BOORSE, G.B.; DENVER, R.J.*: Changes in Hypothalamic Corticotropin-Releasing Hormone (CRH) Content During Environmentally-Induced Metamorphosis in the Western Spadefoot Toad (Spea hammondii)

Western spadefoot toads (Spea hammondii) are desert dwelling amphibians whose tadpoles can accelerate metamorphosis as their ponds dry, thereby escaping desiccation. Corticotropin-releasing hormone (CRH) may transduce the environmental signal into an endocrine response that drives metamorphosis. To further understand the role that CRH plays in environmentally-induced metamorphosis we developed and validated a CRH radioimmunoassay (RIA) to monitor changes in hypothalamic CRH content following exposure to low water in the laboratory. We generated a rabbit polyclonal antiserum against synthetic Xenopus laevis CRH. This RIA detects vertebrate CRH peptides with high sensitivity and shows low cross-reactivity with the CRH-like peptides sauvagine, urotensin and urocortin. Molecular cloning of the S. hammondii CRH cDNA shows that the sequence of the mature peptide is identical to the Xenopus sequence. Thus, this RIA is homologous for both X. laevis and S. hammondii. We validated the CRH RIA for measurement of peptide content in acid hypothalamic extracts. We demonstrate that CRH-binding protein (CRH-BP), which could potentially interfere with the RIA, is present in S. hammondii brain. However, very little biologically active CRH-BP remains after the acid extraction. Addition of varying amounts of recombinant mouse CRH-BP to the standard curve in the RIA shows that, while the BP can interfere at higher concentrations, the amount remaining after acid extraction does not interfere with the CRH RIA. Using this RIA, we show that S. hammondii tadpoles exposed to low water level exhibit brain-region specific changes in CRH content. (Supported by NSF grant IBN9974672 to RJD and a NSF predoctoral fellowship to GCB)