Isolation, purification, and characterization of mouse placental lactogen.

Mouse placental lactogen was purified 1840-fold from BALB/c placentae from days 14-18 of gestation with an overall yield of 29%. The purification procedure included alkaline homogenization and extraction, ammonium sulfate precipitation, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on CM- and DEAE-cellulose, and gel exclusion chromatography on Sephadex G-100. On 10% alkaline polyacrylamide gels, mouse placental lactogen had an Rf of 0.19. Electrophoresis in gels containing NaDodSO4 showed a single band with a mobility corresponding to a Mr of 23,000 +/- 1000. The isoelectric point, determined by isoelectric focusing in 8 M urea/5% 2-mercaptoethanol, was 7.1. When tested in the pigeon crop sac assay, 10 micrograms of mouse placental lactogen produced stimulation comparable with that evoked by 10 micrograms of ovine prolactin. In the rabbit mammary gland radioreceptor assay, mouse placental lactogen was 150% more potent than ovine prolactin in displacing 125I-labeled ovine prolactin from rabbit mammary gland membranes. Iodinated purified mouse placental lactogen could be displaced from rabbit mammary gland membranes by mouse placental lactogen, mouse prolactin, and ovine prolactin. Ovine prolactin was 45% as avid as mouse placental lactogen is displacing 125I-labeled mouse placental lactogen from rabbit mammary gland membranes. Mouse placental lactogen did not crossreact with antisera to mouse prolactin or mouse growth hormone in a radioimmunoassay.     

Inhibition of binding of ovine placental lactogen to growth hormone and prolactin receptors by monoclonal antibodies

From a single cell fusion, five stable hybridomas secreting antiovine placental lactogen (oPL) antibodies were obtained. Three of these secrete immunoglobulin (Ig)G subclass, and the other two secrete IgM class antibodies. Ascites fluids were raised in mice for each clone and were used as the antibody component for the development of solid phase RIA. Three solid-phase RIAs were successfully established using individual IgG subclass monoclonal antibodies, but the IgM class antibodies were ineffective. In all three individual solid-phase RIAs, the binding of [125I]iodo-oPL to the immobilized antibody was inhibited by unlabeled oPL, but not by ovine pituitary PRL (oPRL), ovine GH (oGH), or ovine pituitary extract. Two of the IgG subclass antibodies were able to inhibit the binding of [125I] iodo-oPL to PRL receptors(s) and to GH receptor(s) in rabbit mammary gland and liver, respectively. One of these two IgG subclass antibodies was more effective at inhibiting the binding of oPL to PRL receptor(s) in rabbit mammary gland, whereas the other one is more effective in inhibiting the binding of oPL to GH receptor(s) in rabbit liver. These antibodies, however, could only weakly inhibit the binding of [125I]iodo-oPRL to rabbit mammary gland and were ineffective in inhibiting the binding of [125I]iodo-oGH to rabbit liver. The addition of monoclonal antibodies in both radioreceptor assay (RRA) for PRL (RRA-PRL) and for GH (RRA-GH) did not affect the parallelism of the displacement curve of oPL standard. Our results suggest that oPL might contain two distinct binding sequence(s): one responsible for the binding of oPL to PRL receptor(s) and the other responsible for the binding of oPL to GH receptor(s). These two binding sequences might overlap or be located adjacent to one another. The interaction of monoclonal antibodies with these binding sequences of oPL may block the binding of oPL with PRL and GH receptor(s). Alternatively, our studies suggest that the monoclonal antibodies do not bind to hormone receptor(s)-binding sequence(s) in oPL, but the interaction between oPL and monoclonal antibody might alter the conformational structure of the oPL which will consequently lead to a lower binding of oPL to PRL and GH receptor(s).     

