Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles

Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles

Comparative Biochemistry and Physiology Part C 124 (1999) 109 – 116 www.elsevier.com/locate/cbpc Hormonal profiles correlated with season, cold, and ...

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Comparative Biochemistry and Physiology Part C 124 (1999) 109 – 116 www.elsevier.com/locate/cbpc

Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles Mary L. Wright *,1, Krista L. Proctor, Christina D. Alves Biology Department, College of Our Lady of the Elms, Chicopee, MA 01013, USA Received 25 March 1999; received in revised form 5 July 1999; accepted 6 July 1999

Abstract Bullfrog (Rana catesbeiana) tadpoles are of value to amphibian researchers because of their large size, and year-round availability due to overwintering in many latitudes. Concern over a possible difference in hormonal parameters in tadpoles obtained at different times of the year prompted us to investigate thyroid gland secretion in vitro, plasma and ocular melatonin, and plasma corticosteroids in late pre- to early prometamorphic larvae on 12L:12D. Winter tadpoles exposed to 22°C for 3 weeks of acclimation (winter group) were compared to summer tadpoles kept at 22°C (summer group), as well as to summer tadpoles exposed to cold (12°C) for the 3 weeks (cold group), or kept at 22°C and starved for the last week of acclimation (starved group). Thyroids from the summer group had a significantly higher response to 0.2 mg/ml ovine thyrotropin (TSH) than the other groups, indicating that cold and starvation inhibited subsequent in vitro thyroid sensitivity to TSH. The thyroids of the starved tadpoles had significantly higher baseline (unstimulated) thyroxine (T4) secretion into the culture media, a finding that might be related to starvation-induced acceleration of metamorphosis. Plasma melatonin was lower, and ocular melatonin significantly higher in both summer and starved groups, while the reverse occurred in the winter and cold groups. Thus, seasonal or induced cold brought on a shift in the relationship of plasma to ocular melatonin but starvation had no effect. There were no significant differences among the treatment groups in plasma hydrocortisone (HC) and aldosterone (ALDO) levels, except that HC was lower than ALDO only in the plasma of winter tadpoles. We conclude that seasonal variation needs to be taken into account in endocrine experiments utilizing tadpoles obtained at different times of the year. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Thyroid gland; Melatonin; Seasonal changes; Starvation; Thyroid sensitivity to TSH; Cortisol; Aldosterone; Thyroxine

1. Introduction Rana catesbeiana (bullfrog) tadpoles have an extended larval life that sometimes includes overwintering before transformation to the adult. Overwintering may occur for 1 or more years depending on latitude [5]. During this time, the larvae grow to large sizes mainly during the warmer seasons, with growth interrupted during the winter [16] since larval development is reported to cease at air temperatures of 12.8°C or lower [20]. Although attributed mainly to temperature and the length and severity of winters [5], variation in length of the larval period in R. catesbeiana may also reflect * Corresponding author. Tel.: +1-413-594-2761 Ext. 298; fax: +1-413-592-4871. E-mail address: [email protected] (M.L. Wright) 1 To whom reprint requests should be addressed.

food availability [11]. Up to a critical period in early metamorphosis, associated with early limb bud stages, starvation retards development in R. syl6atica and R. pipiens, whereas after that critical stage, lack of food accelerates transformation to the adult [2]. Other factors, such as crowding and habitat dessication, may also contribute to plasticity in the length of larval life [6]. The thyroid hormones (TH) are the primary inducers of metamorphosis, although other hormones, such as prolactin, adrenal corticoids, and melatonin may synergize with them or antagonize their secretion or action. Early work utilizing amphibian larvae indicated that low temperatures inhibited thyroid activity [1] and tissue responses to TH [12]. However, thyroid secretion was not actually measured, nor were plasma levels of other hormones that might have been involved in growth, development, and metamorphosis. Although

