Ultrastructural evidence for loss of the trophoblastic layer in the chorioallantoic placenta of Australian bandicoots (Marsupialia: Peramelidae)

In most marsupials, placentation involves only the yolk sac; however, in the bandicoot family, Peramelidae, a functional chorioallantoic placentation develops in addition (Hill, 1895, 1897, 1900; Flynn, '22, '23). This duality is viewed as having evolutionary significance because most eutheria have both placentae. Furthermore, the bandicoot trophoblast was reported to vanish from the chorioallantoic site in late gestation (Hill, 1897; Flynn, '23); whereas, the eutherian trophoblast is identifiable throughout later pregnancy and may act as an immunological barrier between maternal and fetal genotypes (Kirby '68). Thus we have re-examined this singular chorioallantoic placenta of the bandicoot in plastic sections with light and electron microscopy. A distinctive feature of the bandicoot placentation is the transformation of the uterine simple columnar luminal epithelium into a highly vascular lining composed almost entirely of discrete syncytial masses (homokaryons). Endometrial blood vessels penetrate among the homokaryons to create a rich network of large diameter capillaries at extremely superficial locations near the maternal surface. In the chorioallantoic placenta (7 mm to 10-11 mm crown-rump embryos) the microvillous surface of the maternal homokaryons interdigitates with the microvillous border of the fetal trophoblast with desmosomal interaction. This trophoblast consists of a single layer of tall columnar undifferentiated cells rich in ribosomes-polysomes, poor in cytoplasmic membranes, and with large nuclei that have distinct clumps of heterochromatin and conspicuous nucleoli. It is thus remarkable that these undifferentiated cells disappear as a recognizable layer later in gestation (12 mm crown-rump embryos). Flynn's hypothesis that the trophoblastic cells disappear by fusing with maternal syncytia gains support from the existence of two populations of nuclei in the syncytial masses only at the chorioallantoic site. One population is comparable to that occurring in the homokaryons pf the yolk sac placenta, i.e., pale staining nuclei with little heterochromatin and small peripheral nucleoli. However, the other nuclei resemble those of the trophoblast cells. Since the trophoblastic cells before their disappearance as a layer possess properties associated with potential for further differentiation, the possibility of fusion between the maternal homokaryons and fetal trophoblastic cells to form heterokaryons composed of two genotypes merits fur     

Specific CSF-1 binding on murine placental trophoblasts and macrophages serves as a link to placental growth

Previous studies have shown that the colony-stimulating factor-1 (CSF-1), stimulates the in vitro proliferation of a fetally-derived adherent, phagocytic and non-specific esterase positive placental cell population which stains positively for cytokeratin and Mac-1. Binding experiments were designed to test whether this is a direct effect of the factor on these cells. Binding/elution as well as autoradiography experiments, show that adherent placental cells specifically bind CSF-1. Based on the expression of the endothelial markers cytokeratin and vimentin three subpopulations of cells were isolated from the murine placenta: labyrinthine-derived trophoblasts (cytokeratin positive, vimentin negative), spongiotrophoblast-derived trophoblasts (cytokeratin positive, vimentin negative) and placental macrophages (cytokeratin negative, vimentin positive). 3H-Thymidine incorporation assays as well as binding experiments, showed that these cells simultaneously respond to and bind the macrophage-specific factor CSF-1. Furthermore, the results indicate that isolated trophoblasts have a low rate of growth and they are very sensitive to mitogenic stimulation, whereas placental macrophages alone have a high rate of growth and therefore are less sensitive to the mitogenic stimulus. These findings are in favour of the existence of an important cytokine regulatory network in the murine placenta, where two major cell populations may collaborate possibly via soluble factors to stimulate placental growth and thus fetal development.     

Transport of maternal LDL and HDL to the fetal membranes and placenta of the Golden Syrian hamster is mediated by receptor-dependent and receptor-independent processes

