Browsing by Subject "thyroid hormone"
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Item Biological Activity of Thyrotropin in Two Teleost Fish, Red Drum (Sciaenops ocellatus) and Goldfish (Carassius auratus)(2012-07-16) Miller, Thomas CharlesThyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the red drum thyroid precluded their use as a bioassay species, the plasma TSH and T4 response to exogenous TSH was next characterized in goldfish. The T4 response in goldfish was stable and repeatable, with T4 levels peaking at 5 hours and remaining elevated for more than 11 hours after bovine TSH injection. Plasma TSH peaked from 2-5 hours following TSH injection with more than 90 percent cleared by 11 hours. The goldfish bioassay was further utilized to evaluate the effects of structural modifications on TSH biological activity. Substitution of four positively charged amino acids at the n-recombinant human TSH, had the same effect in goldfish. The heterothyrotropic potency of mammalian follicle stimulating hormone in goldfish was also enhanced by the same amino acid substitutions. Finally, the importance of oligosaccharides to TSH bioactivity was also examined in goldfish. Deglycosylation abolished TSH bioactivity, even when immunoreactivity persisted in circulation. Furthermore, recombinant canine TSH was less potent when produced in cell lines generating insect-type glycosylation than when produced in a cell line capable of mammalian-type glycosylation. These studies utilizing recombinant mammalian demonstrated conservation of mammalian TSH hormone-receptor interactions in goldfish, suggesting TSH function might likewise be conserved. Thus, I have established goldfish as a sensitive and stable bioassay which can now be utilized to monitor the biological activity of teleost TSH expressed in vitro as well as to evaluate how structural modifications of the TSH molecule influence its vivo biological activity.Item Regulation of Thyrotropin mRNA Expression in Red Drum, Sciaenops ocellatus(2012-10-19) Jones, Richard AlanThe role of thyroid-stimulating hormone (TSH) in the regulation of peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T?), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the sciaenid fish, red drum. Previous research has suggested that the red drum T? cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T? feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with driving this T? cycle, I developed quantitative real time RT-PCR (qPCR) techniques to quantify the daily cycle of expression of the pituitary TSH subunits GSU[alpha], and TSH[beta]. I found that pituitary TSH expression cycled inversely to, and 6-12 hours out of phase with, the T? cycle, consistent with the hypothesis that TSH secretion drives the T? cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, I also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner ring deiodinase (Dio3). Whereas Dio2 was not expressed with an obvious daily cycle, Dio3 was expressed in the pituitary mirroring the TSH cycle. These results are consistent with T? negative feedback on TSH and suggest that TH inactivation by pituitary cells is an important component of the negative feedback system. To further examine the TH regulation of this Dio3 cycle, I developed an immersion technique to administer physiological doses of T? and T? in vivo. Both hormones persist in static tank water for at least 40 hours. Immersion in 200ng/ml T? significantly increased both plasma T? and T? within physiological ranges above control at 4.5 hours. Immersion in 100ng/ml T? increased plasma T? within physiological ranges over control by 22 hours while significantly decreasing plasma T? below control, presumably through inhibition of TSH secretion. T? also significantly inhibited the expression of the TSH [alpha] and [beta] subunits at 4.5 and 22 hours of immersion whereas T? immersion significantly inhibited the expression of the [alpha] and [beta] subunits of TSH by 22 hours. Both Dio2 and Dio3 expression were significantly diminished by T3 and T? at 22 hours. Inhibition of circulating THs with the goitrogen methimazole significantly increased the expression of TSH. These results indicate that both T? and T? are capable of negative feedback regulation of TSH expression in red drum on a time scale consistent with the T? daily cycle, and further support Dio3 destruction of THs in the pituitary, potentially regulated by circulating T?, as a critical component of negative feedback on TSH. This study supports the importance of central mechanisms acting through pituitary TSH secretion in regulating thyroid function in red drum.