The proteomic study of Danio rerio embryos using one-dimensional electrophoresis and mass-spectrometry

Using one-dimensional proteomic mapping (combination of one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry MALDI-TOF-PMF) the protein profile of Danio rerio embryos has been investigated. The fish species Danio rerio is the most effective alternative model of vertebrates used for studies of drug toxicity (e.g. doxorubicin) due to its high degree of homology with human genome. The proteomic profiling resulted in identification of 84 proteins, including 15 vitellogenins. Using the procedure of preparation of homogenates of Danio rerio embryos optimized by ultrasonic treatment promoting removal of yolk basic proteins (vitellogenin) we have registered changes in the proteome profile of D. rerio embryos induced by doxorubicin (DOX). Growth D. rerio embryos in the medium with DOX caused the decrease in the number of vitellogenins, disappearance of cardiac troponins, and induction of caspase-3. All these observations are consistent with the literature data on doxorubicin-induced cardiotoxicity. The proposed method of 1D proteomic mapping may be used not only for protein identification but also for registration of changes in embryonic proteomic profile caused by drugs or any toxic compound for studying the mechanisms underlying induced toxicity.     

The effect of doxorubicin incorporated into a phospholipid delivery nanosystem on HepG2 cells proteome

A phospholipid drug delivery nanosystem with particle size up to 30 nm elaborated at the Orekhovich Institute of Biomedical Chemistry (Russian Academy of Medical Sciences) has been used earlier for incorporation of doxorubicin (Doxolip). This system demonstrated higher antitumor effect in vivo as compared with free doxorubicin. In this study the effect of this nanosystem containing doxorubicin on HepG2 cell proteome has been investigated. Cells were incubated in a medium containing phospholipid nanoparticles (0.5 μg/mL doxorubicin, 10 μg/mL phosphatidylcholine). After incubation for 48 h their survival represented 10% as compared with untreated cells. Cell proteins were analyzed by quantitative two-dimensional gel electrophoresis followed by identification of differentially expressed proteins with MALDI-TOF mass spectrometry. The phospholipid transport nanosystem itself insignificantly influenced the cell proteome thus confirming previous data on its safety. Doxorubicin, as both free substance and Doxolip (i.e., included into phospholipid nanoparticles) induced changes in expression of 28 proteins. Among these proteins only four of them demonstrated different in response to the effect of the free drug substance and Doxolip. Doxolip exhibited a more pronounced effect on expression of certain proteins; the latter indirectly implies increased penetration of the drug substance (included into nanoparticles) into the tumor cells. Increased antitumor activity of doxorubicin included into phospholipid nanoparticles may be associated with more active increase of specific protein expression.     

Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure

The aim of this study was to identify periods in zebrafish (Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE(2)) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE(2) were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE(2)effects was evaluated by exposing zebrafish during the gonad transition stage (43-71 dpf) to 1.67, 3 or 10 ng EE(2)/L. Significant effects of EE(2) on adult reproduction were found with 3 and 10 ng EE(2)/L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE(2) exposure (71 dpf), all individuals in the 3 and 10 ng EE(2)/L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE(2) exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities.     

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.     

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.     

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.     

Effect of triclosan exposure on mortality and behavioral changes of Poecilia reticulata and Danio rerio

Triclosan (TCS), a chemical used for its antibacterial properties, is an ingredient in many detergents, soaps, deodorants, cosmetics, antimicrobial creams, toothpastes, and an additive in various plastics and textiles. The behavioral changes at different TCS concentrations (0.001–0.002–0.005–0.01–0.02–0.05–0.1–0.2–0.5 mg/L) were determined for the each test organisms in the study. The synthetic freshwater has temperature 20 ± 2°C, dissolved oxygen 7.0 ± 2 mg/L, pH 7.0–8.0. All experiments were replicated three times, in 14 h light, 10 h dark incubations, 10 fishes were put to each aquarium. In hourly and daily observations, times of dead and number for Poecilia reticulata, Danio rerio were recorded. Behavioral changes of Poecilia reticulata and Danio rerio were observed, such as fast-moving, uncontrolled swimming, trying to escape out of the water, vertical action to the water surface, loss of balance, respiratory difficulties, chills, and inversion.     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

Subcellular distribution of calcium during spermatogenesis of zebrafish,Danio rerio

Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio ), using a combined oxalate–pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron‐dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.     

