Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro

We investigated matrix–mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix–mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {1 0 4} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {1 0 4} calcite faces. OPN inhibited calcite growth in vitro at the {1 0 4} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.     

八种鸡形目鸟类卵壳及壳膜超微结构观察

８种鸡中,高原山鹑卵壳仅由乳突层和栅栏层构成,缺少护膜层,表面无裂纹,外气孔开放。其它种类由乳突层、栅栏层和护膜层构成,表面有裂纹、外气孔有覆盖。栅栏层都有与飞翔相适应的气泡,飞翔能力强,速度快的种类产卵壳气泡密度高的卵。     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.     

Monoclonal antibodies to mineralized matrix molecules of the avian eggshell.

The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.     

Scanning Microfocus Small Angle X-ray Scattering Study of the Avian Eggshell

Synchrotron microfocus small angle X-ray scattering was used to investigate the nanostructure and microscopic variation of eggshells. It uses a microbeam allowing the ability to probe interactions between the organic and inorganic components at nanometer level and is ideal for mapping over small areas to obtain a detailed analysis of structural variations. Thin sections of eggshells were scanned from the shell membrane （inner） to the cuticle （outer） surface. The data collected was used to produce two-dimensional maps showing microscopic changes within the different layers of the eggshell. The structural alterations ap- parently could have implications at the macroscopic level of the resulting eggshell. As the organic matrix is embedded within the eggshell this may contribute to the variations observed in calcite crystal form and texture, Structural information obtained about a biomaterial at different length scales is important in relating the structure to its functional properties. This knowledge and the principles behind the formation of biomaterials could be used in the attempt of bioengineering new systems.     

Ultrastructure and mineral distribution in the tergite cuticle of the beach isopod Tylos europaeus Arcangeli, 1938

The crustacean cuticle is a hierarchically organised material composed of an organic matrix and mineral. It is subdivided into skeletal elements whose physical properties are adapted to their function and the eco-physiological strains of the animal. Using a variety of ultrastructural and analytical techniques we studied the organisation of the tergite cuticle of the sand burrowing beach isopod Tylos europaeus. The surface of the tergites bear epicuticular scales, sensilla and micro-tubercles. A distal layer of the exocuticle is characterised by a low density of organic fibres and the presence of magnesium-calcite. Surprisingly, the mineral forms regions containing polyhedral structures alternating with smooth areas. Between sub-domains within the distal exocuticle calcite varies in its crystallographic orientation. Proximal layers of the exocuticle and the endocuticle are devoid of calcite and the mineral occurs in the form of amorphous calcium carbonate (ACC). Using thin sections of mineralised cuticle we describe for the first time that ACC forms tubes around single protein-chitin fibrils.     

Ultrastructure of avian eggshell during resorption following egg fertilization

For skeletal mineralization, the avian embryo mobilizes calcium from its calcitic eggshell. This occurs through dissolution of specific interior regions of the shell in a process that also weakens the shell to allow hatching. Here, we have examined eggshell ultrastructure during dissolution occurring between laying of a fertilized egg (with incubation) and hatching of the chick (Gallus gallus). We have focused on changes in shell mammillae where the majority of dissolution takes place. Using scanning electron microscopy, we describe differences in matrix–mineral structure and relationships not observed in unfertilized eggs (unresorbed eggshell). We document changes in the calcium reserve body – an essential sub-compartment of mammillae – consistent with it being an early, primary source of calcium essential for embryonic skeletal growth. Dissolution events occurring in the calcium reserve sac and in the base plate of the calcium reserve body, and similar changes in surrounding bulk mammillae structure, all correlate with advancing skeletal embryonic calcification. The changes in mammillae sub-structures can generally be characterized as mineral dissolutions revealing fine surface topographies on remaining mineral surfaces and the exposure of an extensive, intracrystalline (occluded) organic matrix network. We propose that this mineral-occluded network regulates how shell mineral is dissolved by providing dissolution channels facilitating calcium release for the embryonic skeleton.     

Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle

The avian eggshell is an acellular bioceramic containing organic and inorganic phases that are sequentially assembled during the time the egg moves along the oviduct. As it has been demonstrated in other mineralized tissues, mineralization of the eggshell is regulated by extracellular matrix proteins especially the anionic side chains of proteoglycans. Among them, osteopontin has been found in the avian eggshell and oviduct. However, its precise localization in the eggshell or in different oviduct regions during eggshell formation, nor its function have been established. By using anti-osteopontin antibody (OPN 1), we studied its immunolocalization in the isthmus, red isthmus and shell gland of the oviduct, and in the eggshell during formation. In the eggshell, osteopontin was localized in the core of the non-mineralized shell membrane fibers, in the base of the mammillae and in the outermost part of the palisade. In the oviduct, OPN 1 was localized in the ciliated epithelial but not in the tubular gland cells of the isthmus, in the ciliated epithelial cells of the red isthmus, and in the non-ciliated epithelial cells of the shell gland. The occurrence of osteopontin in each of the oviduct regions, coincided with the concomitant presence of the egg in such region. Considering the reported inhibitory function of osteopontin in other mineralized systems, together with its main occurrence in the non-mineralized parts of the eggshell and at the outermost part of the shell, suggests that this molecule could be part of the mechanism regulating the eggshell calcification.     

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality.     

Fine structure and formation of eggshells in marine Gastrotricha

Summary The fine structure of the gastrotrich eggshell in the hermaphroditic species Turbanella ocellata (Hummon 1974) and the parthenogenetic species Aspidiophorus sp. is described using transmission electron microscopy. The presented evidence strongly suggests that the shell is produced by the egg itself prior to oviposition in both species. The layed egg in Aspidiophorus sp. is provided with a special attachment stalk that is also preformed in the mother animal. Freshly layed eggs of T. ocellata are adhesive all around their surface and lack any specialized structures for attachment. Formation of the spiny eggshell of Aspidiophorus sp. appears to begin with a sudden release of special vesicles containing the preformed spines of the outer eggshell covering. Additional material appears to be secreted by the egg in a more gradual process after the initial vesicle release. The formation of the two fibrous layers in the eggshell of T. ocellata is less well understood and deposition of eggshell material could be seen either as a continuous process or as two separate steps, similar to the events observed for Aspidiophorus sp. For T. ocellata, Tetranchyroderma sp. and Aspidiophorus sp. it is demonstrated that formation of the cuticle occurs as an independent process from that of eggshell formation. This is significantly different from the basic mode of cuticle formation in the annelid line of evolution. The paper argues further that the data support earlier claims of a pronounced difference between the Gastrotricha-Macrodasyida and the Gastrotricha-Paucitubulatina and agree well with the postulated ties of the Gastrotricha and Nematoda. The phylogenetic importance of the eggshell fine-structure is discussed in the framework of present theories on aschelminth phylogeny.Abbreviations cus cuticular spines - cut cuticle - cov coated vesicles - cv cup-shaped vesicles - dp dense particles - ep epidermis - emb embryo - erl lacunae of smooth ER - fgb fibrous and granular bodies - fl fibrous layer - ga Golgi apparatus - gc gut cell - gv Golgi vesicles - im intercellular matrix - isp intercellular space - isl inner shell layer - ld lipid droplet - mdb medium-dense bodies - mvb multivesicular bodies - oc oocyte - od oviduct - osl outer shell layer - o egg - sv spiny vesicles - sh eggshell - st egg-stalk - sl spiny layer - sub substrate - trm trilaminate membrane - yb yolk bodies - yg yolk granule - yoc young oocyte This work was supported by NSF Grant # GB-42211 to R.M. Rieger     

Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116 , osteopontin ( SPP1 ), ovocalyxin-32 ( RARRES1 ), ovotransferrin ( LTF ), ovalbumin and ovocalyxin-36 , as well as key genes in the maintenance and function of the shell gland [ estrogen receptor ( ESR1 ) and carbonic anhydrase II ( CAII )]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.     

Antimicrobial activity of the Anseriform outer eggshell and cuticle

The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.     

