Immunohistochemical demonstration of a hitherto undescribed localization of hemoglobin A and F in endodermal cells of normal human yolk sac and endodermal sinus tumor

In this study of 4 human yolk sacs, the presence of hemoglobin A and F (HbA and HbF) is demonstrated for the first time in epithelial cells (type 1) and erythroid-like cells (type 2) in the endodermal layer by immunoperoxidase technique. Our findings strongly support the hypothesis previously proposed that the red blood cells formed in the yolk sac are of endodermal origin. Tumor with yolk sac differentiation (8 endodermal sinus tumors and 1 embryonal carcinoma with vitelline areas) similarly showed HbA and HbF localisation in endodermal cells. None of 59 germ cell tumors of other types contained these hemoglobins in the neoplastic cells.     

Ultrastructural localization of the 9-kilodalton vitamin D-dependent calcium-binding protein in the murine intraplacental yolk sac

The calcium-binding protein (CaBP) calbindin has been implicated in the molecular mechanism of placental calcium transfer. Previous light microscopic studies have identified CaBP in visceral (but not parietal) endodermal cells of the yolk sac with the most intense immunocytochemical signal observed in the intraplacental yolk sac. In the present studies, electron microscopy was used to study the localization of CaBP in placenta. Placentas of 17-day pregnant mice were fixed by perfusion in 0.5% glutaraldehyde, embedded in low-temperature Lowicryl K4M, and examined in thin section for specific labeling with a polyclonal antiserum. Antibody to CaBP was localized by using protein A-gold particles which were quantified for subcellular compartmentation by using a Videoplan computer system. A high signal for CaBP was found in the visceral endodermal cells of the intraplacental yolk sac. In these cells, gold particles indicating the location of CaBP were observed over 1) the cytoplasmic matrix where the average number of gold particles per micron 2 was 33; 2) the microvilli (17/micron 2); 3) the mitochondria (17/micron 2); and 4) the nucleus (43/micron 2). Sections from antigen-absorbed controls, by contrast, showed few gold particles: cytosol, 2/micron 2; microvilli, 5/micron 2; mitochondria, 5/micron 2; and nucleus, 4/micron 2. Electron-lucent profiles of the Golgi and endoplasmic reticulum contained no particles in the controls and a low particle count (4/micron 2) in the stained sections. Parietal endodermal cells of the intraplacental yolk sac showed a relatively low signal for CaBP compared with the visceral endodermal cells (5 particles/micron 2 vs. 39).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical localization of vitamin D-dependent calcium-binding protein in mouse placenta and yolk sac

A 10,000-dalton calcium-binding protein (10-kd CaBP) has been described in the placentae and yolk sacs of rats and mice. This protein is similar or identical to vitamin D-dependent intestinal CaBP and these proteins have been implicated in the molecular mechanisms of intestinal calcium absorption and transplacental calcium transport. Using an antiserum to the purified 10-kd rat intestinal CaBP, the localization of CaBP in the 16-17-day mouse yolk sac and placenta was studied immunocytochemically with peroxidase-antiperoxidase labelling and quantified by radial immunodiffusion. A high concentration of immunolabelling was observed in the endodermal cells of the intraplacental yolk sac lining the sinuses of Duval. The columnar endodermal cells lining one side of the endodermal sinuses adjacent to fetal vessels contained most of the immunoreactive label. Quantitation by radial immunodiffusion demonstrated a 5.5-fold higher concentration of CaBP in the portion of the placenta containing most of the intraplacental yolk sac than in the maternal half of the placenta. This demonstration of a 10-kd CaBP within the intraplacental yolk sac suggests this protein functions to facilitate placental calcium transport and is the first study which directly supports the hypothesis of a functional role for the sinuses of Duval in calcium transport.     

Localization of rabbit gamma globulins in the mouse visceral yolk sac placenta.

