The purification and characterization of rabbit placental lactogen.

Rabbit placental lactogen, a polypeptide hormone functionally related to the growth hormone/prolactin family, was isolated from placenta by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography on DEAE-and CM-cellulose. The hormone was purified to more than 90% homogeneity, as determined by end-group analysis. On disc gel electrophoresis at pH9.0 it migrates as a pair of closely spaced bands with mobilities of 0.489 (minor band) and 0.511 (major band), and its isoelectric point is 6.1. Its mol.wt. is 20600, as determined by sedimentation--equilibrium centrifugation, and 24200, as estimated by gel electrophoresis in sodium dodecyl sulphate. Its amino acid composition resembles that of rabbit growth hormone and rat prolactin, except for a lower glutamic acid and leucine content. Like the prolactins, rabbit placental lactogen has two tryptophan and six cysteine residues, and its N-terminus, valine, is identical with that for human placental lactogen. By radioimmunoassay, it does not cross-react with antisera to either rat growth hormone or rat prolactin; in addition, it does not cross-react with antisera to bovine placental lactogen by double immunodiffusion. The similarity of the biochemical characteristics of rabbit placental lactogen to the other non-primate placental lactogens lends further support to the hypothesis that these molecules occupy a more central position in the growth hormone/prolactin "tree" than do their primate counterparts.     

Pre-parturitional changes in serum prolactin, placental lactogen, growth hormone, progesterone, and corticosterone in the C3H/HeN mouse

Pre-parturitional changes in serum prolactin, placental lactogen, growth hormone, progesterone, and corticosterone in the C3H/HeN mouse are described. Serum prolactin concentrations display an apparent biphasic pre-parturitional increase. Both serum placental lactogen and growth hormone concentrations are elevated during the second half of pregnancy. Serum placental lactogen concentrations remain elevated until parturition, whereas serum growth hormone concentrations decline on the last two days of pregnancy. Serum progesterone and corticosterone concentrations are elevated during the latter half of pregnancy and decline on the day preceding parturition.     

Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

Solution structure of human prolactin

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.     

Effect of estrogen and placental lactogen on lactogenesis in pregnant rats

The removal of the corpora lutea or ovariectomy on Day 18 of pregnancy induced a rise in serum prolactin 24 h after surgery with a rapid decline to control values 4 h after the surge, only in the ovariectomized group. When hysterectomy was performed in addition to luteectomy or ovariectomy a similar rise in prolactin was obtained. Lactose synthetase activity in mammary tissue was significantly higher in the luteectomized and luteectohysterectomized rats when compared with ovariectomized, ovariohysterectomized rats and the sham-operated group. Estrogen treatment 12 h after ovariectomy increased serum prolactin and lactose synthetase activity to values similar to those measured in luteectomized rats, but this increase was significantly greater when compared with the ovariectomized-nontreated group. Treatment with Tamoxifen did not decrease serum prolactin in the luteectomized rats but lactose synthetase was reduced to values similar to that obtained in ovariectomized rats. Treatment with 2 bromo-alpha-ergocryptine-mesylate (CB-154) prevented the rise in serum prolactin in the ovariectomized, luteectomized and luteectohysterectomized groups, but lactose synthetase activity was lowered to control values (sham-operated rats) only in the luteectohysterectomized rats. According to these findings, rat placental lactogen in the absence of prolactin and progesterone induces lactose synthesis. Estrogen facilitates prolactin but not placental lactogen action on lactose synthetase activity.     

Increase of serum somatomedin C in hyperthyroid patients with pregnancy.

In thyrotoxic women with pregnancy, serum somatomedin C (SmC) concentration was markedly elevated (mean +/- SD 13.57 +/- 4.66 U/ml) compared to thyrotoxic women without pregnancy (1.24 +/- 1.09 U/ml), non-pregnant euthyroid women previously treated for hyperthyroidism (0.87 +/- 0.30 U/ml), normal subjects with pregnancy (6.08 +/- 3.36 U/ml) and pregnant euthyroid women previously treated for hyperthyroidism (5.98 +/- 1.52 U/ml). Since SmC/growth hormone ratio was significantly more in thyrotoxic pregnant women than in normal pregnant women and euthyroid pregnant women previously treated for hyperthyroidism, and since human placental lactogen (HPL), human chorionic gonadotropin (HCG) and prolactin (PRL) do not crossreact with SmC antibody, it is suggested that excess thyroid hormone during pregnancy results in excessive hepatic somatomedin C production.     

