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Effects of temperature, photoperiod, and daily torpor on thyroid function in the white-footed mouse, Peromyscus leucopus
GENERAL
AND
COMPARATIVE
42, 134-140 (1980)
ENDOCRINOLOGY
Effects of Temperature, Photoperiod, and Daily Torpor on Thyroid Function in the W h ite-Footed Mouse, Peromyscus leocopus H. RHODES
DONALD Section of Ecology and Systematics,
Fernow Hall. Cornell
University,
Ithaca, New York 14853
Accepted April 8, 1980 The biological half-life of thyroidal radioiodide and proportions of thyroid iodoamino acids were determined in Peromyscus leucopus after 3 weeks’ treatment with one of several constant ambient temperatures ranging from 7 to 35” and prior exposure to long (16:8 LD) or short (8: 16 LD) photoperiod. Similarly, the biological half-life of radioiodide was determined in mice exposed to temperature cycles of 16 hr at 22”and 8 hr at 7” or 16 hr at 35”and 8 hr at 22” and prior treatment with long photoperiod. The mean radioiodide half-life of mice kept at 7 was 9.4 days which was significantly longer than the pooled radioiodide half-life of 5.6 days exhibited by mice in all other temperature-photoperiod regimes. It is suggested that this long radioiodide half-life was due to the occurrence of daily torpor in the experimental animals. Mice monitored for daily torpor also exhibited significant reductions in the radioiodide release rate. Thyroid activity was not depressed in these animals on days during which they remained euthermic. The daily radioiodide release rate was positively correlated with the minimum body temperature of torpor and negatively correlated with the duration of torpor. It is suggested that this decrease in thyroid activity was a result of a direct effect of body temperature on thyroid function. Percentages of thyroid hydrolysate components were similar throughout all treatments.
It is generally accepted that under laboratory conditions thyroid activity is depressed in m a m m a ls exposed to heat (Collins and W e iner, 1968) and augmented in m a m m a ls exposed to cold (Heroux and Brauer, 1965; Hudson and Wang, 1969). However, Heroux (1969) has questioned the significance of the thyroid during cold acclimation, since laboratory rats exposed to seasonally changing conditions exhibit a decrease or no change in thyroid activity during the winter relative to other times of the year (Heroux et al., 1959; Heroux and Brauer, 1965). One m a jor difference between the laboratory-maintained animals and those m a intained under natural conditions is in the lack of environmental cues available to those animals kept in the laboratory. M u ltiple cues (e.g., temperature, photoperiod) associated with a seasonally changing environment play an important role in the regulation of adaptive responses. For instance, Peromyscus leucopus exposed to a short photoperiod exhibit an in-
crease in hoarding behavior and build larger nests than m ice exposed to a long-day lighting regime, whereas cold exposure results in a substantial increase in nonshivering thermogenesis but has little effect on hoarding behavior or nest construction (Lynch, 1970; Barry, 1976). No study to present examines the influence of m u ltiple cues on thyroid function in a single mamm a lian species. Alterations in thyroid activity may also be important in affecting other adaptive responses in small m a m m a ls. Lynch et al. (1978), basing their conclusions on propylthiouracil-treated animals, report that thyroid block increases the incidence of daily torpor in P. leucopus. Numerous studies have established that thyroid activity decreases prior to or subsequent to the onset of hibernation (Knigge, 1957; Wenberg and Holland, 1973). However, Hudson and W a n g (1%9) and Hudson and Deavers (1976) have presented evidence for several species of ground squirrels which points out
134 00166480/80/090134-07$01.00/0 Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
THYROID
FUNCTION
that thyroid inactivity and hibernation are not directly related. Thus, lack of a consistent relationship between thyroid activity and hibernation makes it difficult to conclude that a decrease in thyroid function is required for hibernation (Hudson, 1973). This does not preclude the possibility that thyroid function is affected by the reduction in metabolism and body temperature associated with h&et-nation. This study is an initial investigation of (1) the influence of temperature and photoperiod on thyroid activity in the whitefooted mouse, Peromyscus leucopus, and (2) thyroid function in P. leucopus which are known to undergo daily torpor. METHODS
AND MATERIALS