The yolk sac placenta has been implicated previously in transmission of passive immunity to the fetus. This work uses an immunohistochemical technique devised by Sternberger et al. ('69) to study this problem. Rabbit serum containing gamma globulins was injected into the uterine lumen of the White Swiss mouse during the last third of pregnancy. Two or four hours later yolk sacs were removed and fixed in 2% paraformaldehyde or freeze-dried and fixed with paraformaldehyde vapors. Finely chopped tissue was treated with (1) sheep antiserum to rabbit serum gamma globulin, (2) an antigen-antibody complex consisting of horseradish peroxidase and rabbit anti-horseradish peroxidase and (3) hydrogen peroxide and 3,3'-diaminobenzidine. Reaction product was heavily concentrated in visceral yolk sac endodermal cells, frequently deposited in endothelial cells of vitelline vessels, and rarely in the serosal basement membrane and mesothelial cells which border the exocoelomic cavity. This supports evidence of other workers that the yolk sac membrane rapidly absorbs substances with which it comes in contact, transport into vitelline vasculature is a route of transfer from mother to fetus, and possible transfer into the exocoelomic cavity and thence to the amniotic cavity may occur in mice.     

Retinoid binding proteins in mouse yolk sac and chorio-allantoic placentas

 In the adult, as well as in the embryo, a number of specific extra- and intracellular binding proteins such as the plasma retinol binding protein (RBP), the cellular retinol binding protein type I (CRBP I), and also the cellular receptors for RBP are thought to regulate transport and metabolism of retinol (vitamin A). Since the regulation of materno-fetal transport of vitamin A is not well understood, we examined the localization of these proteins during the development of the mouse chorio-allantoic and yolk sac placentas. The labyrinthine region of the chorio-allantoic placenta, where exchange of substances can occur between the maternal and fetal circulations, did not contain RBP (mRNA or protein) or antigen(s) similar to the bovine RBP-receptor p63, whereas the visceral endoderm of the yolk sac placenta, the second site for materno-fetal transport, did. Furthermore, only the endodermal cells of the visceral yolk sac appeared to strongly accumulate radiolabelled retinoids. The cellular retinol binding protein (CRBP I) was detected both in the trophoblast layer of the placental labyrinth closest to the fetal endothelium (layer III), and in the visceral endoderm of the yolk sac. Together, these findings suggest that the yolk sac placenta mediates retinol transfer to the embryo/fetus throughout the entire gestation. The chorio-allantoic placenta, on the other hand, does not appear to have this capacity, while the presence of CRBP I does suggest a retinol-metabolizing capability. Accepted: 5 February 1997     

Immunofluorescent Localization of Alpha-fetoprotein Synthesis in the Endodermal Sinus of Rat Placenta

Specific immunofluorescence staining for AFP of the rat yolk sac showed that the synthesis was confined to the visceral layer of the yolk sac endoderm. The AFP synthesis took place not only in the extraplacental yolk sac as demonstrated by Gitlin in 1967, but also in the endodermal sinus structures within the placenta. No immunofluorescence staining was found of Reichert's membrane close to the parietal layer of the yolk sac. These findings are in accordance with the earlier demonstrated AFP synthesis in the visceral layer of the endodermal sinus structures in the tumors.     

An electron microscopic study of absorption of peroxidase conjugated immunoglobulin G by guinea pig visceral yolk sac in vitro,

In the guinea pig and some other animals, passive immunity is conferred on the developing fetus by passage of immunoglobulin from mother to fetus across the yolk sac. In order to examine the cytological pathway involved in immunoglobulin transport, guinea pig visceral yolk sacs from late in gestation were exposed in vitro to peroxidase-conjugated guinea pig immunoglobulin G (IgG-HRP). Tissue was then fixed, incubated to show the site of localization of peroxidase reaction product and prepared for electron microscopy. The results suggested that the first step in the uptake of IgG-HRP by yolk sac is attachment of the protein to the surface coats of endocytic invaginations at the apical surfaces of the endodermal cells. The endocytic vesicles then appear to pinch off from the surface and move deeper into the cytoplasm. Some of the small endocytic vesicles fuse with large apical vacuoles, which often contain large amounts of reaction product. Other small endocytic vesicles pinch off from the surface, move deeper into the cytoplasm and fuse with the lateral plasmalemma; their protein content is emptied into the intercellular space by exocytosis. From the intercellular spaces the protein presumably diffuses across the basement membrane and connective tissue spaces and enters the vitelline capillary bed. It is postulated that the latter cellular pathway, involving small vesicles and the intercellular spaces, is utilized by those immunoglobulins which are transferred intact across the yolk sac endoderm.     

