Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.     

An Instruction on the In Vivo Shell-Less Chorioallantoic Membrane 3-Dimensional Tumor Spheroid Model

The traditional shell chicken chorioallantoic membrane (CAM) model has been used extensively in cancer research to study tumor growth and angiogenesis. Here we present a combined in vivo tumor spheroid and shell-less CAM three-dimensional model for use in quantitative and qualitative analysis. With this model, the angiogenic and tumorigenic environments can be generated locally without exogenous growth factors. This physiological model offers a stable, static and flat environment that features a large working area and wider field of view useful for imaging and biomedical engineering applications. The short experimental time frame allows for rapid data acquisition, screening and validation of biomedical devices. The method and application of this shell-less model are discussed in detail, providing a useful tool for biomedical engineering research.     

Acute effects of prostaglandin E(1) and E(2) on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.     

A scanning and transmission electron microscopic study of the membranes of chicken egg.

Questions regarding the structure of the inner and outer shell membranes of the chicken egg were addressed in this study by correlating observations from light microscopy and scanning and transmission electron microscopy. The egg membrane had a limiting membrane, which measured .9 to .15 microns in thickness and appeared to be a continuous and an impervious layer, but the shell membrane did not. Under the SEM, each membrane was seen to be made up of several fibre layers. In the tear preparations viewed under the SEM two layers were observed in the egg membranes and three to five layers in the shell membrane, with an apparent plane of cleavage between each layer. Each fibre was made up of a central core and an outer mantle layers. The central core was perforated by channels which measured .08 to 1.11 microns in diameter and ran longitudinally along the length of the fibre. Between the mantle layer and the fibre core was a gap or cleft measuring between .03 to .07 microns. The diameter of the fibres of the inner layer of the egg membrane ranged between .08 to .64 microns, whereas those of the outer layer of the same membrane ranged from .05 to 1.11 microns. Fibres in the shell membrane ranged from .11 to 4.14 microns diameter.     

Anatomical arrangement of hypercapnia-activated cells in the superficial ventral medulla of rats.

The anatomical structure of central respiratory chemoreceptors in the superficial ventral medulla of rats was studied by using hypercapnia-induced c-fos labeling to identify cells directly stimulated by extracellular pH or PCO(2). The distribution of c-fos-positive cells was found to be predominantly perivascular to surface vessels. In the superficial ventral medullary midline, parapyramidal, and ventrolateral regions where c-fos-positive cells were concentrated, we found a common, characteristic, anatomical architecture. The medullary surface showed an indentation covered by a surface vessel, and the marginal glial layer was thickened. We classified c-fos-positive cells into two types. One (type I cell) was small, was located inside the marginal glial layer and close to the medullary surface, and surrounded fine vessels. The other (type II cell) was large and located dorsal to the marginal glial layer. c-fos Expression under synaptic blockade suggested that type I cells are intrinsically chemosensitive. The chemosensitivity of surface cells (possible type I cells) surrounding vessels was confirmed electrophysiologically in slice preparations. We suggest that this characteristic anatomical structure may be the central chemoreceptor.     

Irritative effects of some pesticides and a technical component on tissue structure of the chorioallantoic membrane

The chorioallantoic membrane (CAM) is a complete tissue that responds to injury with a complete inflammatory reaction, this process similar to that induced by chemicals in the conjunctival tissue of the rabbit eye. During the study chemicals are placed directly onto the chorioallantoic membrane and the occurrence of vascular injury or coagulation in response to a compound is as an indication of the potential of a chemical to damage mucous membranes. In our study irritant pesticides (Fusilade S, Karathane LC) and a technical pesticide component (Trend) were tested and their effects on the tissue structures of CAM were examined. After treatment with the test materials, first lysis and then haemorrhage were observed macroscopically on CAM. In histological pictures stained with H-E the rupture of the blood vessel wall was seen and blood was observed around the blood vessels in the middle layer. The histological findings correlated well with the macroscopic appearance in this study. In general a good correlation was found between the HET-CAM results and reported data from Draize test. The subjective nature of the evaluation is reduced through the histological examination of treated CAM. The HET-CAM test can be a useful component of a battery of tests needed for replacing the Draize rabbit eye irritation test.     

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.     

