Project description:The impacts of maternal Cd on zebrafish from female to embryo which included fecundity, gametes development, growth, other reproductive functions, and gene expression of 5hpf embryo were up and down regulated after female pretreated with Cd. The mating rate decreased by 30 %. It can be observed in growth delay during 6-somite stage. The ceratohyal head angle of larvae is widest upon 35.6 M-NM-<M maternal Cd treatment and it was showed a dose response. Besides, pericardial edema occurred in some 96hpf larvae. There were showed 33 and 37 target genes appeared significantly down and up regulation after microarray assay, respectively. The major effect on gene up regulation was showed at the functional of transcription (occupied 18.9%) and down regulation at the functional of protein biosynthesis (occupied 33.3%). These results demonstrated that maternal Cd influenced the reproduction ability of female and causes development abnormal of embryo. Sexually mature zebrafish (D. rerio) of both sexes, obtained from the Institute of Cellular and Organismic Biology, Academia Sinica (Taipei, Taiwan), were kept in an aquarium supplied with dechlorinated, circulated, aerated local tap water at 28 M-BM-0C with a 14: 10-h light/dark cycle, and were fed Daphnia and shrimp. Fertilized eggs were incubated in M-bM-^@M-^\zebrafish solutionM-bM-^@M-^\ (E3 solution) which contained 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 1 ppm methylene blue (pH 7.2). Developing embryos of 5 h post-fertilization (hpf) were collected for total RNA extraction, and process of microarray assay. The number of hatched zebrafish larvae at 48 and 72 hpf were recorded. The larvae of 72 hpf larvae were collected for cartilage stain in order to observe the development of pharyngeal arch. Nguyen and Janssen (2001) reported that the LC50 at 96 h of Cd was 0.62~1.33 M-NM-<M for 12-dpf stage zebrafish, and our previous study showed that the zebrafish embryo could induced physiological response with 0.89 M-NM-<M Cd exposure (Wu et al., 2008). Hence, a 10M-CM-^W (8.9 M-NM-<M) , 20M-CM-^W (17.8 M-NM-<M) and 40M-CM-^W (35.6 M-NM-<M) dose of 0.89 M-NM-<M was used in female zebrafish. The Cd medium was prepared using completely dried CdCl2 (Sigma, St. Louis, MO) dissolved in 1 mL concentrated HCl; double-deionized water was used to prepare the 10 mg/L Cd stock solution, which was then diluted with zebrafish solution before being used in the exposure experiments. During all experiments, the test containers were cleaned with HNO3 and thoroughly rinsed with double-deionized water before use.

Project description:Methylmercury (MeHg) is a potent neurotoxin and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. We investigated the gene expression profile in adult female zebrafish whole brain induced by acute (96 hr) MeHg exposure. Fish were exposed by injection to 0 or 0.5 M-BM-5g MeHg/g. Gene expression changes in the brain were examined using a two-color 22,000 feature zebrafish microarray. At a significance level of p<0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism and GABA-A receptors in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to the nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxins and investigate responsive genes as potential biomarkers of MeHg exposure. Wild-type strain AB-1 zebrafish (Zebrafish International Resource Center, University of Oregon, Eugene, OR) were cultured at the Columbia Environmental Research Center (CERC), USGS, for MeHg exposures. Adult female zebrafish were injected with 0 M-NM-<g/g or 0.5 M-NM-<g/g MeHg in 2 M-BM-5L Na2CO3 (pH 6.98)/g body weight. After 96 hr, fish were anesthetized using ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma, St. Louis, MO). Whole brains were removed, flash frozen with liquid nitrogen and stored at 80M-BM-0C. For the microarray experiment, two zebrafish brains were pooled per sample. Four pooled samples were taken from fish treated with 0.5 M-NM-<g/g of MeHg, and the other five were taken from control fish treated with sodium carbonate. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control and treated female brains. Five replicates for the control and four replicates for the treated were analyzed. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA).

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