Influence of microenvironment on mammary epithelial cell survival in primary culture.

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [(32)P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA laddering indicative of apoptosis. A transient increase in laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors.

Autocrine regulation of protein secretion in mouse mammary epithelial cells.

Milk secretion is under autocrine control by an inhibitory milk protein, named FIL (feedback inhibitor of lactation). Lactating mammary acini and epithelial cells cultured on reconstituted basement membrane (EHS matrix) with lactogenic hormones were used to study the characteristics of autocrine inhibition. FIL inhibited milk protein secretion in lactating acini, but not in epithelial cells on EHS matrix. The latter's insensitivity to FIL was due to formation of multicellular structures termed mammospheres, in which cell surrounded a central luminal space. Cell polarization, and the formation of tight intercellular junctions prevented FIL access to the apical cell surface, which faced the mammosphere lumina. When apical access was permitted either by incomplete mammosphere formation or EGTA treatment, FIL inhibited mammosphere protein secretion to the same extent as in lactating acini. The study shows that autocrine inhibition by FIL occurs specifically through interaction with the apical surface of the mammary epithelial cell, and suggests the presence of a FIL receptor on this, but not the basolateral cell membrane.

Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.

Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.

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.

Regulation of mouse mammary cell differentiation by extracellular milk proteins.

Differentiation of mammary epithelial cells prepared from pregnant mice was stimulated by culture on floating collagen gels in the presence of lactogenic hormones. Differentiation was measured by the induction of casein synthesis and fatty acid synthase activity, both of which increased by up to 15-fold during 12 days of culture. Addition of a 10-30 kDa fraction of goat whey proteins to culture medium from day 3 inhibited casein synthesis by greater than 20% compared with control cultures on days 4-12, and prevented further induction of fatty acid synthetase activity. Inhibition of secretory cell differentiation by this fraction, which is known to inhibit milk synthesis acutely in explant culture and lactating goats, suggests that similar autocrine mechanisms may mediate the sequential local effects on milk yield and cell differentiation elicited by manipulation of milking frequency or efficiency.

Metabolic adaptations in goat mammary tissue during long-term incomplete milking.

Lactating goats were milked incompletely in one gland and normally in the other gland twice daily for 24 weeks. Yields of the incompletely milked glands were adjusted so that a calculated 100 ml milk remained in the gland at the end of milking. On average this residual volume was greater by 7.6% of the total milk volume than that left after normal milking and was 25.9% greater than the capacity of the alveoli. After 24 weeks, the weekly milk yield of the incompletely milked gland was 23.8% lower than that of the other gland (P less than 0.001) when expressed in relation to cell number. The metabolic capacity of secretory cells in the two glands was measured in serial tissue biopsies. Two weeks of incomplete milking had no effect on enzyme activities or metabolic fluxes in the incompletely milked gland, but after 24 weeks the activities of several key enzymes were significantly lower in this gland. This was associated with a reduced rate of synthesis of total protein and casein in explants freshly prepared from the tissue. The rate of lactose synthesis also tended to be lower in explants from the incompletely milked gland. Secretory cell number in the two glands was similar at the end of incomplete milking. The data indicate that incomplete milking over a long period caused partial secretory cellular involution via a local chemical feedback mechanism in the gland.

Effect of ionic composition of milk on transepithelial potential in the goat mammary gland.

1. A method is described for altering the ionic composition of the alveolar (residual) milk in the goat mammary gland. Transepithelial (blood-milk) potential difference (PD) was recorded before and after composition was changed. 2. When [Na+] and [K+] were varied while [Na+ + K+] and [Cl-] were maintained constant, there were no significant changes in PD. 3. When [Na+] was varied, with [K+] and [Cl-] constant, there was a linear relation between PD and log [Na+], with a change of 18 mV per tenfold change in concentration. 4. When [K+] was varied, with [Na+] and [Cl-] constant, there was a linear relation between PD and log [K+], with a change of 24 mV per tenfold change. 5. In view of the lack of discrimination between Na+ and K+ in the first experiment, the sum of [Na+] and [K+] was considered. In the experiments in which [Na+] or [K+] were varied, there was a change of 37 and 38 mV, respectively, per tenfold change in [Na+ + K+]. The quantitative effects on PD of changing [Na+] or [K+] when expressed as changes in [Na+ + K+] were not significantly different. 6. When [Cl-] was varied, with [Na+] and [K+] constant, there was a linear relation between PD and log [Cl-], with a change of 22 mV per tenfold change. 7. The results are discussed in relation to the types of ion channel that might be present in the apical membrane of the secretory cell and in relation to hypotheses on the mechanisms of ion transport in the mammary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Feed-back inhibition of milk secretion: the effect of a fraction of goat milk on milk yield and composition.

