Organochlorine pesticides in chorioallantoic membranes of Morelet's crocodile eggs from belize

Recent studies examined the utility of the chorioallantoic membrane (CAM) as a nonlethal, noninvasive indicator of environmental contaminant exposure in oviparous wildlife. The CAM is a highly vascularized extraembryonic membrane that functions as a site for respiration, nutrient transport, and waste storage during embryonic development. After hatching, the CAM is usually discarded with the eggshell and can be used for chemical residue analysis. Chorioallantoic membranes have been used successfully to examine contaminant exposure and predict chemical concentrations in multiple species of birds and reptiles. In this study, we examined organochlorine (OC) pesticide concentrations in CAMs from eggs of Morelet's crocodiles (Crocodylus moreletii) from northern Belize. Multiple OCs were detected in crocodile CAMs, including aldrin, dieldrin, endrin, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene (DDE), heptachlor, lindane, and methoxychlor. Number and concentrations of OC compounds in CAMs were variable. The most prevalent contaminant detected was DDE, which occurred in 69% of CAMs, with concentrations ranging from 0.3 parts per billion (ppb) to 17.0 ppb. The OC burdens in crocodile CAMs confirm contamination of eggs and suggest exposure in embryos and maternal females. These results further support the use of CAMs as qualitative indicators of OC exposure in oviparous wildlife. The efficacy of this sampling technique in the field will depend on the logistics and cost associated with CAM collection and the specific life history traits of the wildlife species.     

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.     

A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein.

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

A Mouse Hepatoma Cell Line which Secretes Several Serum Proteins Including Albumin and α-foetoprotein

A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces α-foetoprotein (AFP).Three clones were isolated from the uncloned culture: BW1, BW2 and B WTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both B W1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least'five serum proteins into the culture medium: albumin, AFP, an a2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 μg/24 h/10⁶ cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.     

Pericytes in experimental MDA-MB231 tumor angiogenesis

The role of pericytes (PCs) during embryonic or tumor angiogenesis is a matter of debate. We studied the expression of cytoskeletal, membrane, and matrix markers in experimental tumors of the human mammary ductal adenoma MDA-MB231 cell line that were grown on avian chorioallantoic membranes (CAMs) from incubation day 10 to 18 (chick) or 8 to 15 (quail). The expression patterns of alpha-smooth muscle actin (alphaSMA) and desmin, of adhesion molecules beta1 integrin and neurothelin, and of fibronectin and laminin were analyzed with conventional and confocal laser scanning microscopy. The CAM arterial wall showed strong alphaSMA signal in all smooth muscle cell layers but the innermost layer, which was desmin positive. Ramified alphaSMA-negative cells with delicate desmin staining accompanied most minor vessels and were also seen basal to the capillary plexus indicating the presence of PCs. In the tumor nodules, a diffuse alphaSMA signal without definite relationship to vascular structures was detected. Strongly desmin-positive, alphaSMA-negative cells were frequent in the zone of contact to the CAM in small nodules, and were scattered in larger tumors. In some regions they were associated with microvessels, and in others appeared detaching from endothelial cells (ECs) or as single migrating cells. We conclude that: (a) the CAM tumor angiogenesis assay is useful for studying PC/EC interactions, (b) PCs are recruited from the CAM into experimental tumor nodules, (c) variability of vasculature in MDA-MB231 tumors may be due to variable PC/EC interactions, and (d) alphaSMA should be used with caution as a general PC marker.     

Enhanced survival of motoneurons in the chick lateral motor column: effects of embryonic skeletal muscle extracts and myoblast-conditioned medium

Muscle-derived factors have shown neurotrophic effects in culture, but their possible effects on the maintenance of embryonic motoneurons have not been demonstrated in vivo. Soluble extracts derived from embryonic chick muscle, or medium conditioned by chick myoblasts, were instilled onto the chorioallantoic membrane (CAM) between the 5th and 11th days of incubation. Counts of the lumbar lateral motor column (LMC) at embryonic day 12 revealed modest but significant increases (12-15%) in motoneuron number for these experimental groups as compared with control treatments. The results suggest that sustaining effects of muscle-derived factors on motoneurons may be demonstrated on the developing LMC by the simple expedient delivery via the CAM, and that these factors can modify the normal program of cell death occurring during this critical period of development.     

25 years of the study of alpha-fetoprotein

alpha-Fetoprotein (AFP) is an embryonic serum protein. Only traces of AFP are present in blood of adult man or animal, but its level substantially increases during liver regeneration and in cases of primary liver carcinoma or embryonic carcinomas. Different aspects of AFP studies (localization of its synthesis, structure and functions, genetic control of its synthesis and its use for cancer diagnostics) are reviewed. The main consideration is given to the cellular mechanisms of regulation of AFP synthesis. Hypothesis of "structural repression of AFP" is substantiated. According to it formation of liver trabeculae leads to suppression of AFP synthesis in hepatocytes, whereas escape of hepatocyte from trabecula results in activation of AFP synthesis. A new experimental model is described: AFP synthesis is activated in primary hepatocyte culture and is suppressed during three-dimensional growth of hepatocytes in collagen gel or mixed culture with nonparenchymal liver cells.     

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.     

Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary In developing and normal adult rat liver the expression patterns of the mRNAs for -fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific³⁵S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.     