COMPARATIVE MORPHOLOGY OF ENDODERMAL SINUS TUMOR (TEILUM) TO HUMAN YOLK SAC and A PROPOSAL OF ENDODERMAL CELL TUMOR

Histological criteria of 8 pure and typical endodermal sinus tumor (EST) were compared with the morphological features of 8 human yolk sacs from 5 to 13 weeks of pregnancy. No common features were found with regard to endodermal sinus structure (ESS) and clear-cell entoblastic pattern (CCEP), but magma reticulare (MR) and eosinophilic hyaline globules (EHG) were detected as common findings. As to the morphology and synthesis of a-feto-protein (AFP), some tumor cells with eosinophilic-granular cytoplasm lining ESS had similarity to the endodermal cell of human yolk sac. Histological features of EST mimicked the morphological structure of endodermal sinus in rodent yolk sac. Pathological study on an ovarian and a gastric adenocarcinoma with high level of AFP concentration demonstrated two cell patterns with eosinophilic-granular and clear cytoplasm showing analogousness to the endodermal cell of human yolk sac. It was suggested that these tumors showed a selective differentiation to the endodermal cell of human yolk sac and might be called ECT. Further study clarified that ovarian embryonal carcinoma or teratocarcinoma with higher AFP concentration had vitelline component of EST or ECT or both.     

Comparative morphology of endodermal sinus tumor (Teilum) to human yolk sac and a proposal of endodermal cell tumor

Histological criteria of 8 pure and typical endodermal sinus tumor (EST) were compared with the morphological features of 8 human yolk sacs from 5 to 13 weeks of pregnancy. No common features were found with regard to endodermal sinus structure (ESS) and clear-cell entoblastic pattern (CCEP), but magma reticulare (MR) and eosinophilic hyaline globules (EHG) were detected as common findings. As to the morphology and synthesis of alpha-fetoprotein (AFP), some tumor cells with eosinophilic-granular cytoplasm lining ESS had similarity to the endodermal cell of human yolk sac. Histological features of EST mimicked the morphological structure of endodermal sinus in rodent yolk sac. Pathological study on an ovarian and a gastric adenocarcinoma with high level of AFP concentration demonstrated two cell patterns with eosinophilic-granular and clear cytoplasm showing analogousness to the endodermal cell of human yolk sac. It was suggested that these tumors showed a selective differentiation to the endodermal cell of human yolk sac and might be called ECT. Further study clarified that ovarian embryonal carcinoma or teratocarcinoma with higher AFP concentration had vitelline component of EST or ECT or both.     

A new cytoplasmic organelle,related to both lipid and glycogen storage materials in the yolk sac of the bat,Tadarida brasiliensis cynocephala

The gestation period of Tadarida is approximately four months long extending from late February to June. During the first half of gestation the yolk sac undergoes a complete collapse bringing two layers of endodermal cells into contact with one another and obliterating the cavity of the yolk sac. Both the endodermal and the mesodermal cells hypertrophy and develop into a glandular-looking organ during the latter half of gestation. Approximately a month before parturition the endodermal cells become progressively laden with lipid and glycogen. Immediately before birth, however, both of these storage materials are depleted. This depletion is preceded by the formation of an extensive array of hexagonal membranous channels within the cytoplasm. Some of the membranes of this organelle are closely applied to lipid droplets and glycogen granules can be observed in linear patterns within the hexagonal channels. Tissues taken at term showed the membranes of the hexagonal channels continuous with a paracrystalline membranous structure. The close morphological association of the membranous organelle to both the lipid and glycogen storage materials indicates that it is involved in their metabolism in the yolk sac of the bat.     

