Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate

J Biol Chem. 2020 Nov 20; 295 (47) :15853-15869

Lilian Stapane1, Nathalie Le Roy1, Jacky Ezagal1, Alejandro B. Rodriguez-Navarro2, Valérie Labas3, Lucie Combes-Soia3, Maxwell T.Hincke4, Joël Gautron1


1 BOA INRAe, Université de Tours, Nouzilly, France

2 Departmento de Mineralogia y Petrologia, Universidad de Granada, Granada, Spain

3 Unité Mixte de Recherches Physiologie de la Reproduction et des Comportements, Université de Tours IFCE, Nouzilly, France

4 Department of Innovation in Medical Education, and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada


Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO[Formula: see text]), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization.


avian eggshell
ACC transport
extracellular vesicle
calcium transport
matrix proteins
protein targeting
calcium-binding protein
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Elsevier, Journal of Biological chemistry