Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc1,3(Fuc1,2)-Gal1,4GlcNAc1-, GalNAc1,6Gal1-, Gal1,4GlcNAc-and Gal1,3GalNAc1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.     

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.     

Bovine trophoblastic cell differentiation on collagen substrata: formation of binucleate cells expressing placental lactogen

The differentiation of trophectoderm in ruminants is marked by the appearance of binucleate cells in cytotrophoblasts. Binucleate cells are produced by the acytokinesis of cytotrophoblasts and undergo endoreduplication. They secrete hormones such as placental lactogen, and exhibit migratory behavior to transfer their hormones into maternal circulations. In this study, we showed that a bovine trophoblastic cell line (BT-1) established from in vitro fertilized blastocysts differentiated into binucleate cells on collagen gel. BT-1 had cytotrophoblastic epithelial characteristics in that it expressed cytokeratin, E-cadherin and interferon-tau. It spontaneously formed multicellular spherical vesicles floating in the medium. We cultured these vesicles on type I collagen substrata. Most vesicles attached to the collagen substrata, and exhibited cell outgrowth and proliferation. We found that after more than 10 days, clusters of binucleate cells appeared in the cell colonies on the collagen gel, but not on the collagen film. These binucleate cells have features characteristic of those in vivo, including an increased nuclear DNA content and the expression of placental lactogen. BT-1 is a useful model with which to study trophoblast differentiation in ruminants.     

Presence of placental lactogen in bovine conceptuses before attachment.

Placental lactogen has been detected by radioreceptor assay in bovine conceptuses colletected between 17 and 25 days post coitum, at or shortly after the time of appearance of binucleate cells in the bovine trophectoderm, but before attachment or implantation.     

Preservation of human chorionic gonadotropin and placental lactogen secretion and normal morphology following long-term culture of normal human placental cells

In order to study the regulation of hCG and hPL secretion during gestation, a system for the preservation of the functional integrity of normal placental cells in long-term culture was established. Normal term placental cells were dispersed with 0.25% trypsin-500 units DNAse I and cultured in a monolayer in Dulbecco's modified Eagle medium with 10% fetal bovine serum. Normal cell morphology, basal hCG and hPL production and hCG responses to dibutyryl cAMP were preserved till 54 days of culture. This model may be useful for the study of long-term regulation of normal placental hCG and hPL synthesis and secretion.     

Progesterone and prostanoid production by bovine binucleate trophoblastic cells

A procedure for preparing highly enriched suspensions of bovine binucleate trophoblastic cells was developed and data showing that these cells produce progesterone, prostacyclin (PGI2), and prostaglandin E2 (PGE2) were obtained. Approximately 200 X 10(6) enzymatically dissociated cells from bovine cotyledons were applied to the surface of a density gradient of 2% to 4% Ficoll-400 using the Wescor CELSEP sedimentation chamber. After 90-120 min of sedimentation at unit gravity, fractions containing binucleate trophoblastic cells were obtained and washed in HEPES-buffered Medium 199. Preparations of 90% to 100% binucleate trophoblastic cells were obtained routinely; viability was 50% to 80%. After incubation at 37 degrees C, concentrations (ng/10(5) cells) of progesterone were greater in those fractions containing binucleate cells than in those containing primarily smaller, mononucleate cells. Total progesterone secreted (mean +/- SEM) after 4 h by 1 X 10(5), 2 X 10(5), 4 X 10(5), 8 X 10(5), and 1.6 X 10(6) binucleate cells was 0.27 +/- 0.03, 1.01 +/- 0.09, 4.02 +/- 0.37, 10.31 +/- 0.92, and 20.96 +/- 2.23 ng, respectively (r = 0.997). Addition of 10% fetal bovine serum (FBS) or normal anestrous cow serum increased (P less than 0.05) production of progesterone by binucleate trophoblastic cells. Luteinizing hormone, follicle-stimulating hormone, prolactin, thyrotropin, and 8-bromo-adenosine 3',5'-cyclic monophosphate had no effect. Binucleate trophoblastic cells also produced PGI2 in relation to number of cells incubated (r = 0.996). Time courses for production of PGI2, PGE2, and progesterone were similar. Aspirin inhibited production of PGI2 and PGE2 by about 50% at a dose of 100 microM; FBS stimulated production of both prostanoids.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.     

Expression of cytokeratin and vimentin in nucleus pulposus cells

With immunohistochemistry using monoclonal antibodies against intermediate filament proteins, nucleus pulposus cells were found to express cytokeratin(s) simultaneously with vimentin in fetal life and childhood. This finding adds to the series of human tissues showing coexpression of cytokeratins and vimentin. Surprisingly remnants of such cells were also found in the nucleus pulposus of adults, and a possible relationship of such cells to chordoma formation is discussed.     

Super-active forms of placental lactogen and prolactin

Both placental lactogen and prolactin can be converted into super-active forms. These super-active hormones, in combination with insulin and hydrocortisone, stimulate accumulation of α-lactalbumin and increase RNA synthesis in explants from mouse mammary glands to an extent greater than the maximal level obtained with the native hormones. Also, they are able to stimulate RNA synthesis by suspensions of mammary epithelial cells which have lost the ability to respond to native lactogen and prolactin.     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.     

Evaluation of the somatogenic activity of bovine placental lactogen with cell lines transfected with the bovine somatotropin receptor

Studies have shown that bovine placental lactogen (bPL) has partial somatogenic activity in vivo even though binding results clearly indicate bPL does not cause homodimerization of the bovine somatotropin receptor (bST-R). To help understand the receptor binding versus biological activity of bovine somatotropin (bST) and bPL we have developed a homologous model system. Full length bST-R was stably transfected into a murine lymphoid cell line, Ba/F3 and a hamster kidney cell line, BHK. From both transfected cell lines, clones were isolated (Ba/F3-C1 and BHK-24) which demonstrated specific binding of bST and, or bPL. Bovine ST stimulated proliferation of the Ba/F3-C1 clonal line over a dose range of 10 to 3000 pM with an EC50 of 100 pM. A bST variant (des 1-4 bST) and porcine ST (pST) which both have approximately 10% of the binding affinity for bST-R as native bST were 1 and 10% as potent as bST in this bioassay, respectively. This suggests that affinity and biological activity are correlated for this system. Proliferation was initiated through the bST-R because addition of a monoclonal antibody which recognizes the extracellular domain of bST-R and inhibits binding of bST to its receptor, inhibited bST-stimulated mitosis. However, even though the affinity of bPL for the bST-R is similar to that of bST, bPL antagonized the proliferative action of bST with an IC50 of 1 nM. Components of the somatogenic signal transduction pathway were also evaluated in both cell lines. Addition of bST to the cell cultures increased phosphorylation of JAK2 in Ba/F3-C1 and BHK-24 cells in a dose-responsive manner but bPL failed to increase phosphorylation of JAK2 in either cell line. In summary, these data support the hypothesis that ST-R homodimerization is necessary for bioactivity in this model system but fail to explain apparent somatogenic activity of bPL in vivo.