Characterization of a sheep trophoblast-derived antigen first appearing at implantation

A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. Lee et al (1985) have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the maternal syncytial layer. Immunoprecipitation of 125I-labelled microvilli by SBU-3, characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.     

Localization and purification of SBU-4--a pregnancy specific protein of the ovine uterus

In order to elucidate the function of molecules of the ovine maternal-fetal interface a monoclonal antibody was produced to intact interplacentomal trophoblast membranes. Extensive immunohistological studies revealed that the monoclonal antibody recognizes a protein designated SBU-4 which originates in the intercaruncular regions of the gravid sheep uterus at about the time of implantation and increases in concentration throughout gestation. The data suggest that SBU-4 is produced by endometrial epithelial cells and that adjacent uninucleate cells of the trophoblast acquire the antigen by endocytosis. Initial biochemical analysis of the purified SBU-4 molecule prepared by monoclonal antibody immunoaffinity chromatography indicates that SBU-4 is high molecular weight glycoprotein complex comprising several sub-units.     

The role of trophoblast binucleate cells in implantation in the goat: a quantitative study

The number of goat trophoblastic binucleate cells, the incidence of their migration and the formation of trinucleate and syncytial cells in the maternal uterine epithelial layer was estimated quantitatively using transmission electron microscopy between 14 and 23 days postcoitum (dpc). Binucleate cells were first observed at 18 dpc and their proportions increased rapidly from less than 1 per cent to 16 per cent by 19 dpc and 22 per cent by 23 dpc. The appearance of trinucleate cells within the maternal uterine epithelial layer coincided with evidence of migration and fusion of binucleate cells with individual uterine epithelial cells, and an increased death rate among the other uninucleate uterine epithelial cells. There was also a slight increase in the incidence of intraepithelial lymphocytes close to the trinucleate cells. The quantitative studies uphold the hypothesis that at implantation in the goat, placental trinucleate cells and their subsequent multinucleate syncytial plaque derivatives are fetomaternal hybrid tissue formed by fusion of a binucleate cell(s) with a single uterine epithelial cell.     

A structural and immunological study of chorionic gonadotrophin production by equine trophoblast girdle and cup cells

Equine chorionic gonadotropin (eCG) production by the fetally derived endometrial cups appears to be necessary for the establishment and maintenance of normal equine pregnancy.Starting at about the 27th day of pregnancy, an equatorial band of trophectodermal cells on the surface of the spherical conceptus forms the chorionic girdle. This girdle consists initially of flat trophectodermal epithelium which corrugates into folds as the cells proliferate. The folds are then pressed against the uterine epithelium by expansion of the conceptus. The cells on the apices of the folds become binucleate before they start to invade the endometrium at days 35-37. Ultrastructural immunogold labelling shows that they begin to synthesize eCG as early as day 32, before they migrate into and through the maternal epithelium. Clusters of the girdle binucleate cells penetrate deep into the endometrium. The mature cup cell has a cytoplasm full of mitochondria, rough endoplasmic reticulum cisternae, a large Golgi apparatus and a strong immunoreactivity for the glucose transporter 1 isoform on its plasmalemma. Immunocytochemistry also demonstrates that eCG is localized in the Golgi cisternae, and in small dense granules similar to those found in the migrating girdle cell and present both in the Golgi region and at the peripheral plasmalemma. Release of eCG would therefore seem to be by the usual exocytotic mechanism as found for other protein hormones. The small size and absence of any significant accumulation of eCG-containing granules are in marked contrast to the numerous large luteinizing hormone (eLH) containing granules in the equine pituitary gonadotroph, although both hormones, eLH and eCG, show complete identity at the amino acid sequence level. These morphological indicators suggest that the cup cell secretes eCG constitutively (that is, continuously), with no requirement for secretagogues, whereas in the pituitary cell the regulated pathway is utilized capable of massive secretion under appropriate stimulation.     

Placental expression of erythropoietin mRNA, protein and receptor in sheep

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta.     

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.     

Oxygen tension directs the differentiation pathway of human cytotrophoblast cells

During placental development, human cytotrophoblast cells can differentiate to either villous syncytiotrophoblast cells or invasive extravillous trophoblast cells. We hypothesize that oxygen tension plays a critical role in determining the pathway of cytotrophoblast differentiation. A highly purified preparation of cytotrophoblast cells from human third trimester placenta was cultured for 5 days in either 20% or 1% oxygen tension. The cells incubated at 20% oxygen formed a syncytium as determined by immunohistochemistry using an anti-desmosomal protein antibody that identifies cell membranes. In addition, the mRNA was markedly induced for syncytin, a glycoprotein shown to be essential for syncytiotrophoblast formation, and for human placental lactogen (hPL), which is a specific marker for syncytiotrophoblast cells. In contrast, the cell incubated at 1% oxygen tension did not fuse by morphologic analysis and did not express syncytin or hPL mRNA. However, these cells expressed abundant amounts of HLA-G, a specific marker for extravillous trophoblast cells, which was not seen in cells incubated at 20% oxygen tension. These results suggest that low oxygen tension directs differentiation along the extravillous trophoblast cell pathway while greater oxygen tension directs differentiation along the villous trophoblast cell pathway.     