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seasonal differences in hormone levels have been studied in adult amphibians [9,13 – 15,17], there is a dearth of information on the larval stages. In order to more precisely define the effect of season, temperature, and starvation, we studied several hormonal correlates associated with winter and summer tadpoles, summer tadpoles supposedly brought to a ‘winter’ condition by exposure to cold for 3 weeks, and summer tadpoles starved for 1 week. Since thyroxine (T4) is at too low a level to detect by radioimmunoassay (RIA) in the plasma of young tadpoles at the stages used here, and because some of our current work utilizes thyroids in vitro, we cultured the thyroids in order to collect 2 days worth of hormone secretion. Both the baseline secretion of T4 in vitro, and the ability of the thyroids to respond to exogenous thyrotropin (TSH), were studied. The plasma levels of aldosterone (ALDO) and hydrocortisone (HC; cortisol), two corticosteroids produced by the interrenal (adrenal) glands, were also monitored. If cold or starvation stressed the animals, the levels of these hormones might rise. Finally, plasma and ocular melatonin levels were determined in the four treatment groups. Melatonin is a hormone whose secretion times and levels depend on the light/dark (LD) cycle and temperature, two environmental factors which differ in summer and winter. Moreover, administration of melatonin has an inhibitory effect on the amphibian thyroid gland [26] and reduces its ability to respond to exogenous TSH [25]. However, as levels of T4 rise at climax of spontaneous metamorphosis, plasma melatonin decreases [22]. Thus, melatonin levels might vary in the different thyroid states of the treatment groups.

2. Materials and methods

2.1. Animals and experimental conditions R. catesbeiana tadpoles were obtained from Charles D. Sullivan Co. (Nashville, TN) and maintained in plastic dishpans on a 12L:12D (L onset 0800 h) cycle at 22°C in 10% Holtfreter’s solution, which was changed three times a week. Tadpoles were fed washed, canned spinach daily in the early photophase. Tadpoles were used at the end of the premetamorphic or early prometamorphic periods, more specifically at stages X – XIV according to Taylor and Kollros [18]. However, in any given experiment, tadpoles within a threestage range were used. Summer tadpoles were obtained in June and used in July. Three treatment groups were formed from summer tadpoles. One group (designated summer) was fed, and acclimated at 22°C for 3 weeks prior to the experiments. Another group of summer tadpoles were fed, but kept at 12°C for the entire acclimation period (cold group). The final group was acclimated at 22°C for 3

weeks but was not fed during the last week (starved group). The tadpoles used were past the critical stage (hind limb bud) noted by D’Angelo et al. [2], so that starvation would be expected to accelerate metamorphosis. Thyroids from tadpoles collected in the summer (the summer, cold, and starved groups) were cultured in a 3-day period in July of 1997, one experiment each day, starting between 0900 and 1030. Blood and eyes were collected from other tadpoles (also summer, cold, and starved groups) in three separate experiments done within a 7-day period in July of 1998, each experiment starting at 1300. Winter tadpoles (winter group) were obtained in January and used in February, 1998. These tadpoles were fed and kept on 12L:12D at 22°C for the 3-week acclimation period prior to the experiments. The thyroid culture, and blood and eye collection, experiments on the winter tadpoles were done 10 days apart from each other. Table 1 summarizes collection season, and the conditions under which the four treatment groups (cold, starved, summer, winter) were kept during acclimation.

2.2. Thyroid gland culture experiments The purpose of these experiments was to determine if there were any differences in baseline T4 secretion or response to TSH of thyroids taken from tadpoles obtained in the winter versus the summer, and from summer tadpoles starved for 1 week or kept in the cold for 3 weeks. After a rinse in a suspension of 0.85% sulfadiazine (Sigma) in 2000 ml 10% Holtfreter’s solution to sterilize the skin, tadpoles were killed by pithing. Paired thyroids attached to the underlying cartilage were dissected out and cleaned of all overlying tissue. All procedures were done under sterile conditions in a tissue culture hood previously sterilized by ultraviolet light. The thyroids were twice rinsed briefly in cold, sterile, 10% Holtfreter’s solution with 300 units ml − 1 penicillin and 300 mg ml − 1 streptomycin, followed by two 3-h rinses in the incubation media, which was

Table 1 Summary of the collection season and conditions under which the four treatment groups were kept during the 3-week acclimation period Designation

Collection season

Temperature (°C)

Nutrition

Cold Starved

Summer Summer

12 22

Summer Winter

Summer Winter

22 22

Fed Not fed last week Fed Fed

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two-thirds strength L-15 medium with 0.1% bovine serum albumin, 50 mg ml − 1 antibiotic/antimycotic solution, and 150 mg ml − 1 gentamycin. Following these rinses, one-half of the thyroids in each group were incubated in 250 ml of L-15, while the other half were cultured in the same volume of L-15 media supplemented by 0.2 mg ml − 1 TSH (NIADDK; o-TSH-12). All culture reagents except TSH were obtained from GIBCO (Grand Island, NY). Each pair of thyroids was cultured in a separate well of a 24-well plate (n=6 thyroid pairs/group) in the dark at 24°C for 2 days, with media collected and replaced after 24 h, and final media collection at 48 h. Collected media were stored frozen at −20°C until RIA for secreted T4 was carried out.

