Egg-shell derived carbon dots for base pair selective DNA binding and recognition.

The development of base pair selective fluorescent binding probes and their interaction mode with nucleic acids have created great interest for sensing and biomedical applications. Herein, we have used chicken egg shell membrane (ESM) as a cost effective easily available protein source for the synthesis of highly fluorescent carbon dots. The detailed characterizations have confirmed the in situ formation of heteroatom doped graphitic carbon nanodots (CDs) from ESM. The intrinsic fluorescence property of the material has been utilized for the label free binding of duplex deoxyribonucleic acid (DNA). The interaction of different natural and synthetic DNAs with carbon dots resulted in the enhancement of fluorescence characteristics of the latter. Analysis of the binding data obtained from steady state fluorescence studies revealed a selective and stronger affinity of CDs to the adenine-thymine (AT) base pair rich double stranded DNA (ds DNA) than that of the guanine-cytosine (GC) pair rich ds DNA. Base pair specific binding was further validated from isothermal titration calorimetry (ITC) and melting temperature data. The thermodynamic profile revealed endothermic binding that was driven by the hydrophobic interaction at the nano-bio interfaces. The results reveal the potential of carbon dots as a new and promising fluorescent probe for base pair selective and sequence specific DNA recognition.

ZP domain proteins in the abalone egg coat include a paralog of VERL under positive selection that binds lysin and 18-kDa sperm proteins.

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm-egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization.

Purification and characterization of avian beta-defensin 11, an antimicrobial peptide of the hen egg.

Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian ß-defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M + H](+) of 9,271.56 Da was obtained, and its N- and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.

Identification of estrogen-responsive vitelline envelope protein fragments from rainbow trout (Oncorhynchus mykiss) plasma using mass spectrometry.

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17ß-estradiol (E2) were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Specifically, plasma peptides of interest were fractionated and subsequently identified via spectra obtained by MALDI QqTOF MS/MS and LC-MALDI TOFTOF MS/MS analysis, de novo sequencing and database matching. The two peptide masses were identified as significant matches for fragments of the C-terminal propeptides from rainbow trout vitelline envelope protein (VEP)α and VEPγ isoforms. Our findings document the presence of the C-terminal propeptides from rainbow trout VEPα and VEPγ proteins in the bloodstream of juvenile male rainbow trout exposed to E2 via MALDI-TOF-MS detection. We provide three possible explanations for the presence of C-terminal propeptides in the bloodstream, as well as compare previously obtained hepatic transcriptomic results with the plasma proteomic results obtained in the present study.

Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs.

To analyze sperm surface molecules involved in sperm-egg envelope binding in Xenopus laevis, heat-solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP-ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP-ML. It was found that SP-ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65-130 and 20-30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose-dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization.

Mechanical properties of zona pellucida hardening.

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP.

Quantitative Comparative Proteomic Analysis of Chicken Egg Vitelline Membrane Proteins during High-Temperature Storage.

To explore the thermally induced alterations in chicken egg vitelline membrane (CEVM) protein abundances, a comparative proteomic analysis of CEVM after 10 days of storage at 30 °C was performed. Altogether, 981 proteins were identified, of which 124 protein abundances were decreased and 79 were increased. Bioinformatic analysis suggested that the altered proteins were related to structure (n = 10), mechanical properties (n = 13), chaperone (n = 15), antibacterial (n = 12), and antioxidant (n = 3). Alterations in abundances of structural proteins, possibly resulting from the disintegration of these complexes, were observed in this study, suggesting a loss in fibrous structure. Several proteins involved in mechanical strength (n = 10), elasticity (n = 3), and chaperone were decreased in abundances, which indicated that deficits in these proteins might affect the CEVM mechanical properties. These findings will extend our understanding of CEVM deterioration during high-temperature storage from a proteomic perspective.

Structural and proteomic analyses of vitelline membrane proteins of blackbird (Turdus merula) and song thrush (Turdus philomelos).

In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird (Turdus merula) and song thrush (Turdus philomelos). We performed structural analyses using scanning electron microscopy. The VM proteins were identified and compared to the best-known chicken VM proteins. According to our results, VM of both species has a typical three-layered structure: the outer layer, inner layer, and the continuous membrane between them. An unusual observation was the finding of "convexity" formed by the inner layer in blackbird. The role of these convex structures is not known, but they can be typical for the species and can be used in their identification. In addition, we identified two proteins in the VM of both species of birds, of which U3KEZ1 FICAL was not previously identified in any other bird species, and the U3JXV8 FICAL protein was confirmed only once in cockatiel parrot VM. The function of these proteins is not exactly known, but their structure shows similarities to the SERPIN proteins that are involved in microbiological defense, i.e., they are immune proteins. This study contributes to the current knowledge about the structure and composition of proteins of VM, especially because similar analyses have never been performed for Turdidae family. Knowledge of the structure and specific proteins of blackbird and song thrush VM can be beneficial in research on ecology and bird biology and also helpful in developing noninvasive and nongenetic identification methods.

Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro.

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEalpha ( approximately 58-60kDa Mr), VEbeta ( approximately 52kDa Mr), and VEgamma ( approximately 47kDa Mr). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEbeta and VEgamma were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEbeta and VEgamma dimers into very large oligomers. Mixtures of VEbeta and VEgamma also give rise to large oligomers. Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.

Organization of the vitelline envelope in ovarian follicles of Torpedo marmorata Risso, 1810 (Elasmobranchii: Torpediniformes).

In Torpedo marmorata, the vitelline envelope (VE), an extracellular envelope surrounding the growing oocyte, consists of fibrils and amorphous materials that are deposited in the perivitelline space starting from the initial steps of oocyte growth. SDS-PAGE analysis of the isolated and purified VE reveals that it consists of different glycoproteins. Furthermore, our investigations showed that the 120 and 66 kDa glycoproteins are positive to an antibody directed against gp69/64 of the Xenopus laevis VE and are synthesized under the control of 17beta-estradiol in the liver, that, together follicle cells and the oocyte, is the biosynthetic site of VE components.

Molecular characterization of egg envelope glycoprotein ZPD in the ovary of Japanese quail (Coturnix japonica).

The egg envelope surrounding avian oocytes exhibits a three-dimensional network of coarse fibers between the granulosa cells and the oocyte. Our previous studies have demonstrated that one of the matrix's components, ZP3, is synthesized in the ovarian granulosa cells. Another component, ZP1, which is critically involved in triggering the sperm acrosome reaction, is synthesized in the liver. We have previously isolated cDNAs encoding quail ZP3 and ZP1, and we now report the isolation of cDNA encoding quail ZPD. By RNase protection assay and in situ hybridization, we have demonstrated that ZPD transcripts are restricted to the granulosa cells of preovulatory follicles. The expression level of ZPD increased progressively during follicular development, and the highest expression was observed in the largest follicles. Western blot analyses using the specific antibody against ZPD indicate that the 40 kDa protein is the authentic ZPD, and the contents of ZPD protein also increased during follicular development. Moreover, we found that the addition of FSH to the culture media enhances the ZPD secretion in the cultured granulosa cells. Two-dimensional gel electrophoresis revealed the presence of several ZPD isoforms with different pI values ranging from 5.5 to 7. Immunohistochemical analyses indicate that the materials recognized with anti-quail ZPD antibody were accumulated in the egg envelope of large yellow follicles. These results demonstrate the presence of ZPD protein in the egg envelope, and that the amount of ZPD in the egg envelope as well as the mRNA in the cells increases at the latter stages of folliculogenesis.

Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein.

Gamete interactions during fertilization exhibit species specificity. In abalone, the sperm protein lysin species-specifically creates a hole in the egg envelope. Lysin evolves rapidly by positive Darwinian selection. Evolution of the egg receptor for lysin provides the selective pressure for lysin's divergence. The egg receptor for lysin is a tandemly repeated sequence that evolves by concerted evolution. Concerted evolution in the egg receptor could explain the rapid, adaptive evolution in sperm lysin and may provide an underlying molecular mechanism that gives rise to species-specific fertilization.

Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.

BACKGROUND INFORMATION: The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. RESULTS: VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. CONCLUSION: From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.

Allorecognition mechanisms during ascidian fertilization.

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.

Automated Macro Approach to Remove Vitelline Membrane Autofluorescence in Drosophila Embryo 4D Movies.

This chapter provides an ImageJ/Fiji automated macro approach to remove the vitelline membrane autofluorescence in live Drosophila embryo movies acquired in a 4D (3D plus time) fashion. The procedure consists in a segmentation pipeline that can cope with different relative intensities of the vitelline membrane autofluorescence, followed by a developed algorithm that adjusts the extracted outline selection to the shape deformations that naturally occur during Drosophila embryo development. Finally, the fitted selection is used to clear the external glowing halo that, otherwise, would obscure the visualization of the internal embryo labeling upon projection or 3D rendering.

Antioxidant and anti-inflammatory properties of chicken egg vitelline membrane hydrolysates.