Early implantation events in the baboon (Papio anubis) with special reference to the establishment of anchoring villi

The development of the baboon anchoring villus has been studied from day 14 to day 48 of gestation, using light and electron microscopy. At day 14, cords of trophoblast could be seen streaming into the endometrium, invading maternal vessels and forming blood-filled lacunae; by 20 days gestation some of these had differentiated into distinctive anchoring villi, with an outer covering of syncytiotrophoblast and inner cytotrophoblast cells which differed from those of floating villi in that a subpopulation detached from the syncytium to form an interconnecting network of cells within the centre of the villus. Subsequent migration of cytotrophoblast into the endometrium formed the cytotrophoblastic shell while fibrillin-like extracellular matrix biosynthesis within the body of the villus provided a firm mechanical support. At the trophoblast-decidual interface, a zone of necrosis and phagocytosis initially developed, which became less extensive with time, so that by 40 days a stable interface was evident with only residual pockets of necrosis. During this period, there was differentiation of decidual cells which by 28 days developed characteristic pedunculated cell processes, and later became surrounded by a basal lamina. The factors that control detachment of cytotrophoblast from the syncytium and the biosynthesis of the specialized, fibrillar extracellular matrix, features that are not apparent in other placental villi, require further investigation, possibly by carefully controlled in vitro experimentation.     

The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia.     

Quantitative analysis throughout pregnancy of intraepithelial large granular and non-granular lymphocyte distributions in the synepitheliochorial placenta of the cow

Intraepithelial lymphocytes are a constant feature of ruminant uterine epithelium. Light microscope quantitation on semithin sections of resin embedded perfused material from pregnant and non-pregnant cows shows that although the proportion of large granular lymphocytes to non-granular lymphocytes in interplacentomal areas increases during pregnancy, the total number of lymphocytes in these areas remains at a similar level. However all types of lymphocytes are eliminated from the caruncular uterine epithelium of the cow by 28 days of pregnancy at the initiation of placentomal development. Despite the subsequent enormous growth in area of this region during pregnancy, no lymphocytes are found in the bovine placentomal cellular uterine epithelium. This pattern is similar to that in the ewe and goat although in these species the placentomal uterine epithelium is modified to maternofetal hybrid syncytial plaques. However, in the deer, a similar cellular placentomal epithelium has numerous intraepithelial lymphocytes and large granular lymphocytes are closely associated with fetomaternal hybrid trinucleate cells formed by binucleate cell migration throughout pregnancy. Cow interplacentomal large granular lymphocytes and trinucleate cells show similar apposition but the formation and fate of cow placentomal trinucleate cells does not involve lymphocytes. Since the caruncular (placentomal) area is 10–20 times that of the intercaruncular this suggests a very different function for intraepithelial lymphocytes in the deer compared with the other ruminant species.     

The cytoplasmic expression of E-cadherin and beta-catenin in bovine trophoblasts during binucleate cell differentiation

Binucleate cells are endocrine cells generated by the acytokinesis and endoreduplication of the trophectoderm in the ruminant placenta. These cells are migratory and secrete hormones into the maternal circulation after fusing with uterine epithelial cells. In this study, we performed immunohistochemistry for E-cadherin and beta-catenin in bovine placenta and a bovine trophoblast cell line (BT-1). We found that E-cadherin and beta-catenin were distributed not only at the cell to cell boundary but throughout the cytoplasm in binucleate cells, although they were concentrated at the cell to cell boundary in epithelial cells in bovine placenta. Moreover, beta-catenin was detected in the nuclei of binucleate cells. Binucleate cells after fusion with uterine epithelial cells (feto-maternal hybrid cells) in the maternal side showed no intracellular expression of E-cadherin and beta-catenin. The transformation into binucleate cells in the BT-1 cell line was also accompanied by the cytoplasmic accumulation of E-cadherin and beta-catenin. We further demonstrated that levels of cytoplasmic beta-catenin were well correlated with the DNA content of binucleate cells in BT-1. The dynamic changes in the distribution of E-cadherin and beta-catenin suggest an important role in binucleate cells, including the rearrangement of cadherin-mediated cell adhesions during cell migration and the onset of endoreduplication probably via the nuclear transfer of beta-catenin.     