Dimeric ("big") human placental lactogen. Immunological and biological activity.

Dimeric ("big") human placental lactogen has been isolated in near homogeneous form from placental tissue. It consists of a disulfide-linked (stable) form and a noncovalently associated (unstable) form of the native hormone. The two forms were separated by exposure to denaturing conditions and resolution by gel exclusion chromatography. Both forms retained immunological activity, ability to bind mammary membranes, and ability to induce mammary N-acetyllactosamine synthetase in vitro. On a molar basis, stable dimeric placental lactogen was more active than placental lactogen in the radioimmunoassay indicating that the immunological determinants on both monomeric units could bind to antibody. On a molar basis, stable dimeric placental lactogen was equally active with monomeric placental lactogen in competing for mammary gland membrane binding sites, indicating that only one active site in the molecule could interact with the membrane at a time. Stable dimeric placental lactogen was also active in an in vitro bioassay using the induction of N-acetyllactosamine synthetase. It is concluded that dimer formation does not alter the biologically active portion of the placental lactogen molecule. Since the carboxyl-terminal region (residues 182-191) is involved in the interchain disulfide bonds of dimeric placental lactogen, this portion of the molecule is probably not necessary for its biological activity.     

Adipose tissue metabolism during early pregnancy in the rat: temporal relationships of changes in the metabolic activity, number of insulin receptors, and serum hormone concentrations

The temporal relationships between changes in rates of fatty acid and acylglycerol glycerol synthesis; the activity of lipoprotein lipase of parametrial adipocytes and their capacity to bind insulin; and the serum concentrations of insulin, progesterone, prolactin, and total lactogenic activity have been examined in rats during the first 15 days of pregnancy. The rate of fatty acid and acylglycerol synthesis showed a transient increase at Days 9 and 12 of pregnancy, whereas there was no change in the activity of lipoprotein lipase activity except for a fall between Days 12 and 15 of pregnancy. The capacity of adipocytes to bind insulin was increased by Day 6 of pregnancy and remained elevated until at least Day 15; no changes in the affinity for insulin were observed. Serum progesterone, insulin, and total lactogenic activity were elevated by Days 3, 9, and 12 of pregnancy, respectively. The results show that progesterone but not placental lactogen could be responsible for the rise in the insulin-binding capacity of rat adipocytes during pregnancy, whereas the fall in lipogenic capacity at about Day 12 of pregnancy coincides with the rise in serum placental lactogen.     

Changes in the lactogenic and stress hormone contents in the blood of pregnant women

The concentrations of prolactin, growth hormone, thyroid-stimulating hormone, insulin, placental lactogen, cortisol, adrenaline, and noradrenaline in the blood plasma of pregnant women were determined by spectrofluorometric and radioimmunological methods. It was shown that the levels of these hormones increased during pregnancy. It is concluded that the adaptive reserves of pregnant women must be increased for the normal function of the mammary glands after childbirth.     

A mutant lactogenic hormone binds, but does not activate, the prolactin receptor

We have generated mutations in mouse placental lactogen II, a hormone in the PRL/GH family that binds to the PRL receptor, to investigate the role of the conserved cysteine residues in hormone function. Disruption of the small C-terminal disulfide loop did not significantly alter hormone activity. Substitution of serine for cysteine-51, which prevents formation of the large disulfide loop, results in a protein equivalent to placental lactogen II in receptor-binding activity; however, this mutant protein is not mitogenic in an assay for lactogenic hormones. These results indicate that PRL receptor occupancy and activation are distinct events.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

Inhibition of nocturnal prolactin surges in the pregnant rat by incubation medium containing placental lactogen

