Embryonic Chorioallantoic Membrane Research Articles

Calcium-transport function of the chick embryonic chorioallantoic membrane. II. Functional involvement of calcium-binding protein, Ca2+-ATPase and carbonic anhydrase.

This study aimed to investigate the mechanism of active calcium transport in the chick embryonic chorioallantoic membrane (CAM) by assessing the functional involvement of three previously identified, putative components of the transport pathway. These components are a calcium-binding protein (CaBP), Ca2+-activated ATPase and carbonic anhydrase. Using specific reagents, including antibodies and enzyme inhibitors in vivo and in vitro in CAM calcium uptake assays, it was shown that these biochemically identified components were all functionally involved. The results of these studies also indicate that active calcium uptake by the CAM requires the presence of the CaBP on the cell surface in a laterally mobile manner, while carbonic anhydrase appeared to be a cytosolic component. We further analysed the subcellular location of the calcium-uptake activity by gel filtration and density-gradient fractionation of cell-free microsomes of the CAM and the results suggest that this activity is associated with the plasma membrane.

Calcium-activated ATPase of the chick embryonic chorioallantoic membrane. Identification, developmental expression, and topographic relationship with calcium-binding protein.

A Ca2+-activated ATPase activity is present in the chick embryonic chorioallantoic membrane (CAM), the placenta-like tissue which translocates eggshell calcium into the embryonic circulation. The enzyme is membrane-bound, ATP-specific, Mg2+-dependent, exhibits dual Km values of 30 microM and 0.3 mM Ca2+, and has a Mr of 170,000. Throughout embryonic development, a single electrophoretic form of the Ca2+-ATPase is found and, furthermore, its specific activity as a function of age follows a bimodal pattern. In particular, from incubation days 14-15 to the end of gestation, a period representing rapid embryonic calcium accumulation, Ca2+-ATPase specific activity increases 6-fold. Cytohistochemistry localized the Ca2+-ATPase exclusively within the CAM ectoderm which lies adjacent to the calcium-rich shell membrane/eggshell. In a parallel study, cleavable bifunctional cross-linking agents were used to characterize the in situ protein topography of the CAM ectodermal surface adjacent to the calcium-binding protein (CaBP), a CAM cell-surface protein associated with calcium transport. We found that the immediate near neighbor of the CaBP is a 170,000 Mr, membrane-bound protein. The 170,000 protein was co-isolated with the CaBP after cross-linkage in situ and subsequent immunoprecipitation with anti-CaBP antibodies. Reductive cleavage of the immune complex released detectable Ca2+-ATPase activity, suggesting that the 170,000 protein is the Ca2+-ATPase of the CAM.

Supplemented eggshell restores calcium transport in chorioallantoic membrane of cultured shell-less chick embryos

It was previously reported (Tuan, 1980a) that the development-specific expression of calcium transport and related functions in the chick embryonic chorioallantoic membrane (CAM) requires the continuous presence of the eggshell, the calcium source of the embryo. To further understand the mechanism of action of the eggshell on the CAM functions, this study reports the effects of eggshell supplementation on chick embryos maintained in shell-less cultures. The cultured embryos were able to accumulate and utilize the exogenous shell calcium, applied directly onto the CAM, for skeletal formation. In the region of the CAM directly adhering to the added shell, calcium transport activity, calcium-binding protein (CaBP) activity, and vitamin K-dependent gamma-glutamyl carboxylase activity were significantly restored. These results strongly suggest that the proximity of shell calcium may regulate expression of calcium transport and related functions in the chick embryonic CAM.

Identification and characterization of a calcium-binding protein from human placenta

The human placenta has been found to contain a specific calcium-binding protein (HCaBP) which increases in amount as a function of gestation. A procedure is described for the purification of the HCaBP to chromatographic and electrophoretic homogeneity. The HCaBP is (a) a high molecular weight protein with a native Mr of 150 000 and appears to be a dimer of subunits of Mr = 70 000; and (b) an acidic protein with a pI of 4.6. Amino acid analysis of the HCaBP revealed an abundance of acidic amino acid residues (27 per cent = Asp and Glu). A specific antiserum has been prepared against purified HCaBP. With Ouchterlony double immunodiffusion, the HCaBP appears to be specific for the human placenta, and is not cross-immunoreactive with the CaBP of the chick embryonic chorio-allantoic membrane.

Biosynthesis of calcium-binding protein of chick embryonic chorioallantoic membrane: In vitro organ culture and cell-free translation

Biosynthesis of the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM) was studied using in vitro organ culture and cell-free translation. The organ culture studies showed: 1) The CaBP is a relatively stable protein ( t 1 2 = 60 h ); 2) Biosynthesis of the CaBP involves microsomes and includes two posttranslational modifications, glycosylation and γ-glutamyl carboxylation; and 3) During embryonic development, a single species of the CaBP is expressed in the CAM. Cell-free translation of total CAM mRNA, including CaBP mRNA, was achieved in a rabbit reticulocyte lysate system using [ 35S]Met as a tracer. Based on the properties of the nascent CaBP polypeptide translated in the presence or absence of microsomal membranes, the early stages of CaBP synthesis appear to be: translation of CaBP mRNA on membrane-bound polysomes, insertion and translocation of the nascent polypeptide across microsomal membranes, and co-translational cleavage of a signal sequence.

Fine structural changes associated with the onset of calcium, sodium and water transport by the chick chorioallantoic membrane

An attempt is made to correlate structure and transport function in the embryonic chorioallantoic membrane. The fine structure of the endoderm and ectoderm in the membrane was examined with particular attention given to the morphological changes that occur when transport is established,in vivo. Two distinctive cells, a granule-rich cell and a mitochondria-rich cell, appear in the endoderm at the time allantoic fluid sodium, chloride and water reabsorption commences. These are indistinguishable from the cells described in toad bladder epithelium. It is suggested that the granule-rich cell is responsible for bulk water movement and the mitochondria-rich cell is specifically engaged in active sodium transport. In the ectoderm, two distinctive cell types are also found to be associated with the onset of active calcium transport. These are referred to as the capillary-covering cell and the villus-cavity cell. The preponderate capillary-covering cell is most likely responsible for transcellular calcium transport. It is postulated that the function of the villus-cavity cell is to secrete hydrogen ions which are necessary, along with carbonic anhydrase, to mobilize Ca(++) from the insoluble calcium carbonate of the eggshell.