The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana (bullfrog) tadpoles is induced by thyroxine

The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana (bullfrog) tadpoles is induced by thyroxine

Comparative Biochemistry and Physiology Part A 129 Ž2001. 653᎐663 The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana ž bullfr...

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Comparative Biochemistry and Physiology Part A 129 Ž2001. 653᎐663

The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana ž bullfrog/ tadpoles is induced by thyroxine Mary L. WrightU , Christina D. Alves Biology Department, College of Our Lady of the Elms, Chicopee, MA 01013, USA Received 29 November 2000; received in revised form 22 February 2001; accepted 22 February 2001

Abstract Melatonin decreases in the plasma of Rana catesbeiana Žbullfrog. tadpoles at the climax of metamorphosis when the thyroxine ŽT4 . level peaks. Since melatonin inhibited thyroid function in vitro, it would be of interest to determine if the decline in plasma melatonin permits greater thyroid hormone secretion, or if the increasing levels of T4 cause the climactic decrease in plasma melatonin. The reciprocal effects of administering T4 or melatonin just prior to metamorphic climax were examined in tadpoles kept at 22⬚C on an 18L:6D cycle. If melatonin functions as a thyroid antagonist at later metamorphic stages, administration of melatonin should decrease plasma T4 , whereas if T4 causes the decline in plasma melatonin, T4 treatment of tadpoles prior to climax should induce the climactic melatonin decrease prematurely. Once daily injection of 40 ␮g melatonin for 5 days at 19.30 h had no effect on metamorphic progress, or on plasma T4 or melatonin levels, except for a transient rise in melatonin just after the injection. Immersion in 2.2= 10y4 M melatonin for 6 days accelerated metamorphosis and decreased plasma melatonin, but had no effect on plasma T4 . Administration of T4 by injection of 0.2 ␮g, or immersion in a 6.3= 10y8 M solution accelerated metamorphosis more than melatonin immersion, raised plasma T4 to climax levels, and induced a decrease in plasma melatonin. We conclude that rapid clearance of exogenous melatonin from the circulation in these experiments did not allow it to affect plasma T4 , and that there is clear evidence that the rise in T4 induces the climax decrease in plasma melatonin. The finding that immersion in a high level of melatonin can lower plasma melatonin and accelerate metamorphosis, whereas a single daily injection does not, provides an explanation for some of the contradictory reports in the literature concerning melatonin’s effect on tadpole metamorphic progress. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Metamorphosis; Plasma melatonin; Thyroxine; Tadpole; Diurnal rhythm; Pineal; Amphibian; Ocular melatonin; Thyroid

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Corresponding author. Tel.: q1-413-594-2761, ext. 298; fax: q1-413-592-4871. E-mail address: [email protected] ŽM.L. Wright.. 1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 3 2 9 - 4

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M.L. Wright, C.D. Al¨ es r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 653᎐663

1. Introduction The metamorphosis of the tadpole into an adult is a developmental phenomenon which is induced by thyroid hormones ŽTH.. After a period of larval growth called premetamorphosis, tadpoles enter prometamorphosis, when the TH rise and adult organs, such as the limbs, undergo marked development. The last stage is metamorphic climax, when the TH peak and larval organs regress. Although a gradual rise of the TH to a peak at climax can alone induce metamorphosis, other hormones, such as adrenal corticoids and prolactin, may synergize with, or antagonize the TH Žreviewed in Kikuyama et al., 1993; Kaltenbach, 1996.. Melatonin might also be a thyroid hormone modulator, since it influenced metamorphic rate ŽDelgado et al., 1984, 1987; Alonso-Bedate et al., 1985; Edwards and Pivorun, 1991; Wright, et al., 1991; Rose and Rose, 1998., antagonized thyroid growth and cell proliferation in premetamorphic stages ŽWright et al., 1996., inhibited T4 secretion in vitro ŽWright et al., 2000., and lowered the in vitro response of the thyroid to exogenous thyrotropin ŽWright et al., 1997a.. Melatonin influenced metamorphic rate during T4stimulated, as well as spontaneous metamorphosis and counteracted the action of exogenous T4 in peripheral tissues ŽWright et al., 1991.. Unlike other proposed TH modulators, which peak at late metamorphosis, melatonin declines in the plasma at metamorphic climax ŽWright and Racine, 1997. and rises again in the froglet after metamorphosis ŽWright et al., 1999a.. Since it is not yet clear how the high levels of TH present at climax occur, it would be useful to know if a decline in plasma melatonin removes thyroid inhibition or, in contrast, if the increasing titers of TH promote the decrease in plasma melatonin. Therefore, we examined the reciprocal effects of administering T4 or melatonin to late prometamorphic tadpoles close to the onset of climax. Exogenous T4 or melatonin was administered by injection in one set of experiments, and immersion in another. Immersion would be expected to produce more constant exposure to the hormone while the perturbation of injection might alter hormone rhythms. If melatonin antagonizes the thyroid at late metamorphosis, administration of exogenous melatonin should decrease plasma T4 . If, on the other hand, the rise in T4 is inducing the decrease in plasma melatonin at climax,

