Lactogenesis induced by ovariectomy in pregnant rats and its regulation by oestrogen and progesterone

Ovariectomy on day 19 of pregnancy augmented galactosyl transferase activity 24 h after surgery preceding by 6 h the significant alpha-lactalbumin accumulation. Progesterone, injected immediately after ovariectomy showed a clear inhibitory effect on both galactosyl transferase and alpha-lactalbumin concentration, measured 30 h after ovariectomy. However, once the synthesis of lactose has been induced, progesterone is no longer inhibitory. Oestrogen induced a significant increase in lactose synthetase activity but no effect was obtained on galactosyl transferase activity. Progesterone, in a time and dose dependent relationship, was capable of preventing the effect of estrogen on lactogenesis. The lactogenic action of oestrogen in ovariectomized pregnant rats might be due to a direct effect at the mammary gland level facilitating the action of prolactin or through an indirect effect mediated via an increase on prolactin release.     

Characteristics of the early effect of prolactin on lactose biosynthesis in mouse mammary gland explants

These studies were carried out to characterize the early effect of prolactin (PRL) on lactose biosynthesis in cultured mammary gland explants derived from 12- to 14-day pregnant mice. The rate of lactose biosynthesis was assessed by the rate of radiolabeled glucose incorporation into lactose. For the rapid isolation of lactose, a new method which involves the use of thin-layer chromatography on cellulose-impregnated plastic sheets was employed. The onset of the PRL stimulation of [3H]glucose incorporation into lactose occurred 6-8 hr after exposing the explants to PRL. The response to PRL was essentially all or none with maximum responses occurring with PRL concentrations above 25 ng/ml. The lowest stimulatory concentration of PRL was 10 ng/ml. The action of PRL on lactose biosynthesis requires both ongoing RNA and protein synthesis since puromycin, cyclohexamide, and actinomycin D abolished the PRL effect.     

Purification and characterization of mouse alpha-lactalbumin from lactating mammary glands

The whey protein, alpha-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14600). Antibodies prepared against these peptides precipitate newly synthesized and secreted alpha-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse alpha-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine alpha-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of alpha-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect alpha-lactalbumin levels as low as 0.25 ng.     

The calcium-dependent electrophoretic shift of alpha-lactalbumin, the modifier protein of galactosyl transferase

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.     

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.     

Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A2 inhibitors

p-Bromphenacyl bromide (BPB) at concentrations of 50 microM and above and quinacrine (50 microM) abolished the actions of prolactin (PRL) on casein and lipid biosynthesis in cultured mouse mammary gland explants. In cultured rabbit mammary gland explants, 100 microM BPB or quinacrine abolished the PRL stimulation of casein synthesis, while 50 microM BPB or 250 microM quinacrine abolished the PRL stimulation of lipid biosynthesis. Since BPB and quinacrine are known to inhibit the enzyme phospholipase A2 (PLA2), it is possible that ongoing PLA2 activity is essential for prolactin to express its actions on at least certain lactogenic processes.     

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.     

Effect of prolactin on phosphate transport and incorporation in mouse mammary gland explants

Inorganic phosphate is present in milk at a concentration that is severalfold higher than in maternal plasma. In cultured mammary tissues from 12- to 14-day-pregnant mice, the intracellular concentration of (32)PO(4) was six times higher than in the culture medium after a 4-h treatment with (32)PO(4). Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated (32)PO(4) uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited (32)PO(4) uptake. All three lactogenic hormones together effected the greatest stimulation of (32)PO(4) uptake. Similar hormone effects were observed with regard to (32)PO(4) incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of (32)PO(4) uptake and incorporation occurred 8-12 h after PRL addition; in dose-response studies, the PRL effect was manifested with PRL concentrations of 50 ng/ml and above. From kinetic studies, the apparent maximal velocity of PO(4) uptake was determined to be approximately 7.7 mM x h(-1) x l cell water(-1); the apparent Michaelis-Menten constant was approximately 3-5 mM. The PRL effect on (32)PO(4) uptake was abolished when sodium was absent from the uptake medium. These studies thus demonstrate a complex interaction of three hormones (I, H, and PRL) in the regulation of (32)PO(4) uptake and incorporation into macromolecules in cultured mouse mammary tissues.     

Stage-specific initiation of milk synthesis in the ovariectomized pregnant mouse

Stage-specific initiation of milk synthesis was investigated in mice ovariectomized on days 7, 9, 11 and 13 of pregnancy, and the lactogenic response of the mammary gland was monitored by using the synthesis of casein, lactose content and PRL binding as parameters. Pregnant mice ovariectomized on day 7 or 9 showed no increase in these parameters 24 h after operation. After day 11 of pregnancy, a clear response was induced by ovariectomy, characterized by a low level of casein synthesis, small amounts of lactose and PRL binding, which increased linearly in a time-dependent manner thereafter. These results indicate that pregnant mice after 11 days are able to initiate synthesis of casein and lactose in parallel. Mammary glands were cultured with insulin, cortisol and PRL. The mammary explants of pregnant mice at 7 and 9 days showed active synthesis of casein after a lag period of 2 days, suggesting that the mammary gland in early pregnancy is less sensitive to PRL, probably due to the absence of an increase in PRL binding after ovariectomy.     

