Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.     

Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling

This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to −196 °C using controlled slow cooling. Stage III oocytes (>0.5 mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2 M DMSO (both prepared in Hank’s medium) for 30 min at 22 °C before being loaded into 0.5 ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10 min, and thawed by immersing straws into a 27 °C water bath for 10 s. Thawed oocytes were washed twice in Hank’s medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 °C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4 MβNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5 min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2 M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to −196 °C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2 M DMSO and 2 M methanol. Trypan blue staining showed that 63.0 ± 11.3% and 72.7 ± 5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30 min at 22 °C, respectively, whilst 14.9 ± 2.6% and 1.4 ± 0.8% stayed intact after freezing in DMSO and methanol to −196 °C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.     

Studies on chilling sensitivity of early stage zebrafish (Danio rerio) ovarian follicles

Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage ovarian follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish ovarian follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) ovarian follicles, which were chilled in KCl buffer and L-15 medium for up to 144 h at −1 °C in a low temperature bath. Ovarian follicles were also exposed to 2 M methanol or 2 M DMSO in L-15 medium for up to 168 h at −1 and −5 °C, respectively. Control follicles were kept at 28 °C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles. These results were also confirmed following in vitro maturation of the chilled ovarian follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for ovarian follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of ovarian follicles at all stages with methanol being the most effective. The study indicated that stage I and II follicles are less sensitive to chilling than stage III follicles, and that early stage zebrafish ovarian follicles may be better candidates for cryopreservation.     

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.     

Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols

Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN₂ plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0 ± 1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2 h incubation at 22 °C the viability of freeze-thawed oocytes decreased to 29.5 ± 5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio

The zebrafish has become a major model system for biomedical research and is an emerging model for the study of behaviour. While adult zebrafish express a visually mediated shoaling preference, the onset of shoaling behaviour and of this preference is unknown. To assess the onset of these behaviours, we first manipulated the early social environment of larval zebrafish subjects, giving them three model shoaling partners of the same pigment phenotype. We then assayed the subjects' preferences using binary preference tests in which we presented subjects with two shoals, one shoal of fish exhibiting the same pigment pattern phenotype as their models and another shoal with a radically different pigment pattern. To determine whether or not the visually mediated preference could be altered once it was established, we further manipulated the social environment of a number of subjects, rearing them with one model shoal and testing them, then changing their social consorts and retesting them. Our results demonstrate that larval zebrafish shoal early in their development, but do not exhibit a shoaling preference until they are juveniles. Moreover, we find that the shoaling preference is stable, as changing the social environment of fish after they had acquired a preference did not change their preference. These data will facilitate investigations into the mechanisms underlying social behaviour in this vertebrate model system.     

Distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

Mitochondria play a vital role during oocyte maturation, fertilization, and embryo development. In this study, confocal microscopy with the mitochondrial membrane potential-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) was used to investigate mitochondria distribution and activity of stage III zebrafish ovarian follicles. To support the mitochondrial origin of the fluorescence obtained by JC-1, a second mitochondrial probe, MitoTracker Green FM, was used. Cryo-scanning and transmission electron microscopy were also used to validate the distribution and localization of mitochondria obtained by mitochondrial staining. The mitochondrial probes were unable to penetrate the oocyte, and as a result it was not possible to observe stained mitochondria in the oocyte cytoplasm. However, mitochondrial staining of the granulosa cell layer surrounding the stage III zebrafish oocyte exhibited a contiguous aggregation pattern of mitochondria. Cryo-scanning electron microscopy studies also showed the oocyte surface to be covered by polygonal patterns of ridges of the same dimensions as the distributional arrangement of mitochondria in the granulosa cells. Though the results suggested the need for defolliculation to assess mitochondrial distribution and activity in the stage III zebrafish oocyte cytoplasm, the findings of this study will contribute to our understanding of oogenesis and folliculogenesis processes in fish.     

Developmental energetics of zebrafish, Danio rerio

Using zebrafish (Danio rerio) as a case study, we show that the maturity concept of Dynamic Energy Budget (DEB) theory is a useful metric for developmental state. Maturity does not depend on food or temperature contrary to age and to some extent length. We compile the maturity levels for each developmental milestone recorded in staging atlases. The analysis of feeding, growth, reproduction and aging patterns throughout the embryo, juvenile and adult life stages are well-captured by a simple extension of the standard DEB model and reveals that embryo development is slow relative to adults. A threefold acceleration of development occurs during the larval period. Moreover we demonstrate that growth and reproduction depend on food in predictable ways and their simultaneous observation is necessary to estimate parameters. We used data on diverse aspects of the energy budget simultaneously for parameter estimation using the covariation method. The lowest mean food intake level to initiate reproduction was found to be as high as 0.6 times the maximum level. The digestion efficiency for Tetramin? was around 0.5, growth efficiency was just 0.7 and the value for the allocation fraction to soma (0.44) was close to the one that maximizes ultimate reproduction.     