2.3. Blood and eye collection experiments These experiments on cold, starved, summer, and winter tadpoles were designed to complement the thyroid culture experiments by providing plasma and eyes for RIA of corticosteroids, plasma melatonin, and ocular melatonin. In each experiment, 15 tadpoles were selected and placed in five subgroups made equivalent by stage. Each subgroup of three tadpoles represented one sample (n=5) for hormone RIA since the blood of the three animals was pooled, as were the eyes from the same tadpoles. Blood and eyes were taken at 1300. It was not thought necessary to sample plasma and ocular melatonin at night, when melatonin might be expected to peak, because studies in progress in which sampling was done throughout the 24-h day indicate that there are no circadian rhythms or significant differences in plasma or ocular melatonin in tadpoles at these young stages. Significant differences among time intervals develop later in metamorphosis (unpublished data). After pithing each tadpole, blood was pipetted from the heart into a separate, siliconized, microcentrifuge tube, and centrifuged at 3300 rpm for 15 min. The supernatants from three tadpoles were then pooled and collected in a fresh tube that was stored at − 20°C until RIA for plasma ALDO, HC, and melatonin. Both eyes were also taken from each tadpole, and the six eyes from the same three tadpoles that had provided the pooled plasma were frozen in a microcentrifuge tube at − 20°C. Prior to RIA for ocular melatonin, the eyes were partially defrosted and cut in half, and 2100 ml of 0.7% saline was added to each tube. The eyes were ground with a glass rod to extract retinal melatonin. Following homogenization, the mixture was centrifuged at 3000 rpm for 20 min to remove all solids, and the supernatant pipetted out. If the extract was not to be assayed that day, it was again frozen at − 20°C until RIA was performed.

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2.4. Radioimmunoassay T4 in the culture media was assayed using a canine total T4 RIA kit (Diagnostic Products Corporation, Los Angeles, CA) employing antibody-coated tubes as previously described [24]. Samples were assayed in duplicate, and counted in a Packard Riastar gamma counter with a counting efficiency greater than 80%. The intra- and inter-assay coefficients of variation (CV) were 4.1 and 4.6%, respectively. Human HC and ALDO coated tube assay kits (Diagnostic Products Corporation) were used to monitor the level of plasma HC and ALDO. the intra- and inter-assay CV’s were 4.1 and 10.1% for HC and 5.8 and 7.1% for ALDO. Crossreactivity of HC for ALDO was 0.03% and for corticosterone (CORTI) 0.94%. ALDO exhibited no crossreactivity for HC and 0.002% for CORTI. Melatonin in the plasma was assayed directly, without extraction, using a human melatonin kit from DiagnosTech, Inc. (Osceola, 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 using a version of the kit designed for salivary melatonin. The melatonin RIA was validated for measurement of plasma and ocular melatonin in amphibian samples by parallelism and dilution studies. Serial dilutions of melatonin-containing samples produced a series that paralleled the standard curve made by the calibrators supplied with the kit. Values for samples obtained after dilution in assay buffer were proportional to the extent of the dilution. The intra- and inter-assay CV’s were 11.7 and 12.2%.

2.5. Statistical analysis The data were evaluated using Student’s t-test, or ANOVA, as appropriate, with the ANOVA followed by Duncan’s multiple range test to isolate differences among the means of the groups. Differences were considered to be significant if PB 0.05.

3. Results TSH treatment in vitro significantly increased the capacity of the thyroids to secrete T4. Each TSH group on each of the 2 days of culture (Fig. 1(B)) had significantly higher T4 in the media than the corresponding group and day in the cultures without TSH (Fig. 1(A)). Baseline, unstimulated, T4 secretion (Fig. 1(A)) was significantly higher in the starved tadpoles than in the summer, cold, or winter groups on day 1, but there were no significant differences among the groups on day 2. The response of the thyroids to TSH in the media (Fig. 1(B)) was greatest in the summer

