A comparative proteomic study of the undeveloped and developed Schistosoma mansoni egg and its contents: The miracidium, hatch fluid and secretions

The schistosome egg is the key agent responsible both for transmission of the parasite from human to molluscan host, and is the primary cause of pathogenesis in schistosomiasis. Characterisation of its proteome is a crucial step in understanding the egg’s interactions with the mammalian host. We devised a scheme to isolate undeveloped eggs from mature schistosome eggs by Percoll gradient and then fractionate the mature egg into miracidial, hatch fluid and secreted protein preparations. The soluble proteins contained within the five preparations were separated by two-dimensional electrophoresis and their spot patterns compared by image analysis. Large numbers of representative spots were then excised and subjected to tandem mass spectrometry to obtain identities. In this way, the principal components of each sub-proteome were established. Chaperones were the most abundant category, with heat shock protein 70 (HSP70) dominant in the undeveloped egg and Schistosoma mansoni protein 40 (Smp-40) in the miracidium. Cytoskeletal proteins were expressed at similar levels in the undeveloped egg and miracidium, with tubulins the most abundant. The proteins of energy metabolism reflected the change from anaerobic to aerobic metabolism as the miracidium developed. None of the above categories was abundant in the hatch fluid but this peri-miracidial compartment was highly enriched for defence proteins such as thioredoxin. Hatch fluid also contained several host proteins and schistosome proteins of unknown function, highlighting its distinct nature and potentially its role. The egg secretions could not be compared with the other preparations due to their unique composition featuring the previously characterised IL-4-inducing principal of S. mansoni eggs (IPSE), Omega-1, egg secreted protein 15 (ESP15), a micro-exon gene 2 (MEG-2) protein and two members of the recently described MEG-3 family. This last preparation contains the subset of egg proteins that probably enables eggs to escape from host tissues and may also initiate granuloma formation, emphasising the need to establish fully the roles of its components in schistosome biology.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida ( ) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida ( Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg hardening

The highly hydrophobic protein aggregate which constitutes the fish eggshell has for the first time been quantitatively solubilized. This study shows that the nonactivated eggshell from cod is composed primarily of only three protein monomers, designated alpha (74 kDa) beta (54 kDa) and gamma (47 kDa). Protein extraction studies of the eggshells before and after egg activation demonstrate that egg hardening is accompanied by a 10-fold decline in total protein solubility, which is due to nonextraction of the alpha, beta, and gamma chains. When present during the egg activation process monodansylcadaverine (MDC-a fluorescent lysine analog) inhibits eggshell hardening and at the same time becomes covalently incorporated into the eggshell. This MDC incorporation is calcium-dependent and suggests the induction of a perivitelline transglutaminase activity after egg activation. (Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the gamma-carbonyl group of glutamine and the epsilon-amino group of lysine with release of ammonia. Crosslinks between proteins are generated when the two amino acid residues are located on different proteins.) Protein solubilization studies and NaDodSO4 gel analysis of the eggshell proteins from eggs subjected to 5 mM MDC during egg activation, reveal that when eggshell hardening is blocked by MDC, the three main eggshell proteins remain extractable even after egg activation. Simultaneously we observed a covalent incorporation of MDC into the gamma protein.     

An ultrastructural study of the egg wall surrounding the miracidium of the digenean Brandesia turgida () (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope

The intrauterine eggs of the pleurogenid trematode Brandesia turgida (Brandes, 1888), exhibiting advanced stages of miracidial differentiation and fully formed, ciliated miracidia, were examined by means of transmission electron microscopy (TEM). Each embryonated egg is composed of a mature miracidium surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate egg-shell; (3) a small remnant of the compact, granular cytoplasm of the outer embryonic envelope (sensu stricto); and (4) a relatively distinct cellular remnant of the inner embryonic envelope. Layers enveloping the egg apparently play an important role in the protection, metabolism and storage of nutritive reserves for the developing miracidium. The outer, anucleate layer, or cocoon, situated externally to the eggshell and composed of a transparent, electron-lucent substance with numerous dense, osmiophilic islands attached to its peripheral membrane, has never previously been seen in TEM studies of the eggs of parasitic platyhelminths. The origin, formation, functional ultrastructure and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. The very small, indistinct remnants of the outer embryonic envelope appear in the form of a very thin, compact, granular cytoplasm closely attached to the inner surface of the eggshell. Conversely, the inner embryonic envelope is frequently apparent at one or both poles of the developed egg as a syncytial envelope formed by the fusion of mesomeres. This envelope, even in eggs containing a fully formed miracidium, still has the features of a metabolically active layer with an energy storage capability. Lysosome-like structures observed in some eggs may be involved in the autolysis of the embryonic envelopes.     

