Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.     

体外培养日本血吸虫成虫生殖器官超微结构的观察

将日本血吸虫成虫于851培养基中培养23天后,对其生殖器官进行透射电镜观察。观察结果显示,雌虫卵巢内卵母细胞出现不同程度的变性、坏死;成熟卵黄细胞的卵黄小滴融合,脂质小滴数量增多、体积增大;培养后期卵壳形成发生障碍,最终导致无活性、无卵壳的畸形卵形成。超微结构观察首次显示,体外初产期虫卵卵壳中有条带状低电子密度区和高电子密度区相间排列。     

Molecular analysis and rescue of a vitelline membrane mutant in Drosophila

The eggshell in Drosophila is produced by ovarian follicle cells during the later stages of oogenesis. Eggshell formation involves the ordered synthesis and assembly of several protein components. Genes encoding the most abundant eggshell proteins have been identified by molecular cloning studies. Morphological examination of eggs produced by females carrying female sterile mutations on the X and third chromosomes have revealed additional loci involved in chorion formation. In this study we screened a collection of female sterile mutants carrying EMS-induced mutations on the second chromosome for eggshell mutants. A class of six mutants with potential vitelline membrane defects was identified on the basis of the response of mutant eggs to hypochlorite solutions. Biochemical analysis showed that one mutant, fs(2)QJ42, failed to produce a major vitelline membrane protein, sV23. The mutation was mapped cytogenetically to 26A, a region previously implicated in vitelline membrane formation by molecular cloning studies. Northern blot analysis using a cloned copy of the sV23 gene as probe showed a 10- to 15-fold reduction of sV23 RNA levels in the mutant. sV23 synthesis and fertility were restored when a normal copy of the sV23 gene was introduced into the mutant via germ line transformation. Transposons carrying the sV23 gene with as little as 147 bp of 5' flanking DNA were capable of restoring fertility and sV23 protein to wild type levels.     

The nudel protease of Drosophila is required for eggshell biogenesis in addition to embryonic patterning

The dorsoventral axis of the Drosophila embryo is defined by a ventral signal that arises within the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer of the eggshell. Production of the ventral signal requires four members of the serine protease family, including a large modular protein with a protease domain encoded by the nudel gene. Here we provide evidence that the Nudel protease has an integral role in eggshell biogenesis. Mutations in nudel that disrupt Nudel protease function produce eggs having vitelline membranes that are abnormally permeable to the dye neutral red. Permeability varies among mutant nudel alleles but correlates with levels of Nudel protease catalytic activity and function in embryonic dorsoventral patterning. These mutations also block cross-linking of vitelline membrane proteins that normally occurs upon egg activation, just prior to fertilization. In addition, Nudel protease autoactivation temporally coincides with vitelline membrane cross-linking and can be triggered in mature eggs in vitro by conditions that lead to egg activation. We discuss how the Nudel protease might be involved in both eggshell biogenesis and embryonic patterning.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

Developmental and age-related changes in proteins in the female reproductive tract of Heligmosomoides polygyrus (Nematoda)

Proteins in the female reproductive tract of Heligmosomoides polygyrus at days 8, 16, 35, 90, and 140 postinfection (PI) were examined using polyacrylamide gel electrophoresis. Sixteen-day-old and 140-day-old worms also were examined histochemically. Egg production of these worms was assessed for each age group. In analyzing proteins using electrophoresis, the reproductive tracts were separated into 3 sections: the tip, or anteriormost part of tract, containing oogonia; the middle region, containing developing oocytes; and the posterior region, containing the uterus with fertilized eggs. Three major reproductive tract proteins were identified as having molecular weights of greater than 140 kDa, 115 kDa, and 82 kDa. These were found in all parts of the reproductive tract from worms of all ages except those at 8 days PI (which are too young to produce eggs) and are believed to be yolk proteins. Four low molecular weight proteins (L1-4) are believed to be nucleoproteins. L4 was absent from the posterior section of the reproductive tracts and L3 was limited to the posterior sections and may be associated with sperm stored in the uterus. Of 5 high molecular weight proteins the second heaviest, designated H2, appeared to be relatively more concentrated in the posterior sections of the reproductive tract. An 85-kDa protein was limited to the tip and middle sections of reproductive tracts. Histochemical tests on sectioned H. polygyrus showed strong positive reactions for protein in cytoplasmic granules in developing oocytes and in eggs of younger worms (16 days) but a reduced reaction in older worms (140 days). Strains for collagen showed a slight positive reaction in and between developing oocytes and a strong reaction in the egg shells. Stains for nucleoproteins particularly reacted with sperm stored in the uterus, and slightly reacted with fertilized eggs and the nucleoli of the intestinal and ovarian epithelium. Egg production by H. polygyrus increased to 123 eggs/female/day by 16 days PI but declined from 121 eggs/female/day at 35 days PI to 64 eggs/female/day in worms 140 days old. Electrophoresis indicated no loss in the different types of proteins in the reproductive tract of older worms, but histochemistry and protein content assays suggest that older worms that produce fewer eggs contain a relatively smaller amount of protein in the female reproductive tract.     