Placental polyploidization: a comparative study in the mouse and the guinea pig

Initially diploid, pure trophectodermal derivatives were dissociated and grown in culture. Overthe 72-hour time course in vitro, uninucleate, binucleate and a small number of multinucleate cells appeared. Moreover, the pattern of binucleation found in these trophoblast cultures resembled that seen during the development of the mouse liver. Thus, the binucleates displayed a progressive increase in nuclear DNA content and the increased DNA values ranged from 2c to 32c. Furthermore, the proportion of uninucleate and binucleate cells changed systematically with growth in vitro and the final binucleate cell population became, on average, 10 to 15 per cent of the total. These results, together with those of other studies, suggest that mouse trophoblast can initially become giant through a binucleate phase.     

顶体反应抑制剂4'-乙酰胺苯基4-胍基苯甲酸酯(AGB)具有阻断HIV-1进入细胞的作用

目的:研究4'-乙酰胺苯基4-胍基苯甲酸酯(AGB)抗HIV-1活性及作用靶点。方法:通过AGB对宿主细胞的毒性实验、合胞体抑制实验、融合阻断实验、对HIV-1感染细胞的保护作用实验和对HIV-1急性感染细胞p24抗原产生的抑制作用等试验,观察AGB对HIV-1复制的影响和作用机制。结果:AGB抑制HIV-1IIIB诱导C8166细胞形成合胞体,EC50为39.5μg/ml;抑制HIV-1感染细胞上清中HIV-1p24抗原的表达,EC50为33.36μg/ml;阻断HIV-1慢性感染H9细胞与正常C8166细胞间融合的作用。结论:AGB具有阻断HIV-1进入宿主细胞的作用,是一种有前景的具杀精子作用的杀微生物剂。     

Effects of continuous calcitonin treatment on osteoclasts derived from cocultures of mouse marrow stromal and spleen cells.

BACKGROUND: Continuous calcitonin (CT) treatment for bone diseases associated with increased bone resorption may be followed by prolonged depression of osteoclast response to CT. The mechanisms of this "escape" phenomenon remain unclear. METHODS: We examined the effects of continuous CT treatment on cell formation, calcitonin receptor (CTR) expression, response to CT, and bone resorption of osteoclasts in a coculture of mouse marrow stromal and spleen cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Cells were cocultured and treated with salmon CT (sCT) for 7, 14, or 21 days. The effects of continuous CT treatment on osteoclast formation was determined by quantitation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs). CTR expression in osteoclasts was determined by binding of [125I]sCT in autoradiography. Bone resorption and CT responsiveness were assessed by examining the formation of resorption pits and by enumerating osteoclast reattachment on dentine slices after sCT rechallenge. RESULTS: TRAP-positive MNCs appeared in cocultures treated with sCT and were similar in number and morphology to those in control cultures, regardless of the concentration and duration of sCT treatment. A decrease in CTR expression was identified as a loss of silver grains from the TRAP-positive cells in cocultures receiving sCT treatment for 14 or 21 days. Partial recovery of CTR expression in TRAP-positive cells was evident in cocultures treated with sCT for only the first 7 days of coculture. TRAP-positive MNCs in cocultures treated with sCT for 14 or 21 days were resistant to the rechallenge with sCT. They attached to dentine slices and caused numerous resorption pits compared with control cells and cells treated with sCT for the first 7 days of coculture (p < 0.01). CONCLUSION: These findings suggest that the escape phenomenon that develops after continuous CT treatment may be due, at least in part to: 1) loss of responsiveness to CT in existing osteoclasts; and 2) development of new osteoclasts that are CTR-deficient and, therefore, refractory to CT rechallenge.     

Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion

Fetal placental tissue from 11 days pregnant mice was dissociated in collagenase and DNase solution and then separated on a 40 per cent Percoll gradient. Trophoblast cells banded at a density of 1.05 g/ml. When cultured on rat tail collagen, these cells formed colonies of mono- and binucleate cells varying in size from 40 to 70 microns. At the time of plating, about 13 per cent of the trophoblast cells secreted mouse placental lactogen II (mPL-II) as determined by reverse haemolytic plaque assay. The ratio of mPL-II-producing cells increased significantly in culture and reached 63 per cent after 48 h. The secretion of mPL-II increased continuously during six days of culture, whereas the total protein release was highest after the first day, declined the second day and then remained relatively constant for the last four days of culture. The DNA content of the cells did not change significantly during the six-day period. When the trophoblast cells were incubated with insulin (1 ng/ml to 5 micrograms/ml), a modest but significant reduction in mPL-II secretion was observed. No change in the mPL-II secretion was seen when epidermal growth factor was administered to the culture in concentrations from 1 ng/ml to 10 micrograms/ml. It is concluded that this in vitro culture system is suitable for studying both mPL-II secretion and the differentiation of mPL-II-producing cells.     