I. Animals. Peromyscus leucopus were live-trapped in the Ithaca, New York, area during the summer (June-August) and the late fag (October-November). Only mice with adult or subadult pelage were used to minimize possible variance in thyroid function due to age differences between mice (Bauman ef al., 1965). The animals were housed individually with food and water provided ad libitum. Fall-caught mice were maintained for 2 months at 7” under a short photoperiod (8:16 LD; lights on 8 AM to 4 PM EST). Cotton was provided as nesting material. Animals caught during the summer were kept at 22” and were exposed to either a long photoperiod (16:8 LD; lights on 7 AM to 11 PM EST) or a short photoperiod (8:16 LD). Wood shavings were supplied in quantities sufficient to cover the cage floor but not to allow nest construction. II. Temperature treatment. Animals were acclimated to laboratory conditions (22”) for at least 3 weeks prior to temperature treatment. Short-photoperiod mice kept at 22” were not introduced into the study until the moult to “winter” pelage and gonadal regression were completed. Gonadal involution was assessed in males by the inability to palpate the testes or, in females, by closure of the vaginal orifice. Groups of 9 or 10 mice containing approximately equal numbers of males and females were exposed for 3 weeks to constant ambient temperatures of 7,10, 15, 22, 30, or 35” and long or short photoperiod. In addition, two groups of mice were transferred to a Lab Line chamber which simulated a day-night temperature-light cycle; temperature and lighting changes occurred simultaneously. One group of animals was exposed to 16 hr at 35” (light) and 8 hr at 22” (dark), while the other was exposed to a cycle of 16 hr at 22” (light) and 8 hr at 7” (dark). All mice employed in temperature acclimation studies were maintained on the same
IN P. leucopus
135
photoperiod to which they had been exposed during the laboratory acclimation period. Subsequent to the 3-week acclimation period, radioiodide half-life and thin-layer chromatography of the thyroid iodoamino acids were assessed for each experimental group. To assess the rate of radioiodide release from the thyroid gland, mice were injected intraperitoneaily with 5 FCi lx51(New England Nuclear Corp., 17 Ci/mg) and 24 hr was allowed for incorporation of the isotope into the thyroid. Neck radioactivity of the mice was determined by holding their ventral neck region against a Lucite platform over an opening 2.27 cm2 in a 3-in. ring of lead which led to a sodium iodide-thallium activated crystal connected to a Nuclear Chicago timer scaler. Three to five I-min counts of the neck area were made to determine the maximum thyroid radioactivity. Neck counts were performed at approximately the same time each day for 5 to 7 days. All values were corrected for isotope decay. Seven to nine days after the fust lz51 injection, the mice were reinjected for 2 consecutive days with 2.5 &i ‘Vday in preparation for thin-layer chromatography of the thyroid hormones. Forty-e&M hours after the second injection the animals were killed by cervical fracture under ether anesthesia. Thyroids were prepared for thin-layer chromatography after the aerobic method of Inoue and Taurog (1968) with one exception: because the thyroid glands were so small, both the thyroid gland and the adjoining trachea were removed. The structure was homogenized in 0.1 ml cold buffered saline (0.03 M Tris-0.11 M NaCI, pH 8.5) and then centrifuged for 15 ruin to remove particulate material. Buffered saline (0.1 ml) containing 0.11 M I-methyl-2-mercaptoimidazole and 0.05 ml of a 10% pancreatin solution (Protease, Sigma Chemicals Co., St. Louis, MO.) was added to the supernatant and digestion was performed for 24 hr at 37” in ParaIilm covered test tubes. At the end of the digestion period a ~-PI sample of the mixture was removed, after being shaken, and then placed on a 20 x 20cm silica gel Eastman Chromagram Sheet (Eastman Kodak, Rochester, N.Y.). Individual samples from each mouse in a group, along with a reference solution containing the four principle iodoamino acids, MIT. DIT, T3, and T., (Sigma Chemical Co.), were run on a single chromatogram sheet. Ascending chromatography was carried out in a solvent consisting of 3: 1 (v/v) ammonia:sec-butanol. Ninhydrin (Sigma Chemical Co.) was used to make the iodoamino acids visible. The labeled areas along with the origin of the chromatogram were then scraped into individual test tubes and suspended in several drops of NCS solution (AmershamSearle, Des Plaines, Ill.). Each tube was counted for 10 miu in a well counter and for each thyroid, the percentage of total counts contributed by the individual iodoamino acids was determined. III. Daily torpor. Fall-caught mice maintained at 7°C were anesthetized with ether and a body-tem-
