Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and alpha-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. These results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

Interactions of insulin, corticosterone and prolactin in promoting milk-fat synthesis by mammary explants from pregnant rabbits

1. The rate of fatty acid synthesis by mammary explants from rabbits pregnant for 16 days or from rabbits pseudopregnant for 11 days was stimulated up to 15-fold by culturing for 2-4 days with prolactin. This treatment initiated the predominant synthesis of C(8:0) and C(10:0) fatty acids, which are characteristic of rabbit milk. 2. Inclusion of insulin in the culture medium increased the rate of synthesis of these medium-chain fatty acids. By contrast the inclusion of corticosterone led to the predominant synthesis of long-chain fatty acids. When explants were cultured for 2-4 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased up to 42-fold, but both medium- and long-chain fatty acids were synthesized. 3. These results show that the stimulus to mammary-gland lipogenesis and the initiation of synthesis of medium-chain fatty acids observed between days 16 and 23 of pregnancy in the rabbit can be simulated in vitro by prolactin alone. 4. When mammary explants from rabbits pregnant for 23 days were cultured for 2 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased fivefold, but there was a preferential synthesis of long-chain fatty acids. Culture with prolactin alone had little effect on the rate or pattern of fatty acids synthesized. 5. The results are compared with findings in vivo on the control of lipogenesis in the rabbit mammary gland, and are contrasted with the known effects of hormones in vitro on the mammary gland of the mid-pregnant mouse.     

Regulation of enzyme turnover during tissue differentiation. Studies on 6-phosphogluconate dehydrogenase in rabbit mammary gland in organ culture.

(1) Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 in the presence or absence of insulin, prolactin and cortisol. (2) Antiserum to 6-phosphogluconate dehydrogenase was raised in sheep and used to titrate the amount of enzyme activity present in explant extracts. Changes in enzyme activity were found to be due to corresponding changes in amount of the enzyme. The greatest increases in the amount of the enzyme were only brought about by culture of explants in the presence of hormones (insulin, prolactin and cortisol) in Medium 199 which contained glucose. (3) The increases in the amount of the enzyme were similar in explants cultured with hormones in Medium 199 which contained 1.39 mM, 5.55 mM or 55.5 mM glucose. (4) When explants were cultured with hormones in Medium 199 which contained glucose (5.55 mM) for 24 h and then cultured with hormones in Medium 199 which contained glycerol (10.9 mM), a decrease in the amount of the enzyme occurred. In contrast, the culture of explants with hormones in Medium 199 which contained glycerol (10.9 mM) for 24 h followed by transfer of the explants to medium which contained glucose (5.55 mM) resulted in an increase in the amount of the enzyme to reach values which were not different from those found in explants cultured throughout with hormones in Medium 199 which contained glucose.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants

1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.     

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland.

Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.     

Thyroid hormone regulation of prolactin binding to mouse mammary glands.

Membranes from mammary glands of mildly hypothyroid mice show a 70–85% reduction in prolactin binding while those from hyperthyroid mice bound 66% more prolactin compared to similar preparations from euthyroid animals. The prolactin binding data for mammary glands correlate well with the ability of the tissue from animals in various thyroid states to respond to prolactin $\text{in}\text{vitro}$ with increased lactose synthetase activity. Binding of prolactin to mammary membranes is enhanced when explants from mid-pregnant mice are cultured overnight in the presence of insulin, hydrocortisone and 10^?9 M L-T₃. This enhancement is not blocked by puromycin. These data suggest that thyroid hormones control the level of prolactin binding in mouse mammary tissue. This may be accomplished, at least in part, by activation of preexisting receptor molecules.     

Hormone-Stimulated Iipid Synthesis in Mammary Culture

Mammary explants from midpregnant mice were cultured for upto 96 hr with various combinations of insulin, prolactin, andcorticosterone. Labeled glucose was added to cultures at 4 hrprior to termination, and explant morphology, glucose uptake,and lipid synthesis were studied in hormone-free and hormone-containingmedia. The results show that without hormones, explants takeup glucose and synthesize lipid at minimal rates. After 48 hrthese activities appear to be primarily those of adipose tissuesince epithelial and connective tissue degenerate without hormones. Insulin increases cell number for 24 hr and maintains survivalfor 96 hr. Its stimulatory effect on lipogenesis precedes itsenhancement of glucose uptake. The addition of prolactin toinsulin-containing cultures has little effect on glucose uptakeand lipogenesis, but stimulates minimal secretion in alveolarlumina. The absence of intracellular vacuoles indicates thatthese products probably contain little lipid. Corticosteroneenhances the effects of insulin on lipid synthesis, but haslittle apparent effect on the secretory morphology of the alveoli. The three-hormone combination has no effect on glucose uptakeabove that obtained with insulin alone; however, it inducesmarked increases in Iipid synthesis as well as maximal morphologicalsecretion by 48 hr. Thus, as for other lactogenic responsesin vitro, insulin, prolactin, and corticosterone act synergisticallyto stimulate lipid synthesis in mammary explants.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

In vitro studies of the influence of prolactin on tail regeneration in the adult newtNotophthalmus viridescens

Summary In vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

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

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

Assessment of DNA methylation changes in tissue culture of Brassica napus

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rapeseed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.     