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Fig. 1. T4 secreted into media on days 1 and 2 of culture by the four treatment groups (see Table 1 for characteristics of each group). Bars represent the mean 9S.E. (n=6). Baseline, unstimulated T4 secretion is shown in A and the response of thyroids in the various groups to exogenous TSH separately in B, since the scales are so different. Both day 1 and day 2 values from the cold, starved, and summer groups were significantly higher in TSH-treated than in unstimulated thyroids (PB 0.01), as were the values from winter tadpoles on day 1 (PB 0.05) and day 2 (PB 0.02). The day 1 data for baseline T4 secretion was significantly different among the treatment groups (ANOVA P B0.005), since the thyroids from starved tadpoles produced significantly (P B0.01) more T4 than those of the summer, winter, or cold groups. The day 2 data for unstimulated thyroids was not significantly different among the four groups. In the TSH-treated, ANOVA was significant on day 1 (PB 0.005) and day 2 (PB0.01). On both days, the summer tadpole thyroids had a significantly greater response to TSH than the thyroids from winter or cold groups (P B0.01) or starved tadpoles (PB 0.05 on Day 1; PB 0.01 on day 2). Day 2 T4 secretion was significantly higher than day 1 in thyroids from summer tadpoles (indicated by * on the graphs) in the unstimulated (P B 0.002) and the TSH-treated (PB0.05) whereas there was no significant difference between day 1 and day 2 data in the other groups.

tadpoles, on both day 1 and day 2. T4 was significantly higher on the second day of culture compared to the first day in both the unstimulated (Fig. 1(A)) and

TSH-treated (Fig. 1(B)) thyroid cultures in summer tadpoles, but not in the other groups. When day 1 and day 2 T4 content in the media was totaled (Fig. 2) for better comparison with melatonin, the baseline level of T4 secreted into the media was greater in starved tadpoles, compared to cold, summer, or winter ones (Fig. 2(A)), and the response to TSH was greatest in summer tadpoles (Fig. 2(B)). Although the large variation in plasma melatonin among individual samples did not allow for any significant differences among the groups (Fig. 2(C)), starved and summer tadpoles tended to have the lowest plasma melatonin, and in these two groups, plasma melatonin was significantly lower than ocular melatonin (Fig. 2(D)), whereas plasma and ocular melatonin were not significantly different in the cold or winter tadpoles. Ocular melatonin (Fig. 2(D)), showed an opposite pattern to plasma melatonin and was significantly higher in starved and summer tadpoles than in cold or winter tadpoles. The winter tadpoles also had significantly lower ocular melatonin than those kept in the cold, indicating that 3 weeks of cold exposure of summer tadpoles did not completely mimic the winter situation. There appeared to be an inverse relationship between in vitro T4 secretion and plasma melatonin on the one hand, and ocular and plasma melatonin on the other. As a result, T4 and ocular melatonin appeared to correspond in that ocular melatonin was highest in the starved and summer tadpoles, which also had the capacity for greatest response to TSH (Fig. 2). There were no significant differences among the groups in the level of plasma ALDO or HC except that HC was significantly lower than ALDO only in the winter tadpoles (Fig. 3).

4. Discussion In the culture experiments, thyroids taken from cold, starved, summer or winter tadpoles were maintained in vitro for 2 days and the media collected at the end of each day to assay for baseline or TSH-stimulated T4 secretion. As the results indicate, the thyroids from starved and summer tadpoles had significantly different T4 secretion levels than the other groups. Previous work indicated that thyroids cultured in vitro retained the circadian rhythmicity of T4 secretion pertinent to the

Fig. 2. Total day 1 and day 2 (individual day data in Fig. 1) thyroid baseline T4 secretion in vitro (A), and total TSH-stimulated secretion (B) compared to levels of plasma (C) and ocular (D) melatonin in the four treatment groups (see Table 1 for characteristics of each group). Values of bars with different letters are significantly different. Bars represent mean 9S.E. (n =6 in A and B and 5 in C and D). Thyroid baseline T4 secretion in vitro was significantly higher (PB 0.01) in the starved group than in the other three groups (ANOVA P B0.01). Response to TSH was significantly higher (PB 0.01) in the summer group (ANOVA P B0.005). Although plasma melatonin was not significantly different among the four groups, it was lower in the starved (PB 0.001) and summer (PB 0.01) groups than ocular melatonin in the same two groups. Ocular melatonin was significantly higher (ANOVA PB 0.005) in the summer than in the cold (P B 0.05) and winter (PB 0.01), and in the starved (P B 0.01) compared to cold and winter tadpoles. The latter also had significantly (P B0.01) lower ocular melatonin than the cold group.

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Fig. 2.

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Fig. 3. ALDO and HC levels in plasma of tadpoles in the four treatment groups (see Table 1 for characteristics of each group). Bars represent mean 9 S.E. (n=3–4 for ALDO and 4–5 for HC). There were no significant differences in plasma ALDO or HC in the four groups. However, as indicated by * on the graph, HC was significantly lower than ALDO in the winter group (PB 0.02).