administration of T4 to late prometamorphic tadpoles should precipitate a fall in plasma melatonin, simulating climax prematurely.

2. Materials and methods 2.1. Animals and experiments The experiments were done in three consecutive summers using late prometamorphic Rana catesbeiana Žbullfrog. tadpoles ŽCharles D. Sullivan Co., Nashville, TN. staged according to Taylor and Kollros Ž1946.. Stage progression, which indicates hindlimb development and is a marker of metamorphic progress, was measured at the beginning and end of hormone treatment in each experiment. The larvae were kept on an 18L:6D cycle ŽL onset 08.00 h. at 22⬚C in light- and temperature-controlled incubators, and fed washed, canned spinach once per day. In the first two experiments, there were eight sampling intervals: 09.00, 13.00, 15.00, 17.00, 21.00, 01.00, 03.00, and 06.00 h. In the last experiment, the 15.00 h interval was omitted. Injections in the first two experiments were given for 5 days intraperitoneally through the tail muscle, alternating sides each day. Tadpoles that were not yet used at injection time on the following day of plasma collection received an additional injection. 2.1.1. Experiment 1 The effect of injected melatonin on plasma T4 and melatonin was examined. Experimental tadpoles were injected with melatonin Ž40 ␮gr0.04 ml 0.7% saline. daily at 19.30 h. Controls were injected at the same time with the same volume of 0.7% saline. 2.1.2. Experiment 2 The effect of injected T4 on plasma T4 and melatonin was studied. Experimental tadpoles were injected with T4 Ž0.2 ␮gr0.05 ml 0.7% saline. daily at 19.30 h. Controls were injected at the same time with the same volume of 0.7% saline. 2.1.3. Experiment 3 T4 and melatonin were administered by immersion in this experiment, and melatonin and T4 groups were compared to a common control group. Tadpoles were immersed in either T4 Ž6.3 = 10y8 M. or melatonin Ž2.2= 10y4 M. in 10%

M.L. Wright, C.D. Al¨ es r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 653᎐663

Holtfreter’s solution for 6 days beginning at 09.00 h on the first day. Control tadpoles were kept in 10% Holtfreter’s solution. The solutions were changed every other day. On day 7, tadpoles were killed at intervals and blood and eyes were collected for assay of plasma T4 and plasma and ocular melatonin. 2.2. Hormone preparation and concentrations Melatonin ŽSigma, St. Louis, MO. was dissolved in alcohol and diluted in 0.7% saline to make the 40 ␮gr0.04 ml melatonin solution for injection in experiment 1. A stock solution of 1 mgrml made in distilled water was diluted in 10% Holtfreter’s solution to make a melatonin immersion solution of 2.2= 10y4 M for experiment 3. The concentration of melatonin in experiment 1 was 40 ␮g because previous work utilizing Rana pipiens tadpoles ŽWright et al., 1991. had indicated that no less than 10 ␮g melatonin had to be administered by injection to affect metamorphic rate, and Rana catesbeiana tadpoles are much larger than Rana pipiens. In addition, preliminary work showed that single injections of up to 50 ␮g of melatonin did not significantly raise the melatonin level in the blood above the control when sampling was done 15᎐45 min after the injection. In the third experiment, melatonin was used at 2.2 = 10y4 M. Melatonin concentrations of 1.5᎐5.4= 10y4 M have been used by other investigators in immersion experiments utilizing Discoglossus pictus ŽAlonso-Bedate et al., 1985. and Xenopus lae¨ is ŽDelgado et al., 1987. tadpoles. L-T4 ŽSigma. was dissolved in 1% sodium hydroxide and distilled water was added to make a stock solution of 10 ␮grml. This stock solution was diluted in 0.7% saline to make the 0.2 ␮gr0.05 ml T4 solution for injection in experiment 2, and in 10% Holtfreter’s solution to make a T4 immersion solution of 6.3= 10y8 M for experiment 3. In experiment 2, the T4 concentration of 0.2 ␮gr0.05 ml saline was chosen on the basis of previous work ŽWright et al., 1990a. and a preliminary experiment which showed that two injections of 0.2 ␮g T4 significantly raised plasma T4 above the control whereas 0.05 ␮g T4 had no effect. In the third experiment, the T4 group was immersed in 6.3= 10y8 M Ž50 ␮grl. T4 . If only 21.7% of TH is taken up by tadpoles immersed in

