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 electron microscopy 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 (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 a vertebrate model of biomineralization.

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