Uptake and storage of thorotrast by the rodent yolk sac placenta: An electron microscopic study

Electron microscopy was used to investigate the uptake and storage of electrondense particulate matter by the rodent yolk sac placenta. Pregnant hamsters were given single intra-uterine injections of Thorotrast on day 13, 14 or 15 of gestation and killed at intervals between 15 minutes and 48 hours thereafter. Electron microscopic examination of yolk sacs removed from the injected animals revealed the rapid and progressive uptake of the tracer particles by the visceral epithelial cells of these fetal membranes. Pinocytic vacuoles (phagosomes) containing Thorotrast were visible in the apical cytoplasm of epithelial cells as early as 15 minutes after injection and became increasingly abundant in these cells at later post-injection intervals up to 18 hours. Epithelial cells which became fully engorged with Thorotrast vacuoles exhibited various pathologic changes, possibly caused by the interference of the metabolically inert metal particles with intracellular digestive mechanisms (the lysosome system). There was no evidence, however, of transport of Thorotrast particles through or between the yolk sac epithelial cells. The connective tissue spaces and blood vessels of the yolk sac, as well as underlying fetal compartments, were free of the tracer particles at all observed intervals after injection.     

Hemopoiesis in the human yolk sac

The endodermal layer of the human yolk sac was examined three-dimensionally with light microscopy on serial sections using scanning electron microscopy and transmission electron microscopy to find the origin of hemo poiesis in the yolk sac. Cell-labelling techniques were also employed using the monoclonal anti-transferrin receptor antibody. Orifices of the endodermal and intracellular tubules facing the yolk-sac cavity were demonstrated on the endodermal surface. Various-sized blood cells in various stages of differentiation and maturation were distributed in the yolk-sac cavity and tubules and were observed also at the orifices of the tubules. The morphological and the immunological findings suggest that blood cells with large nuclei in the endodermal layer are the most immature. The present results suggest that blood cells originate from the endodermal layer and are carried to the embryo through the yolk sac cavity and the vitel-line duct. It is probable that the endodermal and intracellular systems of tubules have an important role in the transport of blood cells, including stem cells.     

Host-parasite interaction and development of infraforms in chicken embryos infected with Coxiella burnetii via the yolk sac.

Two phase I strains of Coxiella burnetii of different virulence were injected into the yolk sacs of chicken embryos, and the yolk sacs and livers were examined at intervals by light, fluorescent, and electron microscopy. The high absorptive and digestive capacities of the yolk endoderm contributed to he entrance of the organisms into endodermal epithelial cells where C. burnetii multiplied. Organisms multiplied not only inside specific vacuoles originating from phagolysosomes but also in the cytoplasm itself. Lysis of the limiting membrane of some phagolysosomes, a normal function of endodermal cells, as well as rupture of vacuoles, provided the release of C. burnetii into the cytoplasm. The C. burnetii strain of greater virulence infected 100% of the endodermal cells, whereas the strain of lesser virulence infected only 60%. Budding of very small particles from the C. burnetii bodies was demonstrated. The particles were regarded as filterable forms of the organism. Despite the enormous multiplication of C. burnetii in the endodermal cells, organisms were only rarely detected in the vitelline blood vessels and liver sinusoids of the embryos. Peculiarities of the infectious process of C. burnetii in chicken embryos and possible mechanisms of limitation of spread of the infection are discussed.     

Morphological and functional features of endodermal cells in rat yolk sac, with special reference to the fetal macrophage differentiation

Morphological and functional features of the yolk sac endodermal cells with special reference to the fetal macrophage differentiation were investigated morphologically under the light and electron microscopes and immunologically with the antigen phenotypic analysis and the phagocytic activity-test, using the syngeneic DA rat-embryos from 8 to 16 days of gestation. Based on the staining property with toluidin blue and the ultrastructural features, the endodermal layer from day 8 to 16-yolk sacs has been known to consist of two kinds of cell type; 10% "clear" cells with clear cytoplasm and 90% "dark" cells with dark cytoplasm. Numerous primary lysosomes, phagolysosomes, lipid droplets and coated vesicles distributed preferentially in the supranuclear portion of endodermal cells. A broad intercellular space was found between "clear" cells and "dark" cells, indicating the loose intercellular binding. It was often found that "clear" cells tend to migrate from the endodermal layer into the mesenchymal layer, where the poor development of basement membrane was seen between them. Cells phagocytosing red blood cells, that resemble morphologically "clear" cells, were also observed in the fetal liver. At ten hours after latex-injection into the yolk sac cavity of 14 days embryos, some cells which phagocytosed latex beads in their cytoplasm were found in the endodermal layer, and also in the liver tissue and loose connective tissue of fetus. These cells were stained positively with monoclonal antibody Mar3 which recognizes preferentially rat-mononuclear phagocyte system. In vitro-latex uptake of separated endodermal cells was also demonstrated by the culture-study of endodermal cell suspension. The present findings indicate that the yolk sac-endodermal layer derived from the proximal endoderm consists of at least two kinds of cell-population with a great similarity to tissue macrophages in morphological and functional senses, and support the concept that some cell-populations of endodermal layer may migrate into fetal tissue and are closely related to the differentiation of fetal macrophages and their precursors.     