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it, as calculated from data in Galton Ž1988., it would give an effective concentration of 1.09 ␮grdl. This level compares favorably with the highest level of plasma T4 Ž0.95 ␮grdl. found at climax in bullfrog tadpoles ŽMondou and Kaltenbach, 1979.. 2.3. Procedures for collecting and processing plasma and eyes At each time interval, tadpoles were pithed, the heart was exposed, and blood was pipetted from the heart into siliconized microcentrifuge tubes. The blood was centrifuged at 3000 rev.rmin for 15 min, after which the plasma was removed and stored at y20⬚C. Eyes were also taken in experiment 3 and frozen for assay of ocular Žretinal . melatonin. Before the assay, the eyes were partially defrosted, cut in half, and the lens removed, The two eyes from the same tadpole were homogenized with a glass rod in 700 ␮l of 0.7% saline. The mixture was then centrifuged at 3000 rev.rmin for 20 min at room temperature to remove solids, and the supernatant pipetted out. If the extract was not to be assayed that day, it was again frozen at y20⬚C. When a collection interval occurred during the scotophase, each tadpole was pithed under a 7.5-W red light bulb before lights were turned on for blood and eye collection. The remaining tadpoles were kept under a light-tight box until time of killing. 2.4. Radioimmunoassay (RIA) procedures T4 was assayed using a canine total T4 RIA kit ŽDiagnostic Products Corporation, Los Angeles, CA. employing antibody-coated tubes as described in Wright et al. Ž1995.. Samples were assayed in duplicate. The detection limit was 0.039 ␮grdl, and the intra- and inter-assay coefficients of variation ŽCV. were 3.2 and 3.1%, respectively. Melatonin in the plasma was assayed directly using a human melatonin kit from DiagnosTech International Inc. ŽOsceola, WI.. The plasma was enzyme-treated prior to the assay to free the hormone from albumin and other binding proteins. Extracted ocular melatonin was assayed without enzyme treatment. The melatonin RIA was validated for measurement of plasma and ocular melatonin in amphibian samples by parallelism and dilution studies as described in Wright et al., 1999b. The detection limit was 0.75 pgrml,

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M.L. Wright, C.D. Al¨ es r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 653᎐663

and the intra- and inter-assay CV values were 8.1 and 14.8%. Tubes were counted in a Packard Riastar gamma counter with a counting efficiency greater than 80%. The calculations producing the standard curve and sample hormone content were performed by computer using the installed counter software.

Table 1 Effect of T4 or melatonin treatment on metamorphosis Exp. Treatmenta

Start of hormone treatment

3. Results In the first experiment, control tadpoles were injected daily for 5 days with 0.7% saline while the experimental animals received 40 ␮g melatonin daily. Injection of melatonin failed to accelerate or retard metamorphosis in this experiment ŽTable 1., and had no effect on plasma T4 ŽFig. 1a.. Plasma melatonin ŽFig. 1b. was significantly above the control only at 21.00 h Ž Ps 0.013. just after the injection at 19.30 h. The treatment difference between the control and melatonin data just missed statistical significance with two-way ANOVA Ž Ps 0.05., while the interaction between treatment and time-of-day was significant Ž Ps 0.004.. The plasma melatonin peaks in the melatonin curve were phase-delayed compared to the peaks of the control curve. The mean of the samples in all intervals Ž24-h mean. was also calculated and separate statistics were done ŽFig. 2a,b., which confirmed that injection of melatonin failed to affect plasma T4 , or to induce a sustained significant rise in plasma melatonin.

End of hormone treatment

1

Control Melatonin-injected

17.4" 0.1 Ž49. 18.4" 0.2 Ž48. 17.3" 0.1 Ž51. 18.3" 0.2 Ž48.

