Factors affecting chilled storage of zebrafish (Danio rerio) embryos

Chilled storage of zebrafish embryos was investigated at a temperature that arrests embryonic development as this technique might offer interesting practical applications. Five parameters played an important role for chilled storage: (a) storage temperature, (b) development stage of embryos, (c) storage solution (extender), (d) postchilling treatment, and (e) inhibition of growth of microorganisms by antibiotics. The optimal chilling temperature was 8 °C. Prim-5 stage (24 h postfertilization [hpf]) and prim-25 stage (36 hpf) embryos had similar high chilling resistance and could be chilled for 33 h without a loss in viability. Five-somite stage (12 hpf) embryos had a lower chilling resistance and could be chilled only for 14 h without a loss in viability. After longer incubation periods, the viability started to decrease. Under these conditions, chilling in physiologic saline solutions was superior to that in water. Fifty percent of the prim-5 stage and prim-25 stage embryos survived for 41 h at 8 °C in water but for 46 h in physiologic saline solution. A similar effect was observed for 5-somite stage embryos (50% survival rate in water, 28 h; 50% survival rate in physiologic saline solution, 35 h). When embryos were incubated in physiologic saline solution instead of water in the postchilling phase, the embryo viability was positively affected, too. Also, supplementation of the storage solution with antibiotics (penicillin and streptomycin) increased the viability of chilled embryos. In summary, the current study shows that chilled storage of zebrafish embryos is possible for sufficiently long periods to synchronize the development of embryos deriving from different spawning dates or to delay the development for experimental purposes. To prolong the storage periods, further development and standardization of the methodology is necessary.     

Thermal biology of zebrafish (Danio rerio)

Zebrafish has become one of the most important animal models in research. Most of the variables studied using zebrafish are influenced by water temperature. The objective of this review was to analyze the published data on the thermal biology of the zebrafish. The paper first provides a brief introduction to zebrafish ecology and thermal tolerance, and continues with a review of the influence of temperature on several physiological variables, including development, growth, metabolism, reproduction, behavior, circadian biology and toxicology. Although a number of papers have already studied the effects of temperature on the zebrafish biology, knowledge in this field is still scarce, especially compared with other model organisms such as the rat, and therefore further research should be encouraged.     

Proteomic analysis of methyl parathion-responsive proteins in zebrafish (Danio rerio) brain

Methyl parathion (MP), an organophosphorus pesticide used worldwide, has been associated with a wide spectrum of toxic effects on organisms in the environment. This study set out to analyze the alteration of protein profiles in MP-exposed zebrafish (Danio rerio) brain and find the proteins responsive to MP toxicity. Zebrafish were subjected to 1, 3 and 5 mg/L MP and the proteomic changes in their brains were revealed using two-dimensional gel electrophoresis. Six protein spots were observed to be significantly changed by MP exposure. Among these, 4 spots were down-regulated, while 2 spots were up-regulated. These altered spots were excised from the gels and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and database searching. The results indicate that these proteins were involved in binding, catalysis, regulation of energy metabolism and cell structure. These data may provide novel biomarkers for the evaluation of MP contamination and useful insights for understanding the mechanisms of MP toxicity.     

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0–2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO, and that recovered PGCs retained ability to differentiate into functional gametes.     

Preference for structured environment in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis)

Information about the welfare and husbandry of pet and laboratory fish is scarce although millions of fish are sold in pet shops and used in laboratory research every year. Inadequate housing conditions can cause behavioural problems also in fish since they are complex animals with sophisticated behaviour. In this study, we investigated the influence of environmental complexity on compartment preference and behaviour in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis). For the preference test, large aquaria were divided by two semi-transparent walls of Plexiglas into an empty compartment, a structured compartment enriched with plants and clay pots, and a smaller compartment in-between, where food was provided. For observation, the empty and structured compartments were divided into six zones of similar size by defining three vertical layers and two horizontal areas (back vs. front area). Seven groups of six to nine zebrafish and seven groups of seven or eight checker barbs were observed on four days each (within a time period of ten days) to assess compartment use and activity, and to assess behavioural diversity and use of zones within compartments. Both zebrafish and checker barbs showed a significant preference for the structured compartment. Nevertheless, in neither species did behavioural diversity differ between the empty and structured compartment. Zebrafish used all zones in both compartments to the same extent. Checker barbs, however, used the structured compartment more evenly than the empty compartment, where they mainly used the lower and middle zones. These results suggest that zebrafish and checker barbs have a preference for complex environments. Furthermore, they indicate that the behavioural and ecological needs of fish may vary depending on species, and recommendations for husbandry should be specified at species level.     

