Light and electron microscopic studies of cellular localization of oPL with monoclonal and polyclonal antibodies.

Accurate knowledge of placental lactogen localization is fundamental to any hypothesis of its synthesis and secretion. We used locally generated monoclonal and polyclonal antibodies from three separate sources to localize ovine placental lactogen immunoreactivity on light and electron microscope Lowicryl K4M sections of ovine placentomes of 97-145 days of gestation, using immunogold techniques. All antibodies demonstrated that immunoreactivity was exclusively localized in the trophoectoderm binucleate cell Golgi body and granules and in granules in the syncytium derived from binucleate cell migration. No evidence was found to support a recent claim that monoclonal antibodies to oPL that were produced in Canada indicated a predominant localization of ovine placental lactogen to uninucleate trophectodermal cells.     

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.     

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.     

Placental growth hormone and lactogen production by perifused ovine placental explants: regulation by growth hormone-releasing hormone and glucose

The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 +/- 0.2 and 353.6 +/- 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.     

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.     

Differential effects of placental lactogen,growth hormone and prolactin on rat liver ornithine decarboxylase activity in the perinatal period

Ovine placental lactogen, (oPL), ovine growth hormone, (oGH), and ovine prolactin, (oPRL) are present in high concentrations in the fetal circulation late in gestation. To determine if these hormones stimulate the activity of ornithine decarboxylase (ODC), an enzyme widely implicated in the control of cellular growth, rat fetuses were injected $\text{in utero}$ with 100 μg of oPL, oGH, oPRL, rat growth hormone (rGH) or rat prolactin (rPRL) and ODC activity in the livers, hearts, and brains of the fetuses was measured 2, 4, and 6 hours after injection. OPL stimulated fetal liver ODC activity by 282 ± 45% (mean ± SEM) as compared to litter mates injected with buffer alone but oGH, oPRL, rGH and rPRL had no effect on fetal liver ODC activity. However, in neonatal rats 24–48 hours old all five hormones significantly increased liver ODC activity. ODC activities in the hearts and brains of the fetuses and neonates were unaffected by any of the five hormones. In other experiments 50 μg of oPL significantly stimulated fetal liver ODC activity while 250 μg of oGH were without effect. However 25 μg of oGH significantly stimulated liver ODC activity in rat pups 1–2 days after birth. These results suggest that oPL, by its stimulation of ODC activity, has somatotropic effects in the fetus and that rat liver ODC activity becomes responsive to growth hormone and prolactin in the perinatal period.     

Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb₂ cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Placental lactogen not growth hormone and prolactin regulates secretion of progesterone in vitro by the 40-45 day ovine corpus luteum of pregnancy

The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL.     

Trinucleate cells and the ultrastructural localisation of bovine placental lactogen

Summary Bovine placental lactogen activity is shown by immunogold electron microscopy to be restricted to (a) the granules and the Golgi body from which they form in the bovine fetal trophectodermal binucleate cell, and (b) granules of similar size and staining reaction in trinucleate giant cells found in the maternal uterine epithelium throughout pregnancy. These results support the hypothesis that a fetal binucleate cell forms a maternal giant cell by migration to and fusion with a uterine epithelial cell.     

The human placental protein 14 (PP14) gene is localized on chromosome 9q34

Summary PP14 protein (placental protein 14) is abundantly secreted by the human endometrium under the influence of progesterone. Human PP14 is homologous to -lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the ABO locus is consistent with the linkage described in bovines between beta-lactoglobulin and the J blood group (homologous to the human ABO group). Offprint requests to: V.C. Nguyen     

Immunolocalization of alpha and beta chains of human chorionic gonadotropin,placental lactogen and pregnancy-specific beta-glycoprotein in term placenta by a touch preparation method

Summary Touch preparations of human placenta yield cells retaining antigenic reactivity to immunoperoxidase stains for and chains of human chorionic gonadotropin, placental lactogen, and pregnancy-specific glycoprotein. This method is a rapid and simple alternative to conventional frozen and paraffin-embedded sections for detection of placental peptides.     

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.     

Immunofluorescence localization of ovine placental lactogen.

Immunoreaction to ovine placental lactogen was found in binucleate and uninucleate cells of the fetal trophoblast.     

Localization of 3H-dihydrotestosterone in the pituitary gland of the rhesus monkey

Summary The uptake and retention of radiolabelled dihydrotestosterone by the pituitary gland was examined in the rhesus monkey. Two animals were given an intravenous injection of 1.0g/kg ³H-dihydrotestosterone (DHT) alone while one monkey received both the labelled androgen and 100g/kg of unlabelled steroid. One and a half hours later, they were sacrificed. The pituitary glands were removed and processed for autoradiography and immunocytochemistry. Autoradiographic localization of DHT was discernible in the partes nervosa, intermedia and distalis, albeit the highest concentration of radiolabelled cells was noted in the pars distalis. Immunocytochemical staining with antibodies to rat PRL, human TSH and ovine LH revealed a population of steroid-concentrating cells that contained TSH and a second group that contained LH. None of the cells that reacted with the anti-PRL serum were radiolabelled.     