2

Control T4-injected

15.5" 0.2 Ž45. 16.0" 0.2 Ž40. 15.5" 0.2 Ž46. 16.8" 0.1 Ž40.c

3

Control 17.0" 0.0 Ž38. 17.4" 0.1 Ž35. Melatonin-immersed 17.0" 0.0 Ž38. 17.7" 0.1 Ž35.d T4-immersed 17.0" 0.0 Ž39. 18.4" 0.1 Ž35.e

2.5. Statistical analysis The data were analyzed using the paired or unpaired Student’s t-test, or one- or two-way ANOVA, as appropriate, with ANOVA followed by the Student᎐Newman᎐Keuls multiple comparison test or Duncan’s Multiple Range test to isolate differences among the means. The 24-h mean represents the mean of all data collected over the experimental period since the samples at each interval came from different animals. The cosinor method to identify significant rhythms was also employed ŽHalberg et al., 1972.. The computer program utilizes the method of least squares for cosine curve fitting, and calculates the acrophase, or time after midnight that the crest of the cosine curve occurs, and the statistical significance of the rhythm. Differences were considered to be significant if P- 0.05.

Stage Žmean " S.E..b

a Hormone treatment lasted for 5 days in experiments 1 and 2, and for 6 days in experiment 3. b Taylor and Kollros Ž1946. stages were changed to Arabic numbers for statistical analysis. The numbers in parentheses indicate the number of samples. c Significantly different from control of same experiment Ž P - 0.001.. d Significantly different from control of same experiment Ž P - 0.05. and from T4 -immersed. e Significantly different from control of same experiment Ž P - 0.01..

In contrast, injection of 0.2 ␮g T4 in the second experiment accelerated metamorphosis ŽTable 1. and had a much more pronounced effect on plasma T4 and melatonin. Exogenous T4 significantly raised plasma T4 above the control Žtwo-way ANOVA: time-of-day, treatment differences, and interaction P- 0.001., with the pronounced peak at 21.00 h occurring shortly after the time of daily injection at 19.30 h ŽFig. 3a.. The high peak after injection was not sustained throughout the day, but nevertheless the level of T4 remained above the control at all sampling intervals Ž P- 0.01.. Injection of T4 also lowered plasma melatonin ŽFig. 3b.. With two-way ANOVA there was a treatment difference Ž Ps 0.037. while time-ofday and interaction were not significantly different. Plasma melatonin in the T4 group was significantly below the control at 13.00 Ž Ps 0.028. and 01.00 Ž Ps 0.021. h. The 24-h mean ŽFig. 2c,d. confirmed that injection of T4 raised plasma T4 and depressed plasma melatonin. In the third experiment, treated tadpoles were immersed in either 2.2= 10y4 M melatonin or 6.3= 10y8 M T4 . Both melatonin and T4 treatment accelerated metamorphosis ŽTable 1., although T4 was more effective than melatonin. In

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order to facilitate comparison with the first two experiments, each hormone treatment was compared separately to the common control group in the presentation of results in the line graphs ŽFig. 4.. Immersion in melatonin failed to affect plasma T4 ŽFig. 4a.. Plasma melatonin decreased Žtreatment Ps 0.018; time-of-day and interaction not significant., mainly at the expense of the peak of melatonin in the dark observed in the control ŽFig. 4b.. The control and melatonin groups were significantly different at 17.00 Ž P- 0.02., 03.00 Ž P- 0.03., and 06.00 h Ž P- 0.01.. Ocular melatonin was not affected ŽFig. 4c.. T4 treatment by immersion had the same result as injection, raising plasma T4 above the control and depressing plasma melatonin. The peak of plasma T4 in the T4 group occurred at 09.00 h ŽFig. 4d., reflecting the start of T4 immersion between 09.00 and 10.00 h on the first day, and the change of solution at the same time on subseFig. 2. Mean of the samples in all intervals compared in control and experimental groups from experiments 1 and 2 Ždata in Figs. 1 and 3.. The effect of injection of melatonin Ža,b. or T4 Žc,d. is shown on plasma T4 and melatonin ŽML. levels. Significant differences between experimental and control are indicated by P values above the bars.

