Experimental increase in temperature affects eggshell thickness, and not egg mass, eggshell spottiness or egg composition in the great tit (Parus major).

Phenotypic effects of global warming have been documented in many different taxa. However, the importance of transgenerational phenotypic plasticity in these adaptations are seldom studied. In birds, temperature could affect egg characteristics. Higher temperatures during egg-laying may reduce maintenance costs for females and allow a higher investment in reproduction. Yet, females may also use temperatures as a cue for the risk of mismatch latter in the season. Thus, higher temperatures may be correlated to an acceleration of embryonic development (e.g. via hormonal manipulation). We performed an experiment in which night-time temperature was increased in the nestbox by approximately 1 °C throughout the entire laying period in great tits (Parus major). We collected one pre-treatment egg (beginning of the laying sequence) and one post-treatment egg (end of the laying sequence). Egg content (yolk androgens and lysozymes in the albumen), eggshell coloration, eggshell mass, egg mass, and shape were not affected by the treatment. However, last-laid eggs in clutches from control nestboxes had a thicker eggshell than last-laid eggs from heated nestboxes, suggesting a putative slight decrease of maternal investment with the experimental increase of temperature. We also observed effects of the laying sequence on egg characteristics. Eggs that were laid late in the laying sequence were heavier, larger, had larger spots and higher yolk androgens than eggs laid earlier. Lysozyme concentration decrease with the laying sequence in late clutches only. Thus, effects of temperature may also change with the laying sequence and it would be interesting in the future to tests the effects on first-laid eggs.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. Hence, we show that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.

Increased collagen accumulation in eggshell membrane after feeding with dietary wood charcoal powder and vinegar.

Collagen in an eggshell membrane is important for egg preservation, medical burn treatment and manufacturing of cosmetics. Because collagen in the membrane is little, it is a need to improve the accumulation in the membrane to develop these applications. Wood charcoal powder with vinegar (WCV) is a natural substance that improves poultry production. In hen fed with WCV, total collagen in the eggshell membrane increased with an increase in dietary WCV and significantly increased in the 1.0% WCV group (p < 0.05). Scanning and light microscopic images revealed that this group had thicker eggshell membranes and a fine mesh structure composed of finer and more densely distributed fibres than in the control. Eggs from WCV group showed slow Haugh unit decrease during egg storage and the decrease correlated with total collagen in eggshell membrane. In intact chicken, type I and type III collagens were found in different specific locations in the oviduct but not in the membrane. The finding that collagen accumulates in the eggshell membrane under WCV feeding suggests that feeding chicken with WCV will permit long-term storage of eggs in poultry production, and the increased volume of total collagen will facilitate its application in medicine and cosmetics.

A new form of Elongatoolithidae, Undulatoolithus pengi oogen. et oosp. nov. from Pingxiang, Jiangxi, China.

A new oogenus and oospecies of the Elongatoolithidae, Undulatoolithus pengi oogen. et oosp. nov., is described on the basis of specimens from the Upper Cretaceous Zhoutian Formation of the Pingxiang Basin, Jiangxi Province, China. The eggs are slightly asymmetrical, paired, and lay radially-oriented in a circular configuration within the clutch, and most suggestive of Macroolithus of the Elongatoolithidae by medium-sized eggs with average polar axis and equatorial diameter of 19.36 and 8.35 cm, and the ornamentation pattern of nodes and ridges on the outer surface. The new oogenus differs from Macroolithus in its prominent ridges 0.67 mm in height, about half of the entire eggshell thickness, gradational boundary between the cone layer and the overlying columnar layer, cone layer-to-columnar layer thickness ratio of 1/8 or 1/4. This discovery adds new data on the morphology and diversification of Late Cretaceous elongatoolithid ootaxa. 

A novel disulfide-rich protein motif from avian eggshell membranes.

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

Preparation of gold nanoparticles on eggshell membrane and their biosensing application.

A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25+/-7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl(4) without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl(4)) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH < or =3.0 and > or =7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO(x)) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO(x). The linear response range of the glucose biosensor is 20 microM to 0.80 mM glucose with a detection limit of 17 microM (S/N=3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field.

Structure-property relationships of a biopolymer network: the eggshell membrane.