Binding of von Willebrand factor and plasma proteins to the eggshell of Schistosoma mansoni

Schistosoma mansoni eggs have to cross the endothelium and intestinal wall to leave the host and continue the life cycle. Mechanisms involved in this essential step are largely unknown. Here we describe direct binding to the S. mansoni eggshell of von Willebrand factor and other plasma proteins involved in haemostasis. Using deletion-mutants, we demonstrated that it is the A1 domain of von Willebrand factor that binds to the eggshell. Our results suggest that binding of plasma proteins to the eggshell promotes binding to the endothelium, initiating the passage of the egg through the blood-vessel wall to be excreted in the end.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Cracks in the shell—zooming in on eggshell formation in the human parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis>

Schistosomiasis, currently the second most common parasitic disease of humans in tropical regions is caused by the eggs of trematode worms of the genus Schistosoma. Understanding egg formation and specifically the synthesis of the eggshell comprises, consequently, a promising starting point to cure and prevent the disease. To shed light on the genetics of the latter process, we analysed the three known S. mansoni eggshell proteins P14, P19 and P48 against the background of the species inferred proteome and of eggshell proteins identified in other trematode species. Our results suggest that eggshell formation in Schistosoma involves a multitude of different proteins organised in currently three distinct protein families (P14, P48 and P34 eggshell protein family). The first two families are of simple structure. Their respective members share a substantial degree of sequence similarity and are, to date, observed only in the genus Schistosoma. In contrast, the P34 family of eggshell proteins is complex. Its in part highly diverged members share only a conserved motif of 67-aa length on average and are detected in various trematode species. The resulting widespread occurrence of this protein motif suggests an important role during eggshell formation in trematodes. Screening more than 7,000 putative proteins of S. mansoni, we could identify six new members of the P34 protein family that are likely to be involved in eggshell formation in this species.Electronic Supplementary Material Supplementary material is available for this article at .     

Understanding control of calcitic biomineralization—Proteomics to the rescue

The avian eggshell is one of the fastest calcifying processes known and represents a unique model for studying biomineralization. Eggshell strength is a crucial economic trait for table egg production, and ensures that a safe egg reaches the consumer kitchen. However, a common toolkit for eggshell mineralization has not yet been defined. In this issue, label‐free MS‐based protein quantification technology has been used by Sun et al. (Proteomics 2013, 13, 3523–3536) to detect differences in protein abundance between eggshell matrix from strong and weak eggs and between the corresponding uterine fluids bathing strong and weak eggs. Proteins associated with the formation of strong eggshells are identified, which are now candidates for further investigations to define the regulatory relationship between specific eggshell matrix proteins and calcite crystal texture.     

Schistosoma mansoni: the egg, biosynthesis of the shell and interaction with the host

The schistosome eggshell is a hardened and tanned structure made from cross-linked proteins. It is synthesized within the female worm from many different kinds of proteins and glycoproteins. Once the egg is released in the circulation, the outer surface of the eggshell is exposed and hence a direct site of interaction between the parasite and the host. The major eggshell protein is p14, but about one third of the eggshell is made from common cellular proteins, some of which are known to be immunogenic. This has many consequences for parasite-host interactions. However, so far, the eggshell has gained little attention from researchers. We will discuss the structure of the eggshell and its role in granuloma formation, host factor binding and egg excretion.     

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF-MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.     

Bemerkenswerte endogene Farbveränderung der Eischale von Chioninia delalandii (Duméril & Bibron, 1839) (Reptilia: Sauria: Scincidae)

The paper describes an unusual endogenous eggshell colouration observed in an egg of the Cap Verde skink Chioninia delalandii.A female specimen, kept in a terrarium, laid three eggs. Two of them were considered as fertilized (oval germ-disk, weakly pink). They were embedded 1 cm deep in a layer of moistened clay granules (substrate/water 2:1) and kept under different temperatures (egg 1 “cool”, 26-27 °C; egg 2 “warm”, 29-30 °C). There was a normal embryonic development in both eggs from their volume-enlargement and characteristic allometric growth (egg wide > egg length). The young hatch after 51 and 56 days.A change in eggshell colour, however, occurred in the “warm” kept egg during the last third of its incubation period. It started with a small spot (2-3 mm) in the outer area of the animal egg pole and spread into a dark-violet colouration over the whole eggshell within 15 days. After hatching of the young the shells of both eggs were examined. In the non-coloured egg there was no great difference in colour between the inner and outer egg layer, while in the coloured egg there was a distinct difference between the inner part, which was dark violet-gray, and the pale gray calcareous deck-layer. From the macroscopic view along the edge of the eggshell it was not identifiable, if the colour pigment was infiltrated into protein fibrils of the condensed surface layer.A possible explanation for the eggshell colouration could be an unusual embryonic pigmentation. This assumption is based on the first appearance of a restricted, point-like coloured area and its further regular extension. It might be that dark pigments (melanophores?) reached the eggshell (membrana testacea) and infiltrated the border-area to the condensed surface layer.     

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca²⁺-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).     