Fig. 1. Experiment 1. Diurnal profile of T4 Ža. and melatonin Žb. in the plasma of control and melatonin-treated tadpoles after 5 days of a single, daily injection of saline or 40 ␮g melatonin ŽML. at 19.30 h Ž n s 6.. The bar shows the time of the scotophase in the 18L:6D cycle, while the arrowhead denotes injection time. Stars indicate a significant difference between control and melatonin-treated groups at that time interval.

quent days. The difference between T4 and control was significant at all time intervals Ž P- 0.001; two-way ANOVA: time-of-day P- 0.003; treatment P- 0.0002; interaction P- 0.006.. Plasma melatonin ŽFig. 4e. was lower than the control at 09.00, 17.00, 03.00, and 06.00 h Ž P- 0.05., and there was a significant treatment difference Ž P0.001. with two-way ANOVA. T4 immersion appeared to raise ocular melatonin ŽFig. 4f. above the control, but a treatment effect with two-way ANOVA just missed statistical significance Ž Ps 0.052.. The 09.00 Ž Ps 0.036. and 13.00 Ž Ps 0.004. h intervals were higher in the T4 group than the controls. The 24-h mean ŽFig. 5a. showed that immersion in T4 significantly raised plasma T4 Ž P- 0.01., while melatonin immersion did not alter plasma T4 . Immersion in either T4 or melatonin lowered plasma melatonin Ž P- 0.01., although T4 had the greater effect ŽFig. 5b.. Ocular melatonin ŽFig. 5c. was higher in the T4 compared to the melatonin group Ž P- 0.05., but there were no significant differences from the control. The 24-h means of plasma versus ocular melatonin in

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the third experiment also had a significant 24-h rhythm with the acrophase occurring approximately 3 h later in the T4-immersed group than in the control. The significant circadian rhythms ŽTable 2. were usually found when the data series showed a significant time-of-day difference with two-way ANOVA Žresults above.. The other significant rhythms were ultradian, or less than 24 h ŽTable 2.. The acrophases of all the ultradian rhythms occurred at approximately the same time, between 04.30 and 06.30 h.

4. Discussion

Fig. 3. Experiment 2. Diurnal profile of T4 Ža. and melatonin Žb. in the plasma of saline-injected control and 0.2 ␮g T4-injected tadpoles after 5 days of a single daily injection at 19.30 h Ž n s 5.. The bar shows the time of the scotophase in the 18L:6D cycle, while the arrowhead denotes injection time. Stars indicate a significant difference between control and T4-treated groups at that time interval. ML s melatonin.

experiment 3 were also compared, and were not significantly different in the control or melatoninimmersed groups. However, plasma melatonin was lower Ž P- 0.001. than ocular melatonin in the T4-immersed group. When the percent difference in stage change between control and experimental groups at the end of hormone treatment was compared to the percent difference in melatonin or T4 levels in the various experiments ŽFig. 6., the degree of development was positively correlated with the T4 , and negatively correlated with the melatonin, concentrations in the plasma, illustrating the inverse relationship between the two hormones. The data of each curve were also analyzed by the cosinor method to detect rhythmicity. T4 rhythms with a period of 24 h occurred in the T4 treated groups in the second and third experiments ŽTable 2., where the acrophase of the rhythm coincided with the onset of treatment. Ocular melatonin in the control and T4 groups of

T4 administration raised plasma T4 above the controls at all intervals to a level that was in the physiological range for bullfrog tadpoles at climax ŽMondou and Kaltenbach, 1979; Wright and Racine, 1997.. As a consequence, metamorphic progress was accelerated. At the same time, exogenous T4 lowered plasma melatonin, initiating the decline in plasma melatonin that occurs at climax of spontaneous metamorphosis ŽWright and Racine, 1997; Wright et al., 1999a.. These findings indicate that the climactic plasma melatonin decrease is induced by T4 , directly or indirectly. Studies of the effect of T4 on the pineal gland might shed light on the mechanism of the plasma melatonin decrease, since T4 might inhibit the synthesis or release of pineal melatonin. T4 treatment by immersion increased ocular melatonin significantly above the control at 09.00 and 17.00 h. If ocular melatonin contributes to circulating melatonin, as has been suggested ŽDelgado and Vivien-Roels, 1989. perhaps the rise in ocular melatonin indicates that T4 suppressed its release. On the other hand, T4 might clear melatonin from the plasma without affecting sites of melatonin release. There is evidence for a T4-induced increase in the uptake of melatonin into tadpole organs ŽWright et al., 1997b.. After simultaneous injection of T4 and w 3 Hx-melatonin, the melatonin content of tail, gills, hindlimb, skin, and intestine was significantly raised over the controls, which received only w 3 Hx-melatonin. The plasma melatonin levels differ in the controls of the three experiments, which were done in the summers of different years. These results reflect the variability among populations which we have noticed in several years of work on plasma melatonin, and also seasonal differences,