The eggshell membrane (ESM) is a biopolymer network that may have potential applications in biomedicine, but it also may reveal important details regarding the behaviour of biopolymer networks. In this paper, we have studied the mechanical and morphological properties of the ESM in order to reveal important structure-property relationships. Light optical microscopy and atomic force microscopy were used to assess the morphology of the ESM. The mechanical properties of membranes and individual fibres were studied by means of tensile tests and nanoindentation tests, respectively. The mechanical behaviour of ESM networks in different environmental conditions showed a non-linear and a linear regime. As for elastomers and other biopolymer systems, the non-linear regime was modelled by the Mooney-Rivlin relation. The Young's modulus in the linear regime of the network was related to the Young's modulus of the individual fibres using Gibson and Ashby analysis for cellular solids. The results of morphological characterization were used to relate the properties of individual fibres to the properties of the whole networks. This enabled us to predict the macroscopical properties of the network based on the properties of the individual fibres. It was found that the ESM networks behaved as both Mooney-Rivlin and Hookean materials in different environmental conditions. This study helps elucidate the properties of the biopolymer networks found in nature and describes important mechanical properties for the use of the ESM as a biomaterial.

Mapping of quantitative trait loci affecting eggshell quality on chromosome 9 in an F(2) intercross between two chicken lines divergently selected for eggshell strength.

Broken and cracked eggshells are major causes of significant economic losses to the egg production industry. The quantitative trait loci (QTL) on chromosome 9 influencing the quality of eggshells were identified by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation conducted over 14 generations. Chromosome-wide highly significant (P < 0.01) QTL associated with egg weight (EW), short length of egg (SLE), long length of egg (LLE) and eggshell weight were mapped to the distal region of chromosome 9. Among the QTL affecting EW, SLE and LLE, ovocalyxin-32 was identified as a potential candidate gene influencing eggshell traits. Marker-assisted selection based on these QTL could be used to develop strategies for reducing the breakage and cracking of eggs in commercial layer houses.

Rab11 is required for synchronous secretion of chondroitin proteoglycans after fertilization in Caenorhabditis elegans.

We previously identified a novel type of caveolin-enriched secretory vesicle in Caenorhabditis elegans oocytes. These vesicles undergo synchronous fusion with the plasma membrane immediately after fertilization, suggesting that they could be cortical granules that have been described in diverse animal species. Here, we report that these vesicles are indeed cortical granules, delivering essential chondroitin proteoglycans and mucin-like glycoproteins to the early embryonic extracellular matrices (ECMs). Furthermore, we have found that the small GTPase RAB-11 and the target-SNARE SYN-4 are required for cortical granule excoytosis after fertilization. In oocytes, SYN-4 localizes mainly to the plasma membrane, whereas GFP::RAB-11 accumulates transiently on the cortical granules during ovulation, immediately prior to fertilization. Importantly, cytokinesis defects in early embryos are commonly observed after depletion of either rab-11 or syn-4, producing a phenotype very similar to that observed after blockade of chondroitin synthesis. Taken together, our results indicate that at least part of the essential role for RAB-11 and SYN-4 in early embryogenesis is in the targeting of cortical granules to the plasma membrane during the precisely regulated secretion of ECM components.

Rab11 is essential for fertility in Drosophila.

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11mo, was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11mo males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11mo/Rab11mo egg chambers, confirming the requirement of Rab11 in these events.

Eggshell membrane as a biodegradable bone regeneration inhibitor.

The efficiency of chicken eggshell membranes combined with a minimally invasive small osteotomy procedure of the ulna to accomplish an efficient release of the radius so that it can continue to grow in an unstressed manner was tested in rabbits. Eggshell membranes were extracted from chicken eggs, rinsed, dried and sterilized with ethylene oxide for 24 h. For reactivity testing, four separate subcutaneous pockets were created in 10 rats in the paravertebral region by blunt dissection and eggshell membranes were implanted in two of them. After 1-16 weeks, the implants were retrieved with the surrounding soft tissues and submitted to histological examination. Subsequently, 10 rabbits were anaesthetized and a complete 0.5 mm wide osteotomy was performed in both the right and the left distal ulna. A piece of eggshell membranes was interposed in the osteotomy site of one ulna. The opposite osteotomized ulna was left as a negative control. The rabbits were injected with oxytetracycline at the time of surgery and this was repeated every 7 days for labelling new bone formation. After 1-16 weeks, ulnar osteotomized regions were histologically examined. After histological, fluorescence microscopy and radiological evaluation, we demonstrate here for the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging. The degradation of the eggshell membrane, due to host reaction, appeared sufficiently late to cause the desirable delay of bone healing that is compatible with the time needed for a corrective response.

