Mammary Gland Explants Research Articles

Temporal sequence of prolactin actions on phospholipid biosynthesis in mouse mammary gland explants.

Studies were carried out to determine the effect of PRL on the metabolic fate of 32PO4 in cultured mammary gland explants derived from 12 to 14-day pregnant mice. Explants were initially cultured for 24-36 h with 10(-7) M cortisol and 1 microgram/ml insulin. PRL (1 microgram/ml) was then added to certain of the cultures and incubation continued for 2-24 h. Tissues were pulse labeled with 5 microCi/ml 32PO4 during the final 2 h of culture. Tissues were fractionated by the method of Bligh-Dyer. Radioactivity was determined in the organic fraction (containing phospholipids), the aqueous fraction, and an insoluble fraction containing macromolecules. In all these fractions, PRL effected a greater than 2-fold increase in radioactivity content; the onset of the PRL responses was 8-12 h after PRL exposure and PRL effected responses at concentrations of 2.5 ng/ml and above. The enhanced rate of 32P incorporation in the macromolecular fraction was found to occur in both the RNA and phosphoproteins in that fraction. As determined by TLC, PRL was also found to increase 32P incorporation into all phospholipid fractions. This was confirmed by observing that [3H]inositol and [3H]choline incorporation into their respective phospholipids was also increased by PRL; the time sequence of response was similar to that when 32PO4 incorporation was determined. The magnitude of the PRL stimulation of 32PO4 incorporation, however, was about 2-fold higher than either the [3H]inositol or [3H]choline incorporation. The magnified response when 32PO4 was employed may reflect a PRL effect on phosphate uptake into the mammary cells; this is supported by the fact that the radioactive content of the aqueous fraction was significantly elevated in the Bligh-Dyer extract. The effect of PRL on phospholipid synthesis probably reflects the initiation of the packaging process involved in the assimilation of milk products.

Possible roles of calcium and calmodulin in mammary gland differentiation in vitro.

In response to insulin, cortisol and prolactin, mammary gland explants from virgin mice will undergo differentiation, as measured by lactose synthetase activity and casein synthesis. W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulphonamide), a calmodulin inhibitor, reduced prolactin-induced differentiation by 80% while inhibiting insulin- and cortisol-induced RNA synthesis only 40%. However, tissue levels of calmodulin did not change during a 3-day culture, with or without prolactin, suggesting that the regulatory component of the calcium-calmodulin system was the intracellular calcium concentration. In support of this hypothesis, verapamil, a calcium channel blocker, also preferentially inhibited prolactin-induced differentiation and prolactin did stimulate calcium accumulation (from a control value of 3.8 +/- 1.3 (S.E.M.) pg calcium/mg wet tissue per 15 min to 61.4 +/- 7.0). On the other hand, A23187, a calcium ionophore which mimicked prolactin actions on calcium accumulation, was not able to induce differentiation in the absence of prolactin; in fact, high concentrations of either A23187 or extracellular calcium actually inhibited lactose synthetase activity, although casein synthesis was relatively unaffected. Therefore, it appears that the calcium-calmodulin system is involved in prolactin-induced differentiation of mammary gland explants but that it cannot be the only mediator of prolactin actions; i.e. it is necessary but not sufficient. There also appears to be another, separate action of calcium on casein synthesis which involves increasing the mammary epithelial sensitivity toward prolactin with respect to casein synthesis but not lactose synthetase activity.

Prolactin receptor: identification of the binding unit by affinity labelling and characterization of poly- and monoclonal antibodies.

The prolactin receptor localized in rabbit mammary gland membranes has been identified by affinity labelling using covalent cross-linking agents such as a unique protein chain of approximately 32,000 daltons. After partial purification (5,000-fold) of these receptors from mammary gland homogenate, polyclonal antibodies, which specifically and completely inhibit prolactin binding in all organs and in all species studied, were raised. These antibodies possessed prolactin-like biological activity (casein synthesis) on rabbit mammary gland explants. Monoclonal antibodies specifically directed against the binding domain of the receptor were also obtained. These antibodies were more species-specific than the polyclonal antibodies. The most potent (M110) possessed higher affinity than prolactin for the receptor and could be a very effective tool to elucidate the structure of the receptor and its immunological detection.

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action.

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

Prolactin-like actions of phospholipase C on RNA and casein synthesis in mouse mammary gland explants.

