Ultrastructural localization of calcium in the chick chorioallantoic membrane as revealed by cytochemistry and X-ray microanalysis

Abstract

The chorioallantoic membrane (CAM) of the chick embryo actively transports calcium from the egg shell into the embryonic circulation. To investigate the intracellular pathway of calcium transport across the CAM, ultrastructural localization of intracellular calcium in cells of the chorionic ectoderm (CE) was determined using cytochemical methods and X-ray microanalysis. Treatment of the CE with potassium oxalate, potassium ferricyanide or potassium pyroantimonate revealed large numbers of electron-dense granules (EDGs) in the ectodermal cells. These measure 30-40 nm in diameter, and are not membrane-bound. These granules were seen in all three cell types of the CE. The presence of calcium in the EDG was directly confirmed by X-ray microanalysis. When strontium or barium ions were applied to the shell membrane side of the CAM, the cells of the CE incorporated these divalent cations and sequestered them in granules (25-40 nm in diameter) in cytoplasm and mitochondria. This study indicates that calcium enters the CE cells by means other than endocytosis, as the EDGs are not membrane-bound, that all three types of the CE cells appear to function in transport of calcium from shell to embryo during embryogenesis, and that the EDG plays important roles in intracellular accumulation of calcium during the process of calcium transport across the chorioallantoic membrane.