Maxwell T. Hincke

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.

Increased expression of BDNF and trkB mRNA in rat facial motoneurons after axotomy.

Motoneurons of the adult survive after axotomy even though they are deprived of putative target derived trophic factors. Alternative sources of trophic support may substitute. In this study we test the hypothesis that the immediate environment of the motoneuronal cell body or the cell body itself increases the production of trophic factors after axonal injury. Using in situ hybridization (ISH) and reverse transcription–polymerase chain reaction (RT‐PCR), we report that after axotomy, rat facial motoneurons increase the expression of mRNA for brainderived neurotrophic factor (BDNF) and its receptor trkB. After transection of the facial nerve, we measured a 2‐to 4‐fold increase in BDNF mRNA expression which had its onset between 3 and 8 h after injury. The BDNF mRNA levels peaked at ∼1–2 days and gradually declined thereafter to return to contralateral levels within 7 days of injury. Western blotting revealed a several‐fold increase in BDNF as early as 24 h, which subsequently reached a maximum in ∼5–7 days and was still sustained at 2 weeks post‐axotomy. Using exon‐specific primers, we determined that the increase in BDNF mRNA is largely due to an increased expression from the promoters of exons IV and III, and to a lesser extent from exons I and II. Analysing the mRNA expression for the BDNF receptor, trkB, we found a 2‐ to 3‐fold increase in full‐length trkB mRNA expression starting 2 days after axotomy which lasted for 2–3 weeks. These findings suggest that BDNF might act locally on axotomized motoneurons in an autocrine fashion, providing support for axotomized motoneurons during the first weeks after axotomy.

Ovotransferrin is a Matrix Protein of the Hen Eggshell Membranes and Basal Calcified Layer

The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

The eggshell: structure, composition and mineralization

The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications.

Ovalbumin is a Component of the Chicken Eggshell Matrix

The protein components of biomineralized structures (matrix proteins) are believed to modulate crystal nucleation and growth, and thereby influence the shape and strength of the final structure. The chicken eggshell contains a complex array of distinct matrix proteins. One of these was found to have similar molecular weight and chromatographic properties as purified egg ovalbumin. A commercially available antibody to ovalbumin was utilized for western blotting to demonstrate that ovalbumin is one of the matrix proteins this is extracted from decalcified eggshell. Immunohistochemistry revealed that ovalbumin is found only in the mammillary bodies of decalcified shell, and is not distributed throughout the shell matrix. These results indicate that ovalbumin is present during the initial phase of shell formation and becomes incorporated into the protein matrix of the mammillary bodies. However, it is not yet clear whether ovalbumin at this site plays a specific role in shell mineralization.

Organic matrix composition and ultrastructure of eggshell: A comparative study

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.

Precursor Matrix Proteins in the Uterine Fluid Change with Stages of Eggshell Formation in Hens

Organic constituents of the uterine fluid, the acellular milieu in which the eggshell is mineralized, were biochemically characterized at initial, mid and final stages of shell calcification in hens. The electrophoretic protein profiles changed at the different stages of shell mineralization. Two major bands (80-kDa and 43-kDa glycoproteins) with calcium affinity were specific to the initial stage. Four protein bands of 180, 150, 116 and 32 kDa, present at the phase of rapid shell formation, coprecipitated with calcium carbonate in vitro. At this stage were also present a calcium-binding glycoprotein of 36-kDa and a 20-kDa protein. Uterine fluid of the final stage was characterized by a darker intensity of the 66-kDa band, which showed calcium-binding ability and by the presence of three additional proteins (72, 13 and 6 kDa). At least seven bands of the uterine fluid showed similar migration patterns to those of eggshell extracts. Western blotting with ovocleidin and ovalbumin antisera demonstrated the presence of these matrix proteins in uterine fluid collected at initial and mid phase, respectively. Total uterine fluid collected at the end of calcification and dialyzed uterine fluid from the various stages delayed the rate of calcium precipitation in vitro. These observations demonstrate the presence of precursors of eggshell matrix in the uterine fluid and support the hypothesis of their involvement in the process of eggshell mineralization.

Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20–25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Biochemical and functional characterisation of eggshell matrix proteins in hens

The eggshell of the hen is a highly ordered, mineralised structure deposited within an acellular milieu – the uterine fluid secreted by the distal oviduct. Spherulitic crystal growth is initiated by deposition of calcium carbonate on aggregates of organic material present on the outer surface of the eggshell membranes. Gel electrophoresis reveals a complex array of proteins in uterine fluid and eggshell extracts. The eggshell matrix proteins can be classified as egg white proteins (lysozyme, ovalbumin, ovotransferrin, clusterin), bone protein (osteopontin), or proteins specific to the uterus and eggshell (ovocleidins-17 and –116; ovocalyxins-32 and –36). Eggshell extracts, uterine fluid and purified fractions are able to modify the morphology of calcite crystals in vitro. In young hens the breaking strength of the eggshell is inversely related to the degree of calcite orientation. Conversely, reduced strength in eggshell from aged hens coincides with a high variability in crystallographic texture. In guinea fowl the exceptional mechanical properties of the eggshell are explained by an increase in the amount of eggshell produced and particular features of the crystallographic texture. These observations suggest that the eggshell matrix influences the process of crystal growth by controlling size, shape and orientation of calcite crystals. This structural control probably contributes in a substantial manner to the mechanical properties of eggshell.

Protein constituents of the eggshell: eggshell-specific matrix proteins

In this article, we review the results of recent proteomic and genomic analyses of eggshell matrix proteins and draw attention to the impact of these data on current understanding of eggshell formation and function. Eggshell-specific matrix proteins from avian (ovocleidins and ovocalyxins) and non-avian (paleovaterin) shells are discussed. Two possible roles for eggshell-specific matrix proteins have been proposed; both reflect the protective function of the eggshell in avian reproduction: regulation of eggshell mineralization and antimicrobial defense. An emerging concept is the dual role (mineralization/antimicrobial protection) that certain eggshell matrix proteins can play.