Phospholipase C stimulated the rate of [3H]-uridine incorporation into RNA in cultured mouse mammary gland explants. This effect was similar to that elicited by prolactin in that the time-course and magnitude of response were the same. In addition, the effects of prolactin and phospholipase C were non-additive when these agents were tested together. Although phospholipase C, by itself, had no effect on the rate of [3H]-leucine incorporation into casein, it was found to stimulate casein synthesis when the explants were simultaneously exposed to 0.5 mM spermidine. These observations are compatible with the idea that at least certain of the actions of prolactin in the mammary gland may be carried out via phospholipase C.

Effect of NDGA, a lipoxygenase inhibitor, on prolactin actions in mouse mammary gland explants

Nordihydroguaiaretic acid (NDGA) at a concentration of 50 μM abolished the actions of prolactin on RNA, casein and lipid biosynthesis in cultured mouse mammary gland explants. NDGA at 100 μM also reduced the magnitude of the prolactin stimulation of ornithine decarboxylase activity. Since NDGA is known to be an inhibitor of lipoxygenase activity, we would conclude that ongoing leukotriene synthesis may be essential for prolactin to express at least certain of its lactogenic actions in the mammary gland.

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro.

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.

Effect of hormones and cyclic AMP on gamma-glutamyltranspeptidase activity of rat mammary gland explants.

gamma-Glutamyl transpeptidase activity was assayed in midpregnant rat mammary gland explants at 14, 22 and 38 h, in the presence and absence of insulin, prolactin and corticosterone. With these 3 hormones the explants attained the characteristics of a secretory gland after 22 h of tissue culture, at which time the enzyme exhibited its maximal activity. The addition of dibutyryl cyclic AMP in the presence of the 3 hormones produced a significant increase in enzyme activity, which was maximal with a 1 mM concentration of the cyclic nucleotide. A similar effect was observed when theophylline or theophylline plus dibutyryl cyclic AMP were added to the culture medium.

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca 2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol- bis-(β-aminoethyl ether) or omission of Ca 2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [ 3H]thymidine into DNA. Addition of calcium to the Ca 2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca 2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-α, the enzyme involved in nuclear DNA replication.

Differential biological activities between mono- and bivalent fragments of anti-prolactin receptor antibodies.

Previous studies have established that antibodies against PRL receptors can mimic PRL effects on casein gene expression and on thymidine incorporation into DNA in the mammary gland. In the present work, bivalent F(ab')2 and monovalent Fab' fragments of the anti-PRL receptor antibodies were prepared. Both inhibited the binding of 125I-labeled PRL to rabbit mammary gland membranes. F(ab')2 as well as the unmodified antibodies were able to enhance casein synthesis and thymidine incorporation into DNA in cultured rabbit mammary gland explants. Moreover, when added to isolated membranes, both were able to induce the generation of the PRL relay which specifically stimulates caseins gene transcription in isolated mammary nuclei. In contrast, monovalent fragments were totally devoid of any of these PRL-like activities. However, bivalent and monovalent antibodies were equipotent in inducing a down-regulation of PRL receptors in mammary explants. These data indicate that the biological PRL-like activity of antibodies against PRL receptors is strictly related to their bivalent structure. This fact indicates a possible crucial role of a microaggregation of PRL receptors in the transmission of the PRL message across the membranes. In addition, these experiments reinforce the idea that internalization and down-regulation are not directly related to PRL action on casein or DNA synthesis in mammary gland.

Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy.

Explants of mammary glands from 60-day pregnant goats showed a mean fourfold increase in fatty acid synthesis from acetate when cultured with insulin+ cortisol. Epithelial cells increased their area by 60% but no secretory activity was induced. In 120-day pregnant goats, fatty acid synthesis and epithelial cell area were greater than at day 60 of pregnancy and were unaffected by hypophysectomy or by daily treatment with bromocriptine from day 60. Neither increased further on culture of mammary explants in insulin + cortisol. Ovine prolactin increased fatty acid synthesis two-fold when added to insulin + cortisol in cultures of mammary tissue from goats on day 60 of pregnancy and secretory activity was induced. On day 120 of pregnancy insulin + cortisol + prolactin sustained or slightly stimulated both fatty acid synthesis and the extensive secretion present in the tissue at the start of culture. Synthesis of medium-chain fatty acids of milk-fat was also sustained by prolactin in one goat. An atmosphere of air was found to maintain normal histological structure of the mammary gland. By contrast, in 95% oxygen, explants from goats which were 60 days pregnant showed epithelial cells filling the lumina of ducts and alveoli in 60% of explants and a poor response to prolactin.

Effects of phospholipases on ornithine decarboxylase activity in mammary gland explants from midpregnant mice.