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Fig. 4. Experiment 3. Diurnal profile of plasma T4 , plasma melatonin ŽML., and ocular melatonin in control tadpoles and those immersed for 6 days in 2.2= 10y4 M melatonin Ža᎐c. or 6.3= 10y8 M T4 Žd᎐f.. The melatonin and T4 data are plotted separately against the common control for clarity of graphing and better comparison with experiments 1 and 2. The bar indicates the time of the scotophase in the 18L:6D cycle. Stars indicate a significant difference between control and melatonin- or T4 -treated groups at that time interval Ž n s 5..

since plasma melatonin is lower in the summer ŽWright et al., 1999b. in these overwintering tadpoles. Animals obtained for research in early summer sometimes have higher plasma melatonin than those collected later in the season. The melatonin controls of the first two experiments also show an injection-induced perturbation of the normal diurnal variation observed on 18L:6D. In contrast, the controls in the immersion experiment, which were not stressed by injection, have a plasma melatonin rhythm similar to that previ-

ously reported ŽWright and Racine, 1997., with a peak in the dark. There is evidence that injection stress can alter hormone levels and rhythms. Whole-body T4 was elevated by handlingrinjection in spadefoot toads ŽDenver, 1997.. Several days of melatonin injections caused major shifts in the T4 release pattern of subsequently cultured thyroid glands ŽWright et al., 1996.. If administration of melatonin had decreased plasma T4 , it would have indicated that a climactic decline in melatonin might permit the rise in

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T4 that occurs at this time. However, melatonin had no significant effect on the T4 level in the plasma. Injection of melatonin also failed to significantly raise plasma melatonin, except for one interval immediately after the injection. Subsequently, although the plasma melatonin rhythm was phase shifted, the level of this hormone was the same as the control and there was no effect of melatonin on metamorphic rate. On the other hand, constant immersion in a high level of exogenous melatonin lowered plasma melatonin sig-

Fig. 6. Graph showing the percent difference in stage change on the last day plotted against the percent difference in the 24-h mean of plasma melatonin ŽML; a. or T4 Žb. in experimental groups relative to controls Ždata from Table 1 and Figs. 2 and 5.. Points from left to right on the x-axis of each graph represent experiment 1, control vs. melatonin; experiment 3, control vs. melatonin; experiment 2, control vs. T4 ; experiment 3, control vs. T4 .

Fig. 5. Mean of the samples in all intervals compared in control and experimental groups of experiment 3 Ždata in Fig. 4. showing the effect of immersion in melatonin ŽML. or T4 on the overall level of plasma T4 Ža., plasma melatonin Žb., and ocular melatonin Žc.. Bars with different letters are significantly different.

nificantly, although not so much as constant immersion in T4 . Although the T4 level did not change, metamorphosis was slightly accelerated. The results of the two melatonin treatments suggest that there is an effective regulation of plasma melatonin in the tadpole which rapidly removes exogenous melatonin from the circulation. Such a regulation might explain the discrepancy between inhibition of T4 secretion by melatonin in vitro at all stages in the life cycle ŽWright et al., 1997a, 2000. and its lack of effect on plasma T4 in vivo in the present work. The antagonistic effect on T4 secretion might be undermined by the rapid clearance of melatonin from the plasma in vivo. There is evidence for rapid clearance of melatonin from the circulation. The half-life of radioactive melatonin has been reported to be between 2 and 4 min after injection into adult mammals, with 35᎐40 min necessary for the next reduction of 50% ŽFiladelfi and Castrucci, 1996.. A 0.5-mg dose of melatonin in the human pro-

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Table 2 Significant rhythms determined by cosinor analysis P Žs.

Acrophasea

8

0.002

Plasma melatonin

9

0.032

Melatonin

Plasma melatonin

11.9

0.025

Control

Plasma T4

8.9

0.002

T4

Plasma T4

24

0.005

Control

Plasma melatonin

12.3

0.033

Ocular melatonin

24

0.0002

Plasma T4

24

0.022

Ocular melatonin

24

0.02

06.20 Ž06.01᎐06.39. 06.16 Ž05.31᎐07.00. 05.32 Ž04.48᎐06.16. 05.04 Ž04.42᎐05.25. 22.09 Ž20.20᎐23.57. 04.31 Ž03.15᎐05.46. 06.52 Ž05.21᎐08.23. 09.11 Ž06.47᎐11.35. 10.03 Ž08.36᎐11.30.

