Project description:Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted endpoints with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 µg BPA caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest. The concentration-response at the ovarian transcriptome level was non-monotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the non-monotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish. Fish were exposed to 0, 0.01, 0.1, 1.0, 10, or 100 µg BPA/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. The design employed six replicate tanks per treatment. Three of the replicates were loaded with three male and three female zebrafish per tank, while the remaining three replicates were loaded with three male and three female fathead minnows. Addition of fish to the exposure tanks was staggered by replicate such that all sampling could be completed within 60 min of the desired 96 h exposure duration. After 96 h of exposure, fish were anesthetized in a buffered solution of tricaine methanesulfonate (MS-222; Finquel; Argent, Redmond, WA, USA) and weighed. Blood was collected from the caudal vasculature using microhematocrit tubes, centrifuged to separate the plasma, and plasma was stored at -80oC until analyzed. Liver, gonad, and brain tissues (including pituitary gland) were snap frozen in liquid nitrogen and stored at -80oC until extracted. Total RNA was isolated from ovary tissue of both fathead minnow and zebrafish using RNeasy kits (Qiagen, Valencia, CA, USA). RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and RNA was quantified spectrophotometrically (Nanodrop). Total RNA samples were stored at -80oC until used for microarray analyses. Ovarian transcripts from 32 fathead minnows (n=5 per treatment, except for control and 1.0 µg BPA/L, n=6) were analyzed using a custom, 15,000 gene microarray (GEO Platform Accession GPL9248) purchased from Agilent Technologies (Palo Alto, CA, USA). Ovarian transcripts from 34 zebrafish (n=6 per treatment, except 0.01 and 0.1 µg BPA/L, n=5) were analyzed using a commercial 44,000 feature microarray (Agilent design 019161; GEO Platform Accession GPL6457). The same hybridization and scanning procedures were used for both platforms. Briefly, microarrays were hybridized following the manufacturer’s guidelines for One-Color Microarray-Based Gene Expression Analysis (version 5.7; Agilent). Hybridizations were conducted with 1 µg of total RNA. Complementary DNA synthesis, cRNA labeling, amplification, and hybridizations were performed following the manufacturer’s kits and protocols (Quick Amp labeling kit; Agilent). Scanning was conducted at 5 µm resolution using an Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc., Concord, Ontario, Canada). Data were extracted from microarray array images using Agilent Feature Extraction Software (Agilent; Palo Alto, CA, USA).

Project description:Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species are complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative PCR and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. Multiple lines of evidence supported the conclusion that variation in the transcriptional profile was primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary tissue. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few putative molecular markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the study, targets including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous spawning fish species. Relative abundance of approximately 15,000 RNA transcripts in 26 ovary samples and 4 expelled oocyte (egg) samples, representing the five different histoclasses defined for the present study, was evaluated using fathead minnow oligonucleotide microarrays. Fathead minnow 15,000 gene arrays were purchased from Agilent (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturerM-bM-^@M-^Ys protocol and recommendations. One ug of total RNA was used for all hybridizations. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Quick Amp Labeling kit; Agilent, Palo Alto, CA). An Axon GenePixM-BM-. 4000B Microarray Scanner (Molecular Devices Inc., city, state) was used to scan microarray images at 5 M-NM-<m resolution.

Dataset Information

Small_fish_comp_tox_FHM_haloperidol_96_h_ovary

Publications

II: Effects of a dopamine receptor antagonist on fathead minnow dominance behavior and ovarian gene expression in the fathead minnow and zebrafish.

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