Small molecules assisting eggshell calcium dissolution for embryonic bone formation

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.