Post-mating change in excretion by mated Drosophila melanogaster females is a long-term response that depends on sex peptide and sperm

Drosophila seminal fluid proteins elicit physiological and behavioral changes in the female after mating. For example, the seminal protein sex peptide (SP) causes females to lay more eggs, reduce receptivity to re-mating, consume more food and produce more concentrated excreta upon mating. It has been reported that SP indirectly increases food consumption as a result of its stimulation of egg production, but its role in producing more concentrated excreta in the mated female was reported to be independent of egg production. Additionally, it has been shown that SP’s effect on food consumption persists for several days after mating, while it is unknown whether this is true for its effect on excretion.     

iTRAQ‐Based Quantitative Proteomic Analysis of Duck Eggshell During Biomineralization

The avian egg is a valuable model for the calcitic biomineralization process as it is the fastest calcification process occurring in nature and is a clear example of biomineralization. In this study, iTRAQ MS/MS is used to detect and study for the first time: 1) the overall duck eggshell proteome; 2) regional differences in the proteome between the inner and outer portions of the duck eggshell. The new reference protein datasets allow us to identify 179 more eggshell proteins than solely using the current release of Ensembl duck annotations. In total, 484 proteins are identified in the entire duck eggshell proteome. Twenty‐eight novel proteins of unknown function that are involved in eggshell formation are also identified. Among the identified eggshell proteins, 54 proteins show differential abundances between the inner, partially mineralized eggshell (obtained 16 h after ovulation) compared to the overall complete eggshell (normally expulsed eggshell). At least 64 of the abundant matrix proteins are common to eggshell of 4 different domesticated bird species (chicken, duck, quail, turkey) and zebra finch. This study provides a new resource for avian eggshell proteomics, and augments the inventory of eggshell matrix proteins that will lead to a deeper understanding of calcitic biomineralization.     

Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.     

PHYLOGENETIC ANALYSIS OF REPRODUCTIVE TRAITS OF MANIRAPTORAN THEROPODS AND ITS IMPLICATIONS FOR EGG PARATAXONOMY

Abstract:  A phylogenetic analysis of reproductive and oological (egg) traits of theropod taxa allows determination of the sequence in which these traits evolved in Theropoda. Our results indicate that several avian reproductive traits, such as adults sitting on eggs, asymmetrical eggs, unornamented eggshell surface, and complex eggshell ultrastructure, were already present in non-avian maniraptorans, and could have evolved in more basal theropods. In addition, non-avian maniraptorans laid two eggs at a time and orientated their eggs subvertically or subhorizontally in their nests, features not retained by neornithine birds. Based on our cladistic analysis it is also possible to infer the phylogenetic affinity of ootaxa of unknown parentage: Protoceratopsidovum was laid by a maniraptoran more derived than oviraptorids, and Parvoolithus probably belonged to a Cretaceous bird. Finally, our analysis reveals that many of the high-level categories of egg parataxonomy (morphotypes and basic types) are unnatural groupings (i.e. non-monophyletic). We recommend that these high-level categories be abandoned because oofamilies are sufficient to categorize egg taxa.     

利用转录组测序筛选鸡蛋褐壳性状相关基因

褐壳鸡蛋在许多国家深受消费者喜爱,消费者通常把蛋壳褐色深浅作为评价鸡蛋品质的重要指标。褐壳鸡蛋蛋壳颜色形成受多基因共同调控,但是具体候选基因及调控机理尚未明确。因此,本研究以纯系褐壳蛋鸡为实验材料,筛选调控褐壳鸡蛋颜色深浅的候选基因。采用转录组测序技术对产深褐壳鸡蛋和浅褐壳鸡蛋的母鸡蛋壳腺组织进行分析,结果显示,共计有8461个基因在蛋壳腺组织表达,其中34个基因在两组之间差异表达。功能分析发现,卵转铁蛋白(ovotransferrin, TF)基因、热休克蛋白70 (heat shock protein, HSP70)基因以及氧化磷酸化通路均与原卟啉Ⅸ合成通路相关,可能影响褐壳鸡蛋蛋壳色素原卟啉Ⅸ的合成和积累。     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.     

Egg envelope synthesis and chorion modification after oviposition in the dragonfly Libellula depressa (Odonata, Libellulidae).

Libellula depressa (Odonata, Libellulidae) is an exophytic dragonfly ovidepositing eggs in clutches on the surface of floating plants and algae. The present work investigates, at ultrastructural level, the gradual differentiation of the egg envelopes and the chorionic changes after egg deposition in water. The ovary of the mature female of L. depressa is composed of numerous strings of panoistic ovarioles, where the eggshell formation takes place gradually throughout the activity of the follicle cells. The present data show that the egg envelopes are constituted of a very thick electrondense vitelline envelope, a thin endochorion and an extremely thick exochorion composed of a fibrillar matrix resting on a thin electrondense layer. After deposition in water, L. depressa eggs, initially white and almost transparent, gradually become brown spots in a semitransparent jelly coat, rich of incorporated debris. The jelly coat enveloping the eggs of L. depressa derives exclusively from the exochorion, constituted of a fibrillar matrix, which swell at contact with water. The jelly-like coat performs an adhesive function and presumably a protective role during egg segmentation and ensuing larval hatching.