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

Summary 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.Supported by NIH Grant AI 13486-01     

Direct yolk sac volume manipulation of zebrafish embryos and the relationship between offspring size and yolk sac volume

Through direct manipulation of yolk sac volume ( V _y), in the zebrafish Danio rerio , V _y and offspring size was positively correlated and the relationship was independent of geographical location, parental size or age and, most importantly, parental genetic factors. Larval survivorship, under a non-feeding regime and up until hatch, was not significantly affected by the manipulation. Decreased V _y significantly decreased maximum standard length ( L _S), maximum body surface area ( A _B), time to yolk sac absorption, L _S and A _B at yolk sac absorption, and the L _S and A _B at starvation. The methodology of V _y adjustment will be useful for the studies of the interaction between early life-history traits and offspring size, egg quality variables and early vertebrate development.     

On the yolk sac of the cat

Summary The phase of primitive erythropoiesis in the feline yolk sac lasts from the 14th to the 20th day after mating. The globular nucleated primitive erythroblasts are formed extravascularly to some extent, but they can be clearly distinguished from the endoderm. They do not undergo a denucleation and are still present in the circulating blood on the 45th day. Aging primitive erythroblasts are characterized by a loss of polysomes, by the appearance of long intracytoplasmic electron-lucent channels, and by a nuclear pyknosis which can turn into a karyolysis. Definitive erythropoiesis begins around the 17th day but, even by the 19th day, it is not particularly prominent. It ends around the 45th day. It is almost exclusively intravascular. The distinction of immature primitive erythroblasts from erythroblasts of the definitive series is difficult, because it is based upon only slight differences in the heterochromatinization, in the nuclear-cytoplasmic ratio, and in the organelle content of the cells. In the definitive series, the nuclear divisions follows the law of the rhythmical halving of the nuclear volume. The cells exhibit more clearly identifiable maturation stages here, and the checkerboard nucleus is more distinct. The vascular endothelium is largely attenuated and moderately fenestrated; it lacks a distinct basement membrane. Organelle-rich adventitial cells are found in close apposition.     

Secretion and glycosylation of alpha-foetoprotein by the mouse yolk sac.

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.     

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 hemopoiesis 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 vitelline 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.     

Ligation of the yolk sac circulation and its effect on the yolk sac ultrastructure and development of the rat fetus]

The effect of an interruption of the yolk sac circulation on the rat visceral yolk sac and the development of the fetoplacental unit was examined in the last third of pregnancy. The yolk sac ischaemia was induced by ligating the blood vessels of the yolk sac stalk which connect the vitelline circulation with that of the fetus. A 3-hour ligature caused an extensive swelling of most cell organelles in the epithelial cells and in the capillary endothelia of the yolk sac. Other structural changes were indicative of a cessation of pinocytosis. A 6-hour ligature resulted in a common increase of cell swelling and in a beginning disintegration of the endothelial cells lining the vitelline capillaries. A 15-hour ligature caused severe ultrastructural cell lesions and macroscopical alterations suggestive of a progressive necrolar finding of a nearly complete loss of the amniotic fluid and the death of the fetus, although the maternal blood flow appeared to be still intact in the placenta.     

Glycans of the early human yolk sac

Summary The pattern of glycan distribution in the early human yolk sac has been investigated using a panel of lectins. Two 6-week and one 8-week human yolk sacs, and one 8-week fetal liver from live, ectopic pregnancies were fixed and embedded in epoxy resin. Lectin histochemistry was carried out on sections of these tissues using 23 biotinylated lectins and an avidin-biotin peroxidase revealing system. Mesothelial surfaces expressed most subsets of N-glycans (other than high mannose types),N-acetyl-lactosamine, sialic acid, andα1,6-N-acetylgalactosamine. Endodermal surface and lateral membranes resembled those of mesothelium, but showed a preponderance ofα2,6-sialyl residues. Most intracellular granules contained N-glycan. There was a marked heterogeneity of granules in the endodermal cells, with different subsets varying in both staining and positional characteristics. The mesenchymal matrix bound most of the lectins used in the study, and expressed fucosyl residues which were also detected in the endothelium. Fetal liver parenchyma showed very similar staining patterns to those seen in the endoderm except for the distribution ofN-acetylglucosamine, which was sparse. Despite some common features, each germ cell layer had a distinct ‘glycotype’, with some saccharides showing extreme topographical restriction.     

