Effects of maternal Cd on the female reproductive functions, gametes development and genes expression of zebrafish

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 μM 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 °C with a 14: 10-h light/dark cycle, and were fed Daphnia and shrimp. Fertilized eggs were incubated in “zebrafish solution“ (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 μM for 12-dpf stage zebrafish, and our previous study showed that the zebrafish embryo could induced physiological response with 0.89 μM Cd exposure (Wu et al., 2008). Hence, a 10× (8.9 μM) , 20× (17.8 μM) and 40× (35.6 μM) dose of 0.89 μM 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.