斑马鱼对无氧运动训练的适应性变化

该研究以斑马鱼为(Danio rerio)对象,研究了四周无氧运动训练对斑马鱼行为、形态、生长、肌肉生化组分及代谢酶活性的影响。旨在探索斑马鱼对无氧运动训练的适应性变化,为进一步了解鱼类适应无氧运动训练的分子机制提供基础数据。结果发现:斑马鱼的日常活跃程度经四周无氧运动训练后显著降低,群聚程度增加;训练组个体体重和体长增长减缓,更利于运动;肌糖原含量显著增加,运动持久能力加强;肌肉乳酸脱氢酶(LDH)活性显著增高,柠檬酸合成酶(CS)活性显著降低,无氧代谢能力加强。即,斑马鱼无氧运动能力和无氧代谢能力在训练后得以明显提升。     

Hatching in zebrafish (Danio rerio) embryos exposed to a 50 Hz magnetic field

Zebrafish embryos were exposed intra ovo to a 50 Hz AC magnetic field of 1000 microT rms, and the progress of asynchronous hatching was monitored. A statistically significant delay was observed when field exposure started 48 h after fertilization. In contrast, when exposure started 2 h after fertilization, no statistically significant effect was seen. When field exposure was administered together with submaximal doses of progesterone at 48 h postfertilization, the two treatments appeared to delay hatching in an additive manner. Evaluating the progress of hatching in zebrafish embryos seems relevant for exploration of EMF effects on reproduction.     

Acclimation to hypoxia increases survival time of zebrafish, Danio rerio, during lethal hypoxia

Survivorship of zebrafish, Danio rerio, was measured during lethal hypoxic stress after pretreatment in water at either ambient oxygen or at a lowered, but nonlethal, level of oxygen. Acclimation to nonlethal hypoxia (pO(2) congruent with 15 Torr; ca. 10% air-saturation) for 48 hr significantly extended survival time during more severe hypoxia (pO(2) congruent with 8 Torr; ca. 5% air-saturation) compared to survival of individuals with no prior hypoxic exposure. The magnitude of the acclimation effect depended upon the sex of the fish: hypoxia pretreatment increased the survival times of males by a factor of approximately 9 and that of females by a factor of 3 relative to controls. In addition, survival time of control and hypoxia acclimated fish depended upon when in the year experiments were conducted. Survival times were 2-3 times longer when measured in the late fall or winter compared to survival times measured during the spring or summer. These results demonstrate a direct survival benefit of short-term acclimation to hypoxia in this genetically tractable fish. The fact that the acclimation effect depended upon the sex of the fish and the season during which experiments were conducted demonstrates that other genetic and/or environmental factors affect hypoxia tolerance in this species. J. Exp. Zool. 289:266-272, 2001.     

Embryonic exposure to cypermethrin induces apoptosis and immunotoxicity in zebrafish (Danio rerio)

Cypermethrin (CYP) is widely used for control of indoor and field pests. As a result, CYP is one of the most common contaminants in freshwater aquatic systems. In the present study, we investigated the effects of CYP exposure on the induction of apoptosis and immunotoxicity in zebrafish during the embryo developmental stage. The mRNA levels of some key genes including P53, Puma, Bax, Apaf1, Cas9 and Cas3 on the mitochondrial pathway of cell apoptosis were significantly up-regulated at the concentration of 3 and 10 μg/l CYP. Correspondingly, the activities of Cas3 and Cas9 increased significantly after exposure to 3 or 10 μg/l CYP. In addition, the mRNA levels of iNOS and the total content of NO were also up-regulated significantly after CYP exposure. Moreover, it was also observed that the mRNA levels of IFN, CXCL-Clc, CC-chem and C3, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to 3 and 10 μg/l CYP, exhibiting CYP's prominent impacts on the innate immune system of zebrafish. Taken together, our results suggest that CYP has the potential to induce cell apoptosis and cause innate immune system disruption in zebrafish during the embryo stage. The information presented in this study will help elucidate the mechanism of CYP-induced toxicity in fish.     

The proteomic study on cellular responses of the testes of zebrafish (Danio rerio) exposed to microcystin-RR

Microcystin-RR (MC-RR) is a commonly encountered cyanotoxin and receives increasing attention due to the risk of its bioaccumulation in aquatic animals like fish. This study investigated the protein profiles of zebrafish (Danio rerio) testes after intraperitoneal injection (i.p.) with 0.5 LD(50) (2000 μg/kg). MC-RR caused a noticeable damage to testicular ultrastructure, showing widened intercellular junction, distention of mitochondria. The testes showed a rapid response of its defense systems to the oxidative stress caused by MC-RR. This is the first to use a proteomic approach to obtain an overview of the effects of MC-RR on the testes of zebrafish. The proteomic results revealed that toxin exposure remarkably altered the abundance of 24 proteins that were involved in cytoskeleton assembly, oxidative stress, glycolysis metabolism, calcium ion binding and other biological functions. In conclusion, MC-RR damaged the testes and was toxic to the reproductive system of male zebrafish mainly through causing oxidative stress.     

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos

Background  

Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.     

Developmental variability during early embryonic development of zebra fish, Danio rerio

Early vertebrate embryos pass through a period of remarkable morphological similarity. Possible causes for such similarity of early embryos include modularity, developmental constraints, stabilizing selection, canalization, and exhausted genetic variability. Supposedly, each process creates different patterns of variation and covariation of embryonic traits. We study the patterns of variation of the embryonic phenotype to test ideas about possible evolutionary mechanisms shaping the early embryonic development. We use the zebra fish, Danio rerio, as a model organism and apply repeated measures of individual embryos to study temporal changes of phenotypic variability during development. In particular, we are looking at the embryonic development from 12 hours post fertilization until 27 hours post fertilization. During this time period, the development of individual embryos is documented at hourly intervals. We measured maximum diameter of the eye, length of embryo, number of somites, inclination of somites, and the yolk size (as a maternal effect). The coefficient of variation (CV) was used as a measure of variability that was independent of size. We used a principal component analysis for analysis of morphological integration. The experimental setup kept environment x genotype interactions constant. Nongenetic parental contributions had no significant effects on interindividual variability. Thus all observed phenotypic variation was based on additive genetic variance and error variance. The average CV declined from 14% to 7.7%. The decline of the CV was in particular expressed during 15-19 h post fertilization and occurred in association with multiple correlations among embryonic traits and a relatively high degree of morphological integration. We suggest that internal constraints determine the patterns of variability during early embryonic development of zebra fish.     

Expression of PKC in the developing zebrafish, Danio rerio

Protein kinase C (PKC) is a family of enzymes involved in a wide range of biological functions. We investigated the expression of PKC-positive cells in zebrafish embryos and larvae within the first week of development to determine the developmental profile of PKC-containing cells. Our other goal was to determine if PKC alpha was associated with Rohon-Beard neurons during the first 5 days of development, when they are reported to undergo apoptosis. First, we confirmed the specificity of the antibodies by Western blotting zebrafish brain homogenates with anti-PKC and anti-PKC alpha, and detected single protein bands of approximately 78-82 kDa in size. Immunohistochemistry showed that several types of neurons were labeled, including neurons in the trigeminal ganglia, the dorsal spinal cord, and the dorsal root ganglia. Double-labeling with anti-PKC alpha and both anti-Islet-1 and zn12 confirmed the identity of the PKC-positive cells in the brain as trigeminal neurons, and in the spinal cord as Rohon-Beard cells. Some Rohon-Beard cells were labeled with anti-PKC alpha up to 7 days post fertilization (dpf). We performed TUNEL labeling and found no correlation between TUNEL-labeled and PKC alpha-labeled Rohon-Beard cells, suggesting that PKC alpha is not involved in Rohon-Beard apoptosis. Only approximately 40% of the approximately 130 Rohon-Beard cells at 24 h postfertilization (hpf) were positively labeled for PKC. Mauthner cells were labeled by anti-PKC, but not anti-PKC alpha, suggesting that the major form of PKC within these cells was not PKC alpha.