136
DONALD
H. RHODES
lease indicates that mice maintained at 7” released radioiodide from the thyroid gland substantially slower (P < 0.01) than did mice in all other treatment groups (Table 1). However, not all mice maintained at 7” had lengthened radioiodide half-lives (longer than 10 days). Within the 7” treatment, 7 of 9 mice (78%) on a short photoperiod and 4 of 10 mice (40%) on a long photoperiod had lengthened radioiodide half-lives. Further, examination of radioiodide half-lives for individual animals throughout all temperature and photoperiod treatments indicates that an additional three mice, two at 10” and one at IS”, exhibited radioiodide half-lives longer than 10 days; these three animals were maintained on a short-day lighting regime. Comparison of means for percentage of radioiodide located in different components of thyroid hydrolysates indicates that the distribution of radioiodide is similar RESULTS throughout all temperature and photoperiod Groups of mice exposed for 3 weeks to treatments (P > 0.05; f(5,107) = 1.27). constant ambient temperatures of 10 to 35” Pooled means (? 1 SE) for Origin, MIT, and daily temperature cycles exhibited a DIT, T3, and T4 are 15.3 + 1.17%, 35.8 t pooled radioiodide half-life of 5.6 days. In 1.28%, 36.2 + 1.24%, 2.2 & 0.14%, and 10.4 contrast, mice maintained at 7” under a long + 0.16%, respectively. Mice which were known to undergo daily or short photoperiod exhibited half-lives of 8.0 and 10.9 days, respectively. Compari- torpor on the basis of body-temperature son of regression slopes for radioiodide re- records exhibited a pronounced decrease in perature transmitter (Minimitter, Model X, Minimitter Inc., Indianapolis, Ind.) was implanted in the abdominal cavity. The incision was closed with silk-thread sutures and the animals were allowed a 4- to 5-day recovery period at 22” on a short photoperiod. The mice were then returned to the 7” chamber housed in cages equipped with an external antenna. The animals were placed on a restricted diet of 2.5-3.0 g laboratory chow per day. Output from the Mimimitter was received on a standard AM receiver and recorded on a cassette recorder. Both the receiver and the recorder were automatically activated for 5 min each hour. Body temperatures were monitored from 3 AM to 1 PM, the period of time within which daily torpor has been observed to occur for this species (Hill, 1975). Any mice which exhibited torpor (Tb less than 30’) were injected intraperitoneally with 5 &i iz51 and release of radioiodide from the thyroid gland was monitored for 4 to 7 days. Neck counts were performed at approximately 1 PM each day; body-temperature records had shown that the animals were euthermic by this time so that measurements did not interrupt periods of torpor. IV. Dafa analysis. Data were subjected to regression analysis and analysis of covariance.
TABLE 1 EFFECTSOFCHRONICTEMPERATURETREATMENTANDPRIORPHOTOPER~OD EXPIWJ~EON RADIOIODIDE RELEASE FROM THE THYROID ~~Peromyscus leucopus
168 LD Temperature
n
7
10
10”
10
IS” 22” 30” 35” 22-7” 35-22
9 9 10
10 10 10
o Biological half-life of lo51 (days). FJP < O.Ol;fl13,50) = 4.49.
Slope
-0.00160” -0.00224 -0.00245 -0.00190
-0.00205 -UO208 -0.80245 -0.00243
8: 16 LD TV,”
n
8.0 5.5 5.2 6.6 5.9 5.9 4.8 5.7
9 9 9 10 10 10
Slope
-0.00117~ -0.00190
-0.00225 -0.00213 -0.00233 -0.00223
Tm
10.9 5.5 5.8 5.5 6.1 5.4
THYROID
FUNCTION
the daily release of radioiodide from the thyroid gland relative to 24-hr release when they remained euthermic (Table 2) (P < 0.001; r(30) = 331). Animals which became torpid exhibited a release of 3 to 7% of the radioiodide present in the gland on the previous day compared to a release rate of 10 to 25% when they did not become torpid. This latter result is similar to the
3.37
IN I’. /eucopu.s
daily release rate of 10 to 21% obtained from Animal A (Table 2) which never became torpid. The 24hr radioiodide release was positively correlated with the m inimum daily body temperature (r = 0.795; P < 0.01; 12= 32) and negatively correlated with the duration of daily torpor (r = -0.762; P < 0.01; n = 32). No correspondence was found between the rate of entry (r =
TABLE 2 ENTRY RATES, AROUSAL RATE, PERCENTAGE lzsI RELEASED FROM THE THYROID PER 24 HR, DURATION OF TORPOR, AND MINIMUM DAILY BODY TEMPERATURE IN SIX INDIVIDUAL P. leucopus MONITORED FOR DAILY TORWR WITH BODY-TEMPERATURE TRANSMITTERS IMPLANTED IN THE ABDOMINAL CAVITY
Minimum T,, (“)
Day
Duration of torpor (hr)
Percentage Pz5 released from gland/24 hr
0.0 0.0 0.0 0.0 0.0 0.0 0.0
21.0 16.0 16.0 18.0 12.0
1.5 4.5 3.5
5.0 5.0
0.0
0.0 6.5 5.0 4.5
0.0
0.0
