Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline

Depletion of proline from insulin, hydrocortisone, and prolactin-containing medium prior to incubating virgin mouse mammary explants prevents both DNA synthesis and functional differentiation in the mammary epithelial cells; however, DNA synthesis in the mammary stroma and total incorporation of radioactive amino acids into total protein appears to continue without hindrance. Removal of glycine instead of proline had no deleterious effect on either DNA replication in the hormone-stimulated epithelium or in its functional differentiation. Functional differentiation was determined by the induction of casein and alpha-lactalbumin synthesis in the insulin, hydrocortisone, and prolactin (IFPrl)-treated explant cultures. As a control, the induction of mouse mammary tumor virus (MMTV) gene expression, a corticosteroid-regulated function, was also measured. Neither the absence of proline or glycine prevented the glucocorticoid stimulation of MMTV gene expression. In contrast to mammary tissue from virgin mice, explants from nonpregnant primiparous mice responded fully to IFPrl stimulation with respect to DNA, casein, and alpha-lactalbumin synthesis in medium depleted of proline. These data suggest that the uncommitted epithelium of virgin mouse mammary glands requires the presence of exogenous proline in order to respond to lactogenic hormonal signals. We have demonstrated earlier that DNA synthesis is a prerequisite of functional differentiation in virgin mouse mammary explants (Smith and Vonderhaar, 1981, Dev. Biol., 88:167-179; Vonderhaar and Smith, 1982, J. Cell Sci, 53:97-114), although cytological differentiation proceeded unencumbered in explants prevented from synthesizing DNA. Here, without proline, neither cytological nor functional differentiation can be induced; this suggests that proline provides an essential metabolic interlock in the acquisition of lactogenic hormone responsiveness in uncommitted mouse mammary tissue.     

The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture.

Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.     

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.     

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.     

The mechanisms by which nitric oxide affects mammary epithelial growth and differentiation

Nitric oxide (NO) enhances prolactin-stimulated DNA synthesis and inhibits prolactin-induced differentiation in mouse mammary epithelium. The molecular pathways used by NO were determined by employing specific inhibitors of the transducers utilized by NO. Inhibitors of the Jun N-terminal kinase (JNK) blocked the effect of NO on DNA synthesis, although this appeared to involve a protein kinase G (PKG)-independent pathway. In contrast, inhibitors of the extracellular signal-regulated kinase (ERK) prevented NO from suppressing alpha-lactalbumin accumulation and this effect was PKG-dependent. NO can also elevate cAMP through the inhibition of phosphodiesterase 3 and cAMP mimicks the actions of NO on both DNA synthesis and differentiation. However, suppression of cAMP levels did not prevent the effects of NO. Therefore, NO uses two separate pathways to affect mammary epithelium: it stimulates growth via JNK and inhibits differentiation through ERK.     

Regulation of milk protein synthesis by progesterone in cultured mouse mammary gland

The effect of progesterone on the synthesis of milk proteins, casein and alpha-lactalbumin was investigated by culturing mammary explants from mid-pregnant mice in serum-free medium. The addition of progesterone at concentrations above 10 ng/ml inhibited both the casein and alpha-lactalbumin accumulation that were induced by the synergistic actions of insulin, prolactin and cortisol. The maximal inhibition was attained at a progesterone concentration of 100 ng/ml. The maximal level of inhibition of the alpha-lactalbumin accumulation was about 90% in the presence of insulin and prolactin or insulin, prolactin and 0.01 microgram/ml of cortisol. The inhibition of the casein accumulation by progesterone was about 80% in the presence of insulin and prolactin, and about 40% in the presence of insulin, prolactin and 1 microgram/ml of cortisol, indicating that cortisol partially antagonized the action of progesterone on the casein synthesis. When the inhibitory effect of progesterone on the accumulation of both alpha-lactalbumin and casein was examined in cultured mammary tissues from virgin, early pregnant, mid-pregnant and late pregnant mice, the degree of inhibition was markedly reduced in tissue from late pregnant mice. This indicates that the susceptibility of mammary gland to the inhibitory action of progesterone varies with the developmental stage of the tissue.     

Impaired development of mammary glands in scorbutic rats unable to synthesize ascorbic acid

The effects of ascorbic acid (AsA)-deficiency on the development of mammary glands were investigated using mutant rats (osteogenic disorder syndrome rats; ODS rats) with hereditary inability to synthesize AsA. Female ODS rats of 21 days old were castrated and divided into two groups. One group was given AsA in their drinking water, and the other was not. All the rats received a daily injection of oestradiol-17 beta and progesterone (EP) from day 28 to day 49 of age. After EP treatment, the concentrations of AsA in the mammary glands of rats not given AsA were less than one tenth of those of rats given AsA and the contents of hydroxyproline in the mammary glands of the former rats were about half of those in the latter. Furthermore, the concentration of serum prolactin in rats not given AsA was reduced to about one third of that in rats given AsA. After EP treatment, whole mounts of mammary glands showed that in rats not given AsA the development of ducts was impaired and there was extensive accumulation of endbuds. Consistent with this finding, EP injections did not increase the area of parenchyma in the mammary glands of rats not given AsA, whereas they increased it about 2-fold in rats given AsA. Moreover, after EP treatment the amount of alpha-lactalbumin was significantly less in the mammary parenchyma of rats not given AsA than in that of rats given AsA. On the other hand, AsA deficiency did not impair the response of the mammary cells to insulin or prolactin in terms of DNA synthesis and alpha-lactalbumin production. These findings indicate that AsA deficiency impaired the development of mammary glands. This effect may be partly attributable to a defect in collagen synthesis in the mammary glands and a decrease in the concentration of serum prolactin.     