LD cycle the tadpoles were on before thyroids were excised [24], and thyroids taken from tadpoles previously injected with melatonin secreted T4 and T3 in vitro at significantly different levels than thyroids from both injected and non-injected controls [26]. Consequently, we believe that in the present work the thyroids secreted T4, or responded to TSH, in a manner consonant with the season or treatment of the tadpoles from which they were taken. Thus, these data provide information about the functional state and responsiveness of the thyroid gland, although they may not reflect T4 levels in the plasma in vivo. However, Kuhn et al. [15] found good correlation between plasma T4 and in vitro thyroid responsiveness to bovine TSH in the adult frog R. ridibunda. There was no significant difference between summer and winter tadpoles in thyroid baseline T4 secretion in vitro, other than that summer thyroids produced significantly more T4 on the second day than on the first, whereas winter tadpoles did not. But the thyroids of summer tadpoles had a significantly higher response to TSH than those of winter tadpoles. This finding indicates that the ability of tadpole thyroids to respond to TSH fluctuates seasonally, and may be more of a factor in the lack of metamorphic progress during the winter than a reduction in the secretion of TSH by the pituitary. Dodd and Dodd [10] suggested that if tissues became unable to utilize THs at low temperatures, higher plasma T4 might result, which could exert negative feedback on TSH. The lowered TSH level would then produce a less active thyroid. The findings here, however, do not support this suggestion. The thyroids of winter tadpoles seemed were as active as the thyroids of summer ones in secreting baseline T4. However, they were unable to respond to the same extent to TSH.

Kuhn et al. [15] found parallel annual variations in plasma TH and in the sensitivity of adult R. ridibunda frog thyroids to exogenous TSH in vitro. Maximal release capacity of the thyroids was found in March, and again in July to August, while minimal sensitivity occurred in winter, and in April to June in a biphasic pattern correlated with reproductive activity as well as season. In adult R. perizi [13], plasma T4 peaked during late spring and summer and declined to a very low level during the winter months. These findings agree with our results and suggest that both adult frogs and larvae have similar summer–winter differences in thyroid activity. Three weeks of cold was enough to induce a ‘winter’ situation in summer tadpoles. The thyroids from tadpoles in the cold at 12°C for the 3 weeks showed a level of response to TSH similar to the thyroids of winter tadpoles and significantly lower than that of summer tadpole thyroids. The winter tadpoles in this study were kept at on 12L:12D at 22°C for 3 weeks during the acclimation period prior to the experiment, nevertheless, they did not take on the hormonal profile of summer tadpoles. Preliminary temperature studies in our laboratory showed that very little metamorphic progress was made by fall tadpoles even when kept for several weeks at 24°C. The temperature had to be raised to 28°C for developmental progress to resume, and for variations in metamorphic rate due to LD cycle differences to occur (unpublished data). Starved tadpole thyroids had the highest level of baseline, unstimulated, T4 secretion on the first day of culture, while on day 2, the baseline T4 was not significantly higher than the other groups. D’Angelo et al. [2] studied thyroid histology in tadpoles that had reached the critical stage to accelerate metamorphosis upon starvation. The thyroids gave evidence of a high level of activity (columnar epithelium, vacuolated colloid), but returned to an inactive state earlier that those of fed tadpoles in normal metamorphosis. They concluded that the accelerated metamorphosis of starved animals is linked more to a sudden burst of thyroid activity than to sustained oversecretion, and our findings support this conclusion. However, the response to TSH of starved tadpole thyroids in the present study was the same as the cold and winter groups and lower than the summer group. Since D’Angelo et al. [2] found that the pituitary glands of starved tadpoles in accelerated metamorphosis were well-differentiated, TSH production may not be decreased in starvation. However, the response to TSH of the starved tadpole thyroids might be hampered by lack of adequate nutrition. Although there was no significant difference when plasma melatonin was compared among the groups, starved and summer tadpoles had lower plasma melatonin compared with cold and winter tadpoles. A difference is suggested since plasma melatonin was

