classes differing in the diversity of their nucleotide sequences and the number of copies per cell. The total amount of the poly(A+)-RNA species in the cells of a lactating cow mammary gland is 9200, i.e., 0.46% of the genome complexity.     

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.     

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.     

Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus

Summary The effects of colchicine on ultrastructure of the lactating mammary cell in the rat and goat were studied by electron microscopy. Changes in tissue of the rat were examined over time (1, 2 and 4 h). The goat gland was evaluated by comparing ultrastructure of tissue at the time of maximum milk flow suppression induced by the drug with that of untreated tissue. Colchicine produced notable changes in the tissue of both species: 1) the secretion of lipid droplets and Golgi vesicle contents (exocytosis) was inhibited and the droplets and vesicles became randomly distributed throughout the cell, 2) the Golgi apparatus was significantly reduced in size, 3) casein and lipid continued to be synthesized as evidenced by greater numbers of secretory vesicles and increased sizes of casein micelles and lipid droplets, 4) secretory vesicles showed a propensity to cluster around lipid droplets, 5) isolated microtubules were found occasionally in the control tissue, ordinarily in the vicinity of the Golgi apparatus, but rarely in the colchicine-treated tissue. These observations indicate that colchicine has two effects leading to suppression of exocytosis in the mammary cell: one involves early interference with capacity of secretory vesicle membranes to fuse and a further effect, related to higher concentrations of colchicine, causes intracellular disorganization and loss of polarity. Microtubules were not seen as directly involved in the mechanisms of exocytosis. The secretion of milk fat globules is coupled to exocytosis and thereby is also inhibited by colchicine.Supported in part by grant HL 03622 of the U.S. Public Health Service     

Is the apocrine milk secretion process observed in the goat species rooted in the perturbation of the intracellular transport mechanism induced by defective alleles at the alpha(s1)-Cn locus?

The structural and quantitative variability of caprine alpha(s1)-casein induced by the extensive polymorphism recorded at the corresponding locus strongly influences the composition (proteins as well as lipids) and the technological behaviour of milk. Immuno-histo-chemistry studies coupled with electron microscopy analysis have shown that a dysfunction exists in the intracellular transport of caseins when alpha(s1)-casein is lacking. Casein accumulation in the endoplasmic reticulum leads to a dilation of the cisternae that could disturb the whole secretion process (including lipids). Despite a long controversy, goat milk secretion is still considered to occur through an apocrine process contrary to the merocrine process described for cow's milk. We suggest that the apocrine pathway of secretion described in the goat could be the consequence of the dysfunction observed in the intracellular transport of caseins when alpha(s1)-casein is lacking. To obtain further clues in the favour of such a hypothesis, we compared the protein and lipid fractions of milks from goats homozygous for different alpha(s1)-casein alleles.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

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.     

Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone- insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.