The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications.

The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

Background: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO₃). Both elements (Ca²⁺, HCO₃ ^-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. Results: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca²⁺/HCO₃ ^- movement) and duodenum (high rates of Ca²⁺/HCO₃ ^- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca²⁺ channel (basolateral uptake of Ca²⁺), 28 kDa calbindin (intracellular Ca²⁺ buffering), the endoplasmic reticulum type 2 and 3 Ca²⁺ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca²⁺ movement across the apical membrane likely involves membrane Ca²⁺ pumps and Ca²⁺/Na⁺ exchangers. Our data suggests that Na⁺ transport involved the SCNN1 channel and the Na⁺/Ca²⁺ exchangers SLC8A1, 3 for cell uptake, the Na⁺/K⁺ ATPase for cell output. K⁺ uptake resulted from the Na⁺/K⁺ ATPase, and its output from the K⁺ channels (KCNJ2, 15, 16 and KCNMA1). We propose that the HCO₃ ^- is mainly produced from CO₂ by the carbonic anhydrase 2 (CA2) and that HCO₃ ^- is secreted through the HCO₃ ^-/Cl^- exchanger SLC26A9. HCO₃ ^- synthesis and precipitation with Ca²⁺ produce two H⁺. Protons are absorbed via the membrane’s Ca²⁺ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl^- ions which are absorbed by the HCO₃ ^-/Cl^- exchanger SLC26A9 and by Cl^- channels (CLCN2, CFTR) and might be extruded by Cl^-/H⁺ exchanger (CLCN5), but also by Na⁺ K⁺ 2 Cl^- and K⁺ Cl^- cotransporters. Conclusions: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca²⁺ and HCO₃ ^- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO₃, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.