The eggshell of the hen is a highly ordered and mineralised structure, which is sequentially deposited within an acellular milieu – the uterine fluid secreted by the distal oviduct. Spherulitic crystal growth of calcite is initiated on organic aggregates on surface of the eggshell membranes, followed by competition between radial crystallites for space to form a compact columnar biomineral. The exceptional mechanical properties associated with the well-defined eggshell ultrastructure and texture arise from the control of crystal morphology and growth by the organic matrix, and, amongst them, proteins specific to the uterus and eggshell (ovocleidins and ovocalyxins). The changes in uterine fluid constituents with stages of egg calcification, their effects on morphology of calcite grown in vitro, and the relationship between eggshell texture and mechanical properties point to this control of eggshell fabric. .
The eggshell is essential for propagation of all avian species; it is a sophisticated structure, whose properties reflect perfectly their crucial functions in reproduction. These functions are basically: (a) to protect the contents of the egg from the microbial and physical environment; (b) to control the exchange of water and gases through pores during the extra-uterine development of the chick embryo; (c) to provide calcium for embryonic development once the yolk stores are depleted. In order to meet these requirements, the eggshell must be a porous ceramic material. It must be as light as possible, and balances the requirement for strength to resist the impact of predators while permitting the hatching embryo to break through from the inner side to escape. For the same reasons, it must be of low chemical and biological activity on the outer surface, but easy to dissolve at the inner surface. This eggshell is rapidly formed at physiological temperatures ≤ 41 °C. All these features are simultaneously present in the remarkable eggshell, which seems to be designed ad hoc, but is certainly the result of an
The aims of this review are to describe the structure and fabric of eggshell and current progress in identification and characterisation of eggshell matrix components, to present evidence supporting their involvement in shell calcification and to propose mechanisms by which their influence results in the unique mechanical properties of this highly regulated crystalline biocomposite ceramic. We focus this review on the chicken (hen) eggshell because most recent studies on eggshell components and their interaction with mineral during fabrication of the eggshell structure have been performed with this domestic specie.
The existence of a perfectly defined structural polycrystalline organization throughout the calcified eggshell has been underlined since the earlier studies of Von Nathusius (1821–1899), whose papers were translated and edited by Tyler
The calcification of the eggshell is the result of a precipitation phenomenon occurring on the eggshell membrane, which takes place during passage of the egg through distinct regions of the oviduct
The weight and size of avian eggs vary within three orders of magnitude, for instance, from the 1.9 kg of the ostrich’s egg to the 0.5 g of the egg of the hummingbird. The weight of the egg of the extinct bird
The main function of the eggshell is to shelter the embryo from external aggression, a function that must be compatible with easy breakage from inside to allow hatching of the embryo. In addition, the eggshell structure must permit exchange of water and gases between the embryo and the environment during extra-uterine development, as well as being a source of calcium for the growing embryo. These requirements are fulfilled by the eggshell, because it is a ceramic material displaying a texture gradient. In the outer zone of the structure, there is a tough structure made of large crystals where the external impacts are absorbed by thin inter-crystalline organic layers that make intra-crystalline crack propagation difficult. However, the inner region of the eggshell is composed of microcrystals of calcite arranged with spherulitic texture, which facilitates the propagation of cracks during piping, when the embryo breaks out of the eggshell with its beak. Moreover, this facilitates the mobilization of calcium to nourish the embryo by dissolution of highly reactive calcite microcrystals. While the defined six-layer eggshell outlined above is a classical description, in fact, the eggshell is a single structure from the viewpoint of its mechanism of formation. It is thought that this high degree of control of size, shape and orientation of the crystals of calcite in avian eggshells, which is responsible for its unique ultrastructure and exceptional mechanical properties (in hen, egg breaking strength is 30 N for a mean eggshell thickness of 0.33 mm), results from competition for crystal growth between crystals belonging to the same and to adjacent nucleation sites
The texture of eggshells can be studied by different techniques. The size and orientation of crystals forming the eggshell can be determined directly by optical microscopy using thin-slices (< 30 μm) of radial sections and cross-polarisers. Information from optical microscopy is visual and accurate, but is also two-dimensional; it is local – lacking statistical meaning – and is limited to about 1-μm resolution. It also requires tedious sample preparation. On the contrary, most X-ray diffraction techniques do not require sample preparation and are suitable for systematic studies. Grain size, mosaicity (which correlates with density of crystalline defects, for instance due to protein incorporation) and crystal orientation (ODF) can be obtained from different X-ray diffraction techniques
Specific differences in shell ultrastructure within the mammillary layer and in crystallographic texture were observed among domestic bird species
Fossils eggs, such as those from the Cretaceous theropod dinosaur
As the model of competition for space shows, the actual ODF and its variation with thickness depend largely on the shape of crystals, i.e., on the relative growth rates of calcite crystal faces, which can be modulated by biomolecules in the crystallization milieu (the uterine fluid). The identification of different macromolecules in the uterine fluid as well as in organic extracts from decalcified eggshell is reported in the next section (3), while the mechanisms and interactions between matrix biomolecules and crystals are reviewed in section 4.