12.0
0.5 3.5
Animal A 34.0 34.0 35.0 32.0 31.0 33.0 32.0
Entry rate (Vmin)
Arousal rate (“imin)
-
-
-
10.0 11.0
Animal B 29.0 20.0 24.5 32.5
Animal C 18.5 18.0 18.5 37.0
0.05 0.08 0.08
0.12 0.16
18.0
-
6.0 3.0
0.13 0.15 0.15
0.18 0.19 0.15
0.11
-
-
14.0
0.05
3.0 13.0 12.0
0.10
0.12 0.14
0.07
0.13
4.5 3.5
6.0 25.0 24.0 25.0 4.0 7.0
0.09 0.08
0.14 0.17
0.0
25.0
5.0
5.0
0.0
25.0
-
0.5 6.5 5.0 3.5
10.0
0.12 0.14 0.12 0.08
Animal D 29.0 25.0 30.5 31.0
0.0 0.0
Animal E 26.0 33.0 35.0 33.5 21.5 25.0
4.5
0.0 0.0 0.0
Animal F 35.0 21.0 35.0 29.5 19.0 19.5 22.0
6.0 6.0 4.0
-
-
-
-
0.17
0.19
0. IO 0.18 0.18 0.14
138
DONALD
-0.301; P > 0.05; n = 16) or arousal (r = -0.172; P > 0.05; n = 16) from torpor and the 24-hr radioiodide release rate.
H.
RHODES
daily torpor in P. leucopus relative to animals exposed to a long photoperiod. (2) Not all cold-exposed mice exhibited altered thyroid function, which concurs with the DISCUSSION finding that daily torpor appears to be charExposure of many small mammals to acteristic of only certain individual P. ambient temperatures outside the limits of leucopus (Hill, 1975; Lynch et al., 1978). (3) their thermal neutral zone induces an in- Mice which were known to undergo bouts crease in radioiodide release from the of daily torpor on the basis of bodythyroid gland during cold exposure and a temperature records exhibited pronounced decrease during heat exposure (Collins and decreases in the release of radioiodide from Weiner, 1%8; Chaffee and Roberts, 1971). the thyroid gland. Thus, although body However, in this study chronic temperature temperatures of mice chronically exposed and photoperiod treatment did not influ- to low ambient temperature were not meaence the rate of radioiodide release from the sured, it is suggested that the observed reductions in thyroid activity were correlated thyroid gland in the majority of Peromyscus leucopus examined. Together with the con- with the occurrence of daily torpor in the stant proportions of thyroid iodoamino experimental animals. acids observed throughout all temperaThe finding that thyroid activity is subture - photoperiod treatments, these lines of stantially depressed in mice known to have evidence suggest that thyroid function does undergone daily torpor suggests two posnot play a critical role in the thermal adap- sibilities with respect to the regulation of tation of this species to long-term temper- thyroid activity: (1) thyroid hormone reature exposure. lease is being “turned off’ prior to and in However, a significant reduction in the preparation for entrance into daily torpor, secretion of radioiodide from the thyroid or (2) thyroid hormone release is decreased gland was observed in 12% of the P. due to the metabolic effect of a reduction in leucopus included in this study. This de- body temperature during the several hours crease in thyroid activity was observed of daily torpor. Since thyroidal radioiodide only in mice exposed to ambient tempera- release was depressed only on days when tures of 15” or less which contrasts with the animals became torpid but did not differ established reports of elevated thyroid from values on days during which the mice function in cold-exposed mammals. remained euthermic, a reduction in body Further, since only a few mice exhibited temperature is the most likely cause of the reduced thyroid activity when exposed to altered thyroid activity. This conclusion is cold, this would not seem to be a consistent supported by the findings that thyroidal adaptive response to low ambient temper- radioiodide release is significantly correature. lated with the minimum body temperature One possible explanation for the ob- reached during bouts of torpor, and negaserved decline in thyroid function is that tively correlated with the amount of time some P. leucopus maintained at low am- spent in torpor (Table 2). Since thyroid hormones are known to bient temperature were undergoing daily torpor. Three observations indicate that stimulate the metabolic rate, it might be this may have been the case. (1) The major- expected that hormone release would deity of mice exhibiting reduced thyroid se- crease preceding entry into torpor or, altercretion were maintained on a short photo- natively, increase during arousal from period. Lynch et al. (1978) have recently torpor. No significant correlation exists demonstrated that a short-day lighting re- between either entry or arousal rate of gime significantly increases the incidence of body-temperature change and the radio-
THYROID
FUNCTION
