Acellular Envelope Research Articles

Dechorionated zebrafish embryos improve evaluation of nanotoxicity.

In response to the growing need to evaluate nanomaterial (NM) toxicity and compliance with the "3Rs" principles (replacement, reduction, and refinement of animal experiments), zebrafish (Danio rerio) embryos have emerged as a promising alternative model for studies on NM toxicity. However, zebrafish embryos are surrounded by an acellular envelope, the chorion, which limits the permeability of NMs. The present study investigated the importance of dechorionated zebrafish embryos for evaluating NM toxicity. We utilized confocal microscopy and field-emission scanning electron microscopy with energy-dispersive spectroscopy to observe the permeability of NMs into the embryonic body using 50-nm fluorescein 5 (6)-isothiocyanate-incorporated silica nanoparticles (FITC-SiO2NPs). We investigated the physiological effects of removing the chorion using pronase on zebrafish embryos. Nanotoxicity was compared depending on the presence or absence of the chorion in zebrafish embryos using the standardized method ISO/TS 22082:2020. The FITC-SiO2NPs were adsorbed onto the embryonic chorion; the Si content was higher in the chorion than in the embryonic body and higher in the intact zebrafish embryos than in the dechorionated ones. Dechorionated zebrafish embryos exhibited no negative physiological effects. The LC50 values of several NMs were lower in dechorionated embryos than those in intact ones. Dechorionated zebrafish embryos exhibited greater sensitivity to NMs than usual. To the best of our knowledge, this is the first study to evaluate NM toxicity using a new standardized test method, ISO/TS 22082:2020, and could contribute towards the increased utility of dechorionated embryos as an alternative model for the evaluation of nanotoxicity.

Comparison of Pronase versus Manual Dechorionation of Zebrafish Embryos for Small Molecule Treatments.

Zebrafish are a powerful animal model for small molecule screening. Small molecule treatments of zebrafish embryos usually require that the chorion, an acellular envelope enclosing the embryo, is removed in order for chemical compounds to access the embryo from the bath medium. For large-scale studies requiring hundreds of embryos, manual dechorionation, using forceps, can be a time-consuming and limiting process. Pronase is a non-specific protease that is widely used as an enzymatic alternative for dechorionating zebrafish embryos. However, whether pronase treatments alter the effects of subsequent small molecule treatments has not been addressed. Here, we provide a detailed protocol for large-scale pronase dechorionation of zebrafish embryos. We tested whether pronase treatment can influence the efficacy of drug treatments in zebrafish embryos. We used a zebrafish model for Duchenne muscular dystrophy (DMD) to investigate whether the efficacies of trichostatin-A (TSA) or salermide + oxamflatin, small molecule inhibitors known to ameliorate the zebrafish dmd muscle degeneration phenotype, are significantly altered when embryos are treated with pronase versus manual dechorionation. We also tested the effects of pronase on the ability of the anthracycline cancer drug doxorubicin to induce cardiotoxicity in zebrafish embryos. When comparing pronase- versus forceps-dechorionated embryos used in these small molecule treatments, we found no appreciable effects of pronase on animal survival or on the effects of the small molecules. The significant difference that was detected was a small improvement in the ability of salermide + oxamflatin to ameliorate the dmd phenotype in pronase-treated embryos when compared with manual dechorionation. Our study supports the use of pronase treatment as a dechorionation method for zebrafish drug screening experiments.

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae.

Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish ( Danio rerio)

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the “naked” embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24 h post-fertilization (hpf) with survival rates of ≥ 90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24 h of development. Dechorionation of younger stages (< 24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~ 400,000 Dalton, but becomes strongly toxic after dechorionation.

Molecular Characterization and Expression Pattern of Zona Pellucida Proteins in Gilthead Seabream (Sparus aurata)1

The developing oocyte is surrounded by an acellular envelope that is composed of 2-4 isoforms of zona pellucida (ZP) proteins. The ZP proteins comprise the ZP1, ZP2, ZP3, and ZPX isoforms. While ZP1 (ZPB) and ZP3 (ZPC) are present in all species, ZP2 (ZPA) is not found in teleost fish and ZPX is not found in mammals. In the present study, we identify and characterize the ZP1, ZP3 and ZPX isoforms of gilthead seabream. Furthermore, by analyzing the conserved domains, which include the external hydrophobic patch and the internal hydrophobic patch, we show that ZP2 and ZPX are closely related isoforms. ZP proteins are synthesized in either the liver or ovary of most teleosts. Only in rainbow trout has it been shown that zp3 has dual transcription sites. In gilthead seabream, all four mRNA isoforms are transcribed in both the liver and ovary, with zp1a, zp1b, and zp3 being highly expressed in the liver, and zpx being primarily expressed in the ovary. However, determination of the ZP proteins in plasma showed high levels of ZP1b, ZP3, and ZPX, with low or non-detectable levels of ZP1a. In similarity to other teleost ZPs, the hepatic transcription of all four ZP isoforms is under estrogenic control. Previously, we have shown that cortisol can potentiate estrogen-induced ZP synthesis in salmonids, and now we show that this is not the case in the gilthead seabream. The present study shows for the first time the endocrine regulation of a teleost ZPX isoform, and demonstrates the dual-organ transcriptional activities of all the ZP proteins in one species.

