Calcium, the cytoskeleton and calpactin (annexin II) in exocytotic secretion from adrenal chromaffin and mammary epithelial cells.
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Adrenal Medulla↗
Biomedical subjects
C J Wilde
Publications and source records attributed to C J Wilde.
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.
Animals↗
Intracellular degradation of newly synthesized casein in perfused rat mammary gland.
Degradation of newly synthesized casein in rat mammary gland perfused in situ was measured by a pulse-chase method using [3H]proline. Casein degradation during secretion was observed in the absence of prolactin, but not with prolactin present. Partial inhibition by chloroquine showed that hormone-dependent degradation occurred intracellularly by a lysosomal mechanism. The study indicates that this post-translational mechanism is a physiological regulator of net casein secretion.
Adenosine Triphosphate↗
Milk yield and mammary function in dairy cows milked four times daily.
Groups of lactating cows and heifers were milked four times daily in two diagonally opposed glands for 4 weeks, and the effects on milk yield studied relative to twice-daily milked glands as controls. Mammary enzyme activities, in vitro synthesis rates of milk constituents and histological scoring were determined in mammary biopsy samples obtained at the end of this period. These were used for assessment of mammary function. Frequent milking increased milk yield only in the treated glands, the contralateral control glands continuing to decline in yield at approximately 2%/week. There was no significant difference in response between cows and heifers; the mean increase in yield was 10.4%. The rate of decline in milk yield tended to decrease with frequent milking, to approximately 1%/week. Consequently the yield of the treated glands continued to be elevated above that of the controls for some time after reversion to overall twice daily milking. Milk protein content was increased slightly by frequent milking. Mammary enzyme activities were approximately 18% higher in the treated glands than in the controls. Synthesis rates of lactose, casein and total protein were unaffected by milking frequency, but were all lower in the gland selected for the second biopsy, reflecting the reduction in milk yield caused by the first biopsy. DNA synthesis was increased by milking frequency, as were the size and number of epithelial cells in histological sections.
Animals↗
Milk yield and mammary function in goats during and after once-daily milking.
Starting in week 5 of lactation, goats were milked unilaterally once daily for 4 1/2 weeks, then twice daily for 12 weeks and finally thrice daily for 2 weeks. The control gland was milked twice daily throughout. During less frequent milking, yield fell by 26%. There was a rapid and essentially complete recovery of milk yield on resumption of twice-daily milking, and five of the six goats subsequently responded to thrice-daily milking with a small increase in milk yield. Mammary function was assessed during each phase by determining enzyme activities and in vitro synthesis rates of lactose and casein in mammary biopsies. There was a trend towards reduced differentiation of secretory cells during infrequent milking, with very little recovery during twice-daily milking but complete recovery during thrice-daily milking. The differentiative state of the control gland remained unchanged throughout.
Animals↗
Hormonal control of protein synthesis in rat mammary explants.
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Animals↗
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.
Acetyl-CoA Carboxylase↗
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.
Animals↗
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.
Adaptation, Physiological↗
Metabolic adaptations in mammary gland during the declining phase of lactation.
Peak milk yield in the ruminant animal is attained relatively early during lactation, and this is followed by a progressive decline so that at the time of "drying off" yield may be reduced to only 50% of its maximum value. Studies in lactating goats indicate that the fall in milk yield after peak was due ultimately to a decrease in the number of secretory cells and that the remaining cells did not lose their metabolic capacity for milk synthesis to any significant degree. Manipulation of milking frequency or efficiency during declining lactation had both acute and long-term effects on milk yield and mammary cell number and activity. Rapid and reversible stimulation of yield by thrice daily or hourly milking was due to more frequent removal of a whey protein, which modulates milk secretion by negative chemical feedback on the secretory cell. Evidence is presented that the feedback inhibitor may act primarily on the secretory process rather than through inhibition of synthesis of individual milk constituents. Long-term thrice daily milking increased milk yield in the later stages of lactation by preventing, at least in part, the fall in secretory cell number after peak. Secretory cell differentiation was also susceptible to manipulation in declining lactation: inefficient milking for 24 wk after peak, i.e., by increasing the residual milk volume in the gland after milking, caused partial involution (dedifferentiation) of the existing secretory cell population. The role of local intramammary mechanisms in modulating milk yield and mammary function in declining lactation is discussed.