Insulin-like growth factor receptor in fetal lamb liver: characterization and developmental changes

Multiplication-stimulating activity (MSA), an insulin-like growth factor (IGF) (rat IGF II), binds to extracts of many tissues from fetal lambs. We now report the presence of high concentrations of a glycoprotein receptor with a high affinity for MSA-II in microsomes prepared from fetal lamb liver. The binding of radiolabeled MSA-II is inhibited by IGF but not by insulin, human, and ovine GH, ovine PRL, ovine placental lactogen, and mouse epidermal growth factor. The relative potencies of human IGF II, human IGF I, and MSA-II in competing with [125I]MSA-II for binding to this receptor are 100:17:3.5 by weight. Binding is pH, time, and temperature dependent. Gel permeation chromatography of the Triton X-100 soluble receptor indicates a hydrodynamic radius of 6.8 nm. Specific binding increases from mid- to late gestation and is associated with changes in both the affinity and concentration of receptors. Receptor concentration increases from 7.95 +/- 3.94 pmol/mg (mean +/- SE) at 78 days gestation to 15.8 +/- 4.3 pmol/mg at 134-140 days (P less than 0.05), whereas receptor affinity decreases from 1.14 +/- 0.34 X 10(9) liter/mol to 0.63 +/- 0.14 X 10(9) liter/mol over this period (P less than 0.05). The presence of very high concentrations of an IGF receptor in fetal lamb liver suggest that this organ may be a major target for IGF action in fetal life. The increase with advancing gestational age of the concentration of IGF receptors which have preferential specificity for IGF II may function to increase the responsiveness of the fetal lamb liver to IGF II stimulation and so compensate for the decline in plasma concentrations of this growth factor which occur near birth.     

Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin

We characterized expression and activity of the bile salt transporters Na(+)/taurocholate (TC) cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep), and the expression of organic anion transporting polypeptides 1 and 2 (Oatp1 and 2) and multidrug resistance associated protein-2 (Mrp2) in pregnancy and throughout lactation in rats. The V(max) for Na(+)/TC cotransport in basolateral liver plasma membrane was increased 1.7-fold in 2 days postpartum relative to control and pregnant rats. This correlated well with an increase in Ntcp messenger RNA (mRNA) and a 2-fold increase in Ntcp protein. Ntcp mRNA remained significantly elevated until 14 days postpartum but had begun to decline by 21 days postpartum. The maximal secretory rate (nmol/min/g liver) for TC in the single pass isolated perfused liver was also increased by 10%, 31%, and 24% at 2, 14, and 21 days postpartum and correlated with increased expression of Ntcp and Bsep mRNA and protein. Infusion of ovine prolactin (oPRL) to ovariectomized rats increased expression of both Ntcp and Bsep mRNA and protein. These data indicate a coordinate increased expression of bile salt transporters postpartum and by PRL. Mrp2 mRNA was stable in pregnancy and postpartum, whereas Mrp2 protein expression decreased significantly in pregnancy, but returned to control levels postpartum. Organic anion transporting polypeptide 2 (Oatp2) mRNA was decreased in pregnancy and increased postpartum, but changes in Oatp2 protein were not significant. Oatp1 mRNA and protein were unchanged in pregnancy and postpartum.     

The glycogenic effects of placental lactogen and growth hormone in ovine fetal liver are mediated through binding to specific fetal ovine placental lactogen receptors

To examine the relative roles of placental lactogen (PL) and GH in fetal metabolism, we have examined the effects of ovine PL (oPL), ovine GH (oGH), and ovine PRL (oPRL) on glycogen metabolism in cultured ovine fetal hepatocytes and have examined the binding of these hormones to hepatic membranes from fetal and neonatal lambs. In ovine fetal hepatocytes, oPL (150 ng/ml-20 micrograms/ml) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%). oGH and oPRL also stimulated glycogen synthesis in fetal hepatocytes, but the potencies of these hormones were only 12% and 4% that of oPL. The dose-response curves of the three hormones were parallel, and their maximal effects were identical, suggesting a common mechanism of action. In hepatic membranes from fetal lambs, the maximal specific binding of [125I]oPL was 26.3% while the maximal specific binding of [125I]oGH was only 0.9-1.5%. The binding of [125I]oPL was saturable and reversible and varied with incubation time and temperature. Unlabeled oPL (1 ng/ml-5 micrograms/ml) caused a dose-dependent inhibition of the binding of [125I]oPL to fetal hepatic membranes, with half-maximal displacement of [125I]oPL by 5-7 ng unlabeled oPL/ml. oGH and oPRL caused parallel displacement of [125I]oPL, but with potencies only 2% and 0.1% that of oPL. Scatchard analysis of oPL dose-response curves indicated that the hormone bound to a single class of receptors with a dissociation constant of 1.1 X 10(-10) M. The maximal specific binding of [125I]oGH to hepatic membranes of neonatal lambs (20.1%) greatly exceeded the binding of oGH to fetal hepatic membranes. In addition, the potency of oGH in competing for [125I]oPL binding sites in neonatal liver greatly exceeded the potency of oGH in competing for [125I]oPL binding sites in fetal liver. Although the biological effects of both oPL and oGH in postnatal subprimate tissues may be mediated through binding to nonprimate GH receptors, the results of these studies suggest that the glycogenic effects of oPL in ovine fetal liver are mediated through binding to specific fetal oPL receptors. The relatively weak biological effects of oGH and oPRL in ovine fetal liver appear to be mediated through the binding of the hormones to fetal oPL receptors. The presence of specific, high affinity PL receptors in ovine fetal tissues provides a mechanism whereby oPL may function as a GH in the ovine fetus.     

Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets

The direct effects of human GH (hGH), ovine pituitary PRL (oPRL), and human chorionic somatomammotropin [placental lactogen (hPL)] on the endocrine pancreas were studied in isolated pancreatic islets maintained in tissue culture. Islets of Langerhans were isolated by collagenase treatment of pancreatic tissue obtained from adult NMRI mice and adult or newborn Wistar rats. The islets were maintained for up to 3 weeks in petri dishes containing tissue culture medium RPMI 1640 supplemented with newborn calf serum or normal human serum. The release of insulin during culture and the islet content of insulin, glucagon, and DNA after culture were determined. The DNA synthesis in the newborn rat islets was evaluated by the incorporation of [methyl-3H]thymidine into islet cell DNA. In mouse islets, 1 micrograms/ml hGH, oPRL, or hPL markedly stimulated insulin release during a 2-week culture period and caused a significant increase in the insulin content in the islets after culture. While hGH did not affect the DNA content in adult mouse islets, an increase was observed in adult rat islets after 2-3 weeks of culture. In islets isolated from 3- to 5-day-old rats cultured for 2 weeks with hGH, there was a 30-40% higher DNA content than that found without hGH. Correspondingly, a significant stimulation of the incorporation of [methyl-3H]thymidine could be demonstrated 24 h after the addition of hGH, oPRL, or hPL. hCG and porcine ACTH had no effect. In conclusion, these results indicate that GH and related hormones have a direct stimulatory effect on both the insulin production and DNA synthesis in isolated islets of Langerhans. Whether the effect is directly on the beta-cell or mediated via locally produced growth factors remains to be determined.     

Evolution of prolactin and placental lactogen receptors in ewes during pregnancy and lactation

The present paper describes a method of membrane preparation from ewe mammary gland using a sucrose cushion (1.3 M) to select smooth membranes; this results in a membrane preparation richer in PRL receptors than the microsomal preparation classically used. This method was used for the characterization and measurement of PRL and ovine placental lactogen (oPL) receptors in three organs of the ewe (mammary gland, liver, and adipose tissue). PRL receptors were measured by competition of iodinated human GH ([125I]hGH) with ovine PRL (oPRL). This hormone, which has both growth and lactogenic activities, appears to interact with PRL receptors with a higher affinity than oPRL itself and is a good probe for the determination of PRL receptors in the ewe. oPL receptors were measured by the specific binding of [125I]oPL. This hormone appears to bind exclusively to a somatogenic site in the ewe, since various GHs compete efficiently for binding, whereas oPRL is without effect. The evolution of PRL and oPL receptors was determined during pregnancy and lactation and at different periods after an estradiol and progesterone treatment, which provokes growth of the mammary gland and milk secretion. During pregnancy, PRL receptors increased in the mammary gland up to day 100. During the last trimester, receptor content remained stable, and a second increase occurred during early lactation. No additional significant changes were observed either for PRL receptors in liver or adipose tissue or for oPL receptors in any of the organs studied (mammary gland, liver, adipose tissue). Injections of large doses of estradiol and progesterone to nonpregnant ewes were able to reproduce effectively the pattern of PRL receptors observed during pregnancy, but had no effect on oPL receptor levels. These studies demonstrate the independence of PRL and PL receptor sites in the ewe and suggest a different hormonal regulation for each type of receptor.     