Rat renal papillary tissue explants survive and produce epithelial monolayers in culture media made hyperosmotic with sodium chloride and urea

The capacity of papillary cells to adapt to elevated osmotic concentrations is unusual among mammalian cells. This capacity was evaluated by using primary tissue culture. Viability and growth of cells in rat renal papillary tissue explants were assessed after culture in media adjusted with urea and sodium chloride to various osmotic concentrations between 300 and 1,500 mOsm/kg water. The survival of cells, including cells resembling those of the collecting ducts and the loop of Henle, was greatest in medium adjusted to 1,000 mOsm with equiosmolar amounts of the two solutes. At 1,500 mOsm only cuboidal tubular epithelium resembling collecting duct epithelial cells survived. In contrast, cells of cortical tissue survived and grew at 300 and 640 mOsm, but not at 1,000 mOsm or above. Epithelial monolayers appeared to proliferate from collecting ducts and spread over the surface of the explants as well as onto the glass surface in the culture dish. Epithelial growth of medullary tissue was most rapid at 300 mOsm and was slower at 700 and 1,000 mOsm. Monolayers did not form at 1,500 mOsm; however, epithelial overgrowth of explants did occur. Hydropenia in the donor animal did not significantly affect the viability or growth of cultured papillary tissue. Explants cultured for 5 days at 300 mOsm followed by a stepwise increase in medium osmolality to 1,100 or 1,500 mOsm and cultured for 3 more days showed low or no survival whereas explants cultured at 700 mOsm survived such increases. Explants cultured for 5 days at 1,500 mOsm survived and grew monolayers when lowered to 300 mOsm. Poor viability and no epithelial proliferation were observed in explants cultured in medium adjusted to 900 mOsm with either urea or sodium chloride alone, suggesting that a mixture of the two solutes in the extracellular space, as found in vivo, may be essential in achieving elevated osmolalities.     

Regulation of enzyme turnover during tissue differention. Studies on the effects of hormones on the turnover of fatty acid synthetase in rabbit mammary gland in organ culture.

1. Explants of mammary gland from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol. 2. Antibodies raised to fatty acid synthetase were used to measure the amount as well as the rate of synthesis and the rate of degradation of the enzyme in the explants over defined periods in organ culture. These measurements were also made after the hormones had been removed from the culture medium. The changes which occur in the activity of fatty acid synthetase are due to changes in the amount of the enzyme present. They are not due to activation or inactivation of the enzyme. 3. The rate of lipogenesis (measured from [1-14C]acetate) in the explants during culture varies independently of the amount of fatty acid synthetase both in the presence and after removal of the hormones. Hence the amount of fatty acid synthetase does not limit lipogenesis. The proportion of medium-chain fatty acids C8:0 and C10:0 (which are characteristic of rabbit milk) synthesized by the explants in the presence of hormones increases at about the same rate as the amount of fatty acid synthetase present. However, when hormones are removed from the medium the proportion of these acids synthesized declines as rapidly as the rate of lipogenesis and not as the amount of fatty acid synthetase presen. 4. The rates of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein in the explants were compared by measuring the incorporation of L-[U-14C]leucine into the protein of the explants. These rates increase by 5-fold and 3.6-fold respectively when explants are cultured with hormones, and they then reach approximately constant rates. When the hormones are removed there is a rapid fall in the rate of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein to values which are similar to those obtained with freshly prepared explanted tissue. 5. In unstimulated explants fatty acid synthetase appears to be degraded with a half-life of 15-21h. During the hormonally stimulated differentiation of the tissue the rate of degradation of the enzyme is considerably decreased or is switched off completely. After the amount of fatty acid synthetase has increased to a maximum the enzyme complex is again degraded with a half-life of 23-29h. The removal of hormones after the explants have been hormonally stimulated for different times results in an increase in the rate of degradation of fatty acid synthetase. However, this increase only occurs if degradation was previously proceeding at a considerably decreased rate. The degradation of the total particulate-free supernatant protein continues throughout the period of differentiation of the explant tissue in culture. It appears to be somewhat decreased during the period of rapid maturation of the tissue during culture.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Prolactin-induced accumulation of casein mRNA in mouse mammary explants: A selective role of glucocorticoid

The role of glucocorticoid in the prolactin-induced accumulation of casein mRNA in mammary explants from midpregnant mice has been studied after an initial 4-day incubation to allow the level of messenger to decline to undetectable levels. Subsequent culture for 3 days: 1) with insulin and glucocorticoid did not result in detectable accumulation of messenger; 2) with insulin and prolactin resulted in a very small accumulation; 3) with insulin, glucocorticoid and prolactin elicited a 20-fold greater accumulation of casein mRNA than the system with only insulin and prolactin. Therefore, although glucocorticoids are not an absolute requirement for casein gene expression in mouse mammary tissue, they are necessary for massive accumulation of casein mRNA induced by prolactin. It appears that this dependence is not a result of either mRNA stabilization or alteration in prolactin receptors. By contrast, stimulation of total epithelial RNA synthesis by prolactin does not have this glucocorticoid dependency.     

Observations on the early actions of prolactin on RNA and casein synthesis in mouse mammary gland explants

The action of prolactin on RNA synthesis in cultured mouse mammary gland explants becomes manifest when the tissues are exposed only briefly (1 h or less) to prolactin. In contrast, the action of prolactin on casein synthesis only becomes apparent when the tissues are cultured for 6 h or more with prolactin. Once the actions of prolactin on RNA and casein synthesis are initiated, however, these effects persist for hours or days, even when the tissues are subsequently cultured in the absence of prolactin.     

Has prolactin an intermedin-like activity on the integument of the Axolotl?

Skin explants of the neotenic form of Ambystoma tigrinum (Axolotl) have been kept in culture during several days. When alpha-MSH or prolactin are added to the medium, a dispersion of melanin in melanosomes is observed. The reaction is reversible in the case of prolactin when the explants are transferred in the medium without hormones. Furthermore with prolactin, expansion of melanin in dermal melanophores is a more permanent and highly reproducible phenomenon, compared with the action of alpha-MSH in the same conditions. The type of action of prolactin could be different of the one of alpha-MSH.