Numerous authors have investigated the nature of the constituents of the shell membrane
Since 1990, numerous efforts have been carried out to identify and characterize the matrix molecules present in the mammillary and palisade layers of the eggshell. Mineral can be removed from the eggshell by decalcification with EDTA or acetic acid. However, the eggshell matrix proteins extracted from such shell exhibits aggregation, limiting their subsequent resolution by liquid chromatography. Use of denaturants alleviates this problem when pure proteins are needed for peptide sequencing, but limit the testing of functional properties of these components. An alternative and complementary source is the uterine fluid that contains the precursors of matrix proteins in their functional and native forms prior to incorporation into eggshell. The identification and characterization of numerous eggshell matrix proteins was initially achieved by amino acid microsequencing of PVDF-blotted bands after SDS-PAGE from eggshell extract and from uterine fluid, as well as generation of specific antibodies against these components after preparative SDS-PAGE
This combination of approaches led to identification of a variety of eggshell matrix proteins that can be subdivided into three groups: proteins that occur in other tissues of the body, egg white proteins, and uterine proteins unique to the process of eggshell formation (
Osteopontin, a phosphorylated glycoprotein present at high concentration in bone and kidney but also in most body secretions is present in the eggshell
Comparison of gene expression by uterine cells when an egg is present or absent in the uterus reveals upregulation of a gene coding for the heparin-sulphate proteoglycan, glypican, previously identified in mouse
Recently, clusterin, a secretory disulphide-bonded heterodimeric glycoprotein was shown to be a component of eggshell matrix
Ovalbumin was the first egg white protein revealed in shell matrix by N-terminal amino acid sequencing and immunochemistry
This group is composed of shell proteins and proteoglycans that are only synthesised by tissues involved in eggshell calcification (red isthmus and uterus). These proteins are novel and specific to the eggshell mineralisation process, and have as yet, with few exceptions, only been identified in the domestic hen.
The presence of glycosaminoglycans (uronic acid, galactosaminoglycan, and hyaluronic acid) in the eggshell has been chemically demonstrated
Ovocleidin 17 was the first matrix protein to be purified using chromatographic techniques and characterized
Ovocalyxin-21 and ovocalyxin-25 have recently been cloned
Ovocalyxin-32 (32kDa) is present in uterine fluid during the growth phase, but is mainly present during the terminal phase of calcification, and consequently is localised in the outer region of the eggshell
Ovocalyxin-36 has also been cloned. Its predicted amino acid sequence corresponds to the N-terminus and internal peptide sequences of a 36 kDa band found in eggshell extracts and in uterine fluid at high levels during the calcification phase of shell formation
Ovocleidin-116
An approach to globally characterize the matrix of various bird species and to contrast common and distinct features by SDS-PAGE and Western blotting across taxonomic groups shows that some matrix proteins are common to the eight domestic birds tested (ovocleidin-17, ovalbumin), and that there is a more restricted distribution for others (ovotransferrin, osteopontin). The distribution of proteoglycans at nucleation sites and within the palisade layer also varies between species
A number of observations support the hypothesis that the eggshell matrix components regulate eggshell mineralisation. The first one is that the composition of the uterine fluid changes at different stages of shell formation: each phase of shell mineralisation (nucleation, rapid crystal growth and the completion of shell formation) is associated with a specific electrophoretic profile of biological macromolecules of the uterine fluid (
The induction time for calcium carbonate precipitation is reduced by the uterine fluid harvested during the formation of mammillary cores, suggesting that the macromolecular cocktail at this stage of the calcification of the egg promotes crystal nucleation. To a lesser extent, the uterine fluid collected during the growth phase also enhances precipitation kinetics. On the contrary, the total uterine fluid harvested at the end of calcification inhibits the precipitation of calcite
In vitro studies have also demonstrated that proteins affect dramatically the morphology of calcite crystals. Calcite grown from pure calcium carbonate solution displays the morphology of cleavage rhombohedra. Neutral to slightly charged proteins have a stronger effect on calcite morphology
The nature of the interaction between matrix molecules and the mineralisation process is poorly understood. Protein adsorption is affected by protein surface properties (electrical charge density, conformation and hydrophobicity) as well as solution conditions (pH, ionic strength, etc.). These parameters affect the protein structure. Adsorption is enhanced by the hydrophobicity of the protein. Also, electrostatic interaction plays an important role, especially with hydrophilic surfaces such as calcite as shown by investigating the effect of a group of globular proteins of similar size and conformation, but with different isoelectric points
If eggshell matrix proteins participate in establishing the morphology of calcite crystals, it would affect the texture of the eggshell and therefore influence its mechanical properties. This hypothesis leads to the prediction that differences in the total amount of eggshell matrix and/or relative composition of the matrix would correlate with variations in eggshell strength. This proposal was tested by micro-extraction, SDS-PAGE electrophoresis and quantification by ELISA of the matrix proteins in eggshell samples. The concentration of three proteins (ovotransferrin, ovalbumin and ovocleidin-17) were analysed in shell from eggs laid at initiation and at the end of the laying year
It is clear from all the above-reviewed results that the matrix components play an active role in the control of growth kinetics and of crystal morphology. Consequently, coupled with competition for crystallisation space, the organic matrix regulates the textural organization within the eggshell. However, additional information is needed to better understand the nature of the interactions between macromolecules and growing calcite crystals, to learn to emulate in vitro actual crystal morphology in eggshells, and to know the relative importance of different matrix components. The eggshell, however, due to its spatial and temporal sequence of formation, as well as to the emerging relationship between textural structure and mechanical properties that is seen between species and at different physiological stages, constitutes a valuable model to better understand the calcitic biomineralisation that is found in diverse organisms.
Transverse section of eggshell viewed in cross-polarized light: (
Fig. 1. Section transversale de coquille, observée en lumière polarisée : (
Cross-section of an eggshell viewed in cross-polarized light. Arrows indicate the orientation of the
Fig. 2. Vue transversale d’une coquille de poule en lumière polarisée. Les flèches indiquent l’orientation des cristaux de calcite. Échelle : 100 μm. (
Electrophoretic profile (SDS PAGE) of the uterine fluid collected at three stages of shell formation and of eggshell organic extract in hens. Coomassie blue staining.
Fig. 3. Profil électrophorétique (SDS PAGE) de fluides utérins collectés aux trois stades de formation de la coquille et d’un extrait organique de coquille de poule.