Concomitant Morphological Modifications of the Avian Eggshell, Eggshell Membranes and the Chorioallantoic Membrane During Embryonic Development

Abstract

The chicken eggshell (ES) consists of 95% calcium carbonate and 3.5% organic matter, and represents the first physical barrier to protect the developing embryo, while preventing water loss. During the second half of development, calcium ions from the inner ES are progressively solubilized to support mineralization of the embryonic skeleton. This process is mediated by the chorioallantoic membrane (CAM), which is an extraembryonic structure that adheres to the eggshell membranes (ESM) lining the inner ES. The CAM surrounds the embryo and all egg contents by day 11 of incubation (Embryonic Incubation Day 11, EID11) and is fully differentiated and functionally active by day 15 of incubation (Embryonic Incubation Day 15, EID15). In this study, we explored the simultaneous morphological modifications in the ES, ESM and the CAM at EID11 and EID15 by scanning electron microscopy. We observed that the tips of the mammillary knobs of the ES remain tightly attached to the ESM fibers, while their bases become progressively eroded and then detached from the bulk ES. Concomitantly, the CAM undergoes major structural changes that include the progressive differentiation of villous cells whose villi extend to reach the ESM and the ES. These structural data are discussed with respect to the importance of ES decalcification in providing the calcium necessary for mineralization of embryo’s skeleton. In parallel, eggshell decalcification and weakening during incubation is likely to impair the ability of the ES to protect the embryo. It is assumed that the CAM could counteract this apparent weakening as an additional layer of physical, cellular and molecular barriers against environmental pressures, including pathogens, dehydration and shocks. However, such hypothesis needs to be further investigated.

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential

“…The capacity of inducing osteogenesis from the CAM may be due to the presence of mesenchymal cells with inherited capacity to differentiate into osteoblasts. The CAM forms during chicken development starting from EDD 5 by the fusion of the allantois and the chorion membrane, which are separated by a mesenchymal layer ( Makanya et al, 2016 ; Halgrain et al, 2022a ). While the external layers mainly consist of epithelial cells, the mesenchymal layer comprises fibroblast-like cells that assume a cuboidal form during maturation ( Gabrielli and Accili, 2010 ).…”

“…Under physiological conditions, the CAM is a dynamic organ whose function is to regulate gas exchange, pH levels, water, and mineral content (via calcium and magnesium solubilization and transfer from the eggshell to the embryo). Starting from EDD 11, the eggshell undergoes significant changes accompanied by a gradual thinning of the shell, and calcium carbonate is gradually resorbed from the shell to contribute to the mineralization of the embryo’s skeleton ( Halgrain et al, 2022a ; Halgrain et al, 2022b ; Biesek, 2023 ). There are different types of cells in the chorionic epithelium during the various stages of calcium resorption, and one of them could be responsible for mineralization ( Halgrain et al, 2022a ).…”

Attaching organic fibers to mineral: The case of the avian eggshell

“…After a fertilized egg is laid, secure attachment of the outer membrane fibers to the mammillae is essential for successful development of the chick. With egg incubation, by embryonic day 15, a fully developed, cellular and vascularized chorioallantoic membrane (CAM) lying immediately beneath the membrane 7 , 25 mediates significant dissolution of the shell to mobilize calcium from mammillary calcite for the growing chick skeleton through acidification mechanisms acting across the membrane. 26 , 27 Secure attachment of the membrane to the shell is therefore required to approximate the underlying CAM close to the shell to facilitate shell dissolution and calcium release.…”

“… 26 , 27 Secure attachment of the membrane to the shell is therefore required to approximate the underlying CAM close to the shell to facilitate shell dissolution and calcium release. 25 , 26 , 27 Toward the end of CAM-mediated shell dissolution, the outer eggshell membrane partially detaches from the shell, 25 , 26 , 28 potentially contributing to shell weakening for the emergence of the chick (pipping). Related to this, an intact eggshell membrane attachment has been implicated as a contributor to shell mechanical strength.…”

Reserves of Calcium, Copper, Iron, Potassium, Magnesium, Manganese, Sodium, Phosphorus, Strontium and Zinc in Goose Egg Yolk during Embryo Development

“…Egg yolk also contains 50% water, 33% fat and 17% protein [ 5 ], all of which can be used by the embryo [ 6 ]. Egg albumin, which provides the protein needed by the embryo, contains about 88% water and is the main source of sodium (Na) and potassium (K); however, it contains low levels of Fe, Cu, Mn and Zn [ 7 , 8 ]. Additionally, eggshell contains high amounts of minerals [ 9 ], including calcium (Ca), and lower amounts of iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), zinc (Zn) and strontium (Sr).…”