Alpha-fetoprotein in the brain of developing rats and pigs. An immunofluorescent study of cell-and-tissue differentiation

Alpha-fetoprotein (AFP) and some other serum proteins have been studied in the developing brain of rats and pigs using the indirect immunofluorescence technique. AFP is shown to be present in the ventricular ependyma, meningeal envelopes, the choroid plexus, blood vessel walls and in a wide scale of differentiating parenchymal cells ever since early embryonic ages of both species. In brain parenchyma the content of AFP is low in immature germinative cells; in both species it starts increasing in postmigratory neuroblasts and reaches a peak at the time of accelerated nerve cell differentiation. In rats, the amount of AFP is highest towards the end of the first postnatal week; then it starts decreasing and disappears towards the end of the 3rd week. In both species AFP is localized in the cytoplasm of nerve cell perikarya and their differentiating processes. Higher concentration of this protein has often been observed at the axonal pole of the cytoplasm of differentiating pyramidal neurons. Immunoglobulin G has been found in non-parenchymal structures, and small amounts also in parenchymal cells of embryonic and early postnatal rats following a pattern of cell-and-tissue distribution similar to that of AFP. In pigs, a low amount of albumin has been shown in differentiating leptomeninges. These data suggest uptake of AFP, and some other serum proteins, from the cerebrospinal fluid into cells of the immature rat and pig brain and its increase (or higher binding) in differentiating neurons.     

Culture of postimplantation rat embryos in rabbit serum for the identification of the growth factor in fractionated rat serum.

The growth factor for postimplantation rat embryos was investigated on the basis of the serum species-specificity in supporting embryonic development in culture. We used rabbit serum as a basal medium for the culture of head-fold stage rat embryos, and examined the effects of various fractions of rat serum on their development. In rabbit serum alone, rat embryos developed poorly. With the rat serum ultrafiltrate of molecular weight (MW) < 300,000, embryonic development improved, but not with the ultrafiltrate of MW < 100,000. With dialyzed rat serum or the globulin fraction of rat serum, embryonic development improved, but the albumin fraction had no effect. It was concluded from these results that some macromolecular growth factor for cultured postimplantation rat embryos was present in the globulin fraction of rat serum. The molecular weight of this growth factor was estimated to be between 65,000 and 300,000. Rabbit serum was considered to be suitable as a medium for the identification of this growth factor.     

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.     

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.     

Adhesion among neural cells of the chick embryo. IV. Role of the cell surface molecule CAM in the formation of neurite bundles in cultures of spinal ganglia

The cell adhesion molecule (CAM) is involved in adhesion among embryonic retinal and brain cells and has been detected in a variety of neural tissues. This paper describes the use of spinal ganglion cultures and specific anti-CAM antibodies to determine the distribution of CAM on plasma membranes of nerve processes, and to assess the results of perturbation of its function during the growth of neurites from ganglia. The results indicate that CAM is distributed over the entire surface of nerve processes, and that specific anti-CAM Fab' fragments alter the morphology of neurite outgrowth. In particular, it was observed that anti-CAM inhibits formation of nerve bundles, so that the ganglion becomes surrounded by a tangled net of fine processes. Growth cone functions, such as neurite elongation, motility, and attachment to the substratum, did not appear to be affected by the antibody. These studies suggest that one of the major functions of CAM is to mediate side-to-side adhesion between neurites to form fascicles, and raise the possibility that this molecule serves a key role in embryogenesis of nerve tissues.     

Lateral transfer of a lectin-like antifreeze protein gene in fishes

Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.     

Alpha-fetoprotein is dynamically expressed in rat pancreas during development

To identify proteins involved in pancreatic development, we used a differential proteomics approach by comparing pancreatic extracts from four biologically significant stages of development: embryonic day (E) 15.5, E18.5, postnatal (P) days 0 and adult. By two-dimensional gel electrophoresis (2D-E) and MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) following database searching and protein annotation, 15 proteins were identified as being differently expressed in the pancreas between the four phases. The expression pattern and the localization of alpha-fetoprotein (AFP), one of significant changed proteins observed, were further determined. Four isoforms of AFP (72 kDa, 60 kDa, 48 kDa and 37 kDa) were found by Western blotting in the pancreas tested, most of them showed a stronger signal in E18.5 followed by a steady decrease and only a 60-kDa isoform was detected in the adult pancreas. Immunolocalization for AFP revealed that a positive reactivity was detectable at E15.5 pancreas, became stronger in the cytoplasm of mesenchyme cells at E18.5, and declined after birth to a nearly undetectable level in adults. The dynamic expression of AFP in rat pancreas from different stages indicates that AFP might be involved in some aspects of pancreatic development.     

RNA-seq analysis of the active chick embryo chorioallantoic membrane reveals genes that encode proteins assigned to ion transport and innate immunity

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology.     

A DNA vaccine against chimeric AFP enhanced by HSP70 suppresses growth of hepatocellular carcinoma

Alpha-Fetoprotein (AFP) is produced principally in fetal liver, gastrointestinal tract and the yolk sac which is temporarily present during embryonic development. AFP is overexpressed in the majority of Hepatocellular Carcinoma (HCC) and thus offers an attractive target for immunotherapy against this neoplasm. Here, we report that anti-HCC effects were achieved in a therapeutic setting with a DNA vaccine encoding mouse AFP and co-expressing Heat Shock Protein 70 (HSP70) gene. We also demonstrated that this vaccine elicited a marked and highly effective AFP specific CTL response against AFP-positive target cells. This vaccine also induced the prolongation of life span in mice bearing the tumor and the eradication of HCC. It is anticipated that vaccine strategies such as this may contribute to the effective future treatment of Hepatocellular Carcinoma. Ying-hua Lan and Yong-guo Li contributed equally to this work.