Proteomic Analysis of Chicken Chorioallantoic Membrane (CAM) during Embryonic Development Provides Functional Insight.

In oviparous animals, the egg contains all resources required for embryonic development. The chorioallantoic membrane (CAM) is a placenta-like structure produced by the embryo for acid-base balance, respiration, and calcium solubilization from the eggshell for bone mineralization. The CAM is a valuable in vivo model in cancer research for development of drug delivery systems and has been used to study tissue grafts, tumor metastasis, toxicology, angiogenesis, and assessment of bacterial invasion. However, the protein constituents involved in different CAM functions are poorly understood. Therefore, we have characterized the CAM proteome at two stages of development (ED12 and ED19) and assessed the contribution of the embryonic blood serum (EBS) proteome to identify CAM-unique proteins. LC/MS/MS-based proteomics allowed the identification of 1470, 1445, and 791 proteins in CAM (ED12), CAM (ED19), and EBS, respectively. In total, 1796 unique proteins were identified. Of these, 175 (ED12), 177 (ED19), and 105 (EBS) were specific to these stages/compartments. This study attributed specific CAM protein constituents to functions such as calcium ion transport, gas exchange, vasculature development, and chemical protection against invading pathogens. Defining the complex nature of the CAM proteome provides a crucial basis to expand its biomedical applications for pharmaceutical and cancer research.

Quantitative proteomics analysis of eggshell membrane proteins during chick embryonic development.

The avian eggshell membrane (ESM) is a meshwork made up of highly cross-linked protein fibers and it is a scaffold upon which biomineralization of the eggshell is initiated. The ESM and associated shell participates in embryonic development by providing physical and chemical protection against pathogen invasion. We performed quantitative proteomic analysis of ESM proteins on multiple days during the three phases of embryonic development. The ESMs were stripped from both fertilized and unfertilized eggs at different days of incubation, and solubilized in a novel manner using TCEP-HCl (Tris (2-carboxyethyl) phosphine hydrochloride). The changes in ESM proteins between occurred during incubation were analyzed. Bioinformatics analysis revealed that of the 12 functional protein clusters identified, protease inhibitors were present at all phases of chick development. A group of proteins involved in calcium binding and oxygen transport were only present during the second phase. Extracellular matrix, cell adhesion proteins related to the vascularization of chorioallantoic membrane (CAM), antimicrobial proteins and proteins involved in the binding and transport of lipids were found in the second and third phases of development. These findings provide insight into the functionality and evolving nature of ESM associated proteins involved in chick embryonic development. BIOLOGICAL SIGNIFICANCE: The eggshell membranes (ESMs) are a fibrous scaffold that consists of highly crosslinked collagens (types I, V but mainly X), glycoproteins and CREMPs (cysteine-rich eggshell membrane proteins). The ESMs aid in the development of the chick embryo and protect it against pathogen invasion. This biopolymeric fibrous net functions as a platform for nucleation of the calcitic eggshell which provides a primary physical barrier against bacterial ingress. Comparative proteomic analyses of proteins in the ESMs from fertilized eggs and unfertilized eggs showed changes in their levels which varied between the specific phases of chick embryogenesis across 19 days of incubation. Bioinformatics characterization of these ESM proteins provides understanding of their evolving nature during chick embryonic development.

Ovocalyxin-36 is a pattern recognition protein in chicken eggshell membranes.

The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).

Recent patents on eggshell: shell and membrane applications.

The chicken eggshell and its membranes are an inexpensive and abundant waste material which exhibit interesting characteristics for many potential applications. The eggshell is formed mainly of calcium carbonate (CaCO3) and is used widely as an animal feed, lime (Ca(OH)2) substitute or a fertilizer. Moreover, the associated eggshell membranes have a high content of bioactive components, as well as properties of moisture retention and biodegradability which have potential use for clinical, cosmetic, nutraceutical and nanotechnology applications. The eggshell membranes have been also used for biosorption of heavy metals and dyes and as a template to synthesize metal nanoparticles. The combination of nanosized calcium phosphate (Ca3(PO4)2) biomaterials synthesized from eggshell and eggshell membrane show promise to develop drug delivery system and nanowires for electronic devices. In addition, a derived product, the soluble eggshell membrane protein (SEP) has applications in tissue engineering. This review discusses the patented applications of eggshell membrane waste: shell and membrane for the last 10 years as well as their future applications.