iodide release, further evidence that changes in thyroid activity associated with the occurrence of daily torpor are the result of a reduction in body temperature and not an active inhibition of thyroid activity. It seems unlikely, however, that the observed depression in radioiodide release from the thyroid gland in conjunction with torpor was due to the effect of a low body temperature on thyroid metabolism alone. In comparison with radioiodide release rates of euthermic mice, radioiodide release rates were reduced by 50% or more on days during which animals underwent bouts of daily torpor. Since torpor seldom exceeded 6 hr in duration, body temperatures remained high for at least 75% of the day. If we assume that hormone release fi-om the thyroid gland may be reduced to a minimum level, or even zero, at the low body temperatures associated with torpor, then radioiodide release should approximate 75% of that observed on days during which the mice did not undergo torpor. Since release rates of radioiodide were always less than the predicted 75%, thyroid function must have been suppressed for longer periods of time than the 6 hr of daily torpor. This suggests that low body temperature may affect thyroid function so as to depress activity subsequent to a bout of torpor, or alter TSH secretion during daily torpor. Tashima (1965) concluded that the observed decline in thyroid activity after the onset of hibernation in the golden hamster resulted from not only a direct effect of temperature on the metabolism of the thyroid gland, but also a reduction in TSH secretion from the anterior pituitary at the low body temperatures of hibernation. Thus, it is possible that the lengthened radioiodide half-lives observed in P. feucopus were the result of a decrease in both TSH secretion and the metabolism of the thyroid gland at the low body temperatures of daily torpor. In summary, thyroid activity in P. leucopus is not directly affected by photo-
IN P. leucopus
139
period or chronic temperature exposure, but the occurrence of daily torpor in this species does result in a substantial decrease in thyroid activity. Further studies are required to determine the mechanism by which this reduction in thyroid activity occurs. ACKNOWLEDGMENTS I thank Dr. M. Richmond for valuable suggestions concerning this manuscript. The research was funded in part by NSF Grant PCM77-21799 to Jack W. Hudson and a departmental research grant to D.H.R.
REFERENCES Barry, W. (1976). Environmental effects on food hoarding in deer mice (Peromyscus). J. Mammal. 57, 731-746. Bauman, T. R., Clayton, F. W., and Turner, C. W. (1965). The L-thyroxine secretion rate, Ltriiodothyronine equivalent, and the biological halflife of L-thyroxine- ‘3’1 in the raccoon (Procyon lotor). Gen. Comp. Endocrinol. 5, 261-266. Chaff=, R. R. J., and Roberts, J. C. (1971). Temperature acclimation in birds and mammals. Annu. Rev. Physiol. 33, 155-202. Collins, K. J., and Weiner, J. W. (1968). Endocrinological aspects of exposure to high environmental temperatures. Physiol. Rev. 48, 785-839. Heroux, 0. (1%9). Catecholamines, corticosteroids, and thyroid hormones in nonshivering thermogenesis under different environmental conditions. In “Physiology and Pathology of Adaptation Mechanisms” (E. Bajusz, ed.), pp. 347-365. Pergamon, New York. Heroux, 0.. and Brauer, R. (1965). Critical studies on determination of thyroid secretion rate in cold adapted animals. J. Appl. Physiol. 20, 597-606. Heroux, O., Depocas, F., and Hart, J. S. (1959). Comparison between seasonal and thermal acclimation in white rats. I. Metabolic and insulative changes. Canad. J. Biochem. Physiol. 37, 474-478. Hill, R. W. (1975). Daily torpor in Peromyscus leucopus on an adequate diet. Comp. Biochem. Physiol. 51, 413-423. Hudson, J. W. (1973). Torpidity in mammals. In “Comparative Physiology and Thermo-regulalion,” Vol. II (G. Causey Whittow, ed.), pp. 98- 158. Academic Press, New York. Hudson, J. W., and Deavers, D. (1976). Thyroid function and basal metabolism in the ground squirrels Ammospermophilus leucurus and Spermophi1u.r spp. Physial. Zool. 49, 425-444. Hudson, J. W., and Wang, L. C. (1969). Thyroid function in desert ground squirrels. In “Physiologi-
140
DONALD
cal Systems in Semiarid Environments” (C. C. Hoff and M. S. Riedesel, eds.), pp. 17-33. Univ. of New Mexico Press, Albuquerque. Inoue, E., and Taurog, A. (1968). Acute and chronic effects of iodide on thyroid radioiodine metabolism in iodine deficient rats. Endocrinology 83, 279-290. Knigge, K. (1957). Influence of cold exposure upon the endocrine glands of the hamster, with an apparent dichotomy between morphological and functional response of the thyroid. Anat. Rec. 127, 157-163. Lynch, G. R. (1970). Effect of photoperiod and cold acclimation on nonshivering thermogenesis in Peromyscus leucopus. Amer. Zool. 10, 308.