Increased Blood Levels of Human S100 in Melanoma Chick Embryo Xenografts’ Circulation

The chorioallantoic membrane (CAM) of the fertilized egg allows grafting of human melanomas for short‐term investigations and offers the opportunity to investigate the behavior of metastasizing cells and the release of S100β into peripheral blood. Tissue from one primary melanoma as well as cutaneous and subcutaneous metastases of 10 melanoma patients with elevated levels of S100 in the peripheral blood before surgery were transplanted onto the CAM of chick embryos at day 5/6 of development. Grafts were nourished by the host blood supply 2 days after transplantation. Histologically, 3 days after grafting, metastasizing melanoma cells could be found near the vessels of the host membrane, penetrating the endothelial layer and entering the blood system. Growth conditions remained stable for 6 days after transplantation. Blood samples were taken from a larger CAM vessel before collecting the xenografts 5 days after grafting. Measurement of human S100 in peripheral blood was performed in a blinded manner. No negative control showed elevated levels of human S100 protein. Samples deriving from melanoma xenografts contained highly elevated levels of S100 protein in 80% of cases. The data strongly support the concept of graft–host interaction concerning adherence of tumors and extravasation of human melanoma cells.     

Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation

The chorioallantoic membrane (CAM) of the fertilized egg allows grafting of human melanomas for short-term investigations and offers the opportunity to investigate the behavior of metastasizing cells and the release of S100beta into peripheral blood. Tissue from one primary melanoma as well as cutaneous and subcutaneous metastases of 10 melanoma patients with elevated levels of S100 in the peripheral blood before surgery were transplanted onto the CAM of chick embryos at day 5/6 of development. Grafts were nourished by the host blood supply 2 days after transplantation. Histologically, 3 days after grafting, metastasizing melanoma cells could be found near the vessels of the host membrane, penetrating the endothelial layer and entering the blood system. Growth conditions remained stable for 6 days after transplantation. Blood samples were taken from a larger CAM vessel before collecting the xenografts 5 days after grafting. Measurement of human S100 in peripheral blood was performed in a blinded manner. No negative control showed elevated levels of human S100 protein. Samples deriving from melanoma xenografts contained highly elevated levels of S100 protein in 80% of cases. The data strongly support the concept of graft-host interaction concerning adherence of tumors and extravasation of human melanoma cells.     

Isolation and biological activity of the proteases released by sea urchin eggs following fertilization.

The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity chromatography on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low pH.The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between 10⁵ and 10⁶ were demonstrated by gel filtration.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

Chick embryo culture using duck egg shell--first successful hatch

The suitability of duck egg shell (DES) for chick embryo culture was investigated. Chick embryos were transferred into DESs with all egg contents after 3 days of normal incubation and cultured. The vessels made of polyethylene cling film were used for shell-less control. Among 35 embryos cultured in DESs, 21 survived until 16 days of incubation (13 days after transfer) and finally 3 newly hatched chicks were obtained at 22 days of incubation. One of them died 4 days later, but remaining two became full-grown cocks showing normal body weight and production of fertile sperms. Among 37 embryos cultured in polyethylene vessels, none survived over the period of 19 days of incubation. It is suggested that DES culture system may be useful for the various experiments using chick embryos.     

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

The Structure and Mineralization of the Carapace of the Crab (Cancer pagurus L.) 3. The epicuticle

Decalcified and undecalcified preparations of the crab Cancer pagurus in the intermoult condition were studied to examine the mineralization and structure of the epicuticle, using light microscopic, electron microscopic, and microradiographic methods. The epicuticle was found to be composed of two layers, one superficial membrane, and one thicker layer, measuring 1-2 μm. From the base layer spines or microtrichia projected. These were approximately 18 μm long and built like the remainder of the epicuticle. The spines and the base layer of the epicuticle contained vertical canals which in undecalcified sections accomodated columns of crystals. These canals were the only location in which minerals occurred in the epicuticle. In decalcified preparations filamentous strands were observed in the canals. Elsewhere in the epicuticular tissue no fibrillar structure was observed. The canals and their contents seemed to extend across the junctional zone between the epicuticle and the exocuticle.     

STUDIES ON AMPHIBIAN YOLK : 2. The Isolation of Yolk Platelets from the Eggs of Rana pipiens

Previous methods which employed simple sucrose or salt solutions to isolate yolk platelets have failed to preserve their superficial layer, and the preparations obtained generally exhibit some contamination when observed with the light or electron microscope. When yolk platelets are suspended in a sucrose-polyvinylpyrrolidinone medium, however, they remain relatively intact and their superficial layer is not lost to the medium. A method, which takes advantage of this fact, is described for the isolation of frog (Rana pipiens) yolk platelets which are free from nuclear contamination and practically free from cytoplasmic contamination. After such platelets are treated with distilled water, the superficial layer is no longer seen and a new dense and granular matrix is frequently found surrounding the crystalline main body. The significance of this and other observations concerning the effects of calcium and ethylenediamine tetraacetate are discussed.     

