Immunogold co-localization of ovine placental lactogen and the antigen recognized by the SBU-3 monoclonal antibody in sheep placental granules

Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function. In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.     

Immunofluorescence localization of ovine placental lactogen.

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

Immunofluorescent localization of ovine placental lactogen

Summary The localization of ovine placental lactogen (OPL) was studied using the immunofluorescence method. The placentome sections were treated by an indirect technique with anti-OPL antibodies obtained from rabbits injected with purified hormone. OPL was located in large cells of the monostratified epithelium of chorionic villi. These cells are mono- or binucleated and PAS-positive. The immunological reaction was inhibited by the specific antigen (OPL, 400g/ml undiluted anti-OPL antiserum) but not by ovine prolactin, bovine growth hormone, human placental lactogen nor by any other polypeptidic hormone tested. Reciprocally, the localization of OPL-secreting cells was unsuccessful with antibodies raised against these control hormones.     

An immunological cryo-ultrastructural study of a sequential appearance of proteins in placental binucleate cells in early pregnancy in the cow

Using the most sensitive immunocytochemical method available, on ultrathin frozen sections, the results in this paper demonstrate that bovine placental lactogen (bPL) is present in the earliest fetal binucleate cells found at 21 days post coitum in the trophectoderm. A second protein, the SBU-3 antigen, which is absent in the early stages of pregnancy appears abruptly in the binucleate cell granules at 30 days post coitum coincident with the start of villus development. Subsequently, the granules contain both bPL and the SBU-3 antigen. This sequential production of unlike proteins indicates that the binucleate cell has different functions depending on the stage of pregnancy and has important roles to play both at implantation and in villus development.     

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.     

Ultrastructural changes in the placenta of the ewe after fetal pituitary stalk section.

Binucleate cells are a normal component of the ovine chorionic epithelium, but are usually separated from the fetal-maternal interface by a thin layer of cytoplasm derived from the principal or uni-nucleate cells of the trophoblast. They are distinguished not only by two distinct and separate nuclei, but also by conspicuous membrane-bound cytoplasmic inclusions in the form of haloed droplets. After fetal pituitary stalk section binucleate cells move up to and participate in the formation of the fetal-maternal interface; furthermore they extend clear blunt-ended pseudopodia into the maternal epithelial syncytium. These activities do not appear to be supppressed by fetal infusion of cortisol or ACTH. The apparent motility of binucleate cells, together with the presence of haloed droplets within the maternal epithelial syncytium, suggests that after fetal pituitary stalk section binucleate cells invade the uterine syncytium, lose their limiting membranes and discharge their contents into the syncytial cytoplasm. Large molecules such as ovine placental lactogen may be transported from fetal to maternal tissues by this mechanism.     

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.     

Control of binucleate cell migration in the placenta of sheep and goats

In the ruminant placenta 15-20% of the trophectodermal epithelium consists of granulated binucleate cells (BNC). In the sheep the granules contain ovine placental lactogen (oPL). These cells migrate from the trophectoderm to form fetomaternal hybrid tissue from implantation to term. The number of BNC, their percentage migration and the potential secretory activity of the syncytium they form were estimated by semiquantitative transmission electron-microscopical techniques after several surgical techniques and hormone or drug infusions. BNC numbers decrease normally just before parturition, and this fall could be eliminated by fetal hypophysectomy or induced early by administration of tetracosactrin to intact or hypophysectomized fetuses. If only one twin was treated with tetracosactrin the placenta of the untreated twin did not show the fall in BNC numbers found in the other unless it died in utero some time before sampling. This indicates fetal control of BNC number and migration. However, fetal catheterization, hypophysectomy, stalk section, adrenalectomy, infusion of mouse epidermal growth factor or bromocriptine had little or no effect on binucleate cell numbers or migration percentages. Maternal carunclectomy, ovariectomy, or epostane or bromocriptine administration also had no consistent significant effect. Previous reports of degeneration of BNC structure plus a decrease in their number (with bromocriptine) or an increase in migration frequency (after adrenalectomy or stalk section) have not been confirmed by this study. The BNC migration delivers the oPL-containing BNC granules close to the maternal circulation but the variation in migration seems only loosely correlated with the reported maternal oPL concentrations. The results indicate that BNC migration is independent of the hormonal milieu, but that BNC production is greatly modified by the hormonal changes just before parturition, with cortisol production by the fetus a possible primary cause.     

Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.     

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.     

Light and electron microscopic immunolocalization of bovine pregnancy-associated glycoprotein in the bovine placentome.

A bovine pregnancy-associated glycoprotein (bPAG) of 67 kDa has previously been isolated from bovine fetal cotyledons. The objective of this study was to determine the cytological localization of that protein in the placentomes and possibly the cells responsible for its production. Highly specific antisera raised against pure bPAG were used to demonstrate the cellular localization of the protein in bovine placentomes by light and electron microscopic techniques. Strong immunostaining was observed exclusively in the cytoplasm of large binucleate cells present predominantly in fetal cotyledonary tissue (villi). Some smaller weakly immunostained cells were also present in caruncular epithelium. By ultrastructural immunogold procedures, the protein was detected only within amorphous cytoplasmic granules. Granules of identical size, but weakly labeled, were found on the maternal side. All cells containing labeled granules were binucleate. These results suggest that bPAG is probably synthesized by trophoblast binucleate cells and stored in granules prior to delivery into the maternal circulation after cell migration.     

Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal antisera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strong TH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest that TH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.     

Blockade of the antigen-antibody reaction using benzil condensation with the guanidyl residue of arginine

Summary Benzil blockade of the guanidyl group of arginine was tried on sections of paraffin-embedded tissue fixed in two different fixatives, in an attempt to evaluate the relevance of this amino acid to the reaction of several proteins with their corresponding antibodies. The two fixatives were 10% formaldehyde, and Bouin's fluid without acetic acid. Both polyclonal and monoclonal antibodies against proteins or peptides (lysozyme, adrenocorticotropic hormone, growth hormone, placental lactogen, and prolactin) were used on human biopsies or material from autopsies. The blockade was effective when monoclonal antibodies were used, whereas no effect or only a small decrease of the intensity of the reaction was observed with polyclonal antibodies. The least definitive result was obtained with prolactin, where a complete blockade was never achieved with monoclonal antibodies. Calcitonin, a peptide that does not contain arginine, was used as a control not susceptible to benzil blockade; no blockade of immunostaining was observed.     

Protective effect of prolactin and placental lactogen on NO-induced Nb2 lymphoma cell apoptosis

Nitric oxide (NO) is an important modulator involved in immune regulation. Here, we describe conditions under which NO-donors induce apoptosis on Nb2 lymphoma cells, as evidenced by decreased cell viability and increased hypodiploid DNA content determined by flow cytometry. In addition, DNA fragmentation typical of apoptosis was shown by agarose gel electrophoresis. This apoptosis was accompanied by a significant increase of caspase-3-like enzymatic activity. Both ovine prolactin (oPRL) and ovine placental lactogen (oPL) exerted a protective effect on the NO-donor-induced apoptosis. Furthermore, dexamethasone (Dex)-induced cell death was also associated with caspase-3-like activity and oPL had the same potency as oPRL in its protective effect on Dex-induced apoptosis of Nb2 cells.