Maternal lipoproteins provide nutrients to the fetus via the placenta, yolk sac, and uterine membrane plus decidua. To determine the transport processes that are responsible for the removal of lipoproteins from the maternal circulation, we measured the clearance rates of maternal LDL and HDL in vivo, as well as the tissue distribution of expression of the LDL receptor, glycoprotein 330 (gp330) and the newly described HDL receptor, SR-BI, in the placenta, yolk sac, and uterine membrane plus decidua at mid- and late-gestation of the hamster. In mid-gestation (day 10.5), LDL clearance rates of the placenta and yolk sac were similar to those in the liver (approximately 100 microl/h per g) and higher than those in the decidua (18 +/- 3 microl/h per g). Clearance rates for HDL-apoA-I and HDL-cholesteryl ether were similar to those of LDL in the placenta and decidua whereas rates in the yolk sac were dramatically higher (>1700 microl/h per g). Additionally, albumin was cleared in the placenta and decidua at approximately 16 microl/h per g whereas the yolk sac cleared the protein at much higher rates (196 +/- 22 microl/h per g). Low levels of LDL receptor were detected by immunoblot analysis in the placenta with trace amounts in the yolk sac. Gp330 and SR-BI were both barely detectable in the placenta but were expressed at high levels in the yolk sac. As gestation progressed to day 14.5, LDL and HDL clearance rates decreased in all three tissues; immunodetectable LDL receptor decreased in the placenta whereas the expression of gp330 and SR-BI in the placenta and yolk sac remained relatively constant. These data suggest that the clearance of maternal lipoproteins by the placenta, yolk sac, and decidua are mediated by receptor-mediated as well as receptor-independent processes.     

Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.     

Endoglin regulates trophoblast differentiation along the invasive pathway in human placental villous explants

Successful invasion of the maternal vascular system by trophoblast cells is a prerequisite for the establishment of a normal hemochorial placenta. Transforming growth factor-beta (TGFbeta) has been implicated in the regulation of trophoblast invasiveness into the uterus. Endoglin is a component of the TGFbeta receptor complex that binds beta1 and beta3 isoforms and is expressed at high levels on syncytiotrophoblast throughout pregnancy and is also transiently up-regulated on extravillous trophoblasts differentiating along the invasive pathway. We investigated the role of endoglin in a serum-free human villous explant culture system that allows the study of trophoblast outgrowth, migration, and invasion and mimics events occurring in anchoring villi during the first trimester of gestation. Addition to explant cultures from 5-8 weeks gestation of a monoclonal antibody to endoglin or of antisense endoglin oligonucleotides significantly stimulated trophoblast outgrowth and migration. These responses were specific, as incubation of explants with nonimmune IgG or sense and scrambled oligonucleotides had no effect. Antisense endoglin-induced trophoblast outgrowth and migration were accompanied by cell division of villous-associated trophoblasts within the proximal region of the forming column and by the characteristic switch in integrins observed in anchoring villi in situ. Treatment of villous explants with antibody and antisense oligonucleotides to endoglin also resulted in an increased fibronectin release into the culture medium. The stimulatory effect of antisense endoglin on fibronectin production was overcome by the addition of exogenous TGFbeta2, but not TGFbeta1 and -beta3. These findings suggest that endoglin expression in the transition from polarized to nonpolarized trophoblasts in anchoring villi is necessary for mediation of the inhibitory effect of TGFbeta1 and/or TGFbeta3 on trophoblast differentiation along the invasive pathway.     

Preference of invasive cytotrophoblast for maternal vessels in early implantation in the macaque

The interaction of cytotrophoblast with maternal endometrium, especially endometrial blood vessels, was examined in macaque gestational stages between 2 and 8 days after the onset of implantation. Serial sectioning of these early implantation sites allowed immunostaining of consecutive sections with a number of different antibodies, facilitating cell identification. In the earliest implantation site, immunostaining showed that antibody to cytokeratin stained cytotrophoblast, syncytial trophoblast, epithelial plaque and endometrial gland cells. However, only those cytotrophoblast cells near the maternal-fetal border and within vessels showed surface staining for neural cell adhesion molecules and only syncytial trophoblast showed SP1 reactivity. Even at this early stage cytotrophoblast filled the lumen of superficial arterioles, whereas dilated venules contained only a few cytotrophoblast cells. In later stages endovascular cytotrophoblast not only plugged many spiral arterioles but also migrated into the walls of these arterioles, and progressed into deeper coils. Displacement of endothelial cells and disruption of vessel walls were illustrated with antibody to factor VIII, TGF alpha, and desmin. Clusters of cytotrophoblast cells at the fetal-maternal interface tended to bypass clusters of epithelial plaque cells and larger clusters of maternal fibroblasts, but readily entered all vascular spaces. Consequently the vascular system constituted a major pathway of invasion, although the arterioles were the only component substantially invaded beyond the trophoblastic-shell/endometrial border.     

