Abstract
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.
Highlights
Biomineralization is a ubiquitous process by which living organisms produce minerals that they use for many different functions [1]
uterine fluid (UF) was examined by transmission EM (TEM) (Figs. 2 and 3), where numerous extracellular vesicle (EV) varying in diameter from 100 to 500 nm were observed (Figs. 2, A and C, and 3A)
We have recently proposed a novel model for avian eggshell biomineralization involving EVs budding from uterine epithelial cells, which transit through the UF and deliver stabilized Amorphous calcium carbonate (ACC) mineral to the eggshell membranes and mineralization front [29]
Summary
Biomineralization is a ubiquitous process by which living organisms produce minerals that they use for many different functions (i.e. protection, gravity sensing) [1]. Both mineral and organic precursors interact to produce the specific eggshell texture and its resulting mechanical properties [17, 23] This process requires transport of large amounts of calcium and carbonate to the site of eggshell calcification; these ions are continuously supplied from the blood across the uterine epithelium [24]. We hypothesized that EDIL3 and MFGE8 bind to EVs budding from uterine cells into the uterine fluid, to guide vesicular transport of stabilized ACC for delivery to the mineralizing site and prevent nonspecific precipitation [27] To test this hypothesis, in the current study we have used transmission EM (TEM) to investigate exocytosis activity at the apical plasma membrane of uterine cells that could be a source of EVs in the uterine fluid. This experimental study is the first to demonstrate vesicular transport of ACC in vertebrates, which we propose supports the rapid eggshell biomineralization process in birds
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