Kinetic and end-point microdensitometry (section biochemistry) of gamma-glutamyl transpeptidase and dipeptidyl peptidase IV in the mature mouse decidua and visceral yolk sac

gamma-Glutamyl transpeptidase (EC 2.3.2.2) and dipeptidyl peptidase IV (EC 3.4.14.5) were measured in mature mouse decidual and visceral yolk sac epithelial cells by means of kinetic (continuous) and end-point (static) microdensitometry (section biochemistry). For continuous measurements a new device for starting the enzyme reaction allowed the first readings to be made already during its very early phase. Since the initial reaction rates of both peptidases were very different in the plasma membrane of decidual and also in the visceral yolk sac epithelial cells, it was difficult to select a sufficient number of cells of the same activity for representative measurements on the basis of kinetic microdensitometry. Static section biochemistry was performed also for statistical reasons, i.e., in order to obtain information about the distribution of the activities of the decidual and visceral epithelial cells and the number of measurements required to guarantee valid data. Various groups of decidual and visceral yolk sac epithelial cells with different gamma-glutamyl transpeptidase and dipeptidyl peptidase IV activities were formed. In this way, different activities of gamma-glutamyl transpeptidase were measured in the plasma membrane of the cells of the antimesometrial, intermediate, and mesometrial decidua. Compared with dipeptidyl peptidase IV, gamma-glutamyl transpeptidase was significantly more active in the plasma membrane of the antimesometrial decidual cells and microvillous zone of the visceral yolk sac epithelial cells.     

Ultrastructure and hydrolase cytochemistry of the developing marmoset yolk sac

Summary Yolk sacs from Callithrix jacchus were investigated light and electron microscopically as well as by qualitative light microscopic enzyme histochemistry on days 35 to 126 of gestation. The thin yolk sac wall of the early stages (day 35–41) consists of the cuboid, endodermal epithelium, the mesothelium of the exocoelom and some interposed blood vessels. The inner endodermal surface is rather smooth. At later stages, the epithelium becomes highly prismatic and forms folds which are lined by a mesenchyme and blood vessels. Microvilli and a small number of endocytotic vesicles are observed at the apices of the epithelial cells, which are interconnected by gap junctions, desmosomes and interdigitations. The cytoplasm of the epithelial cells is characterized by a well-developed rough endoplasmie reticulum, a large Golgi apparatus and glycogen deposits. Four different membrane-bordered types of inclusions can be distinguished in the cytoplasm of the epithelial cells: The type I and II inclusions are considered as secretion granules. Their increase and their localization in the cavities of the endoplasmic reticulum at later stages are ascribed to an inhibition of the intracellular transport at the onset of involution. The type III and IV inclusions may represent lysosomes and related organelles. Bile capillary-like spaces exist between the epithelial cells. The basement membrane is incomplete below the epithelium and absent around the capillaries, the endothelium of which is porous in certain areas. Aminopeptidase M is highly active in the plasmalemma and the bile capillary-like structures of the epithelium, dipeptidylpeptidase IV in the mesothelium and alkaline phosphatase in the blood vessel endothelium. Other membrane hydrolases are absent. Acid proteases, glycosidases, non-specific phosphatases and non-specific esterases can be detected stage-dependently with moderate to high activities in the yolk sac epithelium. Compared with other organs, the yolk sac structure and hydrolase equipment are similar to those of the liver and may, therefore, have similar functions, e.g. synthesis and secretion of proteins. In addition, however, the yolk sac epithelium might also be involved in resorptive processes of material from the lumen followed by lysosomal digestion. The Callithrix jacchus yolk sac starts involution on day 80 of gestation by disintegration of the cells. On day 100, this process is completed. the stage of involution which is late in comparison with other primates, e.g. man and Rhesus monkey, is ascribed to the strongly delayed development of Callithrix jacchus.Supported by the BMFT (Project CMT 35)     