Host-parasite interface between Asellus aquaticus (Isopoda) and larvae of Acanthocephalus anguillae (Acanthocephala)

The present study describes the ultrastructure of the interface between naturally infected isopods, Asellus aquaticus (L.), from the River Brenta (northern Italy) and larvae of Acanthocephalus anguillae (Muller, 1780) Luhe, 1911. A fully developed larva of this helminth occupies the host’s hemocoel and induces displacement of its internal organs. A transparent acellular envelope ranging in thickness from 1.1 to 1.6 µm covers each A. anguillae larva within the hemocoel of the crustacean. This envelope establishes intimate contact with the internal organs and hemocytes of the host. Intact isopod hemocytes, as well as cells in varying degrees of degeneration, were observed mainly on the outermost edge of the envelope of A. anguillae. No melanised acanthocephalan larvae were seen within the hemocoel.

Structure and macromolecular composition of the zebrafish egg chorion.

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43 kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50 kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.

Fine Structure of the Envelope Surrounding the Cystacanth of Acanthocephalus clavula (Acanthocephala) in Its Intermediate Host Echinogammarus stammeri (Amphipoda)

We describe the ultrastructure of the envelope covering each cystacanth of Acanthocephalus clavula within the hemocoel of naturally infected amphipods, Echinogammarus stammeri, from the River Brenta (northern Italy). A fully developed larva of this acanthocephalan occupies a large portion of the host's hemocoel. The envelope bounding the parasite maintains intimate contact with the internal organs and hemocytes of the host. Intact amphipod hemocytes, as well as cells in varying degrees of degeneration, mainly on the outermost edge of the envelope of A. clavula, were observed. No melanized larvae of A. clavula were seen within the hemocoel. This envelope apparently protects the developing acanthocephalan larva from cellular responses of the host. The present paper reports light and transmission electron microscopical observations on this transparent acellular envelope surrounding the cystacanth of A. clavula. In the interface region of the parasite envelope with hemocytes of E. stammeri, cells were found adherent to the outer surface of the larval envelope and also between the inner edge of the envelope and parasite

Fertilization in ascidians: Studies on the egg envelope, sperm and gamete interactions in Phallusia mammillata

Eggs and sperm of the ascidian Phallusia mammillata were studied by light microscopy and scanning and transmission electron microscopy. The egg is enclosed in a rigid acellular envelope, the vitelline coat (vc). The vc consists of a fibrous surface layer, a homogeneous electron dense central layer and a thick inner layer with thin fibers and globular elements. Vacuolated follicle cells are attached to the outer, test cells to the inner surface of the vc. Upon staining with phalloidine-rhodamin both cell types exhibit a distinct pattern of actin filaments. Studies of gamete interactions during fertilization reveal that sperm binding is established between the tip of the sperm and the fibrous layer of the vc at sites which are not covered by follicle cells. Freeze fractures of quick frozen sperm show a cap-like structure at the sperm apex. This structure contains a number of vesicles as detected in sperm prepared for TEM by quick freezing and freeze substitution. In sperm just about to penetrate the vc apical vesicles which may contain the vitelline coat lysins have been found. Wheat germ agglutinin (WGA), which binds to the vc but not to follicle cells and sperm, inhibits fertilization by interfering with sperm-vitelline coat binding. This inhibition was completely abolished by competitive sugars such as N,N′,N″-triacetylchitotriose (1 m M) or N-acetylglucosamine. However, there are indications that WGA binding sites on the vc may not only be N-acetylglucosamine but also sialic acid residues. Preliminary studies reveal similarities between ascidian and mammalian fertilization concerning the phagocytosis-like gamete fusion process.

PERMEABILITY OF THE MOUSE ZONA PELLUCIDA TO IMMUNOGLOBULIN

Reproductive Immunology Group, Department of Pathology, University of Bristol, University Walk, Bristol BS8 1 TD (Received 20th June 1974) During the preimplantation stages of pregnancy, the mammalian embryo is surrounded by an acellular envelope, the zona pellucida. Although little is known of the functions of this membrane it has been suggested that it may be impermeable to antibody molecules (immunoglobulins), thereby protecting the embryo from the potentially adverse effects of maternal immunity (Simmons & Russell, 1966, 1967). In the present study, we have investigated the permeability of the mouse zona pellucida to immunoglobulins by comparing the complementmediated cytotoxic effect of rabbit anti-mouse serum and two of its immunoglobulin components, IgG and IgM, on the development of zona-free and zona-enclosed embryos in vitro. Mouse embryos at the 8-cell stage were recovered in phosphate-buffered saline (PBS) from the oviducts of randomly bred hybrid mice 48 hr after the finding of a vaginal