Rat growth hormone (GH) but not prolactin (PRL) induces both GH and PRL receptors in female rat liver

This study was designed to elucidate which hormone is responsible for the induction of GH and PRL receptors in rat liver. Intact female rats were implanted with osmotic minipumps delivering rat GH (rGH) or ovine GH (oGH) or PRL at various rates from 75 to 800 micrograms/day for 7 days, and binding of radioiodinated bovine GH or ovine PRL (oPRL) tracer was measured on liver microsomal membranes. MgCl2 treatment was used to remove bound hormones from receptors before tracer binding. Infusion of rGH resulted in a significant increase (P less than 0.001) in both GH and PRL binding, the effect being maximal (2.5- to 3-fold for both ligands) at rGH infusion rates from 150 to 400 micrograms/day. Serum rGH levels were elevated 3- to 5-fold in these animals, but somatomedin-C concentrations were not higher than in controls. MgCl2 treatment showed that GH, but not PRL, binding sites in rGH-treated animals were significantly occupied by administered hormone. Analysis of competitive binding curves indicated that receptors for both GH and PRL increased in concentration without changes in binding affinity. In contrast to the rGH effect, oGH infusion from 75 to 400 micrograms/day failed in two experiments to consistently alter either bovine GH or oPRL binding sites. This was not explained by the potency of the preparation at the somatogenic receptor; oGH was in fact more potent than rGH. The effects of rat PRL and oPRL infusion on receptor levels were also assessed. In contrast to previous reports, neither preparation caused induction of either PRL or GH binding sites. oPRL decreased PRL binding by 30-40% when infused between 200 and 400 micrograms/day, whereas rat PRL had a less consistent effect. MgCl2 stripping of membranes suggested that administered PRL preparations did not significantly occupy PRL receptors. GH receptors were unaffected in any PRL-treated group. It is concluded that in intact female rats, rGH regulates the concentration of both GH and PRL receptors. The slight down-regulation of PRL receptors resulting from PRL infusion casts further doubt on the concept that PRL induces its own hepatic receptors.     

A panel of monoclonal antibodies to ovine placental lactogen

A panel of 11 rat monoclonal antibodies (mAbs) has been raised to ovine placental lactogen (PL). By competitive enzyme-linked immunoabsorbent assay (ELISA), confirmed by two-site ELISA, the antibodies were shown to recognize six antigenic determinants on the ovine PL molecule, two of which overlap. One antigenic determinant (designated 1) was shared by other members of the prolactin/growth hormone (GH)/PL family in ruminants, humans and rodents. The binding of (125)I-labelled ovine PL to crude receptor preparations from sheep liver (somatotrophic) or rabbit mammary gland (lactogenic) was inhibited by mAbs recognizing antigenic determinants 2-6. Both types of receptor preparation were affected similarly. In the local in vivo pigeon crop sac assay, mAbs directed against determinants 3 and 6 enhanced the biological activity of ovine PL.     

Measurement of growth hormone and prolactin receptor turnover in rat liver

To study the rate of disappearance of GH and PRL receptors in the livers of rats treated with cycloheximide, a technique has been devised for multiple sampling from the liver of each anesthetized rat. In rats treated with cycloheximide (1 or 5 mg/kg, iv), binding sites for both bovine GH and ovine PRL disappeared following first order kinetics over the 2-h sampling period. The half-time for the GH receptor was 30-40 min, equivalent to a rate constant of approximately 0.02 min-1. The half-time for the PRL receptor was 40-50 min, equivalent to a rate constant of approximately 0.015 min-1. At 0.1 mg/kg cycloheximide, slower disappearance rates were seen for both receptors, and the GH receptor showed a partial recovery. Over the same period, binding sites for insulin were unaltered at any cycloheximide dose. Assuming cycloheximide acts simply to inhibit new receptor synthesis, these rates represent the turnover time for GH and PRL receptors in rat liver.     

Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected.