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Characterization and regulation of growth hormone receptors in goldfish (Carassius auratus L.)



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Recombinant carp (Cyprinus carpio) growth hormone (rcGH) was used to develop a GH receptor binding assay in the goldfish (Carassius auratus). LIGAND analysis indicated a single class of high affinity and low capacity binding sites, with an association constant (Ka) of 1.9 * 1010 M-1 and a maximum binding capacity (Bmax) of 9 fmol mg-1 protein. The hepatic GHR was further studied using a covalent hormone-receptor cross-linking technique. Covalent cross-linking of rcGH or bovine GH (bGH) to goldfish, rabbit and rat liver membrane proteins resulted in the same specifically labeled bands, with apparent molecular weights (Mr) of 88, 142 and >200KD. The cross-reactivity of recombinant teleost GH from carp, rainbow trout (Oncorhynchus mykiss), and sea bream (Sparus aurata ) to mammalian GHR was further studies using GH binding assays and the 3T3-F442A preadipocyte bioassay. LIGAND analysis of 125I-bGH binding to rabbit and rat liver membranes indicated two classes of binding sites: high affinity, low capacity sites, and low affinity, high capacity sites. In contrast, a single class of high affinity and low capacity sites for rcGH and recombinant rainbow trout GH (rtGH) was identified in rabbit and rat liver membranes. A single class of binding sites for recombinant sea bream (rsbGH) was also identified in rabbit liver membranes, but with a much lower affinity. High cross-reactivity of rcGH and rtGH to mammalian GHR also corresponded with a high antimitogenic activity equivalent to that of bGH in the mammalian 3T3-F442A cell line. I also found conclusive evidence for the presence of a specific, high affinity GH binding protein (GHBP) in the serum of goldfish. LIGAND analysis identified a single class of high affinity and low capacity binding sites for rcGH in the goldfish serum, with a Ka of 20.1 * 109 M-1 and a Bmax of 161 fmol ml-1 serum. Ligand blotting revealed multiple forms of GHBP in sera of goldfish, rabbit and rat. In vivo rcGH injection to intact or hypophysectomized (Hx) goldfish resulted in a rapid down-regulation of hepatic GHR, suggesting that endogenous GH may have a role in the regulation of its own receptors in goldfish. The roles of GH, GHR, GHBP and thyroid hormones in endocrine regulation of somatic growth were studied using fasted goldfish. Fasting resulted in significant decreases in body weight and length, liver-somatic index, hepatic GHR, serum GHBP and thyroxin (T4) whereas serum glucose and GH levels increased. Serum triiodothyronine (T3) levels also remained unchanged during fasting. These results suggest that nutrition is also important in the regulation of hepatic GHR and serum GHBP in goldfish. Finally, the influence of GH on goldfish hepatocyte proliferation and hepatic GHR was studied in vitro. All GH displayed antimitogenic effect on the cultured goldfish hepatocytes. In contrast, PRL, T3, insulin, rhIGF-I, and rsbIGF-I had no effect on the growth of cultured goldfish hepatocytes. In vitro administration of rcGH to goldfish hepatocytes resulted in a rapid down-regulation of hepatocyte GHR.





Doctor of Philosophy (Ph.D.)







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