Proteomic analysis of zebrafish (Danio rerio) embryos exposed to cyclosporine A

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes.     

Acute toxicity and gene responses induced by endosulfan in zebrafish (Danio rerio) embryos

Endosulfan has been listed as a persistent organic pollutant, and is frequently found in agricultural environments during monitoring processes owing to its heavy use and persistent characteristics. This study was conducted to understand the effects of endosulfan on the development of zebrafish (Danio rerio) embryos by exposing them to a specific range of endosulfan concentrations. Exposing zebrafish embryos to endosulfan for 96 h yielded no acute toxicity until the concentration reached 1500 μg L^?1, whereas malformed zebrafish larvae developed severely curved spines and shortened tails. About 50% of zebrafish larvae were malformed when exposed to 600 μg L^?1 of endosulfan. Comparative gene expression using real-time quantitative polymerase chain reaction was assessed using endosulfan-exposed zebrafish embryos. CYP1A and CYP3A were significantly enhanced in response to endosulfan treatment. Two genes, acacb and fasn, encoding acetyl-CoA carboxylase b and fatty acid synthase proteins, respectively, were also up-regulated after treating zebrafish embryos with endosulfan. These genes are also involved in fatty acid biosynthesis. The genes encoding vitellogenin and Hsp70 increased in a concentration-dependent manner in embryos. Finally, biochemical studies showed that acetylcholinesterase activity was reduced, whereas glutathione S-transferase and carboxylesterase activities were enhanced in zebrafish embryos after endosulfan treatment. These biochemical and molecular biological differences might be used for tools to determine contamination of endosulfan in the aquatic environment.     

Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples

The zebrafish embryo test is a widely used bioassay for the testing of chemicals, effluents and other types of environmental samples. Oxygen depletion in the testing of sediments and effluents is especially important and may be a confounding factor in the interpretation of apparent toxicity. In order to identify oxygen levels critical to early developmental stages of zebrafish, oxygen consumption of zebrafish embryos between 0 and 96h post-fertilization, minimum oxygen levels required by the embryos for survival as well as the effects of oxygen depletion following exposure to model sediments were determined. No significant effects on zebrafish embryo development were observed for oxygen concentrations between 7.15 and 3.33mg/L, whereas at concentrations between 3.0and 2.0mg/L minor developmental retardations were observed, yet without any pathological consequences. Oxygen concentrations lower than 0.88mg/L were 100% lethal. In the sediment contact tests with zebrafish embryos, native sediments rich in organic materials rapidly developed strongly hypoxic conditions, particularly at the sediment-water interface (0 to 500μm distance to the sediment).     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Cascade effect of cardiac myogenesis gene expression during cardiac looping in <Emphasis Type="Italic">tbx5</Emphasis> knockdown zebrafish embryos

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes. Jen Her Lu and Jenn Kan Lu contributed equally to this work.     

The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.     

Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (Danio rerio)

Bis-(2-ethylhexyl) phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. Its metabolites are known to exert toxic effects on reproduction and development of mammals. The aim of this study was to evaluate the effects of environmentally relevant concentrations of DEHP (0.2 and 20 μg/L) on the reproductive biology of adult male zebrafish (Danio rerio). The effects of DEHP and 17β-ethynylestradiol (a positive control) were determined after one or three weeks of exposure by TUNEL assay, histomorphometric analysis and evaluation of reproductive performance. DEHP impaired reproduction in zebrafish by inducing a mitotic arrest during spermatogenesis, increasing DNA fragmentation in sperm cells and markedly reducing embryo production (up to 90%). In conclusion, relatively short-term exposure to environmentally relevant concentrations of DEHP is able to alter spermatogenesis and affect reproduction in zebrafish.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Developmental toxicity of triclocarban in zebrafish (Danio rerio) embryos

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 μg/L TCC from 4‐hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development–related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.     

Developmental expression of aquaporin-3 in zebrafish embryos (Danio rerio)

Fish embryos have never been successfully cryopreserved because of the low permeability of cryoprotectants into the yolk. Recently, we used aquaporin-3 fused with a green fluorescent protein (AQP3GFP) to modify the zebrafish embryo, and demonstrated that the pores functioned physiologically. This increased the water and cryoprotectant permeability of the membranes. We have continued our work on AQP3-modified embryos and here we report their developmental expression of AQP3, the success of various culture media on their survival and development, and their reproductive success. The AQP3GFP expression begins within 30 m after the mRNA AQP3GFP injection into the yolk of the 1- to 4-cell embryo. This expression is distributed in the membranes throughout the blastoderm and the yolk syncytial layer within 24 h. It diminishes after 96 h. We found no difference in the survival or normal development of embryos from AQP3GFP or wild-type adults. Additionally, zebrafish embryos did not require special culture medium to survive after AQP3GFP modification. In fact, they survived best in embryo medium (ca. 40 mOsm). Embryos reared entirely in embryo medium had a higher percent survival and a higher percent normal development than those exposed to a high osmolality sucrose culture medium (ca. 330 mOsm). The mechanism whereby these embryos can maintain their internal osmolality in a hypoosmotic solution with water channels in their membranes is unknown.     

Induction of apoptosis and inhibition of cell growth by <Emphasis Type="Italic">tbx5</Emphasis> knockdown contribute to dysmorphogenesis in Zebrafish embryos

Background  

The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.     

Studies on chilling sensitivity of zebrafish (Danio rerio) oocytes

Human activity in the last few decades has had a devastating effect on the diversity of fresh water and marine fish. Further decline of fish population may have serious economic and ecological consequences. One of the most promising techniques to preserve fish population is to cryopreserve their germ cells. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryo cryopreservation and fish oocyte cryopreservation has never been studied systematically. The aim of this study is to investigate the chilling sensitivity of fish oocytes. Experiments were conducted with zebrafish stage III (vitellogenic) and stage V (mature) oocytes, which were chilled at 10, 5, 0, -5 or -10 degrees C for 15 or 60 min using a low temperature bath. Control oocytes were kept at room temperature at 22 degrees C. Oocyte viability was assessed using three different methods: trypan blue staining (TB), thiazolyl blue tetrazolium bromide (MTT) staining and observation of germinal vesicle breakdown (GVBD). The results showed that zebrafish oocyte are very sensitive to chilling and their survival decreased with decreasing temperature and increasing exposure time periods. Normalised survivals assessed with TB staining after exposure to 0, -5 or -10 degrees C for 15 or 60 min were 90.1+/-6.0, 77.8+/-7.6, and 71.2+/-9.3%, and 60.2+/-3.8, 49.6+/-6.7, and 30.4+/-3.0%, respectively. The study found that the sensitivity of viability assessment methods increase in the order of MTT < TB < GVBD. It was found that stage III oocytes were more susceptible to chilling than stage V oocytes, and that individual female had a significant influence (p < 0.0001) on oocyte chilling sensitivity. Zebrafish oocyte chilling sensitivity may also be one of the limiting factors for development of protocol of their cryopreservation.