Exp.

Treatment

Hormone

1

Control

Plasma T4

2

3b

T4

a b

Period of rhythm Žh.

Hours after midnight of the first day. The 95% confidence limits are given in parentheses. The melatonin group had no significant rhythms.

duced supraphysiological melatonin levels in the plasma for only 3 h after administration ŽMatsumoto et al., 1997.. Melatonin is enzymatically degraded in the liver, or deacetylated and deaminated in some tissues ŽFiladelfi and Castrucci, 1996.. Studies are underway to determine the fate of injected melatonin. Preliminary findings indicated that considerable melatonin appeared in the 10% Holtfreter’s solution in which the tadpoles were kept when supraphysiological concentrations were injected. Moreover, 30 min after a single injection of 30 ␮g of melatonin into bullfrog tadpoles, the concentration of melatonin increased in peripheral tissues. Although very high doses of melatonin have usually been used in both injection and immersion experiments in the literature Žcited above., plasma melatonin levels in bullfrog tadpoles are less than 400 pgrml at their peak on an 18L:6D cycle ŽWright and Racine, 1997 and this paper.. The present findings also suggest that more physiological doses of melatonin should be tried in studies of the effect of melatonin on tadpole development. Lower doses might not precipitate a plasma melatonin decrease and thus allow a thyroidal effect. Experiments along these lines are presently in progress in our laboratory.

The findings showed that plasma melatonin does not always increase after exogenous melatonin treatment, and highlight the importance of the mode of administration of this hormone. Any set of conditions that results in lowered plasma melatonin might drive the system toward climax and thus accelerate metamorphosis, while a treatment such as injection, which raises the melatonin level slightly, might have no effect, or retard metamorphosis, depending on the dose. This interpretation and the present results might explain some of the discrepancies in the literature on the effect of melatonin on tadpole metamorphic progress, although dosage, time of administration, and LD cycle also have to be taken into account. Injection of melatonin into tadpoles often retarded growth or metamorphosis, or had no effect, Že.g. Delgado et al., 1984; Gutierrez et al., ´ 1984; Wright et al., 1991. as might be expected if it produced a transient plasma melatonin increase. On the other hand, immersion in melatonin, which decreases plasma levels of the hormone, accelerated development ŽEdwards and Pivorun, 1991; Rose and Rose, 1998.. The differences in the rate of development on various LD cycles ŽDelgado et al., 1984, 1987; Gutierrez et al., ´ 1984; Wright et al., 1990b, 1991. could also depend on whether the light regimen fostered an

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increase or decrease in plasma melatonin in a particular species. Melatonin inhibited the thyroid of premetamorphic Rana pipiens tadpoles ŽWright et al., 1996.. As the TH level rises toward metamorphic climax, a decrease in plasma melatonin is induced by T4 , either through uptake into tissues, reduced secretion of melatonin by the pineal or other sites of melatonin production, or some other mechanism. Increased melatonin in the tissues might modulate the uptake or action of T4 , or its conversion to T3 . The T4 content of young Žlate premetamorphic. tadpole intestine in vitro was not affected by simultaneous injection of T4 and melatonin, but melatonin reduced the T4 content of older Žlate prometamorphic. tadpole intestine ŽWright et al., 1997b.. Additional studies on peripheral tissues and the effect of T4 on sites of melatonin production might clarify further the interaction of melatonin and TH in anuran tadpole metamorphosis.

Acknowledgements The authors are grateful to Carol Racine, Karen Cuthbert, Catharine Guertin, Julie Duffy, Agata Pikula, and Robert Weir for technical assistance in some experiments. This research was supported by NSF grants IBN-9222504 and IBN9723858. References Alonso-Bedate, M., Delgado, M.J., Nava, P., Gutierrez, ´ P., 1985. Further studies on morphogenesis, growth and regeneration in Discoglossus pictus ŽOtth. tadpoles immersed in a melatonin solution. Acta Embryol. Morphol. Exp. n.s. 6, 167᎐175. Delgado, M.J., Vivien-Roels, B., 1989. Effect of environmental temperature and photoperiod on the melatonin levels in the pineal, lateral eye, and plasma of the frog, Rana perizi: importance of ocular melatonin. Gen. Comp. Endocrinol. 75, 46᎐53. Delgado, M.J., Gutierrez, P., Alonso-Bedate, M., 1984. ´ Growth response of premetamorphic Rana ridibunda and Discoglossus pictus tadpoles to melatonin injections and photoperiod. Acta Embryol. Morphol. Exp. 5, 23᎐39. Delgado, M.J., Gutierrez, P., Alonso-Bedate, M., 1987. ´ Melatonin and photoperiod alter growth and larval development in Xenopus lae¨ is tadpoles. Comp. Biochem. Physiol. 86A, 417᎐421.

