Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit.

BACKGROUND: 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 during eggshell biomineralization. Main Body. 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. CONCLUSION: 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.

Increased expression of fibroblast growth factor 23 is the signature of a deteriorated Ca/P balance in ageing laying hens.

The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.

Possible roles of parathyroid hormone, 1.25(OH)2D3, and fibroblast growth factor 23 on genes controlling calcium metabolism across different tissues of the laying hen.

This study provides an integrative description of candidate gene expression across tissues involved in calcium (Ca) metabolism during the egg laying cycle, using the well-defined model of Ca supply as fine or coarse particles of calcium carbonate (CaCO3). Plasma and tissue samples were collected from hens at the peak of laying at 0 to 1, 9 to 10, and 18 to 19 h postovulation (PO). After mRNA preparation from the parathyroid gland, medullary bone, liver, kidney, duodenum, and jejunum, gene expressions were quantified using RT-qPCR. The highest levels of parathyroid hormone (PTH) mRNA in the parathyroid gland (P < 0.05), and of the active form of vitamin D3 1.25(OH)2D3 in the plasma (P < 0.01) were observed at 18 to 19 h PO. During this active phase of eggshell formation, bone resorption was attested to high levels of plasma inorganic phosphorus (iP) and the receptor activation of nuclear factor-κB expression in the bone (P < 0.001 and P < 0.05, respectively). At this stage, 5 genes of the transcellular and the paracellular Ca absorption pathways in the intestine (P < 0.05) and the Ca channel transient receptor potential cation channel subfamily V member 5 (P < 0.05), involved in its reabsorption in the kidney, were overexpressed. At 0 to 1 h PO during the subsequent daylight period, 2 candidates of the transcellular and the paracellular Ca pathways (P < 0.05) remained at high levels in the intestine, while calbindin D 28K expression was the highest in the kidney (P < 0.05). As PTH mRNA and 1.25(OH)2D3 were low, bone accretion was likely active at this stage. The phosphaturic hormone fibroblast growth factor 23 (FGF23) was overexpressed at 18 to 19 h PO (P < 0.05) in the bone when plasma iP was high, which suggested a role in the subsequent reduction of P reabsorption in the kidney, as attested to the decreased expression of P cotransporters, leading to iP clearance from the plasma at 0 to 1 h PO (P < 0.05). The low levels of 1.25(OH)2D3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO3, higher plasma levels of 1,25(OH)2D3 and higher expression of several genes involved in bone turnover reflected a stronger challenge to Ca homeostasis. Altogether, these data support the hypothesis that FGF23 could drive vitamin D metabolism in the laying hen, as previously documented in other species and explain the tight link between P and Ca metabolisms.

Candidate genes of the transcellular and paracellular calcium absorption pathways in the small intestine of laying hens.

To meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.