Phospholipases A2 (PLA2) and C (PLC) stimulate ornithine decarboxylase (ODC) activity in mouse mammary gland explants preincubated with insulin and cortisol. PLC concentrations of 0.1 micrograms/ml produced responses that were close in magnitude to that of PRL, whereas PLA2 at concentrations of 5-50 micrograms/ml was 35-40% as efficacious as 1 microgram/ml PRL in stimulating ODC activity. The time courses of PLC, PLA2, and PRL actions on ODC activity were not different from one another. When PRL and PLC were tested together, a response greater than the sum of the responses of each of these agents alone was observed; PLA2 (25 micrograms/ml) when tested with PRL produced a nonadditive response. The action of PRL on ODC activity was significantly attenuated by quinacrine, an inhibitor of PLC and PLA2 activities. These results suggest that PRL, PLA2, and PLC stimulate ODC activity via similar mechanisms in the mammary gland and make tenable the idea that the action of PRL on ODC activity may be carried out via an action of PRL on PL activity.

Extracellular calcium ion concentration required for prolactin to express its actions on casein, ribonucleic acid, and lipid biosynthesis in mouse mammary gland explants.

The depletion of extracellular calcium ions by EGTA abolishes the PRL stimulation of [14C]acetate incorporation into lipids in mouse mammary gland explants. This abolition is reversed by the addition of calcium, but not magnesium. The chelation of calcium ions was similarly shown in earlier studies to abolish PRL's actions on RNA and casein synthesis. When the actions of PRL on lipid, RNA, and casein biosynthesis were tested when the extracellular calcium ion concentration was varied, it was observed that 5-10 microM calcium was required for PRL to manifest its actions. The calcium ionophore A23187 was unable, however, to mimic the PRL stimulation of lipid, RNA, or casein synthesis. Trifluoperazine, an inhibitor of calmodulin action had no effect on the PRL-induced synthesis of lipids, RNA, or casein, except at pharmacological levels.

Early Action of Prolactin on Ornithine Decarboxylase Activity Is Not Essential for the Subsequent Actions of Prolactin on Casein and Lipid Biosynthesis

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

Characteristics of the action of prolactin on [3H]-thymidine incorporation into DNA in mammary gland explants from virgin mice.

The prolactin stimulation of the rate of [3H]-thymidine incorporation into DNA in mammary gland explants from virgin C3H mice was studied. The onset of this effect occurred between one and two days after adding prolactin to the culture medium. Prolactin effected an enhanced rate of [3H]-thymidine incorporation at all concentrations from 10 ng/ml to 10 micrograms/ml. The response is essentially an "all or none" phenomenon since the effect at 10 ng/ml was not different from that at 10 micrograms/ml. Hydrocortisone was not essential from the prolactin response, but it did significantly increase the basal rate of [3H]-thymidine incorporation. Both quinacrine (an inhibitor of phospholipase A2 activity) and indomethacin (an inhibitor of prostaglandin biosynthesis) abolished the action of prolactin on [3H]-thymidine incorporation into DNA.

Effects of transglutaminase or phospholipase A 2 inhibitors on down-regulation of prolactin receptors and stimulation of casein and DNA synthesis in mammary gland explants

A number of compounds have been found to inhibit the internalization of α 2-macroglobulin and/or epidermal growth factor into fibroblasts. Using the same inhibitor we tried to block the down-regulation of prolactin receptors, supposed to mirror internalization of PRL receptors, in order to investigate the possible role of internalization in the mechanism of prolactin action. In rabbit mammary cells it appeared that bacitracin and ethylamine completely block prolactin receptor down-regulation, but dansylcadaverine, the most potent inhibitor of transglutaminase, was without effect. The blockage of phospholipase A; by chlorpromazine or bromophenacyi bromide (BPB) was without effect on the down-regulation of prolactin receptor. Subcellular distribution of [ 125I]oPRL has been studied after incubation with or without these various inhibitors after fractionation of mammary gland homogenate on sucrose gradient. Any of these compounds was able to increase the labelling of prolactin receptors located in plasma membrane fractions, suggesting that the rate of internalization of prolactin was not modified. In addition, none of these compounds inhibited the stimulation of β-casein and DNA synthesis by prolactin. These results suggest that both transglutaminase and phospholipase A 2 are not involved in the mechanism of prolactin-induced down-regulation of prolactin receptors, although bacitracin and ethylamine are able to block this phenomenon probably by different mechanism. In all cases, inductions of mitogenesis and β-casein synthesis by prolactin in the rabbit mammary cells were not modified by the various compounds utilized. We conclude that neither transglutaminase nor phospholipase A 2 are involved in the internalization of prolactin-receptor complexes, although bacitracin, ethylamine and quinacrine are able to block the down regulation of prolactin receptors by other means.