Rats hysterectomized on Day 7 or 8 of pregnancy continued to have nocturnal prolactin surges 1 day later. Conditioned medium obtained from incubation of Day 11 placentas infused via the jugular vein completely blocked this nocturnal surge, indicating a negative feedback of placental secretions on prolactin. Infusion of an ultrafiltrate of the conditioned medium which only contained molecules with Mr above 10,000 also blocked the prolactin surge. Next, it was determined whether this feedback of placental secretions on prolactin may work by way of hypothalamic dopamine. Levels of dopamine in hypophysial stalk blood from pregnant rats on Day 12, a time when secretion of placental lactogen is high, were not different from those in rats in which placental lactogen was absent. It is concluded that termination of prolactin surges at midpregnancy may be due to feedback of placental secretions, possibly placental lactogen, on the hypothalamus and/or pituitary. However, these experiments do not support the hypothesis that this inhibition is mediated by alteration in hypothalamic dopamine secretion.     

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.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Gamma-glutamyltransferase activity in mammary gland of pregnant rats and its regulation by ovarian hormones, prolactin and placental lactogen.

Ovariectomy and ovariectomy plus hysterectomy on day 18 of pregnancy increased gamma-glutamyltransferase activity in the mammary gland. The withdrawal of progesterone and the subsequent release of prolactin are responsible for the rise in enzyme activity. Rat placental lactogen in the absence of prolactin and progesterone is able to induce gamma-glutamyltransferase activity.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

Maternal hormone levels during Na 872 infusion in late pregnancy

In order to assess the effects of Na 872 infusion on maternal endocrinology during late pregnancy, we measured serum estradiol, estriol, progesterone, human placental lactogen, follicle simulating hormone, prolactin, cyclic adenosine monophosphate, cortisol and insulin before, during and after Na 872 infusion (bromhexine 2 mg/min for one hour) in ten mothers. As compared to six control mothers, no changes could be observed. This suggests that the claimed maturing effect of Na 872 on the fetal lungs is not mediated by the hormonal changes measurable in maternal blood.     

Hormonal control of Luteal 20alpha-Hydroxy steroid dehydrogenase and delta5-3beta-hydroxy steroid dehydrogenase during luteolysis in the pregnant rat.

Treatment of pregnant rats with human chorionic gonadotrophin, luteotrophin (luteinizing hormone), luteotrophin-releasing hormone, prostaglandin F2alpha, aminoglutethimide, or by foetoplacental removal or hysterectomy achieved a common multiple-response pattern, namely increased activity of luteal 20alpha-hydroxy steroid dehydrogenase with decreased activity of delta5-3beta-hydroxy steriod dehydrogenase and release of delta4-3-oxo steroids in vitro. 2. Similar effects of foetoplacental removal are noted in pregnant mice. 3. Gonadotrophin induced lower activities of 20alpha-hydroxy steroid dehydrogenase, except at the very end of pregnancy, and partly inhibited the induction caused by foetoplacental removal. 4. The results suggest that existence of a placental factor that restrains these changes until the end of normal pregnancy, which is produced in amounts proportional to the number of placentae and is conveyed to the ovary via the blood. 5. This factor was not replaced by prolactin. 6. It is argued that neither placental lactogen nor pituitary luteotrophin participate in the induction of 20alpha-hydroxy steroid dehydrogenase at late pregnancy in the rat. 7. Aminoglutethimide induced 20alpha-hydroxy steroid dehydrogenase only in late pregnancy. This was partly reversed by progesterone, wholly reversed by progesterone plus oestrogen, and did not involve the pituitary.     

The regulation of ovine placental lactogen the role of the fetal hypothalamic-pituitary axis

The possible r?le of the fetal hypothalamic-pituitary axis in regulating the secretion of ovine placental lactogen (oPL) was investigated in chronically-catheterised ewes and fetuses in late pregnancy. Intravascular administration of agents to fetuses that significantly increased fetal prolactin concentrations (chlorpromazine 6.25 mg;thyrotrophin releasing hormone, 10 micrograms), significantly reduced fetal prolactin concentrations (bromocriptine, 0.033 mg/h), or significantly reduced fetal growth hormone (GH) concentrations (somatostatin, 2.5 micrograms/min), had no effect on maternal or fetal oPL concentrations. Mean fetal levels of prolactin or GH in late gestation could not be correlated with oPL concentrations, although fetal hypophysectomy prevented the normal prepartum fall in oPL concentrations.