Evolution of the Avian Eggshell Biomineralization Protein Toolkit – New Insights From Multi-Omics

Abstract

The avian eggshell is a remarkable biomineral, which is essential for avian reproduction; its properties permit embryonic development in the desiccating terrestrial environment, and moreover, are critically important to preserve unfertilized egg quality for human consumption. This calcium carbonate (CaCO3) bioceramic is made of 95% calcite and 3.5% organic matrix; it protects the egg contents against microbial penetration and mechanical damage, allows gaseous exchange, and provides calcium for development of the embryonic skeleton. In vertebrates, eggshell occurs in the Sauropsida and in a lesser extent in Mammalia taxa; avian eggshell calcification is one of the fastest known CaCO3 biomineralization processes, and results in a material with excellent mechanical properties. Thus, its study has triggered a strong interest from the researcher community. The investigation of eggshell biomineralization in birds over the past decades has led to detailed characterization of its protein and mineral constituents. Recently, our understanding of this process has been significantly improved using high-throughput technologies (i.e., proteomics, transcriptomics, genomics, and bioinformatics). Presently, more or less complete eggshell proteomes are available for nine birds, and therefore, key proteins that comprise the eggshell biomineralization toolkit are beginning to be identified. In this article, we review current knowledge on organic matrix components from calcified eggshell. We use these data to analyze the evolution of selected matrix proteins and underline their role in the biological toolkit required for eggshell calcification in avian species. Amongst the panel of eggshell-associated proteins, key functional domains are present such as calcium-binding, vesicle-binding and protein-binding. These technical advances, combined with progress in mineral ultrastructure analyses, have opened the way for new hypotheses of mineral nucleation and crystal growth in formation of the avian eggshell, including transfer of amorphous CaCO3 in vesicles from uterine cells to the eggshell mineralization site. The enrichment of multi-omics datasets for bird species is critical to understand the evolutionary context for development of CaCO3 biomineralization in metazoans, leading to the acquisition of the robust eggshell in birds (and formerly dinosaurs).

Thicker eggshells are not predicted by host egg ejection behaviour in four species of Australian cuckoo

“…Thus, it is possible that there is insufficient variation in the phenotype for evolution to act effectively upon. However, we suggest that this is not the case due to the wealth of other studies that have demonstrated evidence for the evolution of eggshell thickness in avian and non-avian egg laying species 50 – 52 . Another point to consider is whether the co-evolutionary interactions between hosts and cuckoos in our study are sufficiently strong to select for changes to eggshell thickness.…”

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species

“…It is also expressed in hen digestive tract (172). Chicken OCX-36 is encoded by the BPIFB3 gene; its protein sequence shares 20-25% sequence homology to mammalian proteins associated with the innate immune response, such as lipopolysaccharide-binding proteins (LBP), bactericidal permeability-increasing protein (BPI) proteins and palate, lung and nasal epithelium clone (Plunc) family proteins and is involved in lipid binding functions (173,174). It displays lipopolysaccharide and lipoteichoic acid binding activity, suggesting its role as a pattern recognition molecule, which binds bacterial endotoxins in order to eliminate pathogens such as Staphylococcus aureus (31).…”

“…Ovocleidins (OC-17, OC-116) are mainly secreted in hen uterine fluid during the active calcification stage of shell formation (14,168). Ovocleidin-116 (OC-116; MEPE, matrix extracellular phosphoglycoprotein, is the mammalian ortholog) is a major component of the chicken uterine fluid and is the most abundant matrix protein in the eggshell (14,174). Ovocleidin-17 is a phosphorylated protein with a C-type lectin (CTL) domain, which exhibits bactericidal activity against both Gram-positive and Gram-negative bacteria including Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa (175).…”

“…This protein is dispersed throughout the calcified matrix with greater abundance in the mammillary layer (5). CTL proteins that are homologs of OC-17 have been identified in eggshells of many bird species, and it is thought that OC-17-like/XCA-2 and XCA-1 are eggshell-specific proteins restricted to vertebrates that produce a calcitic shell (174). Phylogenetic analysis indicates that these CTL family members have been duplicated multiple times during avian speciation (176).…”

Myostatin Mutation in Japanese Quail Increased Egg Size but Reduced Eggshell Thickness and Strength

“…On the surface of an uncalcified eggshell membrane, calcified and mineralized shell layers are formed in the order of the mammillary layer, the palisade layer, the vertical crystal layer, and the outermost cuticle layer [ 12 ]. There are several factors affecting eggshell strength across different avian species, such as the egg weight and the crystallographic orientation of the mineralized eggshell [ 13 , 14 ]. Because the same species have the same crystallographic orientation, however, eggshell thickness is a main factor affecting eggshell strength [ 15 ].…”