Animals↗
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.
Anestrus↗
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.
Animals↗
Exogenous GnRH induces ovulation in seasonally anoestrous lactating goats (Capra hircus).
A specific sheep LH radioimmunoassay was validated for the measurement of goat LH, and used to monitor luteal-phase LH episodes and the preavulatory LH surge in progestagen sponge-synchronized cycling goats. No luteal-phase LH episodes were detected during 12 h of frequent (15-min) blood sampling in 2 goats. A preovulatory LH surge was recorded in 5/5 goats, with a mean amplitude of 45.4 +/- 7.2 ng/ml and a mean time of onset of 38.4 +/- 1.2 h after removal of a progestagen-impregnated sponge. In anoestrous goats, single i.v. injections of 1000 and 2000 ng GnRH induced LH episodes with a mean amplitude of 2.04 +/- 0.11 and 3.67 +/- 0.06 ng/ml respectively, but injections of 250 or 500 ng did not consistently elevate LH concentrations. Progestagen-primed, seasonally anoestrous lactating goats were treated with repeated injections of 1500 ng GnRH (every 2 h for 52 or 78 h) in May 1985 or 1986. All 10 had kidded in March of the same year, and were consequently at peak lactation at the time of GnRH treatment. A preovulatory LH surge was detected in 9 goats with a mean time of onset of 59.5 +/- 2.9 h (1985) or 39.6 +/- 3.3 h (1986) after vaginal sponge removal. All animals displayed oestrus and ovulated, and 9 of the goats were mated: in 5 of these animals pregnancies were successfully carried to term. The results show episodic LH release in response to GnRH and indicate that ovulation can be induced in seasonally anoestrous goats, even at peak lactation, and normal pregnancies may result.
Anestrus↗
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.
Animals↗
Transferrin receptor activity in rat mammary epithelial cells.
The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.
Animals↗
The effect of goat milk fractions on synthesis of milk constituents by rabbit mammary explants and on milk yield in vivo. Evidence for autocrine control of milk secretion.
Lactose and casein synthesis by rabbit mammary explants in organ culture was inhibited when fractions of goat milk were included in the culture medium. Inhibition was dose-dependent, and readily reversed when milk fractions were removed. The pattern of effects obtained with various fractions of milk indicated that inhibition was caused by a protein of 10,000-30,000 Da, which was present in the milk serum or whey fraction. The inhibitor fraction decreased milk accumulation when injected into lactating rabbit mammary glands via the teat ducts, whereas other milk proteins had no effect. Results are discussed in terms of autocrine regulation of milk synthesis through negative feedback by milk constituents.
Animals↗
Metabolic adaptations in mouse mammary gland during a normal lactation cycle and in extended lactation.
The development and involution of mouse mammary tissue was measured in terms of changes in the cell population and the synthetic capacity of cells during the course of pregnancy, lactation and natural weaning, and after lactation was extended by introduction of foster litters of younger pups. Synthetic capacity was assessed by measuring the activities of several key enzymes closely concerned with the synthesis of milk constituents together with other constitutive enzymes. The capacity of the tissue to synthesize lactose and casein was also measured using short-term cultures of freshly prepared mammary explants. Results showed that the mammary cell population began to increase and differentiate around the time of parturition. Cell numbers increased up to day 5 of lactation, whereas cellular differentiation continued through lactation until day 15, around the time of peak milk yield. After day 15 there was a sharp drop, followed by a gradual decline in synthetic capacity and cell population, as pups were weaned. Substitution of younger pups for the natural litter at day 15 prevented the normal fall in milk secretion. In this case, mammary cell number remained at day 15 levels; cellular differentiation was also maintained to a considerable degree, although several markers of differentiation decreased during extended lactation.
Adaptation, Physiological↗
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.
Animals↗