H. RHODES Lynch, G. R., White, S. E., Gnmdel, R., and Berger, M. S. (1978). Effects of photoperiod, melatonin administration and thyroid block on spontaneous daily torpor and temperature regulation in the white-footed mouse, Peromyscus leucopus. J. Comp. Physiol.
125, 157-163.
Tashima, L. S. (1%5). The effects of cold exposure and hibernation on thyroidal activity of Mesocricetus
auratus.
Gen.
Comp.
Endocrinol.
5,
267-277. Wenberg, G. M., and Holland, J. C. (1973). The circannual variations of thyroid activity in the woodchuck, Marmota monax. Comp. Biochem. Physiol.
44, 775-780.
AND
COMPARATIVE
42, 134-140 (1980)
ENDOCRINOLOGY
Effects of Temperature, Photoperiod, and Daily Torpor on Thyroid Function in the W h ite-Footed Mouse, Peromyscus leocopus H. RHODES
DONALD Section of Ecology and Systematics,
Fernow Hall. Cornell
University,
Ithaca, New York 14853
Accepted April 8, 1980 The biological half-life of thyroidal radioiodide and proportions of thyroid iodoamino acids were determined in Peromyscus leucopus after 3 weeks’ treatment with one of several constant ambient temperatures ranging from 7 to 35” and prior exposure to long (16:8 LD) or short (8: 16 LD) photoperiod. Similarly, the biological half-life of radioiodide was determined in mice exposed to temperature cycles of 16 hr at 22”and 8 hr at 7” or 16 hr at 35”and 8 hr at 22” and prior treatment with long photoperiod. The mean radioiodide half-life of mice kept at 7 was 9.4 days which was significantly longer than the pooled radioiodide half-life of 5.6 days exhibited by mice in all other temperature-photoperiod regimes. It is suggested that this long radioiodide half-life was due to the occurrence of daily torpor in the experimental animals. Mice monitored for daily torpor also exhibited significant reductions in the radioiodide release rate. Thyroid activity was not depressed in these animals on days during which they remained euthermic. The daily radioiodide release rate was positively correlated with the minimum body temperature of torpor and negatively correlated with the duration of torpor. It is suggested that this decrease in thyroid activity was a result of a direct effect of body temperature on thyroid function. Percentages of thyroid hydrolysate components were similar throughout all treatments.
