Effects of metamorphosis and captivity on the in vitro sensitivity of thyroid glands from the tiger salamander, Ambystoma tigrinum, to bovine thyrotropin

The sensitivity of thyroid glands from the tiger salamander, Ambystoma tigrinum, to bovine thyrotropin (bTSH) was tested in vitro. Thyroids were taken from subjects representing metamorphic stages I (premetamorphic larvae), II (onset of climax), and VII (completion of gill resorption), as well as from captivity control larvae. Exogenous TSH reduced the cumulative uptake of 125I in vitro by thyroids from stage I larvae after 24 and 48 hr. The capacity of thyroids to release thyroxine (T4) in vitro was used subsequently as a measure of their responsiveness to TSH. Baseline levels of T4 release in vitro were variable but did not differ significantly among developmental stages. A low dose of bTSH (5 X 10(-6) IU/ml) did not increase in vitro T4 release compared with that of controls. A larger dose (5 X 10(-4) IU/ml) caused greater increases in T4 release from thyroids of stage II and VII subjects than from those of controls. This dose produced only a small response by thyroids from captivity-control subjects. The results suggest that the thyroids of Ambystoma increase in their capacity to respond to TSH during the process of metamorphosis.     

Synthesis and storage of prolactin in the pituitary gland of bullfrog tadpoles during metamorphosis

Changes in prolactin (PRL) levels and PRL-producing activities in the pituitary gland of tadpoles during metamorphosis were studied. PRL concentration in the pituitary gland as measured by a homologous radioimmunoassay was relatively low during pre- and prometamorphosis and early climax (2.5-3.5 micrograms/100 micrograms protein). PRL levels increased thereafter and reached maximum (7.3 micrograms/100 micrograms protein) at late climax (stage 24). It was also demonstrated that the pituitary cells which immunologically reacted with the antiserum to bullfrog PRL increased in number as metamorphosis proceeded. Measurement of incorporation of [3H]leucine into pituitary PRL in vitro revealed that elevation of PRL synthesis took place in the tadpoles which had reached climax. Radioimmunoassay and bioassay of PRL in the pituitary glands of adult frogs and tadpoles indicated that there was no difference in immunological and biological properties between adults and larvae.     

Elevation of plasma aldosterone levels of tadpoles at metamorphic climax

Plasma aldosterone concentrations in Rana catesbeiana tadpoles during metamorphosis were determined by radioimmunoassay. Aldosterone levels were relatively low (0.7-1.4 ng/ml) during pre- and prometamorphosis. At the onset of climax, aldosterone concentration increased markedly and reached a peak (10.9 ng/ml) at stage XXII. Subsequently, aldosterone levels declined and at the end of metamorphosis (stage XXV), the average concentration was 3.9 ng/ml plasma. Administration of ACTH elevated plasma aldosterone levels in hypophysectomized tadpoles. When hypophysectomized animals were kept in thyroxine (T4), plasma aldosterone concentrations also increased. Combined ACTH and T4 treatment caused a marked increase in plasma aldosterone concentration. The possible involvement of thyroid hormone in the elevation of plasma aldosterone during metamorphosis is discussed.     

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 μg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.     

Regulation of pituitary thyrotropin gene expression during Xenopus metamorphosis: negative feedback is functional throughout metamorphosis

Several hypotheses have been proposed to explain the increase and sustained expression of pituitary thyrotropin (TSH) in the presence of elevated plasma thyroid hormone (TH) concentrations at metamorphic climax in amphibians. It has been proposed that the negative feedback of TH on TSH is inoperative until metamorphic climax, and that it is established at this time by the upregulation of pituitary deiodinase type II (DII); DII converts thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)). However, earlier investigators, using indirect measures of TSH, reported that TH negative feedback on TSH was functional in premetamorphic tadpoles. In an effort to understand pituitary TSH regulation during amphibian metamorphosis, we analyzed multiple pituitary genes known or hypothesized to be involved in TSH regulation in tadpoles of Xenopus laevis. Tadpole pituitary explant cultures were used to examine direct negative feedback on TSH mRNA expression. Negative feedback is operative in the early prometamorphic tadpole pituitary and both T(3) and T(4) can downregulate TSH mRNA expression throughout metamorphosis. The expression of both DII and TH receptor betaA mRNAs increased during development and peaked at climax; however, these increases coincided with similar increases in deiodinase type III, which inactivates TH. Moreover, corticotropin-releasing factor (CRF) receptors, CRF binding protein and thyrotropin-releasing hormone receptor type 2 mRNA expression also peaked at climax. Our data suggest that the regulation of TSH is more complex than the timing of DII expression, and likely involves a balance between stimulation of TSH synthesis and secretion by neuropeptides (e.g. CRF) of hypothalamic or pituitary origin, increased pituitary sensitivity to neuropeptides through upregulation of their receptors, and intrapituitary TH levels.     

Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog,Rana catesbeiana

Diel variation in plasma thyroxine (T(4)), and plasma and ocular melatonin was studied in Rana catesbeiana tadpoles and postmetamorphic froglets on 12:12 and 6:18 light/dark (LD) regimens. A progressive rise in plasma T(4) initiates metamorphosis while melatonin can modulate metamorphic progress. Changes in the phase of the rhythms of these two hormones during development might influence the hormonal regulation of metamorphosis. The hormones studied exhibited LD cycle-specific diel fluctuations except in froglet plasma T(4) and all hormones at prometamorphosis on 6L:18D. On 12L:12D, plasma T(4) and ocular melatonin peaked during the scotophase at prometamorphosis and early climax, whereas the plasma melatonin acrophase shifted from the light to the dark at climax. A nocturnal peak of plasma melatonin closely correlated with the onset and offset of dark appeared in the froglet, while the peak of ocular melatonin shifted to the light. Compared to 12L:12D, the peaks of the diel fluctuations on 6L:18D occurred later than on 12L:12D in synchrony with an earlier onset, and increase in length, of the scotophase. The phase of the hormone rhythms changed during metamorphosis in such a way that the peaks of melatonin had a different relationship to the T(4) peaks as development proceeded. On both LD cycles, the 24-h mean of plasma T(4) rose at climax and fell in the froglet whereas plasma melatonin decreased at climax and then rose to a high level in the froglet. After only minor changes during metamorphosis, froglet ocular melatonin levels decreased on 12L:12D and increased on 6L:18D. The findings indicate that the hormonal flux during metamorphosis has circadian aspects, which might explain variations in the response to exogenous hormone treatment at different times of the day and LD cycle-specific timing of development. A fall in plasma melatonin at climax appears to be as much a part of the hormonal changes of metamorphosis as a rise in plasma T(4).     

Plasma thyroxine and triiodothyronine levels in spontaneously metamorphosing Rana catesbeiana tadpoles and in adult anuran amphibia.

We have developed sensitive and reliable radioimmunoassays for T4 and T3 in amphibian plasma and have used these procedures to measure plasma T4 and T3 levels in spontaneously developing Rana catesbeiana tadpoles at various stages of metamorphosis. During premetamorphosis circulating levels of both T4 and T3 were below the limits of detection of the RIA procedures (T4 less than 50 ng/100 ml, T3 less than 5 ng/100 ml). A gradual rise in plasma T3 and T4 became apparent during prometamorphosis, and at the onset of metamorphic climax the levels of both T4 and T3 increased sharply. Peak levels for both hormones were observed in the middle of metamorphic climax (stage XXIII). The circulating T3 level reached a mean peak of 78 ng/100 mg, at least 15 times greater than the level during premetamorphosis. The peak T4 level was 0.5 microgram/100 ml, about a 10-fold increase over the premetamorphosis level. The surge in thyroid hormone secretion lasted only for several days, and during the latter half of metamorphic climax there was a fairly rapid decrease in plasma T4 and T3. By 2 days post-climax the levels had declined to about 20% of their peak values. Free T4 and T3 levels in plasma followed the same general pattern as the total hormone levels during the various stages of tadpole development. In adult R. catesbeiana, plasma T4 and T3 levels were surprisingly low. Similarly low values were observed in Bufo marinus and in Rana pipiens. The very low levels of circulating T4 and T3 both in premetamorphosis tadpoles and in adults suggest that thyroid hormones in anuran Amphibia may be of importance only during the period of metamorphosis.     

Inhibition of the response of mouse thyroid to tryrotropin induced by chronic triiodothyronine treatment.

