A new model for vertebrate mineralization via stabilized amorphous calcium carbonate for avian eggshell formation

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 mineralization takes place. Herein, we report further experimental evidence for this model. We confirmed the presence of extracellular vesicles (EVs) that contain ACC in uterine fluid using transmission electron microscopy and elemental analysis. We also demonstrate high levels of expression of vesicular markers in the oviduct segments where eggshell is formed. 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 HCO3-, 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 vertebrate biomineralization. These results could give insight to understand the mineralization of otoconia, which are calcium carbonate biomineralized structures present in all vertebrates and necessary for balance and sensing linear acceleration.