A milk fraction containing whey proteins of 10-30 kDa was injected into one mammary gland of lactating goats via the teat canal. This fraction produced a temporary dose-dependent reduction in milk yield in the treated gland; the milk yield of the other gland, which received an equal volume of carrier solution, was not affected. Injection of a second fraction, containing whey proteins of greater than 30 kDa, affected milk secretion only at high doses, and this effect was not wholly specific to the treated gland. The 10-30 kDa fraction and the greater than 30 kDa fraction produced similar transient changes in the concentrations of several ions and lactose in milk of the treated gland, but not in that of the untreated gland. These data indicate that a milk constituent present in the 10-30 kDa whey inhibits milk secretion in a temporary and reversible manner. The results are discussed in relation to regulation of milk secretion through local feedback inhibition.

Distribution of milk in the goat mammary gland and its relation to the rate and control of milk secretion.

Changes with time in the distribution of milk between different storage regions of the mammary gland were studied in goats. Milk was allowed to drain from the gland through a catheter (catheter milk); the gland was then milked by hand, before (hand milk) and after the administration of oxytocin (alveolar or residual milk). The hand-milk fraction was of negligible volume. There was an essentially linear increase in the catheter-plus hand-milk fraction from 0 to 16 h. The alveolar fraction increased up to 6 h, but thereafter remained constant to 16 h. There was considerable but consistent variation between animals in the alveolar fraction as a proportion of total milk volume. The proportion of residual milk was inversely related to milk secretory rate per unit weight of mammary tissue. The hourly administration of oxytocin without milking had no apparent effect on the distribution of milk in the gland 1 h later.

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.

The effects of long-term thrice-daily milking on milk secretion in the goat: evidence for mammary growth.

Goats were milked twice daily for 2-4 weeks after parturition. For the remaining 9 months of lactation, the right mammary gland was milked thrice daily while the left gland was milked twice daily. In the first and second weeks of thrice-daily milking the milk yield of the right gland increased relative to the left gland. Subsequently there was only a slight increase in the difference in milk yields between the glands, which reached a maximum after 10 weeks. Total yield of the right gland for the period of thrice-daily milking was significantly higher, by 30%, than that of the left gland. The peak yield of the right gland was significantly greater, by 29%, than that of the left gland. There was no significant difference between the rates of decline of yield (persistency) from peak lactation. There were no significant differences in the composition of the milk from the two glands with respect to Na+, K+, Cl-, lactose, protein, citrate, fat or calcium. At 40 weeks of lactation the weight of the right gland was significantly higher than that of the left gland and the weight of the parenchyma of the right gland was also significantly higher, by a similar percentage. At this stage of lactation, milk yield of the right gland was 43% higher than the left gland, while the weight of tissue was 34% higher. Thus over three-quarters of the difference in yield was accounted for by the greater gland size, and the remainder by greater milk secretion per unit weight of tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic nucleotides in goats' milk: changes with physiological state.

Concentrations of cAMP and cGMP were measured in mammary secretions of goats. [cAMP] and [cGMP] increased during late pregnancy. At the onset of copious milk secretion, [cGMP] decreased while [cAMP] increased further. After the cessation of milking in late lactation, [cGMP] increased and [cAMP] tended to decrease. During withdrawal of food for 48 hr, the rate of milk secretion fell, [cGMP] increased and [cAMP] decreased; the changes were reversed on re-feeding. During temporary inhibition of milk secretion by intramammary treatment with colchicine, [cGMP] increased while [cAMP] decreased. It is concluded that cyclic nucleotide concentrations in milk change with physiological state.