Placental villous stroma as a model system for myofibroblast differentiation

Different subtypes of myofibroblasts have been described according to their cytoskeletal protein patterns. It is quite likely that these different subtypes represent distinct steps of differentiation. We propose the human placental stem villi as a particularly suitable model to study this differentiation process. During the course of pregnancy, different types of placental villi develop by differentiation of the mesenchymal stroma surrounding the fetal blood vessels. In order to characterise the differentiation of placental stromal cells in the human placenta, the expression patterns of the cytoskeletal proteins vimentin, desmin, alpha- and gamma-smooth muscle actin, pan-actin, smooth muscle myosin, and the monoclonal antibody GB 42, a marker of myofibroblasts, were investigated on placental tissue of different gestational age (7th-40th week of gestation). Proliferation patterns were assessed with the proliferation markers MIB 1 and PCNA. Additionally, dipeptidyl peptidase IV distribution was studied in term placenta and the ultrastructure of placental stromal cells was assessed by electron microscopy. Different subpopulations of extravascular stromal cells were distinguished according to typical co-expression patterns of cytoskeletal proteins. Around the fetal stem vessels in term placental villi they were arranged as concentric layers with increasing stage of differentiation. A variable layer of extravascular stromal cells lying beneath the trophoblast expressed vimentin (V) or vimentin and desmin (VD). They were mitotically active. The next layer co-expressed vimentin, desmin, and alpha-smooth muscle actin (VDA). More centrally towards the fetal vessels, extravascular stromal cells co-expressed vimentin, desmin, alpha- and gamma-smooth muscle actin, and GB 42 (VDAG). Cells close to the fetal vessels additionally co-expressed smooth muscle myosin (VDAGM). Ultrastructurally, V cells resembled typical mesenchymal cells. VD cells corresponded to fibroblasts, while VDA and VDAG cells developed features of myofibroblasts. Cells of the VDAGM-type revealed a smooth muscle cell-related ultrastructure. In earlier stages of pregnancy, stromal cell types with less complex expression patterns prevailed. The media smooth muscle cells of the fetal vessels showed a mixture of different co-expression patterns. These cells were separated from extravascular stromal cells by a layer of collagen fibres. The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessels in the centre of the stem villi. The spatial distribution of the various stages of differentiation suggests that human placental villi could be a useful model for the study of the differentiation of myofibroblasts.     

Immunohistochemical localization of carboxypeptidases E and D in the human placenta and umbilical cord

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.     

Spatiotemporal expression of C-CAM in the rat placenta

We investigated the expression of the immunoglobulin superfamily cell adhesion molecule, C-CAM, in developing and mature rat placenta. By immunohistochemical staining at the light microscopic level, no C-CAM-expression was seen before Day 9 of gestation, when it appeared in the trophoblasts of ectoplacental cones. On Day 10.5, spongiotrophoblasts and invasive trophoblasts around the maternal vessels of the decidua basalis were stained positively. On Day 12.5, C-CAM was detected in the spongiotrophoblasts of the junctional layer, but labyrinth trophoblasts and secondary giant trophoblasts were not stained. On Day 17.5, C-CAM was found only in the labyrinth and lacunae of the junctional layer. At this stage, both the labyrinth cytotrophoblasts of the maternal blood vessels and the endothelial cells of the embryonic capillaries were strongly stained. Placental tissues from gestational Days 12.5 and 17.5 were analyzed by immunoelectron microscopy to determine the location of C-CAM at the subcellular level. On Day 12.5, positive staining of the spongiotrophoblasts was observed, mainly on surface membranes and microvilli between loosely associated cells. On Day 17.5, staining was found primarily on the microvilli of the maternal luminal surfaces of the labyrinth cytotrophoblasts, and both on the luminal surface and in the cytoplasm of endothelial cells of the embryonic vessels. RT-PCR analysis and Southern blotting of the PCR products revealed expression of mRNA species for both of the major isoforms, C-CAM1 and C-CAM2. Immunoblotting analysis of C-CAM isolated from 12.5-day and 14.5-day placentae showed that it appeared as a broad band with an apparent molecular mass of 110-170 kD. In summary, C-CAM was strongly expressed in a specific spatiotemporal pattern in trophoblasts actively involved in formation of the placental tissue, suggesting an important role in placental development. In the mature placenta, C-CAM expression was confined to the trophoblastic and endothelial cells lining the maternal and embryonic vessels, respectively, suggesting important functions in placental physiology.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

The ultrastructure of the integument of the American eel, Anguilla rostrata

The yolk sac of the little brown bat is unusual in that during the course of gestation both the inner endodermal cells (bordering the yolk sac cavity) and outer mesothelium (facing the exocelom) form simple columnar epithelia which persist throughout gestation. These endodermal cells develop an extensive system of agranular endoplasmic reticulum, numerous lipid droplets and unusual "giant" mitochondria. During development the Golgi apparatus changes position from the apical to the basal side of the nucleus, reversing the polarity of the cells. In general, the endodermal cells have cytological features suggestive of synthetic or secretory activity. The mesothelial cells develop an extensive "absorptive apparatus" in their apices, while large crystalloid-containing granules become numerous in their basal cytoplasm. The mesothelial cells have large deposits of glycogen, especially during mid-gestation, but few mitochondria and little granular endoplasmic reticulum. Endodermal cells do not absorb exogenous protein (peroxidase) even if it is injected directly into the yolk sac cavity. However, placement of peroxidase either in the exocelom or in the maternal vascular system results in the appearance of this protein in the "absorptive apparatus" of mesothelial cells as well as in macrophages in the stroma of the yolk sac. While evidence of absorption was clear, no direct evidence of transport of tracer to fetal blood vascular system was obtained. It is postulated that a major function of the hypertrophied mesothelial cells during gestation is the absorption of proteins and possibly other substances from the exocelomic fluid. The major function of the hypertrophied endodermal cells may be synthesis and secretion of substances into the fetal circulation.     