It is generally accepted that under laboratory conditions thyroid activity is depressed in m a m m a ls exposed to heat (Collins and W e iner, 1968) and augmented in m a m m a ls exposed to cold (Heroux and Brauer, 1965; Hudson and Wang, 1969). However, Heroux (1969) has questioned the significance of the thyroid during cold acclimation, since laboratory rats exposed to seasonally changing conditions exhibit a decrease or no change in thyroid activity during the winter relative to other times of the year (Heroux et al., 1959; Heroux and Brauer, 1965). One m a jor difference between the laboratory-maintained animals and those m a intained under natural conditions is in the lack of environmental cues available to those animals kept in the laboratory. M u ltiple cues (e.g., temperature, photoperiod) associated with a seasonally changing environment play an important role in the regulation of adaptive responses. For instance, Peromyscus leucopus exposed to a short photoperiod exhibit an in-
crease in hoarding behavior and build larger nests than m ice exposed to a long-day lighting regime, whereas cold exposure results in a substantial increase in nonshivering thermogenesis but has little effect on hoarding behavior or nest construction (Lynch, 1970; Barry, 1976). No study to present examines the influence of m u ltiple cues on thyroid function in a single mamm a lian species. Alterations in thyroid activity may also be important in affecting other adaptive responses in small m a m m a ls. Lynch et al. (1978), basing their conclusions on propylthiouracil-treated animals, report that thyroid block increases the incidence of daily torpor in P. leucopus. Numerous studies have established that thyroid activity decreases prior to or subsequent to the onset of hibernation (Knigge, 1957; Wenberg and Holland, 1973). However, Hudson and W a n g (1%9) and Hudson and Deavers (1976) have presented evidence for several species of ground squirrels which points out
134 00166480/80/090134-07$01.00/0 Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
THYROID
FUNCTION
that thyroid inactivity and hibernation are not directly related. Thus, lack of a consistent relationship between thyroid activity and hibernation makes it difficult to conclude that a decrease in thyroid function is required for hibernation (Hudson, 1973). This does not preclude the possibility that thyroid function is affected by the reduction in metabolism and body temperature associated with h&et-nation. This study is an initial investigation of (1) the influence of temperature and photoperiod on thyroid activity in the whitefooted mouse, Peromyscus leucopus, and (2) thyroid function in P. leucopus which are known to undergo daily torpor. METHODS
AND MATERIALS
I. Animals. Peromyscus leucopus were live-trapped in the Ithaca, New York, area during the summer (June-August) and the late fag (October-November). Only mice with adult or subadult pelage were used to minimize possible variance in thyroid function due to age differences between mice (Bauman ef al., 1965). The animals were housed individually with food and water provided ad libitum. Fall-caught mice were maintained for 2 months at 7” under a short photoperiod (8:16 LD; lights on 8 AM to 4 PM EST). Cotton was provided as nesting material. Animals caught during the summer were kept at 22” and were exposed to either a long photoperiod (16:8 LD; lights on 7 AM to 11 PM EST) or a short photoperiod (8:16 LD). Wood shavings were supplied in quantities sufficient to cover the cage floor but not to allow nest construction. II. Temperature treatment. Animals were acclimated to laboratory conditions (22”) for at least 3 weeks prior to temperature treatment. Short-photoperiod mice kept at 22” were not introduced into the study until the moult to “winter” pelage and gonadal regression were completed. Gonadal involution was assessed in males by the inability to palpate the testes or, in females, by closure of the vaginal orifice. Groups of 9 or 10 mice containing approximately equal numbers of males and females were exposed for 3 weeks to constant ambient temperatures of 7,10, 15, 22, 30, or 35” and long or short photoperiod. In addition, two groups of mice were transferred to a Lab Line chamber which simulated a day-night temperature-light cycle; temperature and lighting changes occurred simultaneously. One group of animals was exposed to 16 hr at 35” (light) and 8 hr at 22” (dark), while the other was exposed to a cycle of 16 hr at 22” (light) and 8 hr at 7” (dark). All mice employed in temperature acclimation studies were maintained on the same
IN P. leucopus
135