Effects of frequent milking on milk secretion and mammary blood flow in the goat.

When goats were milked every hour for 8 h without the use of exogenous oxytocin, milk ejection each hour appeared to be more complete than had previously been reported. Moreover, the rate of milk secretion increased by 15% over the period of hourly milking. There were no major changes in the composition of the aqueous phase of milk. Mammary blood flow was not apparently changed when goats were milked hourly: there was no significant effect of time of day (between 09.00 and 17.00 h) on blood flow. These results are discussed in relation to the mechanism by which frequent milking increases the rate of milk secretion.

The effects of milking thrice instead of twice daily on milk secretion in the goat.

Milk yields of goats milked twice daily at 16/8 h intervals or 12/12 h intervals were equal. In goats normally milked twice daily at 16/8 h intervals, one mammary gland was milked thrice daily for one week while the other continued to be milked twice daily. This was done at 3, 7, 16, 24 and 33 weeks of lactation. Feeding level was maintained during these experiments. At every stage of lactation, thrice-daily milking increased milk yield (by about 8%). Stage of lactation had no effect on the size of the response. Yields of each gland increased by as much in an experiment involving thrice-daily milking both glands. Milk yield increased as soon as thrice-daily milking was started, and decreased towards normal values as soon as twice-daily milking was resumed. Milk composition during thrice-daily milking was unaffected with respect to concentrations of Na+, K+, Cl-, lactose, protein, fat and citrate. There was no change in milk yield of the gland which continued to be milked twice daily. The stimulatory effect was entirely local to the gland receiving the extra milking. Milk secretion rate during the 16 h period which included the extra milking was significantly higher during thrice-daily than during twice-daily milking; secretion rate during the rest of the day was unaffected. There were significant diurnal variations in secretion rate during thrice-daily milking such that the secretion rate during the 23.00-07.00 h period was highest and the secretion rate during the 07.00-15.00 h period was the lowest.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of decreased feed intake on the response of milk secretion to frequent milking in goats.

During declining lactation in goats, dietary intake was decreased and the animals switched from positive to negative energy balance. Milk yield and empty udder volume decreased. Goats on the reduced diet were milked hourly with the aid of oxytocin for 8 h. No stimulatory effect of hourly milking was observed. The results are discussed in relation to the factors which limit the rate of milk secretion in the declining phase of lactation.

Effects of frequent milking on milk secretion during lactation in the goat: relation to factors which limit the rate of secretion.

Goats were milked hourly with the aid of oxytocin at different stages of lactation. Udder volume and milk yield were also measured. The marked variation between goats in the time after parturition at which peak milk yield is attained and in the rate of decline after peak is illustrated. Hourly milking had a stimulatory effect on the rate of milk secretion in early lactation (before peak) and in declining lactation (after peak), in both cases at previous milk yields of 1.1 - 1.48 g/ml udder volume . d. There was no stimulatory effect of hourly milking on milk yield at or near peak lactation (yield before the experiment greater than 1.48 g/ml volume . d) or in late lactation (less than 1.1 g/ml . d). The responses of milk yield to hourly milking are discussed in relation to the factors which limit the rate of secretion. In particular, it is concluded that a stimulatory response indicates that before the experiment the rate of secretion could not have been limited directly by the arterial supply of one or more substrates for milk synthesis. It is stressed that the results were obtained under one dietary regime only.

Blood flow and catecholamine concentration in bovine and caprine skin during thermal sweating.

1. Blood flow and the concentrations of noradrenaline, adrenaline and dopamine were determined in the skins of cattle and goats, before, at the onset of and 3 hr after commencement of sweating induced by heat exposure (40 degrees C). 2. The onset of sweating in both cattle and goats was associated with a rise in cutaneous blood flow, which was thus independent of sweat pattern. Cutaneous blood flow was also higher at 40 degrees C than at 15 degrees C. 3. The predominant catecholamine in the skin of both species was dopamine, which in the goat increased in concentration in the warm environment. 4. There was no clear evidence of a change in the amount of any of the cutaneous catecholamines during exposure to 40 degrees C, although there was a consistent tendency for the concentrations of adrenaline in the calf and noradrenaline in the goat, to fall during the onset of sweating.