Congenital mesoblastic nephroma: an immunohistochemical and lectin study

Eight cases of congenital mesoblastic nephroma (CMN) were examined. Three CMNs were of the classical (typical) variant, two were cellular (atypical), and three showed a mixed pattern. A panel of nephron segment-specific tubular epithelial markers (the lectins Tetragonolobus purpureas, Phaseolus vulgaris erythroagglutinin, and Arachis hypogaea and antibodies to epithelial membrane antigen, cytokeratin, and Tamm-Horsfall protein) were used to differentiate epithelial structures within the tumor. Antibodies against vimentin, desmin, and muscle-specific actin were used as mesenchymal markers. A monoclonal antibody to the long (embryonic) form of polysialic acid (PSA) on the neural cell adhesion molecule was used as a putative renal oncodevelopmental marker. An antibody to proliferating cell nuclear antigen also was applied, which revealed increased proliferative rate in cellular CMNs. In addition to clearly entrapped native renal tubules, CMNs contain tubular structures with immature, dysplastic epithelium and occasional epithelial cell clusters embedded deep within the tumor. These immature tubules and clusters express distal nephron, including collecting duct markers and, occasionally, vimentin and PSA. We propose that these primitive tubules and epithelial structures may originate from the ureteric bud. An epithelial differentiation of the tumor cells also is possible. In one pure cellular CMN and two mixed CMNs the cellular component showed diffuse staining for PSA. The PSA (neural cell adhesion molecule) expression of the cellular component suggests that CMN may originate from the uninduced nephrogenic mesenchyme.     

An in vitro morphological study of the mouse visceral yolk sac and possible yolk sac immunocyte precursors

Mouse visceral yolk sac has been organ cultured from 9 days of gestation, a time prior to the thymus being lymphoid, until 12 days of gestation, a time after which the thymus is lymphoid. During the culture period the endodermal epithelial cells survived well, erythropoiesis diminished, endothelial-lined cavities formed in the mesodermal mass, and cells developed which have been classified as large, medium and small immunocyte precursors. The cytoplasm of the immunocyte precursors contains polysomes, spherical mitochondria, a few profiles of rough endoplasmic reticulum, occasional granules and a large Golgi complex. This study offers morphological support for the yolk sac origin of immunocyte precursors in the mouse which may seed the thymus and liver.     

Diagnosis of lumbar disc herniation by three-dimensional MRI

Trophoblasts cells which are derived from the outer layer of the blastocyst have developed mechanisms by which they can invade the uterus and tap into the maternal circulation. In contrast to tumor cell invasion trophoblast invasion is precisely regulated, being confined spatially to the uterus and temporally to early pregnancy. The invasive properties manifested by trophoblasts are made possible by the secretion of proteolytic enzymes which can degrade components of the extracellular matrix (ECM). A number of investigators have shown that the matrix metalloproteinases (MMPs) are important mediators of trophoblast invasion. The two type IV collagenases, MMP-2 and MMP-9, which specifically degrade type IV collagen and gelatins have been of particular interest in this respect. In this paper we examine the expression and regulation of MMPs and their inhibitors in a series of trophoblast continuous cell lines. These cell lines, ED27, ED31, ED77, and a choriocarcinoma cell line, BeWo, were initially characterized with respect to various properties, including cytokeratin, hCG, and hPL expression. We have looked at the expression of MMPs and their inhibitors in these cell lines and their in vitro invasive behavior. Using zymography and RT-PCR we show that the trophoblast cell lines produce both MMP-2 and MMP-9, while the BeWo produce only MMP-2. Using an in vitro invasion assay the trophoblast cell lines were shown to be capable of invading while the BeWo were unable to invade. These results suggest that expression of MMP-9 in these cells is crucial for invasion. We have also examined the regulation of MMP expression by cytokines and found that MMP-9 expression could be modulated by IL-1 beta in these cell lines. The data presented in this paper suggest that these trophoblast cell lines present an ideal model system to investigate the regulation of metalloproteinases in trophoblast invasion.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.