Prolactin,cortisol, and insulin regulation of nucleoside uptake into mouse mammary gland explants

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent Km for uridine uptake was calculated to be 312 microM, and the Vmax 2.90 micromol/hr/L cell water; PRL had no effect on the Km but increased the Vmax to 5.88 micromol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.     

Inhibition by progesterone of the lactogenic effect of prolactin in the pseudopregnant rabbit.

The effects of progesterone on lactose synthesis activity and changes in mammary gland cells were studied in pseudopregnant rabbits simultaneously treated with prolactin. The injection of progesterone alone on Days 15 and 17 of pseudopregnancy decreased the activity of lactose synthetase (LSA) and galactosyl transferase (GTA), while the administration of prolactin for 2-4 days increased their activities. Th e simultaneous administration of progesterone and prolactin decreased the increase in LSA observed with prolactin alone by 70% on the 4th day of treatment, and decreased GTA by 30%. Progesterone completely suppressed the polyribosome profile and the ratio of endoplasmic reticulum bound polyribosomes to free polyribosomes induced by prolactin. The increase in RNA content in the mammary gland induced by prolactin was also suppressed by progesterone. The results suggest that progesterone inhibits the lactogenic action of prolactin without interfe ring with its mammogenic role.     

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.     

Cross-linking of the components of lactose synthetase with dimethylpimelimidate.

The cross-linking of the two components of lactose synthetase, alpha-lactalbumin and a galactosyltransferase, with dimethylpimelimidate was examined. The extent of the cross-linking at pH 8.1 was found to be dependent upon the presence of substrates or inhibitors for the galactosyltransferase. N-acetylglucosamine and mixtures of either N-acetylglucosamine, Mn-2+ and UDP, or UDP-galactose and Mn-2+ promoted the formation of cross-linked species. Glucose or a mixture of UDP and Mn-2+ were much less effective in promoting cross-linking. Two types of intermolecularly cross-linked species of alpha-lactalbumin and the galactosyltransferase were obtained. Each was a 1:1 cross-linked complex of alpha-lactalbumin and either of the two forms of the transferase with molecular weights of about 42,000 and 48,000, respectively. Cross-linked complexes were not observed with more than 1 molecule each of alpha-lactalbumin and the transferase. The cross-linked complexes were obtained in homogeneous form by gel filtration on Sephadex and absorption of uncross-linked enzyme by affinity chromatography on alpha-lactalbumin-Sepharose in the presence of N-acetylglucosamine. They migrated on gel electrophoresis in sodium dodecyl sulfate with mobilities in accord with their predicted molecular weights as 1:1 complexes of alpha-lactalbumin and the transferase. The amino acid composition of the cross-linked complex was in reasonable agreement with the expected composition of a 1:1 mixture of alpha-lactalbumin and galactosyltransferase. The enzymic properties of the cross-linked and uncross-linked enzymes were compared. The cross-linked complex had a much higher intrinsic lactose synthetase activity than did uncross-linked enzyme although only about 1% of the potential activity of uncross-linked enzyme in the presence of optimal concentrations of alpha-lactalbumin. The lactose synthetase activity of the cross-linked complex, however, was unaffected by exogenous alpha-lactalbumin. In addition, the complex readily catalyzed the transfer of galactose from UDP-galactose to xylose in the absence of exogenous alpha-lactalbumin. The N-acetyllactosamine synthetase activity of the complex was low compared to its activity with other monosaccharides. Ovalbumin, which is a good acceptor for the uncross-linked transferase, was not an acceptor for the cross-linked complex. Kinetic studies of the complex suggest that its modified catalytic activity is not the result of the modification by dimethylpimelimidate but reflects the expected effects of is provided, and that     

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.     

Saponin effects of prolactin-like stimulation of ornithine decarboxylase activity in mouse mammary gland explants.

Saponin, a naturally occurring plant glycoside, was found to elicit a prolactin-like stimulation of ornithine decarboxylase (ODC) activity in mouse mammary gland explants. A dose-response activation of ODC was observed with saponin at concentrations between 2 and 10 micrograms/ml. At concentrations of 10 and 15 micrograms/ml, saponin effected a response similar to that of PRL; when tested in concert, PRL and saponin caused a nonadditive response. The time-course of the saponin and PRL effects on ODC activation were not different; a maximum response occurred 2-4 hours after addition of saponin. The saponin and PRL responses were abolished by antibiotics (puromycin and cyclohexamide) that inhibit protein synthesis, but not by actinomycin D which inhibits RNA synthesis. Finally, saponin, by itself, did not affect the rate of milk product formation, but at higher concentrations (above 0.5 microgram/ml) impaired the PRL stimulation of lipid and casein synthesis.     

Studies on the particulate lactose synthetase of mouse mammary gland and the role of α-lactalbumin in the initiation of lactose synthesis

1. Some of the kinetic properties of the particulate lactose synthetase of mouse mammary gland were investigated and shown to resemble those that have been reported for the soluble enzyme. Typical values for intact preparations were 2.3mum for the apparent K(m) for alpha-lactalbumin at 40mm-glucose and 1.7mm for the apparent K(m) for glucose at the endogenous concentration of alpha-lactalbumin. 2. Digitonin treatment increased total assayable activity approximately twofold but almost eliminated the endogenous activity found in the absence of alpha-lactalbumin, these findings being consistent with the location of endogenous activity within Golgi vesicles. 3. From the properties of the particulate fraction from lactating mice it was deduced that the effective endogenous alpha-lactalbumin concentrations were in the range 1-10mum. 4. The concentration of alpha-lactalbumin was not significantly different in particles isolated at various stages of pregnancy and early lactation. 5. The implications of these results for the control of lactose synthetase in vivo are discussed.     

Progesterone and the metabolic control of the lactose biosynthetic pathway during lactogenesis in the rat

1. Lactogenesis was initiated in pregnant rats by ovariectomy, thereby causing progesterone withdrawal, after which the mammary tissue was analysed for contents of enzymes and metabolites concerned with the biosynthesis of lactose. 2. Lactose synthesis increased about 126-fold with little or no accompanying change in the contents of most metabolic intermediates or in the adenine nucleotide energy charge. 3. Comparison of mass-action ratios with equilibrium constants showed that phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose epimerase (EC 5.1.3.2.) catalysed reactions close to equilibrium. Nucleoside diphosphokinase (EC 2.7.4.6.) activity was very high and probably equilibrates the UTP-UDP and ATP-ADP couples. Lactose synthetase and hexokinase (EC 2.7.1.1) appeared to catalyse rate-limiting reactions. 4. Large increases were seen of UDP-glucose pyrophosphorylase (5-fold), lactose synthetase A protein (3.8-fold) and alpha-lactalbumin (28-fold), but not of hexokinase, phosphoglucomutase, UDP-glucose epimerase, nucleoside diphosphokinase or glucose 6-phosphate dehydrogenase (EC 1.1.1.49) activities. 5. It appeared that the increased lactose synthesis was largely accounted for by the increased lactose synthetase A protein activity and alpha-lactalbumin.     

Early action of prolactin on ornithine decarboxylase activity is not essential for the subsequent actions of prolactin on casein and lipid biosynthesis

The actions of prolactin (PRL) on casein and lipid biosynthesis in cultured mouse mammary gland explants require the ongoing synthesis of the polyamines. This is supported by the fact that (MGBG) methylglyoxal bis(guanylhydrazone), a drug that inhibits the conversion of putrescine to spermidine, abolishes the effects of PRL on casein and lipid biosynthesis; the inhibitory effects of MGBG are reversed by the addition of spermidine to the culture medium. alpha-Difluoro methyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase activity, reduces the PRL-stimulated ornithine decarboxylase (ODC) activity by more than 95%, and yet does not suppress the effects of PRL on RNA, casein or lipid synthesis. These observations suggest that PRL's early action on ODC activity is not essential for the subsequent actions of PRL on the synthesis of certain of the components of milk.     

Effects of Glucose Availability on Expression of the Key Genes Involved in Synthesis of Milk Fat,Lactose and Glucose Metabolism in Bovine Mammary Epithelial Cells

As the main precursor for lactose synthesis, large amounts of glucose are required by lactating dairy cows. Milk yield greatly depends on mammary lactose synthesis due to its osmoregulatory property for mammary uptake of water. Thus, glucose availability to the mammary gland could be a potential regulator of milk production. In the present study, the effect of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in vitro was investigated. Bovine mammary epithelial cells (BMEC) were treated for 12 h with various concentrations of glucose (2.5, 5, 10 or 20 mmol/L). The higher concentrations of glucose (10–20 mmol/L) did not affect the mRNA expression of acetyl-CoA carboxylase, diacyl glycerol acyl transferase, glycerol-3 phosphate acyl transferase and α-lactalbumin, whereas fatty acid synthase, sterol regulatory element binding protein-1 and beta-1, 4-galactosyl transferase mRNA expression increased at 10 mmol/L and then decreased at 20 mmol/L. The content of lactose synthase increased with increasing concentration of glucose, with addition of highest value at 20 mmol/L of glucose. Moreover, the increased glucose concentration stimulated the activities of pyruvate kinase and glucose-6-phosphate dehydrogenase, and elevated the energy status of the BMEC. Therefore, it was deduced that after increasing glucose availability, the extra absorbed glucose was partitioned to entering the synthesis of milk fat and lactose by the regulation of the mRNA expression of key genes, promoting glucose metabolism by glycolysis and pentose phosphate pathway as well as energy status. These results indicated that the sufficient availability of glucose in BMEC may promote glucose metabolism, and affect the synthesis of milk composition.     

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.