藏马鸡卵壳的扫描电镜观察

利用扫描电镜对我国特有珍禽──藏马鸡的卵壳进行了超微结构观察。电镜下显示：藏马鸡卵壳从内向外由壳膜层、锥体层、海绵层和表层等组成。壳膜层内层致密、含少量纤维,外层为纵横交错成网状的纤维结构,锥体层由许多乳头状突起密集排列组成,海绵层为似沉积岩层的层状结构,表层在卵壳最外面,上由具保护性的透明蛋白质薄膜覆盖。与同属的褐马鸡的卵壳进行比较,其超微结构存在差异。     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.     

Proteomics Analysis of Avian Eggshell Matrix Proteins: Toward New Advances on Biomineralization

Eggs are widely consumed all over the world. The eggshell is its protective barrier whose original function is to protect the embryo during development. Avian eggshells are made of calcium carbonate with a small amount of organic matrix (proteins and proteoglycans). During eggshell formation, the mineral precursors interact with matrix proteins to regulate the calcification of this highly resistant biomineral. In order to better characterize the functions of matrix proteins in eggshell biominerals, many proteomics studies have been performed during the last 15 years. The chicken eggshell is the main model studied in birds, but there is a need for comparative approaches in order to determine whether there is a general protein toolkits associated with calcitic biomineralization, and to determine its components. The study by Zhu et al., reported in article number 1900011, volume 19, issue 11, is a major step forward as it is the first shell proteomics survey performed on duck. Thus, it will contribute to improved knowledge of the eggshell mineralization process and will provide new insight for shell quality improvement and to guide biomimetic efforts in material sciences.     

Amorphous and crystalline calcium carbonate distribution in the tergite cuticle of moulting Porcellio scaber (Isopoda, Crustacea)

The main mineral components of the isopod cuticle consists of crystalline magnesium calcite and amorphous calcium carbonate. During moulting isopods moult first the posterior and then the anterior half of the body. In terrestrial species calcium carbonate is subject to resorption, storage and recycling in order to retain significant fractions of the mineral during the moulting cycle. We used synchrotron X-ray powder diffraction, elemental analysis and Raman spectroscopy to quantify the ACC/calcite ratio, the mineral phase distribution and the composition within the anterior and posterior tergite cuticle during eight different stages of the moulting cycle of Porcellio scaber. The results show that most of the amorphous calcium carbonate (ACC) is resorbed from the cuticle, whereas calcite remains in the old cuticle and is shed during moulting. During premoult resorption of ACC from the posterior cuticle is accompanied by an increase within the anterior tergites, and mineralization of the new posterior cuticle by resorption of mineral from the anterior cuticle. This suggests that one reason for using ACC in cuticle mineralization is to facilitate resorption and recycling of cuticular calcium carbonate. Furthermore we show that ACC precedes the formation of calcite in distal layers of the tergite cuticle.     

The Structure and Calcification of the Crustacean Cuticle

The integument of decapod crustaceans consists of an outer epicuticle,an exocuticle, an endocuticle and an inner membranous layerunderlain by the hypodermis. The outer three layers of the cuticleare calcified. The mineral is in the form of calcite crystalsand amorphous calcium carbonate. In the epicuticle, mineralis in the form of spherulitic calcite islands surrounded bythe lipid-protein matrix. In the exo- and endocuticles the calcitecrystal aggregates are interspersed with chitin-protein fiberswhich are organized in lamellae. In some species, the organizationof the mineral mirrors that of the organic fibers, but suchis not the case in certain cuticular regions in the xanthidcrabs. Thus, control of crystal organization is a complex phenomenonunrelated to the gross morphology of the matrix. Since the cuticle is periodically molted to allow for growth,this necessitates a bidirectional movement of calcium into thecuticle during postmolt and out during premolt resorption ofthe cuticle. In two species of crabs studied to date, thesemovements are accomplished by active transport effected by aCa-ATPase and Na/Ca exchange mechanism. The epi- and exocuticular layers of the new cuticle are elaboratedduring premolt but do not calcify until the old cuticle is shed.This phenomenon also occurs in vitro in cuticle devoid of livingtissue and implies an alteration of the nucleating sites ofthe cuticle in the course of the molt.