The role of thyroid-stimulating hormone (TSH) in the regulation of peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T4), 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 T4 cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T4 feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with driving this T4 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 T4 cycle, consistent with the hypothesis that TSH secretion drives the T4 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 T4 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 T3 and T4 in vivo. Both hormones persist in static tank water for at least 40 hours. Immersion in 200ng/ml T4 significantly increased both plasma T4 and T3 within physiological ranges above control at 4.5 hours. Immersion in 100ng/ml T3 increased plasma T3 within physiological ranges over control by 22 hours while significantly decreasing plasma T4 below control, presumably through inhibition of TSH secretion. T4 also significantly inhibited the expression of the TSH [alpha] and [beta] subunits at 4.5 and 22 hours of immersion whereas T3 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 T4 at 22 hours. Inhibition of circulating THs with the goitrogen methimazole significantly increased the expression of TSH. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH expression in red drum on a time scale consistent with the T4 daily cycle, and further support Dio3 destruction of THs in the pituitary, potentially regulated by circulating T4, 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.