Ca/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio

Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca²⁺/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP). The isolated cDNA clone possessed an open reading frame of 1272 bp encoding 424 amino acids and shared 47% and 48% amino acid identity with rat and human CaMKP, respectively. Interestingly, zCaMKP lacks the Glu cluster corresponding to residues 101-109 in the rat enzyme, and was not activated by polycations such as poly-l-lysine. The recombinant zCaMKP required Mg²⁺ rather than Mn²⁺ for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, α-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca²⁺ signaling pathways in zebrafish. Cotransfection of zCaMKP with mammalian CaMKI significantly decreased phospho-CaMKI in ionomycin-stimulated 293T cells. During embryogenesis, the expression of zCaMKP increased gradually after 48 h post-fertilization, as demonstrated by Western blotting using an anti-zCaMKP antibody. The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides resulted in an increased incidence of embryos with severe morphological and cellular abnormalities, i.e., a significant increase in the number of round-shaped embryos and apoptotic cells in the whole body. A marked decrease in zCaMKP expression was observed in the antisense- but not control oligo-injected embryos. Embryonic death was rescued by coinjection with recombinant rat CaMKP but not with phosphatase-dead mutant (D194A). These results clearly show the significance of zCaMKP during zebrafish embryogenesis.     

Ontogeny of cardiovascular control in zebrafish (Danio rerio): Effects of developmental environment

The goal of this symposium paper was to identify and quantify developmental plasticity in the onset of cardiovascular responses in the zebrafish. Developmental plasticity was induced by altering the developmental environment in one of three ways: (1) by developing zebrafish in a constant current of 5 body lengths per second, (2) by developing zebrafish at a colder temperature (20 °C), and (3) by developing zebrafish in severe hypoxia (DO = 0.8 mg/L). Early morphological development was significantly affected by each of the treatment environments with hypoxia slowing development the most and producing the highest variation in measurements. Development in constant water current did not significantly affect the timing onset of cardiovascular responses to the pharmacological agents applied. Development at 20 °C significantly delayed the onset of all cardiovascular responses measured by 2–3 days. Development in hypoxia, however, not only delayed onset of all cardiovascular responses, but also shifted the onset relative to the developmental program. Hypoxia clearly has a profound affect on the onset of cardiovascular regulation and it will take many more studies to elucidate the mechanisms by which hypoxia is having its effect. Furthermore, long term studies are also needed to assess whether the plasticity measured in this study is adaptive in the evolutionary sense.     

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.     

Mitochondrial Ca transport and permeability transition in zebrafish (Danio rerio)

We have studied mitochondrial Ca²⁺ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca²⁺ uptake system that, like the Ca²⁺ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca²⁺-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].     

Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.     

Development and validation of a homologous zebrafish (Danio rerio Hamilton–Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals

Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17α-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30–85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5±2.7 and 4.9±1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) ≤1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.     

Functional differences in the cytochrome P450 1 family enzymes from Zebrafish (Danio rerio) using heterologously expressed proteins

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.     

Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.     

Effect of methanol and Me2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish (Danio rerio)

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me₂SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2 M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5 h post-exposure incubation. Higher concentrations of methanol (3 and 4 M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4 M treatments resulted in a decrease in viability assessed by Fluorescein diacetate–Propidium iodide (FDA–PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5 h incubation following methanol exposure, indicating a delayed effect. The use of Me₂SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1 h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA–PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development.     

Knockdown of a galectin-1-like protein in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) causes defects in skeletal muscle development

We previously identified and characterized four galectin-1-like proteins in zebrafish, Drgal1-L1, Drgal1-L2, Drgal1-L3, and one splice variant of Drgal1-L2, of distinct ontogenic expression. Drgal1-L1 is maternal; Drgal1-L2 is zygotic and strongly expressed in the notochord, while Drgal1-L3 is both maternal and zygotic. Knockdown experiments in zebrafish embryos using a morpholino-modified antisense oligo targeted to the 5’-UTR sequence of Drgal1-L2 resulted in a phenotype with a bent tail and disorganized muscle fibers. This effect was dose-dependent as follows: 62–66% at 17 ng, 29–35% at 5.7 ng, 21–28% at 1.9 ng, and 14–17% at 0.6 ng. However, no (or a negligible number of) Drgal1-L1 knockdown embryos showed similar morphological defects, indicating that the observed effects are sequence-specific, and not due to the toxicity of the morpholino-modified oligos. Further, ectopic expression of native Drgal1-L2 specifically rescued the phenotype, as co-injection of the full-length sense Drgal1-L2 mRNA with Drgal1-L2-MO yielded 60–62% normal embryos. As the notochord serves as the primary source of signaling molecules required for proper patterning of adjacent tissues, such as neural tube, somites, and heart, these results suggest that galectins produced by the notochord play a key role in somitic cell differentiation and development.