Influence of cortisol on the larval bullfrog thyroid axis in vitro and in vivo and on plasma and ocular melatonin.

Adrenal (interrenal) steroids have an important role in amphibian development, antagonizing the metamorphic changes induced by the thyroid at first and then synergizing with the thyroid hormones as their level rises during metamorphosis. Because most of the studies of corticoids at metamorphosis have focused on peripheral tissues, we investigated the effect of cortisol (hydrocortisone; HC) in vitro and in vivo on the thyroid of Rana catesbeiana (bullfrog) tadpoles on 12:12 light/dark (LD) cycles. Plasma and ocular melatonin, which is altered by changes in thyroxine (T(4)) levels, were also assayed in some experiments. Thyroids from premetamorphic tadpoles secreted less T(4) into culture media when incubated with 10 micrograms/ml HC and 0.2 micrograms/ml ovine thyrotropin (TSH) than with TSH alone and when cultured in the absence of TSH following 5 days of 10-micrograms HC injections, indicating that HC inhibited the thyroid at young stages. The effect of 10 micrograms/ml HC at older stages was investigated by culturing thyroids and pituitaries separately on the first day in control or HC media and then incubating the thyroids on the second day in homologous pituitary-conditioned media as a bioassay for pituitary TSH. HC had no effect on baseline T(4) secretion by the thyroids of prometamorphic or climax tadpoles on the first day but increased T(4) secretion over the control on the second day. Thyroids cultured with TSH and HC showed no increase in T(4) secretion over the control TSH group on the second day, indicating that, in the previous experiments, HC had enhanced pituitary secretion of TSH, rather than the response of the thyroid to TSH. In vivo, 5 days of injections of 10 micrograms HC increased plasma T(4) at prometamorphosis and decreased it at climax. There was no marked effect of HC on plasma or ocular melatonin levels. The findings showed that the nature of the effect of HC on the thyroid axis changes during metamorphosis from inhibition at early stages to a positive influence at prometamorphosis and finally to a negative effect on the T(4) level in the plasma at climax.

Distribution and reciprocal interactions of 3H-melatonin and 125I-thyroxine in peripheral, neural, and endocrine tissues of bullfrog tadpoles.

Tissue distribution of 125I-thyroxine (T4) and 3H-melatonin and the effect of each hormone on the tissue content of the other were studied because previous work indicated that melatonin antagonized metamorphosis through peripheral, as well as thyroidal effects. Late pre- to prometamorphic Rana catesbeiana tadpoles on an 18 light:6 dark cycle were used for injection of hormones in vivo or to supply tissues for in vitro hormone administration. Labeled melatonin uptake was highest in intestine, ventral skin and pituitary; lowest in thyroid and brain and intermediate in hindlimb, tail and gills. The tissue content of labeled T4 was distributed in nearly the same way, except that the thyroid level was relatively higher, and pituitary lower, than that of labeled melatonin. The pineal, studied only in the tracer T4 experiments, had the highest content of labeled T4 of all tissues. Simultaneous injection of either 0.007 or 0.2 microgram T4 increased 3H-melatonin uptake into peripheral tissues that undergo major metamorphic changes but not into neural or endocrine organs. In contrast, 0.033, 3.75 or 15 micrograms melatonin had no significant influence on the content of 125I-T4 in any tissue studied in vivo. Results of in vitro labeling of selected tissues were generally in agreement with the in vivo work except that the 125I-T4 content of intestinal segments from late prometamorphic larvae was lower in melatonin-treated than in control groups. The results suggest that peripheral tissues are a major site for T4-melatonin interactions and that T4 may modulate its own action through influencing melatonin levels in target tissues and perhaps in the thyroid. Because melatonin had no effect on tissue T4 content in young tadpoles, retardation of metamorphic events by melatonin does not seem to involve modulation of T4 availability to the tissues.

Effect of melatonin on the response of the thyroid to thyrotropin stimulation in vitro.

Thyroidal-melatonin interactions are of particular importance to amphibian development since the thyroid controls the progress of metamorphosis while melatonin may coordinate its rate with prevailing environmental conditions. Melatonin antagonized thyroxine (T4) action at the tissue level and directly inhibited baseline T4 secretion in culture, so the present work sought to determine if it antagonized the response of the thyroid to thyroid stimulating hormone (TSH) as well. A preliminary experiment showed that, in Rana pipiens, the concentration of TSH (0.2 microg/ml) used in the culture of tadpole thyroids stimulated T4 secretion as much as frog pituitaries, but more than late premetamorphic tadpole pituitaries. There was no significant effect of 1 to 15 microg/ml melatonin in TSH-containing thyroid cultures of various Rana species of tadpoles and frogs in experiments with media collected once every 24 or 48 hr, although 15 microg/ml melatonin tended to depress T4 secretion. In a final experiment, a higher melatonin concentration was used as well as more frequent media collections. Thyroids from prometamorphic Rana catesbeiana tadpoles were cultured in L-15 media with periodic stimulation by 0.2 microg/ml TSH, or TSH and 10 or 100 microg/ml melatonin. Media were collected at the end of two 3-hr TSH pulses, and every 8 hr thereafter for the next 3 days. Melatonin was administered until the end of Day 2 while TSH was given only on Day 2 in addition to the original 3-hr pulses. The secretion of T4 was inhibited significantly by 10 microg/ml melatonin at only two of the early media collections. In contrast, 100 micro;g/ml melatonin significantly depressed T4 secretion in response to TSH at all but one interval and completely inhibited the thyroidal response to TSH reintroduced into the media on Day 2. The findings suggest that a high concentration of melatonin is inhibitory to the thyroidal response to TSH, but that lower concentrations do not significantly overcome the TSH stimulus.