Schistosoma mansoni: eggshell formation is regulated by pH and calcium

The protein precursors of the schistosome eggshell are synthesized and packaged into secretory vesicles in the vitelline cells. These vesicles appear to contain an emulsion of eggshell precursor material. Evidence is presented to show that these secretory vesicles are acidic as in other systems and that this acidity stabilizes the emulsion and prevents the eggshell cross-linking reactions from occurring. Alkalinizing treatments trigger eggshell formation within the secretory vesicles as shown by (1) the induction of autofluorescence and (2) by electron microscopy which shows that the eggshell precursors have aggregated within the secretory vesicles into spherical particles bearing microspines. These aggregates formed in the secretory vesicles were isolated and shown to have the same protease resistance and amino acid composition as authentic eggshell. The calcium ionophore A23187 induces scattered autofluorescence in intact female worms which electron micrographs show to be due to exocytosis of eggshell material. Based on these observations we propose a model for the formation of schistosome eggshell and suggest that it may apply to all trematodes in which the eggshell precursors are present as stable emulsions in the secretory vesicles of the vitelline cells.     

7C female sterile mutants fail to accumulate early eggshell proteins necessary for later chorion morphogenesis in Drosophila

Seven noncomplementing female sterile mutations that affect eggshell assembly in Drosophila have been mapped to the 7C1-3 region of the X-chromosome. TEM of the mature eggshell of one of the alleles, fs(1)410, shows a lack of organization within the endochorion and an accumulation of electron dense material in the vitelline membrane of stage 14 eggchambers. SDS-PAGE of radiolabeled eggshell proteins shows that two proteins, s67 and s85, fail to accumulate in the fs(1)410 eggshell. In wild-type flies s85 is produced during stage 10 of oogenesis and then processed to s67 in stages 13 and 14. Neither s85 nor an additional stage 10 specific follicle cell protein (s130) are detected in fs(1)410 or four of the mutant alleles. Short-term labeling studies, analyses of in vitro translation products, and the simultaneous occurrence of s85 and s130 as electrophoretic variants in geographic fly strains indicate s85 is derived from s130. Although major biochemical differences appear in stage 10, mutant and wild-type eggshells are morphologically indistinguishable until stages 13-14. These results suggest that follicle cell proteins synthesized during the time of vitelline membrane deposition (stage 10) are important for proper assembly of the chorion layers during stages 13 and 14.     

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.     

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 .     

The proteome of the insoluble Schistosoma mansoni eggshell skeleton

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold’s layer, von Lichtenberg’s envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.     

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.     

Heterodera glycines: eggshell ultrastructure and histochemical localization of chitinous components

The eggshell in most nematodes consists of an outer vitelline layer, a middle chitinous and an inner lipid layer. Earlier work with eggs of Heterodera glycines suggests the presence of two chitinous layers but the vitelline layer was not observed. From our observation the outer chitin layer described in past literature is actually a vitelline layer. Histochemical analysis has demonstrated that chitin is absent from the outer envelope. Electron microscope observations of the eggshell show a waxy appearance and osmium staining consistent with that of the proteinaceous vitelline layer found in other nematodes. Lectin localization also shows that the eggshell continues to develop past fertilization with the delivery and integration of eggshell precursors. Contrary to previous reports, we propose that the ultrastructure of the eggshell H. glycines follows the common three-layer structure observed in other nematodes.     

Presclerotized eggshell protein from the liver fluke Fasciola hepatica

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.     

A unique nuclear receptor direct repeat 17 (DR17) is present within the upstream region of Schistosoma mansoni female-specific p14 gene

The eggs produced by sexually mature female Schistosma mansoni are responsible for the pathogenesis of the disease. The eggshell precursor gene p14 is expressed only in the vitelline cells of sexually mature female worms in response to a yet unidentified male stimulus. Herein, we report the identification of a novel nuclear receptor response element in the upstream region of the p14 gene. This element contains the canonical hexameric DNA core motif, 5′-PuGGTCA, composed of an atypically spaced direct repeat (DR17). Schistosome nuclear receptors SmRXR1 and SmNR1 specifically bound to the p14-DR17 element as a heterodimer. SmRXR1, but not SmNR1, bound to the motif as a monomer. Introduction of mutations in the TCA core sequence completely abolished the binding by SmRXR1/SmNR1 heterodimer. This finding supports our hypothesis that the expression of Schistosoma mansonip14 gene is regulated through the nuclear receptor signaling pathway.     

Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms

Control of morbidity associated with schistosomiasis via chemotherapy largely relies on the drug praziquantel. Repeated therapy with praziquantel has created concerns about the possible selection of resistant worms and necessitated the search for novel drugs to treat schistosomiasis. Here, a murine model was infected with Schistosoma mansoni and treated with oral 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89), which caused a significant reduction in fecundity and egg burden and reduced morbidity when administered at 5-weeks post-infection.The analysis showed that the mode of action occurred through the ingestion of activated N-89 by the worms, and that there was no direct external effect on the S. mansoni worms. Ultrastructural analysis of the treated worms showed disruptions in the gut lumen and the presence of large volumes of material, suggestive of undigested blood meals or red blood cells. In addition, there were reduced vitelline cells in female worms and damage to sub-tegmental musculature in male worms. Eggs recovered from the treated mice showed both damage to the eggs and the production of immature eggs. Expression of mRNA responsible for gut and digestive function and egg production was also significantly affected by N-89 treatment, whereas control genes for musculature showed no significant changes.Thus, N-89 drastically affected the total digestive function and egg production of S. mansoni worms. Physiological processes requiring heme uptake such as egg production and eggshell formation were subsequently affected, suggesting that the compound could be a possible therapeutic drug candidate for schistosomiasis control.     

An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle

We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages -15 to -1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N-terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.     

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.