Selective enhancement of the induction of alpha-lactalbumin activity in rat mammary explants by epidermal growth factor

Epidermal growth factor (EGF) enhances the induction of alpha-lactalbumin in mammary explants from pregnant and virgin rats in the presence of insulin (I), hydrocortisone (F) and prolactin (P). EGF also enhances the prolactin-independent induction of alpha-lactalbumin in tissue from pregnant rats and evokes prolactin-independent induction of alpha-lactalbumin in mammary tissue from virgin rats in the presence of I and F. Casein synthesis and galactosyltransferase activity are unaffected by EGF in the IFP-system, and are not induced in the IF-EGF-system. Multiplication stimulating activity, nerve growth factor, fibroblast growth factor and platelet-derived growth factor do not mimic the selective effects of EGF on rat alpha-lactalbumin. These influences of EGF on the differentiation of isolated rat mammary tissue are compared with those on mouse and rabbit tissue studied previously.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

The interaction of cortisol and prostaglandins on the phenotypic expression of the alpha-lactalbumin gene in the mouse mammary gland in culture

Addition of cortisol at concentrations above 300 nM selectively inhibited the synthesis of alpha-lactalbumin and the accumulation of its mRNA in the mouse mammary gland cultured in the presence of insulin and prolactin, whereas the same treatment augmented casein synthesis and the accumulation of casein mRNA. Prostaglandin E2 or F2 alpha reversed the inhibitory effects of cortisol in a dose-dependent manner, without affecting casein production. The levels of prostaglandin E2 or F2 alpha in tissue explants cultured with insulin and prolactin increased about 2.6-fold over those in uncultured tissue, and the addition of cortisol decreased these levels approximately 2-fold. These results indicate the ability of prostaglandins to counteract the inhibitory effect of cortisol on the alpha-lactalbumin gene expression in the mouse mammary gland.     

The presence of the milk protein, alpha-lactalbumin and its mRNA in the rat epididymis

alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.     

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.     

Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis,inconsequent of mitosis

Virgin mouse mammary gland in explant culture will differentiate and synthesize casein and α-lactalbumin when insulin, hydrocortisone, and prolactin (IFPRL) are present in the culture medium. Explants whose DNA synthesis has been blocked differentiate cytologically, mobilize lipid, synthesize RNA, and incorporate ³H-amino acids into proteins to the same extent as unblocked tissue. Nevertheless, casein synthesis as measured by immunoprecipitation with casein-specific antiserum remains at the zero-time level in blocked explants while unblocked explants produce casein at five- to eightfold greater levels. Electrophoretic analysis of immunoprecipitated radioactive proteins showed that the IFPRL-treated virgin tissue made all four size classes of mouse casein. Immunoperoxidase studies of explants revealed that the number of mammary epithelial cells positive for casein was 2–8% in blocked and 24–31% in unblocked, in good agreement with the radioimmunoprecipitation results. Immunoelectron microscopy demonstrated the accumulation of casein within the cisternae of the granular endoplasmic reticulum and in Golgi vacuoles in the unblocked epithelial cells. Similar accumulation did not occur in blocked cultures despite the secretory appearance of the cells. Autoradiographic analysis of blocked and unblocked explants, incubated in the presence of IFPRL and [³H]thymidine for 72 hr, showed that 53–57% of the epithelial cells synthesized DNA in unblocked explants, whereas only 2% incorporated the label in the presence of cytosine arabinoside. When explants were incubated with IFPRL and various concentrations of colchicine, only 5–6% of the epithelial cells were found to enter mitosis. Since cell duplication cannot account for the severalfold increase in casein-producing cells in the unblocked explants, the results suggest that the requirement for DNA synthesis in this system may involve either polyploid cells or the augmentation of specific sequences necessary for the facilitation of terminal differentiation. Similar requirements for DNA synthesis were not observed in mammary explants from pregnant and primiparous (but nonpregnant) mice.     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii)

Lactating tammars can provide two different milks simultaneously from adjacent glands to a young newborn (phase 2 of lactation) and an older animal at heel (phase 3 of lactation). Evidence that the two glands are controlled independently is shown by the capacity of mammary explants from these glands to synthesize different whey proteins and DNA and RNA at different rates. Prolactin is essential for the maintenance of milk synthesis, but its role in differential responses of the individual mammary glands remains to be established. Potential mechanisms for the control of milk synthesis are discussed.     

Super-active forms of placental lactogen and prolactin

Both placental lactogen and prolactin can be converted into super-active forms. These super-active hormones, in combination with insulin and hydrocortisone, stimulate accumulation of α-lactalbumin and increase RNA synthesis in explants from mouse mammary glands to an extent greater than the maximal level obtained with the native hormones. Also, they are able to stimulate RNA synthesis by suspensions of mammary epithelial cells which have lost the ability to respond to native lactogen and prolactin.     

Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.     

Role of the cytoskeleton in laminin induced mammary gene expression

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.     

Pituitary-induced lactation in mammary gland explants from the pregnant tammar (Macropus eugenii): a negative role for cyclic AMP

1. alpha-Lactalbumin and casein have been isolated from tammar milk. 2. alpha-Lactalbumin was induced in mammary explants by culture with anterior pituitary. 3. Casein was induced maximally in the presence of a physiological concentration of prolactin alone. 4. Progesterone did not inhibit the prolactin-induced synthesis of casein, alpha-lactalbumin, galactosyltransferase or fatty acids. 5. Both dibutyryl cAMP and a combination of cholera toxin and IBMX did significantly inhibit the induction of casein and alpha-lactalbumin. 6. Progesterone withdrawal is not a component of the lactogenic trigger in this marsupial but cAMP may be a common intracellular signal for negative control of lactogenesis in both marsupials and eutherians.