The pig yolk sac II

Summary Since previous morphological studies have revealed abundant rough endoplasmic reticulum in the yolk sac endoderm, pig yolk sac explants from 30 day old embryos were incubated for 3–12 h with [³H]-l-leucine in order to study their protein biosynthesis. They were fractionated into a 12,000×g-pellet, 105,000×g-pellet, and supernatant. Newly synthesized proteins in these tissue fractions, and proteins discharged into the culture medium, were analysed with the aid of scintillation technique and identified by column chromatography, SDS-polyacrylamide gel electrophoresis with urea, isoelectrofocusing, and 2D-electrophoresis. Most of the radioactivity incorporated into the tissue fractions was regained from the coarse pellet and was located in the molecular weight region between 70,000 and 45,000 daltons, indicating that most of the newly synthesized proteins are membrane bound and include albumin. Albumin, an acid protein of a MW around 80,000 daltons, and many neutral and basic peptides were present in the culture medium. The yolk sac endodermal cells of the pig synthesize less proteins than those of the cat, although the pig cells display much larger amounts of endoplasmic reticulum.     

The pig yolk sac I

Summary Yolk sacs from pig embryos ranging between 18 mm and 55 mm in length were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and histochemistry. The organ was no longer present in embryos of 70 mm length. The endoderm proliferates in embryos of about 20 mm length with gland-like endodermal cell columns and finally becomes stratified, representing over 90% of the yolk sac mass. The endodermal cells show a high activity of oxidoreductases and lysosomal enzymes; their luminal surface bears few absorptive specializations. The mesothelium is inert, as judged from its surface ultrastructure, organelle composition and enzyme content. TEM reveals the endodermal cells to be polarized even in stratified areas. They resemble liver parenchymal cells with respect to their basal villi, which are exposed to capillaries with discontinuous or fenestrated endothelium. Giant mitochondria with crystalline inclusions in the mature endodermal cytoplasm are outnumbered by large stacks of the rough ER, which can amount to 60% of the cytoplasm. This conspicuous RER is suspected to be the production site of serum proteins which are discharged into the vascular bed. Close to the time of the organ's regression, an unusual storage of material in terminal buds of the ER was found. Intercellular canaliculi and the endocytic apparatus of the endoderm are thought to serve regression.     

The human foetal yolk sac

Summary Specimens of human foetal yolk sac from conceptuses of 8 and 10 weeks menstrual age were studied with the electron microscope. At 8 weeks columns of endodermal cells projected into the underlying mesenchyme. Several types of endodermal cell were identified; some contained much granular endoplasmic reticulum and abundant glycogen; others resembled the haemocytoblasts present in the mesenchyme and yet others contained membrane-bounded channels similar to those seen in megakaryocytes. It was suggested that the endoderm is the site of origin of the blood cells but that, while the platelets may be formed within the endoderm, the normal development of the red cells is conditional upon their early release into the mesenchyme and possibly the attainment of an intravascular position. Intravascular macrophages were identified and their role in determining the nature of the blood picture during the period of functional acitvity of the sac discussed. The morphology of the epithelium on the external surface of the sac was discussed in relation to the possibility of its playing a part in the exchange of materials between the yolk sac and the chorionic cavity.Supported in part by grant no. 5-T01-GM-00582-08 from the U.S. Public Health Service.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

In vitro studies on the effect of yolk sac antisera on functions of the visceral yolk sac: I. Pinocytosis and transport of small molecules