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significantly lower than ocular melatonin in the starved and summer groups, but not in the other groups. At the same time, starved and summer tadpole thyroids had the highest baseline T4 secretion or response to TSH in vitro. An antagonistic relationship between thyroid activity, or TH levels, on the one hand, and melatonin on the other, has been found frequently in studies of T4-melatonin interactions. Melatonin inhibited the response of the thyroid to TSH in vitro [25]. At metamorphic climax, when circulating T4 peaked, plasma melatonin decreased [22,23], and the melatonin decrease at climax could be induced prematurely by exogenous T4 [21]. This reciprocal relationship between T4 and melatonin is not necessarily related to the morphological changes of later stages of metamorphosis, as indicated by the present work utilizing young tadpoles, and shown in the finding that R. catesbeiana froglets after metamorphosis have low plasma T4 and high plasma melatonin [23]. Comparison of the plasma and ocular melatonin data also indicates that there is an inverse relationship, since cold and winter tadpoles have high plasma and low ocular melatonin and starved and summer tadpoles have the reverse. The meaning of this reciprocal relationship is not yet clear, but it might be due to retinal melatonin being released into the circulation, so that when melatonin is high in the plasma, it is comparably lower in the eye, and vice versa. Delgado and VivienRoels [3] suggested that ocular melatonin might contribute to circulating melatonin when they found higher levels of melatonin in the retina of R. perizi adult frogs than in the pineal or plasma. Ocular melatonin in winter tadpoles was significantly lower than in cold tadpoles, although both the cold and winter tadpoles had significantly less ocular melatonin than starved or summer tadpoles. Evidently the extreme lowering of ocular melatonin associated with winter takes more than 3 weeks of cold to accomplish. Unusually low ocular melatonin in winter tadpoles at older stages has been noted in other work in our laboratory. The finding that cold lowers ocular melatonin in anuran larvae is in accord with the observations of Delgado and Vivien-Roels [3] and Delgado et al. [4] on adult R. perizi frogs where nocturnal ocular melatonin was greatly depressed at low temperatures. However, Valenciano et al. [19] recently observed that melatonin production by cultured eyecups of R. perizi was higher at cold (15°C) than warm (25°C) temperatures. These findings are not really contradictory since melatonin production by eyecups in vitro could increase in the cold, whereas in vivo, more ocular melatonin could be released into the circulation at low temperatures, leaving less to be extracted from the eyes. Higher levels of corticoids might have been expected in the summer and starved tadpoles where baseline T4 secretion, or the response of the thyroids to TSH, was

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also highest, since activation of the thyroid axis is linked to an increase in corticosteroids. Corticotropin releasing hormone from the hypothalamus is believed to control both the thyroid and the adrenal axes through stimulating both pituitary TSH and adrenocorticotropin [8]. Although ALDO was more than twice as high in the plasma of starved than cold or winter groups, however, there were no significant differences among the treatment groups in plasma ALDO or HC. CORTI is also an important corticoid in amphibia, but not enough plasma was available to assay for it in this study. Plasma HC was significantly lower than ALDO in winter tadpoles, an effect not mimicked by 3 weeks of cold. Heat or starvation, rather than cold, might be stressful to the tadpoles. Habitat dessication accelerates metamorphosis [11] just as starvation does after a critical stage of development [2]. Experimental water volume reduction in the western spadefoot toad resulted in higher levels of whole body T4, triiodothyronine, and CORTI, manifestations of activation of both thyroid and adrenal axes [7]. In the present work, experimentally-induced starvation elevated thyroid baseline T4 secretion in vitro and reduced the response to TSH. It had no significant effect on melatonin or corticosteroid levels. Thus, starvation does not seem as drastic as habitat dessication, or else it is a stress that requires a longer response period. Only 1 week of starvation was used here because of observed instances of cannibalism in R. atesbeiana tadpoles. Perhaps it was not long enough to elicit a stress response. In summary, hormonal correlates of summer tadpoles included low plasma melatonin, high ocular melatonin, and a high sensitivity of the thyroid to TSH. Both winter tadpoles and cold-induced summer ones were characterized by high plasma melatonin, low to moderate ocular melatonin, and a lowered sensitivity of the thyroid to TSH. Winter, but not cold-induced, tadpoles were also marked by significantly lower HC than ALDO in the plasma. Starvation in summer tadpoles did not change the melatonin or corticosteroid profiles but increased baseline secretion of T4 and reduced the response of the thyroid to TSH in vitro. The findings indicate that seasonal differences in secretion patterns and responsiveness of endocrine glands need to be taken into account in hormonal studies utilizing bullfrog larvae.

Acknowledgements The authors thank Karen Cuthbert, Catharine Guertin, and Julie Duffy for technical assistance. TSH was a gift of the National Hormone and Pituitary Program of the NIH. This research was supported by NIH grant DK46535 and NSF grant IBN-9723858.

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