Effects of interferon-gamma and tumor necrosis factor-alpha on survival and differentiation of oligodendrocyte progenitors

OBJECTIVE: There is strong evidence from recent clinical studies that ascending intrauterine infection is associated with an increased incidence of periventricular leukomalacia in very premature fetuses. Periventricular leukomalacia is characterized by disrupted myelination from a loss of oligodendrocyte progenitors. We investigated the effects of proinflammatory cytokines on the survival and differentiation of this cell type. METHODS: Cultures of more than 90% A2B5-positive progenitors were prepared from neonatal rats and kept for 3 days in medium supplemented with factors that stimulate cell proliferation. After 1 day in proliferation medium, cells were treated with interferon-gamma (100 U/mL) and tumor necrosis factor-alpha (100 ng/mL) for 48 hours triggering an increase in apoptotic A2B5 progenitor cells from 3.2 +/- 2.3% to 11.0 +/- 2.6%. After cytokine treatment cultures were transferred to medium containing factors to promote differentiation of progenitors into the myelinating phenotype. RESULTS: In cytokine pretreated cultures, only 2.6 +/- 1.1% of total cells survived after a total of 9 days in vitro, whereas in untreated cultures most cells differentiated as shown by expression of myelin basic protein, myelin-associated glycoprotein, 2,3-cyclic nucleotide 3-phosphodiesterase, and myelin oligodendrocyte-specific protein. Using ten-fold reduced concentrations of combined interferon-gamma (10 U/mL) and tumor necrosis factor-alpha (10 ng/mL) pretreatment resulted in a survival to 11.2 +/- 4.9% of total cells with 36.3 +/- 11.6% A2B5-positive cells at day 9. This indicates a major enrichment of undifferentiated cells compared with untreated controls which harbored only 1.0 +/- 0.3% A2B5-positive cells. CONCLUSION: Inflammatory cytokines not only induced apoptotic cell death but also prevented the differentiation of immature A2B5 oligodendrocyte progenitors into the myelinating phenotype.     

Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal cows and in cattle with retention of fetal membranes

Matrixmetalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a key role in tissue re-modelling in the placenta. In the present study, distribution of MMP-2, MMP-9 and TIMP-2 was demonstrated immunohistochemically in the bovine placenta and interplacentomal tissue. Specimens representing the whole gestation until parturition were processed. Additionally, materials from cows with and without retention of fetal membranes were compared. MMP-2 expression was abundant in the maternal septae of the placentome in early gestation, with ongoing pregnancy immunoreactivity was restricted to the stromal tissue at the openings of maternal crypts. The chorionic epithelium opposite to these regions was also positive for MMP-2. MMP-9 expression was observed in the chorionic epithelium, except in the giant binucleate cells. In addition, the maternal epithelium and stroma showed immunoreactivity for MMP-9. No differences in MMP-2 and MMP-9 distribution could be observed between cows with proper release of fetal membranes and cows with retained fetal membranes. Giant binucleate cells expressed TIMP-2 during the whole gestation. Immunostaining for alpha-smooth muscle actin revealed contractile elements in the bovine placentome. Balance between proteolytic enzymes and their activators and inhibitors is essential for regular development of the placenta. The expression of TIMP-2 in the giant binucleate cells indicates an essential role of inhibitory factors during gestation. It is likely that less TIMP-2 is produced at the end of pregnancy as the number of binucleate cells is diminished.     

The role of galectin-1 in trophoblast differentiation and signal transduction

Galectins are proteins with the ability to bind β-galactosides through a conserved carbohydrate recognition domain. Galectin-1 exerts its biological effects by binding glycan ligands on proteins involved in cell adhesion and growth regulation. Galectin-1 inhibits trophoblast cell proliferation and induces syncytium formation. Its down-regulation in the syncytiotrophoblast has been associated with early pregnancy loss. In the choriocarcinoma-derived BeWo cells the galectin-1 induced growth inhibition is apoptosis-independent, but rather appears to be mediated by binding to cell surface receptors, such as the receptor tyrosine kinases REarranged during Transfection (RET) and Janus Kinase (JAK) 2 as well as vascular endothelial growth factor receptor 3. On the syncytiotrophoblast and extravillous trophoblast galectin-1 binds the Thomsen-Friedenreich disaccharide on mucin-1. The cell differentiation processes induced by binding to these receptors ultimately lead to the inhibition of proliferation and syncytium formation.