Vitelline membrane (VM) is a multilayered structure that surrounds the egg yolk serving to separate the yolk and the white. Due to its poor solubility in aqueous-based media, VM proteins and their biological properties have not been fully defined. In the current study, VM was hydrolyzed using different enzymes under the optimum hydrolysis conditions. Antioxidant and anti-inflammatory properties were evaluated in chemical and cellular models. Flavourzyme- and trypsin-treated samples showed the highest radical scavenging and ferric ion reducing effect (31% and 20 µM of Trolox equivalents/mg, respectively). In cellular studies, all VM hydrolysates were cyto-compatible and inhibited nitric oxide production by RAW264.7 macrophage cells significantly. Lipopolysaccharide-stimulated up-regulation of pro-inflammatory cytokines in RAW264.7 cells was suppressed by flavourzyme-treated VM. These results revealed that enzymatic hydrolysis of VM is a promising approach to produce peptides with several bioactivities (free radical scavenging, metal chelation, and anti-inflammatory) as valuable ingredients for cosmeceuticals and nutraceuticals.

Tracking down the ZP domain: From the mammalian zona pellucida to the molluscan vitelline envelope.

Oocytes from virtually all organisms are surrounded by at least one coat. This specialized extracellular matrix, called the zona pellucida (ZP) in mammals and the vitelline envelope (VE) in nonmammals, has a structural function and plays essential roles in oogenesis, fertilization, and early development. During the last 15 years, compelling evidence has accumulated that all ZP/VE subunits polymerize using a conserved sequence, the ZP domain, so that the basic structural features of egg coat matrices have been maintained through evolution. Moreover, ZP domains have been identified in many other polymeric extracellular proteins from eukaryotes. This review compares the ultrastructure and molecular composition of egg coats from mollusc to human, suggests a common mechanism for assembly of ZP/VE proteins, and discusses alternative models of how these could be arranged within filaments.

Isolation and Characterization of Chicken Yolk Vitelline Membrane Lipids Using Eggs Enriched With Conjugated Linoleic Acid.

The vitelline membrane (VM) encloses the chicken egg yolk, separating it from albumen. The VM weakens during storage, and dietary lipid modification significantly affects its strength. However, no studies have characterize the fatty acyl residue (FA) composition of the VM, and reports of VM isolation and quantified lipid content are inconsistent. Therefore, the objectives of this study were: (1) to develop a washing and isolation method that removes residual yolk from VM without damage; (2) to determine the FA and lipid composition of CLA-rich egg yolk VM, relative to controls; (3) to determine the effect of 20 days of refrigeration on VM FA and lipid composition. To determine VM FA and lipid composition, 36 hens received either a corn-soybean meal-based control diet ("Control"), or the Control supplemented with either 10 % soy oil ("Soy control"), or 10 % CLA-rich soy oil ("CLA") for 30 days. VM were analyzed the day of collection ("fresh"), or after 20 days of refrigeration ("refrigerated"). There were no differences in FA compositions of fresh and refrigerated membranes within a treatment. CLA-rich yolk VM contains CLA, greater SFA, and significantly greater DHA relative to controls. Direct MALDI-TOF-MS identified 15 phosphatidylcholines, three phosphatidylethanolamines, one sphingomyelin, and 15 triacylglycerols in VM. Lipid species that showed significant differences among egg types included nine phosphatidylcholines and six triacylglycerols. MALDI analysis indicated significant differences in nine lipid classes on the VM inner layer. After refrigeration, five lipid classes on the inner layer and seven lipid classes on the outer layer had statistically significant differences among VM types.

Crystallization and preliminary crystallographic data of vitelline membrane outer layer protein I, VMO-I.

The vitelline membrane outer layer protein I (VMO-I), which is isolated from the vitelline membrane outer layer of hen's eggs, has been crystallized from an acetate buffer solution by the hanging-drop method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 62.42 A, b = 110.52 A, c = 44.15 A. There are two molecules (M(r) = 18,000) per asymmetric unit. The crystals diffract to at least 2.2 A Bragg spacings.

Exocytosis and proteomic analysis of the vesicle content of granular hemocytes from a crayfish.

The circulating blood cells (hemocytes) of invertebrates are important in cellular immune reactions and to deliver immune factors synthesized in these cells to the external milieu. Previously, we have shown that release of vesicle contents is involved in a regulated exocytosis and here we show which proteins in the vesicles are most abundant and which are released by triggering of exocytosis using a calcium ionophore, lipopolysaccharides-peptidoglycan and peroxinectin, a cell adhesion and degranulation factor from the hemocytes. The ionophore caused release of nine proteins and six of them were characterized and found to be a masquerade-like protein, a masquerade-like serine proteinase, a mannose receptor protein, a vitelline membrane outer layer protein I, and two anti-microbial peptides. The released protein band with a mass of 76 kDa is more likely pro-phenoloxidase and/or peroxinectin. When peroxinectin was used as a trigger of exocytosis, seven proteins could be identified and for the lipopolysaccharides-peptidoglycan six proteins could be identified and all of them were also released by the ionophore treatment. Interestingly, several anti-microbial peptides were the most abundant proteins and were efficiently released by all treatments as were two masquerade-like proteins one of which is functioning as an opsonic protein.