Glutaraldehyde activated eggshell membrane for immobilization of tyrosinase from Amorphophallus companulatus: application in construction of electrochemical biosensor for dopamine.

Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. K(m) and V(max) values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min(-1), respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 degrees C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 degrees C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at -0.19 V versus Ag/AgCl (3M KCl). Linearity was observed within the range of 50-250 microM with a detection limit of 25 microM.

The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo.

BACKGROUND: Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage. RESULTS: We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization. CONCLUSION: Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.

In situ characterisation of a microorganism surface by Raman microspectroscopy: the shell of Ascaris eggs.

Intestinal nematodes are very common human parasites and a single species, Ascaris lumbricoïdes, is estimated to infect a quarter of the world's population. A sticky external layer covers their eggs. This work shows that Raman vibrational confocal spectroscopy is able to give information on the biochemical composition of the shell of Ascaris eggs. The biochemical localised characterisation of Ascaris eggs was performed directly on the eggs in their aqueous environment. The studied parasites came from two origins: dissections of adult females and extractions from biosolid sludges. The presence of mucopolysaccharides, proteins and chitin in the shell was demonstrated. The presence of ascaroside compounds was shown particularly via the narrow and intense bands from the organised long CH2 chains. To the best of our knowledge, this is the first time that the latter have been observed in Raman vibrational spectra of microorganisms. Hydration of the shell was different depending on the intensity of the colour of the sludge eggs. Knowledge of the biochemical structural properties of egg surfaces would be useful to understand the egg adhesion phenomena on vegetables contaminated by reused wastewater.

Domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis.

Null-mutation in Drosophila importin-alpha2, such as the deficiency imp-alpha2(D14), causes recessive female sterility with the formation of dumpless eggs. In imp-alpha2(D14) the transfer of nurse cell components to the oocyte is interrupted and the Kelch protein, an oligomeric ring canal actin organizer, is normally produced but fails to associate with the ring canals resulting in their occlusion. To define domains regulating Kelch deposition on ring canals we performed site-directed mutagenesis on protein binding domains and putative phosphorylation sites of Imp-alpha2. Phenotypic analysis of the mutant transgenes in imp-alpha2(D14) revealed that mutations affecting the Imp-beta binding-domain, the dimerization domain, and specific serine residues of putative phosphorylation sites led to a normal or nearly normal oogenesis but arrested early embryonic development, whereas mutations in the nuclear localization signal (NLS) and CAS/exportin binding domains resulted in ring canal occlusion and a drastic nuclear accumulation of the mutant proteins. Deletion of the Imp-beta binding domain also gave rise to a nuclear localization of the mutant protein, which partially retained its function in ring canal assembly. Thus, we propose that mutations in NLS and CAS binding domains affect the deposition of Kelch onto the ring canals and prevent the association of Imp-alpha2 with a negative regulator of Kelch function.

Characterization and adsorption properties of eggshells and eggshell membrane.

The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of fibrous proteins. From the isotherm data of methylene blue at 25 degrees C, the Freundlich model yielded a somewhat better fit than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0mg/g in aqueous medium, which was attributed to their poor pore properties.

Enzymatic and microbiological inhibitory activity in eggshell membranes as influenced by layer strains and age and storage variables.

