Calcium carbonate in avian eggshell is used as a calcium source for bone formation during embryonic development. We studied ostrich eggshells as a representative avian egg and found that the calcium reserve assembly (CRA) region, eventually dissolving for bone formation, consists of nano-granules comprising amorphous calcium carbonate (ACC) by careful microscopic observation. ACC is unstable phase of calcium carbonate with high solubility and is stabilized by additives. As such a stabilizer, we found small molecules from eggshell internal region. Particularly, the ACC was recreated by the formation of calcium carbonate in the presence of D-myo-inositol 4,5-bisphosphate (Ins(4,5)P2), isolated from ostrich eggshell for the first time in this study. Further mass spectrometric imaging technique revealed that Ins(4,5)P2 is localized at the CRA region, suggesting that this compound could play a major role that makes it easier to dissolve and internalize eggshell calcium during embryogenesis.
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