Laminin in testicular germ cell tumours. An immunohistochemical study

Thirty-five testicular germ cell tumours comprising 16 yolk sac tumours, 15 embryonal carcinomas and 13 seminomas were examined for the presence and distribution of laminin using an indirect immunoperoxidase technique. In addition, nine normal yolk sacs and 23 carcinomas of the lung were studied. All the yolk sac tumours were positively stained for laminin. Both extra- and intracellular staining were found. Hyaline, eosinophilic material present within the tumours was positively stained, although with varying intensity. In 12 out of 15 embryonal carcinomas, laminin was found as a membrane staining but cytoplasmic staining also occurred. In 10 out of 13 classical seminomas, a membrane staining of many tumour cells was found, while cytoplasmic staining occurred in only a few seminomas. In all but one of the yolk sacs, laminin was present in the membrane beneath both the mesoblastic outer cell layer and the visceral endoderm. Intracellular staining was seen in some of the cells in both cell layers. In nine out of 23 carcinomas of the lung, laminin occurred extra- as well as intracellularly. Thus, this study showed that in normal yolk sacs the presence of laminin was not found to be particularly associated with any of the cell layers. Likewise, demonstration of laminin within yolk sac tumours did not define different patterns or subtypes of the yolk sac tumour. In addition, demonstration of laminin was not found to be useful in differentiating either between yolk sac tumours and embryonal carcinomas or between seminomas and non-seminomatous germ cell tumours. The findings add, however, interesting knowledge to histogenesis and embryogenesis.     

Protein absorption and transport by the guinea pig visceral yolk sac placenta

The cytological basis for protein transport across the guinea pig visceral yolk sac at 36–44 days of gestation was studied by means of electron microscopy following injection of horseradish peroxidase and ferritin. These results were compared with those obtained after administration of colloidal thorium dioxide. Distribution of the tracer molecules was studied at 2, 10, 20, 40, and 160 minutes after injection into the uterine lumen. All three tracer molecules were rapidly absorbed by endoderm cells. Although most of the protein appeared to be retained in droplets in endoderm cells, some protein was transmitted. Peroxidase was found to be rapidly transmitted across the yolk sac, ferritin somewhat more slowly, and colloidal thorium was not transmitted at all. Protein which had exited from the endoderm cells followed any of three pathways: (1) it crossed the visceral basement membrane and entered the vitelline capillaries; (2) it crossed the mesodermal compartment, crossed the mesothelial cells and entered the exocoelomic cavity; or (3) some of the protein was sequestered by macrophages in the splanchnic mesoderm. The pathways observed are consistent with those suggested by previous authors for the passage of maternal antibodies and serum proteins to the guinea pig fetus.     

Expression of heme oxygenase-1 in rat ontogeny

Heme oxygenase (HO), the heme-degrading enzyme, plays an important role in heme catabolism. Among three isozymes, HO-1 is an inducible form expressed mainly in macrophages. In rat ontogeny, HO-1 immunoreactivity was detected in mononuclear cells in the yolk sac at 10 days of gestation. HO-1-expressing cells were then detected in the fetal liver and their numbers increased during the gestational period. The numbers of HO-1-positive cells and HO-1 mRNA levels in the liver peaked at 18 days of gestation. Most of the macrophages expressed both HO-1 and a macrophage scavenger receptor. Macrophages in the fetal liver showed marked hemophagocytosis. Macrophages in the lung, spleen, bone marrow, and other tissues also expressed HO-1. HO-1 immunoreactivity was also observed in syncytial cells of the chorionic villi, the endodermal layer of the yolk sac, and renal tubules of the fetus. Intestinal mucosal epithelial cells expressed HO-1 after birth. These findings imply that HO-1 is crucial for macrophages in heme catabolism from an early stage of ontogeny. HO-1 expression in non-macrophagic cells may be required for other purposes such as protection from oxidative stress and various stimuli.