The production of congenital malformations by the administration of teratogenic antisera to pregnant animals has been reported from many laboratories. This work has focused our attention on the importance of the yolk sac placenta in supporting the rat embryo during early organogenesis and the significance of yolk sac dysfunction in rodent teratogenesis. The studies reported in this article deal with the effect of teratogenic antisera on the process of yolk sac transport; specifically pinocytosis (as measured by 14C-sucrose uptake) and small-molecule transport utilizing 14C-alpha-aminoisobutyric acid (AIB) and 3H-2-deoxyglucose (DOG). We sought to determine whether several different yolk sac localizing antibodies interfere with these transport processes, and, if so, which transport processes were most affected. The results of the experiments indicated that teratogenic antisera interfered with the process of pinocytosis in the yolk sac and that pinocytosis can be reduced as much as 40%. Nonteratogenic antisera, even when they localized in the yolk sac, did not interfere with the process of pinocytosis. Furthermore, the teratogenic antisera did not interfere with the transport of small molecules (either AIB or DOG) in the yolk sac. These results indicated that while fluorescent localization of an antiserum in the yolk sac did not invariably indicate the potential for teratogenicity, it is likely that the reduction in pinocytosis may directly correlate with the teratologic and embryopathic events. This work reaffirms the view that the yolk sac in important during rodent organogenesis and that yolk sac dysfunction can play an important role in the development of congenital malformations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.     

Experimental manipulation of the rodent visceral yolk sac

The visceral yolk sac (VYS) is an especially important placental organ in the rodent because it is the primary source of exchange between the embryo and mother during early organogenesis before the chorioallantoic placenta circulation is established. The VYS is involved with nutritional, endocrine, metabolic, immunologic, secretory, excretory, and hematopoietic functions. The VYS also plays a role in steroid metabolism and interacts with a variety of blood-borne factors: parathyroid hormone, glucocorticoids, insulin, and vitamin D metabolites. The importance of the VYS during development is emphasized by the embryotoxicity resulting from exposure to agents which cause VYS dysfunction when administered to the pregnant animal during organogenesis. Several experimental procedures have provided useful information concerning a variety of VYS functions from early organogenesis to term: Culture of the Embryo, Fetal Incubation, Culture of the Fetus, Giant Yolk Sac, Short- and Long-Term Culture of the Yolk Sac, Modified Ussing's Chamber, Single or Double Diffusion Chamber, and the use of Heterologous Rodent Visceral Yolk Sac Antibodies. Since human yolk sac pathology has been associated with developmental toxicity and spontaneous abortion, it is important to discover whether there are some common functional roles among different mammalian species and to determine if other experimental animal models can be used to study the possible contribution of human yolk sac dysfunction to some human reproductive problems.     

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.     

alpha 1-Fetoprotein mRNA of rat yolk sac and hepatoma.

Rat alpha 1-fetoprotein mRNA was isolated and purified to apparent homogeneity by means of immunoadsorption and oligo (dT) cellulose affinity chromatography. Purified AFP mRNA migrated as a 21S peak in 2.5% SDS-polyacrylamide gels. The translation product of this mRNA in micrococcal nuclease treated reticulocyte lysate was identified as AFP by specific immunoprecipitation, SDS-gel electrophoresis and tryptic digestion analysis. DNA complimentary to AFP mRNA was synthesized with avian meyloblastosis virus RNA-dependent DNA polymerase. This AFP cDNA was used as a probe to quantitate AFP mRNA in the developing rat liver and to compare the complexity and diversity of AFP mRNA derived from the normal rat liver and Morris hepatoma 7777. We found that the amount of functional AFP mRNA is decreasing during liver development. There is very little, if any, AFP mRNA in the adult rat liver. A high degree of homology between the AFP mRNA sequences of yolk sac and hepatoma was also found.     

Alpha-fetoproteins produced by the yolk sac and the liver are glycosylated differently.

Chromatography of mouse alpha-fetoprotein (AFP) on concanavalin A (con A)-Sepharose gave two main AFP variants, one nonreactive with con A and the other bound and eluted with α-methylmannoside. The nonreactive variant showed partial reactivity with Ricinus communis agglutinin I indicating that it is at least partly glycosylated. The nonreactive fraction greatly diminished after birth when the yolk sac is lost. Internally labeled AFP was almost completely nonreactive with con A when obtained from cultures of visceral yolk sac, while more than 90% of AFP from the liver bound to con A. These results show that a developmental change takes place in the glycosylation pattern of AFP as its synthesis shifts from the yolk sac to the liver.