Denver, R.J., 1997. Environmental stress as a developmental cue: corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis. Horm. Behav. 31, 169᎐179. Edwards, M.L.O., Pivorun, E.B., 1991. The effects of photoperiod and different dosages of melatonin on metamorphic rate and weight gain in Xenopus lae¨ is tadpoles. Gen. Comp. Endocrinol. 81, 28᎐38. Filadelfi, A.M.C., Castrucci, A.M.dL., 1996. Comparative aspects of the pinealrmelatonin system of poikilothermic vertebrates. J. Pineal Res. 20, 175᎐186. Galton, V.A., 1988. The role of thyroid hormone in amphibian development. Am. Zool. 28, 309᎐318. Gutierrez, P., Delgado, M.J., Alonso-Bedate, M., 1984. ´ Influence of photoperiod and melatonin administration on growth and metamorphosis in Discoglossus pictus larvae. Comp. Biochem. Physiol. 79A, 255᎐260. Halberg, F., Johnson, E.A., Nelson, W., Runge, W., Sothern, R., 1972. Autorhythmometry-procedures for physiologic self-measurements and their analysis. Physiol. Teach. 1, 1᎐11. Kaltenbach, J.C., 1996. Endocrinology of amphibian metamorphosis. In: Gilbert, L.I., Tata, J.R., Atkinson, B.G. ŽEds.., Metamorphosis. Academic Press, Inc, New York, pp. 403᎐431. Kikuyama, S., Kawamura, K., Tanaka, S., Yamamoto, K., 1993. Aspects of amphibian metamorphosis: hormonal control. Int. Rev. Cytol. 145, 105᎐148. Matsumoto, M., Sack, R.L., Blood, M.L., Lewy, A.J., 1997. The amplitude of endogenous melatonin production is not affected by melatonin treatment in humans. J. Pineal Res. 22, 42᎐44. Mondou, P.M., Kaltenbach, J.C., 1979. Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen. Comp. Endocrinol. 39, 343᎐349. Rose, M.F., Rose, S.R., 1998. Melatonin accelerates metamorphosis in Xenopus lae¨ is. J. Pineal Res. 24, 90᎐95. Taylor, A.C., Kollros, J.J., 1946. Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94, 7᎐23. Wright, M., Racine, C., 1997. Inverse relationship between plasma T4 and plasma and ocular melatonin in prometamorphic and climax bullfrog Ž Rana catesbeiana. tadpoles. In: Kawashima, S., Kikuyama, S. ŽEds.., Proceedings of the XIIIth International Congress of Comparative Endocrinology. Monduzzi Editore, Bologna, Italy, pp. 403᎐407. Wright, M.L., Pathammavong, N., Basso, C.A., 1990a. DNA synthesis is unaffected but subsequent cell division is delayed in tadpole hindlimb epidermis when thyroxine is given in the dark. Gen. Comp. Endocrinol. 79, 89᎐94.

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Wright, M.L., Blanchard, L.S., Jorey, S.T., Basso, C.A., Myers, Y.M., Paquette, C.M., 1990b. Metamorphic rate as a function of the lightrdark cycle in Rana pipiens larvae. Comp. Biochem. Physiol. 96A, 215᎐220. Wright, M.L., Cykowski, L.J., Mayrand, S.M. et al., 1991. Influence of melatonin on the rate of Rana pipiens tadpole metamorphosis in vivo and regression of thyroxine-treated tail tips in vitro. Dev. Growth Differ. 33, 243᎐249. Wright, M.L., Blanchard, L.S., Pikula, A., Labieniec, K.E., 1995. Circadian rhythms of thyroid secretion, morphometry, and cell division in prometamorphic and climax Rana tadpoles. Gen. Comp. Endocrinol. 99, 75᎐84. Wright, M.L., Pikula, A., Cykowski, L.J., Kuliga, K., 1996. Effect of melatonin on the anuran thyroid gland: follicle cell proliferation, morphometry, and subsequent thyroid hormone secretion in vitro after melatonin treatment in vivo. Gen. Comp. Endocrinol. 103, 182᎐191.

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