The studies described in this thesis were designed to address the hypothesis that stress and glucocorticoids modify the levels of corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) mRNA in the rainbow trout ( Oncorhynchus mykiss) brain. Standard cloning techniques provided the full-length cDNA sequences coding for trout NPY and two CRF paralogues. Using a ribonuclease protection assay (RPA), a tissue distribution demonstrated that both CRF paralogues are primarily abundant in the preoptic area of the trout brain, whereas NPY mRNA is more abundant in the telencephalon. The effects of social stress on CRF1 and NPY mRNA levels were examined with pairs of sized-matched juvenile female trout. After 72 h of interaction, CRF1 and NPY mRNA levels were respectively 51% and 32% higher in the preoptic area of subordinate trout. These results suggest that CRF and NPY may be involved in the control of the stress axis in trout. However, my experiments could not distinguish between the effects of stress and food deprivation. The levels of plasma cortisol in isolated trout indicated that confinement in small boxes was more stressful than isolation in a large tank. The elevation in CRF1 mRNA levels was greater and more persistent in confined fish. CRF1 mRNA levels were also elevated in trout physically disturbed to exhaustion but only after repeated chasing events. Together, these results suggest that the duration and intensity of stress are important factors determining the magnitude and persistence of the elevation in CRF mRNA levels. The effects of glucocorticoids on CRF and NPY mRNA levels were examined by exposing trout to cortisol, a glucocorticoid receptor antagonist (RU-486) or a cytochrome P450 inhibitor that blocks cortisol synthesis (metyrapone). My results suggest that glucocorticoids modify CRF1 and NPY mRNA levels but only part of the results support the presence of a glucocorticoid-induced negative feedback loop. Further studies are required to clarify the role of glucocorticoids in the regulation of the stress axis activity in fish.

Amphibian metamorphosis is a dramatic larvae-to-adult transformation regulated by temporal changes in thyroid hormones (TH) as well as interactions with other hormone systems. This developmental process is extremely responsive to changes in environmental conditions and susceptible to aquatic contaminants that interfere with hormone-dependent processes. While many of these endocrine disrupting compounds (EDCs) have been studied for their estrogenic effects on reproduction, the plasticity of amphibian metamorphosis provides a unique model for examining the thyroid-mediated effects of developmental exposure to estrogenic EDCs. The first objective of this thesis was to assess the toxicological response and developmental effects of estrogenic EDCs during the larval life-stages of amphibian development. After establishing baseline toxicity data for the test species, Rana pipiens, a developmental exposure was conducted to determine the estrogen-sensitivity of distinct periods during metamorphosis. Stage-specific exposures altered tadpole growth parameters and resulted in lasting effects such as delayed metamorphosis and female-biased sex ratios. Therefore, the second objective was to identify potential mechanisms by which xenoestrogens alter TH-dependent metamorphosis using a targeted gene expression approach. Development of multiplex and simplex real-time PCR assays and subsequent analysis established that thyroid- and estrogen-responsive transcripts in the tadpole brain are differentially regulated by exogenous TH and upon exposure to an estrogenic EDC. A short-term "challenge" assay confirmed that estrogenic exposure suppresses and in some cases blocks the ability of TH to induce the expression of target genes involved in mediating tissue-specific TH sensitivity during metamorphosis. The results of this research (1) provide evidence of cross-regulation between thyroid and estrogen-regulated genes during amphibian development and (2) indicate that developmental exposure to estrogenic EDCs may affect normal TH-responsiveness necessary for proper timing of metamorphosis.

An avian embryo is ectothermic, with body temperature determined by environmental temperature. Upon hatching, the neonate begins a conversion so that endothermic capacity becomes feasible and body temperature becomes independent of environment. Whole animal metabolic rate and ventilation response, cardiovascular development, and maturation of muscle mitochondrial flux were the focus of this dissertation because of the direct role in shivering thermogenesis. Precocial ducks and altricial Double-crested Cormorants exhibit increasing hematocrit and disproportionate increases in fractional heart mass resulting in greater oxygen delivery capacity and increased capacity of muscles to utilize oxygen compared with ectothermic American Alligator and Common Snapping turtles. By selecting for faster growth and higher meat yield in the domestic chicken, differences in whole-animal, tissue, cellular, and regulatory responses are evident between broiler and layer type birds. In the altricial red-winged blackbird, despite appearance of a whole animal endothermic response sometime after 7 dph, capacity of skeletal muscles involved in shivering thermogenesis peaks prior to that time. Thus, full development of endothermy is delayed in this species, allowing the altricial nestling to allocate energy towards growth rather than metabolic maintenance. Hypothyroidism in neonate red-winged blackbirds results in delayed maturation of the cardiovascular system and mitochondrial oxidative capacity of skeletal muscle. Such deficiencies were quickly recovered once the animals returned to a normothyroid state, apparently at the cost of increasing body mass. Insights into onset of thermoregulation provide a more thorough understanding of metabolic and physical transitions a hatchling bird must undergo to reach the adult endothermic phenotype. Endothermic capacity will continue to be at the forefront of physiological research because of the significance of changes between the energetic relations of an animal that must occur with its environment.

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