The use of molecular biological methods to assess the effects of endocrine disrupting chemicals and natural hormones on growth in the sheepshead minnow (Cyprinodon variegatus) Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/k930c235x

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  • The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T₃, E₂, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T₃ and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E₂, suggesting that pharmacological levels of E₂ may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E₂ or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E₂ grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E₂ were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases.
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