Identification of a nonimmunoreactive but highly bioactive form of prolactin in the mouse pituitary by gel electrophoresis.

Polyacrylamide gel electrophoresis of fresh mouse pituitary extracts in 10% acrylamide reveals three closely situated bands of protein in the area where prolactin usually migrates. The fastest migrating band constitutes only 10–30% of the total, but it is twice as active in the pigeon crop-sac bioassay as the major band and has little or no immunologic cross-reactivity against an antiserum to the major constituent. This newly recognized band may be a hitherto unrecognized molecular variant of prolactin.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.     

Iodinated bovine prolactin. Characterization and binding to mammary gland cells.

Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of 125I to give a specific activity of 0.041 muCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing 125I-prolactin from antibody. High specific activity 125I-prolactin (1 iodine atom/molecule; 70 muCi/microgram) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro 125I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

Summary The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion. This investigation was supported by Grant No. CA 05388 awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California.     

Characterization of cell-surface glycopeptides from mouse cerebral cortex that inhibit cell growth and protein synthesis.

The occurrence of multiple forms of rat prolactin with different molecular weights (size heterogeneity) was studied with anterior pituitary extracts, purified rat prolactin and 125I-labelled rat prolactin. In each case, three main forms of the hormone were detected by gel filtration on Sephadex G-100: a major one (80--90%) corresponding to monomeric prolactin (mol.wt. 22000--25000), a peak (8--20%) that could be a dimer (mol.wt. 45000--50000) and a small quantity (1--5%) of a component of much greater molecular weight. On freezing and thawing of 125I-labelled rat prolactin, there was little interconversion of monomer and 'dimer' peaks, but both were converted substantially to very high-molecular-weight material. All three peaks of 125I-labelled rat prolactin could be precipitated by anti-(rat prolactin) serum and all three gave similar patterns of radioactive peptides after digestion with chymotrypsin followed by high-voltage paper electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the monomer peak of 125I-labelled prolactin migrated as a single component of mol.wt. 22000, the very high-molecular-weight peak largely dissociated to a component running in the same position as the monomer, and the 'dimer' peak migrated partly as a component of mol.wt. 45000 and partly as a component migrating with monomeric prolactin. No treatment was found that could dissociate the 'dimer' peak completely to monomeric prolactin.     

Isolation and partial characterization of secreted mouse pituitary prolactin.

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Synthesis of two lactogenic proteins by the mouse placenta in vitro.

The culture medium of mouse placental tissues was analyzed on acrylamide gel electrophoresis to localize lactogenic substances. Placental explants from 12- or 14-day-pregnant BALB/cHe mice were organ-cultured for 6 days in leucine-free Way-mouth's medium supplemented with 3H-leucine (10 mu/Ci/ml) and insulin (0.12I.U./ml). The medium was collected every other day and subjected to acrylamide gel electrophoresis. The electrophoretic pattern of radioactive leucine incorporated into proteins was examined on stained 7% gels. Five protein bands were associated with high radioactivity. The location of lactogenic activity on acrylamide gel was when investigated by the technique of organ culture of mouse mammary tissues. Placental explants from 12- to 14-day-pregnant BALB/cHe mice were organ-cultured in Waymouth's medium supplemented with insulin for 2 days. After electrophoresis, proteins were eluted by keeping the segment of acrylamide gel in phosphate buffer, dialyzed and dissolved in tissue culture medium 199 supplemented with insulin and cortisol. Mammary tissues from 8-day-pregnant KA2 mice were cultured for 3 days in the medium containing each eluate. Mammary glands always responded to eluted proteins from two positions of 7% gel, as judged in histological sections. The data suggest the presence of two lactogenic substances in the mouse placenta.     

Prolactin binding in rat mammary gland during pregnancy and lactation.

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Lactogenic response of mouse mammary explants from different days of pregnancy to placental lactogen and pituitary prolactin

The lactogenic response of mouse mammary gland explants to human placental lactogen (hPL) and ovine pituitary prolactin (oPRL) was examined on days 10 to 18 of pregnancy by measuring 3H-amino acid incorporation into calcium-rennin precipitable casein. To determine the lactogenic response of the explants, the mean slopes of dose-response curves were calculated for each hormone treatment. Slope means of dose-response curves for oPRL and hPL did not differ from each other on any day of pregnancy examined. A triphasic pattern of response was suggested when slope means of dose-response curves for both hormones were plotted as a function of day of gestation. Peak responses were observed on days 10, 13 and 17-18. Combinations of oPRL and hPL, in ratios of oPRL:hPL = 2:1 and oPRL:hPL = 1:2, also produced a triphasic pattern of sensitivity very similar to that produced by either hormone alone. These results suggest that mouse mammary explants may be more sensitive to oPRL and hPL on days 10, 13 and 17-18 of pregnancy.     