Administration of 1 mU bovine TSH iv to mice resulted, within 1 hour, in the increase of the serum T4 level from 32 +/- 1.4 ng/ml to 53 +/- 2.6 ng/ml (Mean +/- SE, n = 24). Treatment with 1 mug triiodothyronine (T3) per day, for 10 days, abolished the responsiveness of the thyroid to TSH, as measured by thyroxine (T4) release. Thyroidal response to TSH was measured also in vitro. The basal hormonal release was 4.66 +/- 0.55 ng T4 and 0.98 +/- 0.15 ng T3 per thyroid per 3 h (n = 30). In the presence of bovine TSH (0.2 mU/ml) the hormonal secretion increased 3-fold for T4 and 2.5-fold for T3. Thyroids from mice pretreated with T3 for 10 days showed almost no response to TSH. Partial refractoriness to TSH was already significant 5 days after T3 pretreatment. Responsiveness to TSH was restored 3 days after T3 withdrawal or after 3 daily injections of 10 mU bovine TSH, concomitant with the last 3 days of T3 pretreatment. These results indicated that the prolonged absence of an adequate level of trophic hormone may be the cause of thyroidal unresponsiveness to acute TSH treatment. With 20 mU of TSH, cAMP levels rose from 4 +/- 0.5 picomoles to 80 +/- 9.3 picomoles per thyroid (n = 6). In mice subjected to 10 days of T3 pretreatment the response was markedly reduced: 20 +/- 3 picomoles/ thyroid. Thyroids of the T3-treated mice responded normally to 1 mM DBcAMP in vitro. From these results it was concluded that the impaired responsiveness of the thyroids to TSH occurs at a step prior to cAMP accumulation.     

Expression of the Xenopus laevis prolactin and thyrotropin genes during metamorphosis.

The cDNAs encoding Xenopus laevis prolactin (PRL) and the alpha and beta subunits of thyroid-stimulating hormone (TSH alpha and TSH beta, respectively) have been cloned from a pituitary library. Results of developmental RNA blot analysis contradict the long-held biological role for PRL as a juvenilizing hormone in amphibia. The pituitary gland of a premetamorphic tadpole expresses PRL mRNA at very low levels. The abundance of PRL mRNA increases late in metamorphosis as a response to thyroid hormone (TH), suggesting that PRL is more likely to have a function in the frog than in the tadpole. TSH alpha and -beta mRNA levels increase through prometamorphosis; this rise does not appear to be regulated directly by TH. At climax, both TH and TSH mRNA levels drop. The sequential morphological changes that characterize prometamorphosis depend upon the gradual increase of endogenous TH, which peaks at climax. This increase in TH in turn depends upon the lack of a traditional thyroid-pituitary negative-feedback loop throughout prometamorphosis.     

Metabolism of thyroxine in Rana catesbeiana tadpoles during metamorphic climax

Previous studies have indicated that premetamorphic tadpoles do not convert T4 to T3 to a measurable extent (1). The present study was performed to determine whether a T4 5'-monodeiodinating system is acquired at later stages of development. [125I]T4 metabolism in vivo was determined in tadpoles at most stages of prometamorphosis and metamorphic climax and, for comparison, in premetamorphic tadpoles. The conversion of [125I]T4 to [125I]T3, as indicated by the presence of an 125I-labeled product in serum and liver preparations that cochromatographed with carrier T3, was sometimes observed in tadpoles near the end of prometamorphosis and was always evident in tadpoles that were either undergoing or had completed metamorphic climax. However, during this phase, no correlation could be drawn between the extent of T3 production and morphological development. The formation of T3 from T4 in vivo was significantly decreased in tadpoles pretreated with propylthiouracil. The T45'-monodeiodinating system could be induced in premetamorphic tadpoles by injecting them with either T4 or T3. This finding together with the observation that normal acquisition of this system occurs at the time when endogenous T4 and T3 levels are rising rapidly suggest that its development is under the control of the thyroid hormones.     

Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis

We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHβ in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs. Finally, in vivo exposures of tadpoles to THs were conducted to confirm the results of the ex vivo experiments. When pituitaries from climactic tadpoles were removed from the influence of endogenous hormones, TSHβ mRNA expression increased late or not at all whereas the rate of TSHβ secreted into media increased dramatically, suggesting that TSH secretion, but not TSH mRNA expression, is under the negative regulation of an endogenous signal during the climactic stages of metamorphosis. Pituitaries from pre- and prometamorphic tadpoles were more sensitive to TH-induced inhibition of TSHβ mRNA expression and secretion than pituitaries from climactic tadpoles. The observed decrease in sensitivity of pituitary TSHβ mRNA expression to negative feedback by THs from premetamorphosis to metamorphic climax was confirmed by in vivo experiments in which tadpoles were reared in water containing THs. Based on the results of this study, a model is proposed to explain the seemingly paradoxical, concurrent rise in serum TH concentrations and pituitary TSH mRNA expression during metamorphosis in larval anurans.     

Thyroxine surge in metamorphosing flounder larvae

The tissue concentration of thyroxine (T4) in larval flounder (Paralichthys olivaceus) was studied at various stages of their metamorphosis using a specific radioimmunoassay developed for samples obtained by quantitative extraction of the hormone from fish eggs and larvae. T4 concentrations were below the limit of detection in extracts of fertilized eggs and larvae until the end of prometamorphosis. Several dorsal fin rays continued growing from premetamorphosis until the end of prometamorphosis. The hormone became detectable at the onset of metamorphic climax when the elongated dorsal fin rays stopped growing. A sharp increase in tissue T4 concentration was observed in midclimax of metamorphosis, and the high level of T4 (11-13 ng/g body weight) was maintained until the end of the climax. The period of elevated T4 levels coincided with resorption of the elongated dorsal fin rays. The right eye completed translocation from the right to the left side of the body during metamorphic climax. The tissue T4 level declined to about a half of the peak value during postclimax, and then increased moderately in later adult-type juveniles. The results of an additional experiment, in which fertilized eggs and newly hatched larvae were treated with T4 and/or thiourea, suggested the presence of negative feedback regulation of pituitary-thyroid axis, implying an involvement of thyroid hormones even in early premetamorphic larvae. These results suggest that metamorphic climax is induced by a surge of thyroid hormone, and that thyroid hormone may also regulate development before and after the metamorphosis.     

Putative nuclear triiodothyronine receptors in tadpole liver during metamorphic climax

It has been shown previously that the maximum binding capacity (MBC) of the putative T3 receptors in tadpole red blood cells (RBCs) is increased during development and can be stimulated by treatment with thyroid hormone (TH). The present study was performed to determine if the MBC of tadpole liver nuclei is also increased during development or after treatment with TH. Because of the relatively high levels of endogenous TH in tadpoles during climax, the use of an in vivo saturation assay employing [125I]T3 was not feasible. Thus, MBC was determined by measuring by RIA the amount of T3 bound to the liver nuclei in tadpoles pretreated with sufficient T3 to saturate the receptors. Values were then corrected for the nonsaturable fraction using data obtained in tadpoles given a large dose of T3 (10 nmol). After this dose, essentially all of the T3 in the nucleus was bound to nonsaturable sites. MBC values estimated by this method and by Scatchard analysis were comparable. In contrast to the observations in tadpole RBCs, no significant change in the MBC of liver nuclei occurred as the tadpole progressed from early prometamorphosis to metamorphic climax; in tadpoles at stages XII-XIV and XIX-XXIII, MBC values were 0.308 +/- 0.024 (+/- SE) and 0.260 +/- 0.035 pmol/mg DNA, respectively. Furthermore, treatment of tadpoles with T4 (1 nmol T4; 14 days before study), which resulted in a marked increase in receptor number in RBCs, had no effect on MBC in hepatic nuclei. The amounts of nucleus-bound endogenous T3 in liver and RBCs were also determined. From these data and the MBC values, it was calculated that hepatic and RBC nuclear receptors were, respectively, 80% and more than 90% occupied with T3. These findings indicate that there is tissue specificity in the response of receptor MBC to TH during metamorphosis, and that most of the TH on the receptor during climax is T3.     