Eggshell membranes (ESM) have been shown to exhibit antibacterial activity. The purpose of this study was to evaluate the enzymatic and biological [decimal reduction times (D-values)] activities of ESM as a function of bird breed, age, and ESM stabilization treatments. Younger White Leghorn (WL) hens produced ESM with 28% higher lysozyme activity than Rhode Island Red (RIR) layers. In contrast, older WL layers produced ESM with 17% less lysozyme activity than ESM from RIR layers. Similarly, beta-N-acetylglucosaminidase (beta-NAGase) ESM activities differed by hen age within breeds with younger hens yielding 14 to 16% more enzyme activity. D54 degrees C-values of Salmonella Typhimurium cells preexposed to WL ESM did not differ as a function of bird age (33, 50, and 81 wk). The ESM Lysozyme and beta-NAGase activities varied somewhat over a 6-mo storage study after treatment with 1 of 5 stabilization methods [i.e., storage at 4 degrees C, -20 degrees C, or ambient air storage after freeze drying, air drying (23 degrees C), or forced-air drying (50 degrees C)]. Both air and forced-air drying yielded significant reductions in beta-NAGase and lysozyme ESM activity (ca 12 to 30%) after the initial 24 h and then remained fairly stable during the extended storage. Freeze-dried samples retained the most enzymatic activity (95%) throughout the 6-mo trial, whereas refrigerated ESM lost 20 and 18% of the beta-NAGase and lysozyme activities, respectively. Frozen ESM lost 22% of the beta-NAGase activity, whereas lysozyme was nearly unaffected after 6 mo. The ESM biological activities against S. Typhimurium were not adversely impacted by layer breed or age. No significant loss in biological activity of ESM was detected 24 h after processing or after 6 mo of storage for refrigerated, frozen, and freeze-dried membranes, whereas significant reductions were observed for air- and heat-dried ESM. These findings demonstrate that ESM enzyme and biological activities are relatively constant across layer breeds and over extended storage. Based on these and other findings, ESM may have potential commercial value as a processing adjuvant in food and pharmaceutical product applications.

Immunohistochemical localization of the progesterone and oestrogen receptors in the shell gland of sexually immature ostriches (Struthio camelus) with active or inactive ovaries.

The immunohistochemical localization of progesterone and oestrogen receptors was studied in the shell gland of the immature ostrich (Struthio camelus) during periods of ovarian activity and inactivity. In birds with active ovaries moderate to strong immunostaining for the progesterone receptor was observed in the surface epithelium and tubular glands. In contrast faint progesterone receptor immunostaining was observed in the surface epithelium of the shell gland in ostriches with inactive ovaries. In addition, bud-like invaginations of the surface epithelium, which signaled tubular gland development, were negative for the progesterone receptor. Oestrogen receptor immunostaining, which was seen only in birds with active ovaries, was weak and restricted to nuclei of the surface epithelium. These results suggest that steroid hormones secreted by the active ovary regulate the differentiation of the shell gland. Furthermore, the influence of these hormones on the shell gland appears to be mediated predominantly through the activation of the progesterone receptor.

Electron miscroscopy and histochemical studies on four Egyptian helminthes eggs of medical importance.

By SEM the Fasciola gigantica egg is ovoid with a small knob like operculum, while the egg of Heterophyes heterophytes is broad oval with the operculum more tapering. The egg shell of fertilized Ascaris lumbricoides has interconnected ridges and peak-like projections, while the egg of Enterobius vermicularis is flattened with a thicker margin at the curved side. By TEM, Fasciola egg shell consists of fine reticulum fibrils of three layers. The outer lipoprotein of perivitelline membrane beneath which 2 membranes separated by inclusions, middle of protein globules and inner lipoprotein layer with minute electron-dense granules of melanin or polymer origin, in some parts of the shell giving the egg its brown coloration. The Heterophyes egg shell is more or less similar to that of Fasciola but lacking the minute electron-dense granules. The egg shell of Ascaris has outer ulterine layer with three consecutive layers, basal lipoprotein layer and the inner lipid or ascaroside layer which is the most resistant layer. The Enterobius egg shell consists of five layers, external uterine, internal uterine, vitelline, chitinous and lipid layer. Histochemically, Fasciola egg shell consists of nine amino-acids, and that of Heterophyes consists of ten amino acids. In Ascaris, the lipid layer characteristically consists 25% protein and 75% lipid. The histochemical examination of Enterobius as a detailed example, showed different degrees of reactions with mercuric bromophenol blue, diazotization coupling, Sakaguchi reaction, Sudan black and Mallory's triple stain. Temperature showed marked effect on eggs survival. Eggs of Fasciola and Heterophyes withstand more low temperatures but those of Ascaris and Enterobius withstand more high ones. There are marked correlations between the egg shell constitution, histochemical compositions on one hand and water permeability and egg dryness on the other hand. The results were photographed and discussed.