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Distribution of carnosine-like peptides in the nervous system of developing and adult zebrafish (Danio rerio) and embryonic effects of chronic carnosine exposure

Carnosine-like peptides (carnosine-LP) are a family of histidine derivatives that are present in the nervous system of various species and that exhibit antioxidant, anti-matrix-metalloproteinase, anti-excitotoxic, and free-radical scavenging properties. They are also neuroprotective in animal models of cerebral ischemia. Although the function of carnosine-LP is largely unknown, the hypothesis has been advanced that they play a role in the developing nervous system. Since the zebrafish is an excellent vertebrate model for studying development and disease, we have examined the distribution pattern of carnosine-LP in the adult and developing zebrafish. In the adult, immunoreactivity for carnosine-LP is specifically concentrated in sensory neurons and non-sensory cells of the olfactory epithelium, the olfactory nerve, and the olfactory bulb. Robust staining has also been observed in the retinal outer nuclear layer and the corneal epithelium. Developmental studies have revealed immunostaining for carnosine-LP as early as 18 h, 24 h, and 7 days post-fertilization in, respectively, the olfactory, corneal, and retinal primordia. These data suggest that carnosine-LP are involved in olfactory and visual function. We have also investigated the effects of chronic (7 days) exposure to carnosine on embryonic development and show that 0.01 μM to 10 mM concentrations of carnosine do not elicit significant deleterious effects. Conversely, treatment with 100 mM carnosine results in developmental delay and compromised larval survival. These results indicate that, at lower concentrations, exogenously administered carnosine can be used to explore the role of carnosine in development and developmental disorders of the nervous system. This research was supported in part by an Intramural Research Grant Program Award from Michigan State University (no. 06-IRGP-899 to M.C.S.) and a grant from the National Institutes of Health (no. DC-03112 to F.L.M.).     

富马酸二甲酯对斑马鱼胚胎早期发育的影响

为研究富马酸二甲酯对斑马鱼(Danio rerio)胚胎早期发育的影响,选取不同发育阶段的斑马鱼胚胎,用富马酸二甲酯进行染毒处理,观察胚胎形态发育的异常,计算其对不同发育时期胚胎的24 h、48 h半数致死浓度(LC50)和胚胎72 h孵化率,并考察富马酸二甲酯对胚胎血管发育的影响。结果表明,富马酸二甲酯影响斑马鱼胚胎的早期发育,呈剂量依赖性特点,并与开始处理的时间点有关。富马酸二甲酯引起2 hpf(受精后2 h,2 hours post-fertilization)、10 hpf、24 hpf斑马鱼胚胎死亡的24 h LC50值分别为:13.33μmol/L、17.98μmol/L、32.50μmol/L,48 h LC50值分别为:13.31μmol/L、16.35μmol/L、22.50μmol/L;长期低浓度富马酸二甲酯(≥6μmol/L)作用引起胚胎72 h孵化率下降。27.5μmol/L富马酸二甲酯作用后会显著降低胚胎血管内皮生长因子受体2(VEGFR2)的表达水平。     

Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Unlike mammalian GRIFIN, the zebrafish homologue (DrGRIFIN) represents a functional carbohydrate-binding galectin

Galectins, a family of β-galactoside-binding proteins, participate in a variety of biological processes, such as early development, tissue organization, immune regulation, and tumor evasion and metastasis. Although as many as fifteen bona fide galectins have been identified in mammals, but the detailed mechanisms of their biological roles still remain unclear for most. This fragmentary knowledge extends to galectin-like proteins such as the rat lens crystallin protein GRIFIN (Galectin-related inter fiber protein) and the galectin-related protein GRP (previously HSPC159; hematopoietic stem cell precursor) that lack carbohydrate-binding activity. Their inclusion in the galectin family has been debated, as they are considered products of evolutionary co-option. We have identified a homologue of the GRIFIN in zebrafish (Danio rerio) (designated DrGRIFIN), which like the mammalian equivalent is expressed in the lens, particularly in the fiber cells, as revealed by whole mount in situ hybridization and immunostaining of 2 dpf (days post fertilization) embryos. As evidenced by RT-PCR, it is weakly expressed in the embryos as early as 21 hpf (hour post fertilization) but strongly at all later stages tested (30 hpf and 3, 4, 6, and 7 dpf). In adult zebrafish tissues, however, DrGRIFIN is also expressed in oocytes, brain, and intestine. Unlike the mammalian homologue, DrGRIFIN contains all amino acids critical for binding to carbohydrate ligands and its activity was confirmed as the recombinant DrGRIFIN could be purified to homogeneity by affinity chromatography on a lactosyl-Sepharose column. Therefore, DrGRIFIN is a bona fide galectin family member that in addition to its carbohydrate-binding properties, may also function as a crystallin.     

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.     

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO₄) (59.4%), ΔUA-1→3-GalNAc(6SO₄) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism. The contributions of Aline R.C. Souza and Eliene O. Kozlowski should be considered equal.