Putative nuclear triiodothyronine receptors in tadpole erythrocytes during metamorphic climax

The characteristics of the nuclear T3 receptors present in red blood cells (RBCs) of Rana catesbeiana tadpoles undergoing metamorphic climax have been investigated with a T3 saturation technique. Because there were significant amounts of receptor-bound endogenous hormone, prolonged incubation in vitro (48 h at 21 C) was necessary to achieve binding equilibrium. Receptor number, which averaged 783 +/- 35 (+/- SE) sites/nucleus during early prometamorphosis (stages XIV-XVI), increased rapidly during the subsequent stages of this phase. By stage XIX, receptor number had reached a maximum of 2464 +/- 152. During climax, receptor number decreased steadily, and by stage XXV, it was lower than that observed during premetamorphosis (stages V-X). This decrease was attributed to the replacement of larval RBCs, which have a high receptor content, by adult RBCs, which possess significantly fewer sites per nucleus. At midclimax, adult and larval RBCs were separated on a Renografin continuous density gradient. Adult cells constituted more than 65% of the total cell population and were shown to contain only 126 +/- 46 sites/nucleus. Although the striking increase in receptor number preceded the substantial increases in plasma T4 and T3 levels that occurred during climax, it was associated with a measurable increase in plasma thyroid hormone levels. Furthermore, receptor number in stage XIV tadpoles was increased markedly after immersion for 14 days in water containing sufficient T3 to raise plasma T3 only to levels normally observed in late prometamorphosis. These findings strongly support the hypothesis that the increases in receptor number and plasma thyroid hormone levels that occur before climax in this species are causally related.     

Effects of bovine TSH on the tissue thyroxine level and metamorphosis in prometamorphic flounder larvae

Bovine thyroid-stimulating hormone (TSH) was microinjected (0.2 microliter/fish) into prometamorphic flounder larvae and the effects on metamorphosis as well as the tissue thyroxine (T4) and triiodothyronine (T3) levels were studied. After a single injection of TSH (5 mIU/g), the tissue T4 concentration increased markedly after 5 hr, reached a peak after 10 hr, and decreased subsequently. T4 concentration after 24 hr was still higher than in saline-injected fish but returned to the control level 48 hr after the injection. On the other hand, tissue T3 concentration was kept lower than the detectable level (0.2 ng/g) throughout the experimental period of 72 hr after a single injection of TSH (5 mIU/g). TSH treatment also accelerated the process of metamorphic climax, such as shortening of the second fin ray and eye migration. These results suggest that an increased secretion of TSH from the pituitary stimulates the thyroid, resulting in a surge of the tissue T4 concentration which induces the climax of the flounder larvae.     

The pars distalis nerves and metamorphosis in Rana temporaria.

The length of prometamorphosis in the tadpoles of Rana temporaria is influenced by different treatments: (1) thyroidectomy, (2) propylthiouracil, (3) 1-thyroxine, (4) low temperature. In normally developing tadpoles the fluorescent pars distalis fibers persist until climax. Even when prometamorphosis is artificially prolonged, irrespective of the reason, the fibers still persist until climax. When the prometamorphic period is artificially shortened, or when a prolongation of prometamorphosis is interrupted by thyroxin-induced climax, the fibers disappear. Thus the disappearance of the aminergic nerves is a metamorphic event associated with climax.     

Thyroid hormones inhibit frog corticotropin-releasing factor-induced thyrotropin release from the bullfrog pituitary in vitro

Due to the lack of a radioimmunoassay (RIA) system for amphibian thyrotropin (TSH), no direct evidence that thyroid hormone suppresses the release of TSH from the amphibian pituitary has been obtained. However, we recently developed an RIA for bullfrog (Rana catesbeiana) TSH and thus were able to study the effect of thyroid hormone on the release of TSH from the bullfrog pituitary. Enzymatically dispersed pituitary cells of larval, juvenile, and adult bullfrogs were cultured in the absence or presence of 100 nM corticotropin-releasing factor of bullfrog origin (fCRF), which is known to be a potent stimulator of the release of TSH. The amount of spontaneously released TSH was higher in late prometamorphic and climactic tadpoles than in early prometamorphic larvae and juvenile and adult frogs. Pituitary cells from tadpoles at metamorphic climax responded to fCRF to release much more TSH than those from early and late prometamorphic tadpoles and juvenile and adult frogs. In all cases, the fCRF (100 nM)-induced, but not the basal, release of TSH was significantly suppressed by 1 nM triiodothyronine (T(3)) and 1000 nM thyroxine (T(4)), when examined using adult pituitary cells. The suppressive effect of thyroid hormones was revealed to be dependent on their concentrations.