Effects of cyclic nucleotides on lipid biosynthesis in mouse mammary gland explants.

The effects of dibutyryl cAMP, 1-methyl-3-isobutyl xanthine (MIX), cGMP, dibutyryl cGMP, and 8-bromo cGMP on the rate of lipid synthesis in mouse mammary gland explants were studied. Dibutyryl cAMP at 10(-4) M selectively inhibited the effect of prolactin on the rate of [14C]acetate incorporation into lipids. At 10(-3) M, dB-cAMP inhibited the effects of insulin, insulin plus cortisol, and prolactin. The phosphodiesterase inhibitor, MIX, inhibited both basal and prolactin-stimulated incorporation rates in a concentration-dependent fashion. These data suggest an inhibitory role for cAMP in the regulation of lipogenesis in the mammary gland. Cyclic GMP, db-cGMP, and 8-bromo cGMP were all without effect on either basal or prolactin-stimulated incorporation rates. Therefore, it appears that cGMP, by itself, is not involved in the regulation of lipogenesis in the mammary gland.

Persistent alterations in hormonal sensitivities of mammary glands from parous mice.

Mammary gland explants from virgin mice required supraphysiological concentrations of insulin, cortisol, and PRL for the induction of casein synthesis and lactose synthetase activity; dose-response curves for any one hormone were identical for each parameter of differentiation. Tissue from midpregnant mice was 5-7 times more sensitive to hormonal induction, responding to near-physiological concentrations of insulin, cortisol, and PRL; again, both casein synthesis and lactose synthetase activity required the same amounts of any individual hormone. Induction of lactose synthetase activity in glands from nonlactating nonpregnant parous mice required as little hormone as tissue from pregnant animals; however, induction of casein synthesis required a higher concentration of hormones, as was observed with glands from virgins, leading to a discrepancy between the optimal hormonal concentrations required for these two markers of differentiation in parous mice. Similar data were found in the time courses. Induction in explants from midpregnant animals was 1-2 days faster than that in virgins; in parous mice, induction of lactose synthetase activity was as rapid as in midpregnant mice, but induction of casein synthesis was slower and shifted toward that for the tissue from virgins. These data suggest that those mechanisms responsible for the rapid induction and increased hormonal sensitivity of the mammary gland during pregnancy are still operative after involution of the gland with respect to at least one parameter of differentiation.

Effects of cyclic nucleotides on ornithine decarboxylase activity in mammary gland explants from mid-pregnant mice

Dibutyryl cAMP and prolactin stimulated ornithine decarboxylase activity inmouse mammary gland explants which had been preincubated with insulin and cortisol for 1 day; maximally stimulatory concentrations of dibutyryl cAMP and prolactin produced a response which was greater than the sum of the responses of prolactin and dibutyryl cAMP when tested alone. 8-Bromo-cGMP inhibited ornithine decarboxylase activity whereas other derivatives of cyclic nucleotides were without effect. Cortisol concentrations were found to be important for optimizing the dibutyryl cAMP and prolactin responses. Optimal prolactin responses were obtained with cortisol concentrations greater than 10 −7 M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10 −7 M. Despite the differing optimal cortisol concentrations for the prolactin and dibutyryl cAMP responses, it is concluded that prolactin and dibutyryl cAMP probably stimulate ornithine decarboxylase activity in the mammary gland via the same mechanism.

Effects of prostaglandins on the prolactin stimulation of lipid biosynthesis in mouse mammary gland explants

The effects of prostaglandins E 1 and E 2, indomethacin, arachidonic acid, and 8,11,14-eicosatrienoic acid on the rate of ( 14C)-acetate incorporation into lipids in mouse mammary gland explants were studied. Prostaglandins El and E 2, as well as their precursors 8,11,14-eicosatrienoic acid and arachidonic acid, inhibited the rate of ( 14C)-acetate incorporation into lipids, possibly through increased cAMP levels or through end product inhibition of acetyl CoA carboxylase and fatty acid synthetase by long chain fatty acids. Indomethacin at concentrations of 50 ug/ml and above significantly reduced the basal rate of 14C-acetate incorporation into lipids, but it did not abolish the prolactin response. Since the prostaglandins, at low concentrations, have prolactin-like actions on RNA synthesis and ornithine decarboxylase activity, and since indomethacin attenuates the actions of prolactin on RNA synthesis, casein synthesis, and ornithine decarboxylase activity, it seems apparent that all of the metabolic actions of prolactin in the mammary gland may not occur via the same primary mechanism.