Distributional characteristics of lymphatic vessels in normal human nasal mucosa and sinus mucosa

An immunohistochemical staining technique with the D2-40 antibody was undertaken to examine the functional and morphological features of lymphatic networks in tissue sections and whole-mount preparations of normal nasal mucosa and ethmoid sinus mucosa. In normal nasal mucosa, most lymphatic vessels were found in the superficial mucosa beneath the epithelial layer. Some of these vessels were dilated, whereas others were compressed and had a slit-like lumen. Whole-mount preparations revealed the extent of lymphatic vessels in normal ethmoid sinus mucosa. A network of lymphatic vessels was mainly found in the subepithelial layer, where lymphatic vessels represented rich networks, possessing antler-like branches and typical blind ends. However, these lymphatic networks were not arranged in the form of lymphangion chains, with each lymphangion consisting of a contractile compartment and valve. Thus, recognition of the distinctive features of the lymphatic network in normal nasal and sinus mucosa might aid investigations of lymphatic involvement in sinonasal diseases, such as rhinitis, sinusitis, and malignancy. This work was supported by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea.     

Observations on the Formation and Ultrastructure of the Egg Membrane in the fern Ceratopteris thalictroides (Parkeriaceae)

The mature egg of ferns possesses a layer of egg membrane in the periphery. However, the fine trastructure and the formation of the egg membrane in the egg of ferns are still unclear. The present paper described the formation and the ultrastructure of the egg membrane in the egg of the fern Ceratopteris thalictroides using transmission electron microscopy (TEM). The results show that the egg membrane begins to form at the stage of the maturing egg. The egg membrane in the upper side of the egg is prominent. It is formed by attaching of osmiophilic sheets of endoplasmic reticulum to the inner surface of the plasmalemma . When mature the egg membrane in the upper side of the egg is much thick in the central region and it becomes thinner gradually towards the margin. Some osmiophilic materials are filled in the spaces between the sheets of endoplasmic reticulum . On the contrary, the egg membrane in the lower side of the egg is thin . It is composed of twoosmiophilic membranes which are associated with each other closely. The formation and the ultrastructure of the egg membrane are described for the first time . Some functions of the egg membrane are also discussed .     

寒武纪宽川铺生物群中球状胚胎化石表面刺状结构及其意义*

以宽川铺生物群中获取的磷酸盐化立体保存的胚胎化石为研究材料,对胚胎化石表面超微结构和内部解剖结构进行观察并对比分析。结果显示,大部分胚胎化石表面被一单层光滑卵膜结构所包裹,卵膜之下胚胎形态多样;刺状结构位于卵膜之下,为原生质胚胎分泌而成的保护层,出现在不同大小和不同类型的胚胎中。结果表明:1)单一依靠胚胎大小或者表面结构不能作为胚胎的分类依据;2)与现生海葵等动物胚胎表面微绒毛相似,Olivooides表面刺状结构可能具有保护胚胎和生态扩散的功能;3)带刺Olivooides很可能发育成为动物演化早期最简单的带刺幼虫形式,是动物间接发育的证据。     

On the infiltration of Golgi bodies from the follicular epithelium to the egg

Summary 1. In a well advanced oocyte of the tortoise the egg membranes besides the theca and the single-layered epithelium consist of a zona pellucida often differentiated into zona striata and a homogeneous layer; underlying these two layers is a layer of cortical fibrillae or fibrillar layer, Next to this layer, is the limiting membrane of the egg which is not present in all stages and generally disappears in a well developed oocyte. In certain animals either the homogeneous layer or fibrillar layer is absent. 2. In certain animals,Golgi bodies seem to be extruded into the follicle cells from the theca cells. 3. At a particular stage of development the follicle cells become very active and produce a large number of smallGolgi bodies. TheseGolgi granules filter through canalicular passages of the zona radiata into either the homogeneous layer and from thence into the fibrillar layer or where a homogeneous layer is not present directly to the fibrillar layer. Where a fibrillar layer is not present they are transferred directly to the limiting membrane and from thence to the egg. 4. In certain cases e. g. in Fowl, Calotes and Uromastix, fairly large lumps ofGolgi bodies are extruded from the follicle cells through the zona pellucida into the egg. Here the fine canilicular passages do not seem to form a vehicle for the passage of these comparatively larger bodies. 5. The fine canalicular passages in the zona radiata ofTestudo graeca andKachuga smithii and the fibrillar prolongation of the cytoplasm which we have called the fibrillar layer are marked features of the egg membranes at certain stages of development of the egg. During the period when infiltration ofGolgi bodies through these passages takes place slides prepared by silver nitrate and osmic methods show black beaded chains ofGolgi granules in various stages of descent. 6. It is claimed that the extrusion and infiltration ofGolgi bodies from the follicular epithelium to the egg are established phenomena at least in the Vertebrates.