Ultrastructural localization of thyrotropin (TSH) in the porcine anterior pituitary

Summary Two different immunocytochemical techniques based on specific antibodies against -subunits of porcine, rat and bovine TSH were applied at the ultrastructural level to identify the TSH cells in the porcine anterior pituitary and to compare the subcellular localization of the hormone.The post-embedding method on serial ultrathin sections revealed the localization of TSH in the granules of a specific cell type, negative for the other hormones. TSH was found in polyhedral cells characterized (i) by their content of granules that were the smallest of all the cell types examined, and (ii) by their flattened or slightly dilated RER cisternae. The pre-embedding method applied to isolated cells permitted a good penetration of antisera and the maintenance of antigenicity in sites inaccessible to the post-embedding method. Thus, immunoreactivity of TSH was detected in the secretory granules, the cytoplasmic matrix and in portions of the rough endoplasmic reticulum, in association with some membranes and inside some saccular structures.     

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.     

Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb(2) cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Regulation of cloned prolactin-inducible genes in pigeon crop

Polyadenylated RNA from PRL-stimulated pigeon (Columba livia) crop was used as template to produce a cloned cDNA library in plasmids. The library was screened by differential hybridization against labeled nucleic acid populations representative of both unstimulated and PRL-stimulated crop tissue. By this method four independent clones coding for PRL-inducible mRNAs were identified. The regulation of these four genes ranged from modest (2- to 3-fold) to major (greater than 70-fold). A clone designated DA4 was complimentary to the most markedly stimulated crop mRNA. This mRNA encoded a polypeptide with a molecular weight of 35,500 which corresponds with the major induced protein synthesized in vivo. Messenger RNADA4 stimulation was dose dependent showing maximal induction by ovine PRL systemic injections in the 200 micrograms/day range. Above this dose PRL was less effective. The onset of mRNADA4 accumulation after a single PRL injection was rapid with statistically significant levels occurring by 3 h. Several lactogenic type hormones, but not an ungulate GH, were potent inducers of mRNADA4. The receptor responsible for mRNADA4 stimulation responds to mammalian lactogens (ovine PRL, human GH, human placental lactogen, bovine placental lactogen) and also can be blocked by an antibody to rabbit mammary gland PRL receptors. These results argue that regulation of pigeon crop gene expression (specifically mRNADA4 may be a relatively simple model of lactogenic hormone mechanisms.     

Placental Lactogen Is Expressed but Is Not Translated into Protein in Breast Cancer

Introduction

Several studies reported that the pregnancy-specific hormone placental lactogen (hPL) is expressed at both mRNA and protein levels in breast cancer. The overall objective was to establish hPL, the product of the CSH1 and CSH2 genes, as a biomarker for breast cancer.

Methods

CSH expression was determined at the mRNA level in breast cancer cell lines (BCC) and primary carcinomas by real-time and conventional PCR and the products verified as CSH1 by sequencing. Expression of hPL protein was examined by western blots and immuno-histochemistry, using commercial and custom-made polyclonal and monoclonal antibodies.

Results

Variable levels of CSH mRNA were detected in several BCC, and in some primary tumors. We detected a protein, slightly larger than recombinant hPL by western blotting using several antibodies, leading us to postulate that it represents an hPL variant (‘hPL’). Furthermore, some monoclonal antibodies detected ‘hPL’ by immunohistochemistry in breast carcinomas but not in normal breast. However, further examination revealed that these antibodies were non-specific, as efficient suppression of CSH mRNA by shRNA did not abolish the ‘hPL’ band. Custom-made monoclonal antibodies against recombinant hPL detected hPL of the correct size in placental lysate and hPL-overexpressing BCC, but not in unmodified cells or primary carcinomas. hPL protein was detected only when mRNA was increased several thousand fold.

Conclusions

We call into question previous reports of hPL expression in breast cancer which relied on mRNA levels as surrogates for protein and/or used improperly validated antibodies to measure hPL protein levels. Our data suggests that an inhibitory mechanism(s) prevents translation of CSH mRNA in breast cancer when not highly expressed. The mechanism by which translation of CSH mRNA is inhibited is intriguing and should be further investigated.     

Detection of gonadotrophic hormones on isolated thecal interstitial cells of the domestic hen, Gallus domesticus, by an immunohistochemical method

Summary An immunohistochemical method was used to demonstrate the presence of gonadotrophins in isolated ovarian interstitial cells. The cells were obtained by collagenase digestion of large ovarian follicles after removal of the yolk and the granulosa layer. Using a peroxidase-labelled anti-rabbit serum with anti-chicken follicle stimulating hormone (FSH) serum raised in rabbits, a strong positive reaction was obtained. Anti-human FSH serum also produced a positive result but the reaction was weaker. There was no apparent difference in the staining reaction of cells which had been preincubated with ovine FSH serum. Treatment with anti-ovine luteinizing hormone (LH) resulted in a faintly positive reaction.The viability of the cells was tested by the Trypan Blue method and they were identified as steroid-producing cells by the histochemical demonstration of their 3-hydroxysteroid dehydrogenase activity.