Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells.

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

Effects of supplementing pregnant heifers with methionine or melatonin on the anatomy and other characteristics of their lateral hind claws.

Three groups of four primiparous Holstein-Friesian heifers were fed throughout pregnancy either a control diet or that diet supplemented with either 5 to 6 g per day of rumen-protected intestinally available methionine or 25 mg melatonin. They were euthanased three days after calving. The dietary supplements had no effect on the impression hardness or the concentrations of cysteine and methionine in samples of claw horn collected from a range of sites, or on the areas of erosion in the sole and heel. Significant differences were recorded for the hardness of the horn in the order wall >sole >heel. These differences were associated with higher concentrations of cysteine and lower concentrations of methionine in samples of horn from the dorsal wall than in samples from the prebulbar region of the sole. There were no significant differences attributable to the dietary supplements in the soft tissue anatomy of the solear dermis and epidermis.

Protein degradation in mammary gland explants. Evidence for limited heterogeneity of protein degradation rates.

Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that isotope ratios for each fraction are grouped predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The results also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly related to these molecular properties of the proteins.

Regulation of intracellular casein degradation by secreted milk proteins.

Intracellular degradation of newly synthesised casein was measured by a pulse-chase method in freshly prepared goat mammary explants. After incubation in medium containing L-[5-3H]proline, explants were washed and cultured again in unlabelled medium containing 5 mM proline; at intervals up to 24 h the amount of radiolabel incorporated in casein was measured. Tissue was obtained in week 33 of lactation after goats had been milked incompletely in one gland (the test gland) for 24 weeks; the contra-lateral (control) gland was milked normally. In explants from the control gland, casein was not degraded during or after secretion: L-[5-3H]proline incorporated in casein increased to a maximum value which was maintained through the chase period. For four out of five goats, explants from the test gland showed a decrease in total [3H]casein radiolabel at 0-4 h of the chase, indicating that a proportion of casein was degraded during secretion. Intracellular casein degradation was also observed when control gland explants were cultured in chase medium containing a goat whey fraction known to inhibit casein production and milk secretion (Wilde, C.J. et al., (1987) Biochem. J. 242, 285-288). This suggests that the greater volume of residual milk left by incomplete milking reduced secretory efficiency, rendering casein susceptible to intracellular degradation, and that this occurred through the action of a secreted milk constituent, which acts as a chemical feedback inhibitor of milk secretion.

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.

[How to recognize POAD? The diagnosis of peripheral occlusive arterial disease in the physician's office]. / Diagnostik der arteriellen Verschlusskrankheit: Mit Stethoskop, Doppler und Laufband der pAVK auf den Fersen.

Peripheral occlusive arterial disease (POAD) encompasses all stenotic and occlusive changes affecting the aorta and arteries supplying the extremities, and in 90% of the cases is due to atherosclerosis. In the majority of patients the lower extremities are involved, andonly every third patient has symptoms. Apart from its significance as an independent disease entity, POAD is also an important coincidence marker, in particular for coronary heart disease. For the diagnostic clarification of POAD, a stepped strategy is recommended, including clinical function testing, such as an exercise treadmill test. Doppler sonography and determination of the Doppler pressure index (ankle-brachial index) make possible a noninvasive accurate staging. Color-coded duplex sonography and imaging procedures provide further information, and are indispensable for establishing the indication for interventional measures.

The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion.

We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH-deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland.

Local control of the mammary gland.

Studies on increasing the frequency of milking in dairy animals have led to the uncovering of the mechanism by which tactical control of the rate of milk secretion is achieved locally within each mammary gland, against a strategic, systemic control by the hormones that maintain all glands in the secretory condition. Experiments in vivo established that the response is local, and were compatible with the hypothesis that milk contains an inhibitor of its own secretion which accumulates during storage within the lumen of the mammary gland and which acts in an autocrine manner on the secretory cells. Isolation of a protein, initially from goats' milk, called FIL (feedback inhibitor of lactation) has enabled, and is enabling, further studies to be done from the whole-animal down to the molecular level. Examples at the whole-animal level are: the effects of immunization against FIL on the rate of secretion; the concentration of FIL and the kinetics of its formation and breakdown; the importance of the internal structure of the mammary gland and the capacities of the alveolar and ductular storage regions in determining feedback inhibition; differences between individuals and species influencing the degree of control exerted by FIL in matching supply of milk to demand by the young. Other local control mechanisms at the onset and cessation of lactation, including mammary distension, are also discussed.

Autocrine regulation of milk secretion.

Mammary development and the rate of milk secretion are regulated by frequency and completeness of milk removal. This regulation occurs through chemical feedback inhibition by a milk constituent. Novel, immunologically related milk proteins able to perform this function have been isolated from caprine, bovine and human milk, based on their ability to inhibit milk constituent synthesis in mammary tissue and cell cultures, and to decrease temporarily milk secretion when added to milk stored in the mammary gland. Inhibition is concentration-dependent, suggesting that milk accumulation and removal is accompanied by cyclical changes in inhibitor accretion and depletion in milk. Feedback inhibition is an autocrine mechanism: the caprine inhibitor, termed FIL (feedback inhibitor of lactation) is synthesized by mammary epithelial cells in primary culture. Inhibition is by reversible blockade of the secretory pathway, an effect which, by down-regulating cell-surface hormone receptors, has longer-term consequences on epithelial cell differentiation. Treatment of goat mammary epithelial cell cultures with caprine FIL initially decreased milk protein secretion and subsequently reduced milk protein messenger RNA abundance. Thus the actions of a single milk constituent can bring about both the effect of milking frequency on milk secretion rate and a sequential modulation of cellular differentiation which acts to sustain the secretory response. Long-term regulation, through changes in galactopoietic hormone receptors, also provides an efficient mechanism for integrating acute intramammary regulation of lactation with strategic endocrine control of mammary tissue development.

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii).

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of beta-lactoglobulin and beta-casein gene expression increased, whereas there was no change in the levels of expression of alpha-lactalbumin and alpha-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

Autocrine inhibition of milk secretion in the lactating tammar wallaby (Macropus eugenii).

The lactating tammar wallaby progressively alters the rate of secretion and composition of its milk to provide appropriate nutrition for the developing offspring, whose needs are signalled by changes in the pattern and efficiency of its sucking. Tammars are also capable of asynchronous concurrent lactation, when the mother provides a dilute milk for a newborn young permanently attached to the teat (phase 2A of lactation), and a concentrated milk from an adjacent mammary gland for a young-at-heel (phase 3). The relationship between suckling behaviour and milk secretion, and the ability of adjacent glands to function independently, suggests that milk secretion is controlled locally, within each mammary gland, by a mechanism sensitive to frequency and completeness of milk removal. To determine if tammar milk contains a factor able to control milk secretion, milk fractions have been screened in tissue and cell culture bioassays. A 6-30 kDa fraction of phase 3 whey was found to inhibit milk constituent synthesis and secretion in vitro, and inhibitory activity was associated with two discrete fractions obtained by anion exchange chromatography, which contained protein bands migrating anomalously at 66 kDa and 63 kDa in SDS-PAGE. These bands were recognised in Western blotting by antiserum raised against a bovine autocrine inhibitor of milk secretion. By the same criteria, milk secreted in phase 2B of tammar lactation, when milk secretion is low and suckling intermittent but less vigorous than phase 3, also contained a feedback inhibitor of milk secretion. The results indicate that, as in dairy animals, marsupial milk secretion is under local control through feedback inhibition by a milk protein, and raise the possibility that autocrine feedback may influence the transition from phases of low milk secretion (phase 2A, 2B) to a high rate in the final third phase of lactation.

Galactopoietic and mammogenic effects of long-term treatment with bovine growth hormone and thrice daily milking in goats.

Starting in mid-lactation, goats were treated daily for 22 weeks with 0.15 mg recombinant bovine GH (bGH)/kg, or an equivalent volume of vehicle. One gland of each goat was milked thrice daily throughout treatment, the other twice daily. Mammary differentiation was studied in biopsy samples obtained before treatment and after 3 and 22 weeks of treatment, by determination of in-vitro synthesis rates of milk constituents and measurement of enzyme activities. Mammary growth was measured using a whole-body imaging technique (magnetic resonance imaging; MRI). bGH caused an immediate and sustained increase in milk yield of approximately 23% overall, whilst the glands milked thrice daily produced approximately 14% more than the control glands milked twice daily. The effects of the combined treatment were additive, but not synergistic. A synergistic effect of the combined treatment resulted in a significant improvement in lactation persistency. A stimulatory effect of milking frequency on mammary enzyme activities was evident only in bGH-treated goats at 3 weeks, but in both groups at 22 weeks. Synthesis rates of casein and lactose were increased at 3 weeks only by the combined treatment. Thus bGH accelerated or augmented the differentiative response to thrice daily milking. Mammary parenchyma volume, estimated by MRI, increased significantly during the first 12 weeks of bGH treatment and remained higher throughout the rest of the treatment period. Cell number was estimated from parenchyma volume and DNA concentration; this decreased significantly in the controls between weeks 1 and 22, but remained constant in the bGH group. In nine of the ten goats, parenchyma volume and cell number increased in the gland milked thrice daily relative to the control gland milked twice daily during the course of the experiment. Thus bGH stimulated growth of the mammary gland over and above that induced by the frequent milking. The absence of any detectable increase in thymidine incorporation suggests that this growth consisted of cellular hypertrophy rather than hyperplasia.

Autocrine regulation of casein turnover in goat mammary explants.

Local feedback control of milk protein secretion was investigated in goat mammary explants by measuring degradation of newly synthesized casein in the presence of a goat milk whey fraction. Reduced net synthesis of [3H]casein in the presence of the milk fraction was due, at least in part, to its degradation during secretion, suggesting that this process is under autocrine regulation.

Extracellular matrix and mouse mammary cell function: comparison of substrata in culture.

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveolus-like structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominantly in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.

Extracellular matrix and mouse mammary cell function: Comparison of substrata in culture.

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.

Effects of long-term thrice-daily milking on mammary enzyme activity, cell population and milk yield in the goat.

In two separate experiments, lactating goats were milked unilaterally thrice daily instead of twice daily for periods of 13 and 37 wk, starting at 2 to 5 wk after parturition. The other gland was milked twice daily throughout. In both experiments thrice-daily milking increased milk yield significantly. In the first experiment, after 37 wk the amounts of RNA and DNA, rate of cell proliferation and activities of several enzymes per cell were greater in the thrice-milked gland (P less than .05). In the second experiment, after 9 or 10 d of thrice-daily milking the activities of several key mammary enzymes per cell measured in biopsy samples had increased in the thrice-milked but not the twice-milked gland. After 13 wk of thrice-daily milking, the DNA content of the glands and the activity per cell of the key enzymes was the same in both glands, showing that the synthetic capacity of the gland was being used more effectively in the treated gland. These results indicate that several mechanisms contributed to the increased milk yield induced by thrice-daily milking.

Effects of time of year and reproductive state on the proliferation and keratinisation of bovine hoof cells.

Cell proliferation and protein synthesis (keratinisation) were measured in vitro in hoof biopsy samples taken from two groups of seven heifers, the first calving in the winter and the second in the summer. Both parameters were significantly higher in summer than in winter irrespective of the heifers' reproductive state. The mean (se) measure of the rate of protein synthesis was 199 (27) dpm/microg DNA/hour in summer and 4 (1) dpm/microg DNA/hour in winter, and the equivalent values for cell proliferation (measured as DNA synthesis) were 375 (56) dpm/microg DNA/hour and 17 (4) dpm/microg DNA/hour. Changes around parturition depended on the time of the year. In the winter-calving heifers, the rates of proliferation and keratinisation increased significantly after calving from 22.3 (7.2) to 70.4 (16.6) and from 2.1 (0.7) to 12.4 (2.8) dpm/microg DNA/hour, respectively. In the summer-calving heifers, proliferation decreased from 388.2 (91.0) to 66.7 (9.6) dpm/microg DNA/hour but the rate of keratinisation did not change. Lesion scores and locomotion scores deteriorated after parturition, especially in the winter-calving group. The hooves were harder in summer than winter but their hardness was not affected by the heifers' reproductive state.

Mammary growth during lactation: implications for increasing milk yield.

Milk yield is greatly influenced by the size of the mammary secretory cell population. In rats, proliferation of cells during early lactation may account for as much as three-quarters of the increase in daily milk yield between parturition and peak lactation; the remainder is due to increased activity of existing cells. Conversely, in goats, all the initial decline in milk yield after peak lactation can be attributed to loss of secretory cells. Increased frequency of milking enhances milk yield and reduces secretory cell loss, whereas goats hemimastectomized at peak lactation undergo compensatory changes in the remaining gland, which include a complete maintenance of cell number for at least 18 wk. Cell proliferation is increased in both cases, showing that mammary growth can occur during established lactation. The significance of this observation is discussed in relation to ways in which the milk yield of dairy animals might be increased.

Milk production in concurrently pregnant and lactating goats mated out of season.

Five lactating goats which had kidded normally in March were mated during seasonal anoestrus in May, at the time of peak milk production, after ovulation had been induced using gonadotrophin-releasing hormone (Knight et al. 1988). Milk yield was unaffected by the hormone treatment, and decreased at the same rate as that of control (non-pregnant) goats for the first 8 weeks of the pregnancy. Thereafter yield declined more quickly in the test goats and just before parturition (in October) was 57% of the control value. Following parturition in the test animals, yield rose rapidly as the second lactation was established. None became 'dry' at any stage. Yield continued to decline with advancing lactation in the controls, which were mated normally in October or November and dried-off in December. During their second ('extra') lactation in the winter the test animals produced 12% less than in a normal second lactation in summertime; during the year the extra lactation meant that the test animals produced 73% more milk than the controls. In some, a second concurrent pregnancy was established during the extra lactation, with the result that three lactations were obtained in the time normally taken for two. Mammary cell number and proliferation rate were both higher in the pregnant animals than in the controls in week 23 of the first lactation.