Radioimmunoassay for ovine, caprine and bovine prolactin in plasma and tissue extracts

1. A radioimmunoassay for ovine prolactin is described based on the inhibition of the reaction between (131)I-labelled ovine prolactin and guinea-pig or rabbit antiserum to ovine prolactin. The extent of the reaction after a 4-day incubation period is determined by chromatoelectrophoresis or by adsorption of unchanged (131)I-labelled ovine prolactin on charcoal. The sensitivity is equal to 5.9ng. of prolactin/ml. of plasma with chromatoelectrophoresis, or 0.2ng. of prolactin/ml. of tissue extracts with the charcoal separation. 2. A complete cross-reaction demonstrated between ovine prolactin and caprine pituitary extracts allows the assay to be used to measure caprine prolactin. The partial cross-reactions between ovine prolactin and bovine prolactin and between ovine prolactin and bovine pituitary extract differ, and an alteration in the immunological activity of bovine prolactin during its isolation is suggested. Bovine prolactin in plasma may be measured against a bovine pituitary extract as standard. No cross-reactions were demonstrated with pituitary extracts from a number of other species. The extent of the contamination of ovine and bovine growth hormone preparations by their respective prolactins is shown. 3. Dilutions of ovine and caprine plasma inhibit the reaction between (131)I-labelled ovine prolactin and antiserum with the same characteristics as ovine prolactin. 4. The immunoreactive material in plasma fractionates on Sephadex G-200 and in sucrose density gradients as a single peak similar to that shown by freshly dissolved ovine prolactin. There is no evidence that ovine prolactin is bound to a plasma protein. 5. By suppressing prolactin secretion and assaying serial samples of plasma thereafter it is shown that the immunological activity of the surviving hormone becomes progressively altered with time. It is suggested that this alteration is usually not detected but introduces an element of uncertainty into the quantitative but not the qualitative value of the measurements obtained by reference to standard ovine prolactin.     

Characterization of a sheep pituitary-derived growth factor for rat and human mammary tumor cells

A growth factor for rat and human mammary tumor cells (MTGF-Pit) was isolated from lyophilized powders of whole sheep pituitaries by a rapid four-step procedure utilizing acetic acid extraction, heating at 93 degrees C, and sequential chromatography in 0.10 M acetic acid on sulphopropyl Sephadex and Sephadex G-50. From 10 g of pituitary powder, 8-10-mg amounts of MTGF-Pit were isolated. By 8 M urea, 0.1% SDS-12.5% polyacrylamide gel electrophoresis analysis followed by Coomassie blue staining, this preparation was shown to be one major stained band. When assayed for growth effects on cells maintained in serum-free medium, 5.1-19.2 nM MTGF-Pit half replaced the growth of MTW9/PL rat and MCF-7 and T-47D human mammary tumor cells in response to 2% to 10% serum. MTGF-Pit shows mitogenic activity toward normal human diploid fibroblasts only at concentrations in excess of 2.5 X 10(-4) M, while rat fibroblasts are unresponsive even at this high concentration. From data available, we conclude that a mitogenic activity for epithelial-type mammary cells has been isolated, and this growth factor appears to be a previously undetected acid- and heat-stable activity that is highly abundant (estimated at 0.16% or more of the total dry weight of the pituitary powder). The isolated ovine MTGF-Pit (3,900 +/- 200 daltons) does not share the molecular weight of native prolactin (24,000 daltons), "cleaved" prolactin (16,000 daltons), or growth hormone (22,000 daltons), and by all tests applied cannot be replaced with other known hormones and purified growth factors. We conclude a potent new mammary tumor cell mitogenic activity has been identified from sheep pituitaries.     

A random-walk model for retardation of interacting species during gel electrophoresis: implications for gel-shift assays.

We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the triplex is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration "cyclic capture and dissociation" (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation.     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice.

Prolactin receptors were monitored by measuring 125I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (Kd) of 0.99 . 10(-9) M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydrocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (Kd) of the receptors.     

A receptor site for prolactin in lactating mouse mammary tissues.

Prolactin iodinated by lactoperoxidase method showed immunologically, electrophoretically and biolo9gically similar properties to native prolactin and possessed enough specific radioactivity for receptor studies. 1251-prolactin was incubated with mouse mammary tissues at 8 days of lactation. Both binding and release of 1251-prolactin depended on incubation time and temperature and were maximal at 37 degrees C. Michaelis constant was estimated to be 1.4 X 10(-9) M from Lineweaver-Burk plot and to be 1.2 X 10(-9) M from id-value of the dose-response curve for displacement with native prolactin. Total number of binding sites for prolactin was 1.38 X 10(-15) mole per mg weight of tissue. Ovine prolactin, human growth hormone and human placental lactogen complete with 1251-prolactin and dose-response curves for these three hormones were all parallel. These results suggest the existence of a specific receptor site with high affinity for prolactin in lactating mouse mammary glands.     

Identification of prolactin receptors in hepatic nuclei.

Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Subcellular localization of cryptic prolactin receptors in mammary tumor cells

Primary cultures of carcinogen-induced rat mammary tumors incubated at 37 degrees C with 125I-labeled ovine prolactin (5 ng/ml) accumulate intact prolactin. A steady state is reached at 24--48 h and loss of accumulated prolactin is slow t1/2 24 h). Accumulated prolactin is rapidly released when cryptic prolactin receptors are unmasked by energy depletion, suggesting that accumulated prolactin and cryptic receptors reside in the same cellular compartment. Under normal conditions, the accumulated prolactin is released slowly and is partially degraded. Subcellular fractionation on discontinuous sucrose gradients indicates that cryptic receptors reside in vesicle fractions (p less than or equal to 1.16). After energy depletion, the unmasked receptors are in cell surface membrane fractions (p = 1.18-1.20). Prolactin accumulation within receptor-containing vesicles in mammary tumor cells may account for their increased growth sensitivity (compared with normal mammary cells) to low physiologic levels of prolactin.     

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)