photoperiod to which they had been exposed during the laboratory acclimation period. Subsequent to the 3-week acclimation period, radioiodide half-life and thin-layer chromatography of the thyroid iodoamino acids were assessed for each experimental group. To assess the rate of radioiodide release from the thyroid gland, mice were injected intraperitoneaily with 5 FCi lx51(New England Nuclear Corp., 17 Ci/mg) and 24 hr was allowed for incorporation of the isotope into the thyroid. Neck radioactivity of the mice was determined by holding their ventral neck region against a Lucite platform over an opening 2.27 cm2 in a 3-in. ring of lead which led to a sodium iodide-thallium activated crystal connected to a Nuclear Chicago timer scaler. Three to five I-min counts of the neck area were made to determine the maximum thyroid radioactivity. Neck counts were performed at approximately the same time each day for 5 to 7 days. All values were corrected for isotope decay. Seven to nine days after the fust lz51 injection, the mice were reinjected for 2 consecutive days with 2.5 &i ‘Vday in preparation for thin-layer chromatography of the thyroid hormones. Forty-e&M hours after the second injection the animals were killed by cervical fracture under ether anesthesia. Thyroids were prepared for thin-layer chromatography after the aerobic method of Inoue and Taurog (1968) with one exception: because the thyroid glands were so small, both the thyroid gland and the adjoining trachea were removed. The structure was homogenized in 0.1 ml cold buffered saline (0.03 M Tris-0.11 M NaCI, pH 8.5) and then centrifuged for 15 ruin to remove particulate material. Buffered saline (0.1 ml) containing 0.11 M I-methyl-2-mercaptoimidazole and 0.05 ml of a 10% pancreatin solution (Protease, Sigma Chemicals Co., St. Louis, MO.) was added to the supernatant and digestion was performed for 24 hr at 37” in ParaIilm covered test tubes. At the end of the digestion period a ~-PI sample of the mixture was removed, after being shaken, and then placed on a 20 x 20cm silica gel Eastman Chromagram Sheet (Eastman Kodak, Rochester, N.Y.). Individual samples from each mouse in a group, along with a reference solution containing the four principle iodoamino acids, MIT. DIT, T3, and T., (Sigma Chemical Co.), were run on a single chromatogram sheet. Ascending chromatography was carried out in a solvent consisting of 3: 1 (v/v) ammonia:sec-butanol. Ninhydrin (Sigma Chemical Co.) was used to make the iodoamino acids visible. The labeled areas along with the origin of the chromatogram were then scraped into individual test tubes and suspended in several drops of NCS solution (AmershamSearle, Des Plaines, Ill.). Each tube was counted for 10 miu in a well counter and for each thyroid, the percentage of total counts contributed by the individual iodoamino acids was determined. III. Daily torpor. Fall-caught mice maintained at 7°C were anesthetized with ether and a body-tem-
136
DONALD
H. RHODES
lease indicates that mice maintained at 7” released radioiodide from the thyroid gland substantially slower (P < 0.01) than did mice in all other treatment groups (Table 1). However, not all mice maintained at 7” had lengthened radioiodide half-lives (longer than 10 days). Within the 7” treatment, 7 of 9 mice (78%) on a short photoperiod and 4 of 10 mice (40%) on a long photoperiod had lengthened radioiodide half-lives. Further, examination of radioiodide half-lives for individual animals throughout all temperature and photoperiod treatments indicates that an additional three mice, two at 10” and one at IS”, exhibited radioiodide half-lives longer than 10 days; these three animals were maintained on a short-day lighting regime. Comparison of means for percentage of radioiodide located in different components of thyroid hydrolysates indicates that the distribution of radioiodide is similar RESULTS throughout all temperature and photoperiod Groups of mice exposed for 3 weeks to treatments (P > 0.05; f(5,107) = 1.27). constant ambient temperatures of 10 to 35” Pooled means (? 1 SE) for Origin, MIT, and daily temperature cycles exhibited a DIT, T3, and T4 are 15.3 + 1.17%, 35.8 t pooled radioiodide half-life of 5.6 days. In 1.28%, 36.2 + 1.24%, 2.2 & 0.14%, and 10.4 contrast, mice maintained at 7” under a long + 0.16%, respectively. Mice which were known to undergo daily or short photoperiod exhibited half-lives of 8.0 and 10.9 days, respectively. Compari- torpor on the basis of body-temperature son of regression slopes for radioiodide re- records exhibited a pronounced decrease in perature transmitter (Minimitter, Model X, Minimitter Inc., Indianapolis, Ind.) was implanted in the abdominal cavity. The incision was closed with silk-thread sutures and the animals were allowed a 4- to 5-day recovery period at 22” on a short photoperiod. The mice were then returned to the 7” chamber housed in cages equipped with an external antenna. The animals were placed on a restricted diet of 2.5-3.0 g laboratory chow per day. Output from the Mimimitter was received on a standard AM receiver and recorded on a cassette recorder. Both the receiver and the recorder were automatically activated for 5 min each hour. Body temperatures were monitored from 3 AM to 1 PM, the period of time within which daily torpor has been observed to occur for this species (Hill, 1975). Any mice which exhibited torpor (Tb less than 30’) were injected intraperitoneally with 5 &i iz51 and release of radioiodide from the thyroid gland was monitored for 4 to 7 days. Neck counts were performed at approximately 1 PM each day; body-temperature records had shown that the animals were euthermic by this time so that measurements did not interrupt periods of torpor. IV. Dafa analysis. Data were subjected to regression analysis and analysis of covariance.
TABLE 1 EFFECTSOFCHRONICTEMPERATURETREATMENTANDPRIORPHOTOPER~OD EXPIWJ~EON RADIOIODIDE RELEASE FROM THE THYROID ~~Peromyscus leucopus
168 LD Temperature
n
7
10
10”
10
IS” 22” 30” 35” 22-7” 35-22
9 9 10
10 10 10
o Biological half-life of lo51 (days). FJP < O.Ol;fl13,50) = 4.49.
Slope
-0.00160” -0.00224 -0.00245 -0.00190
-0.00205 -UO208 -0.80245 -0.00243
8: 16 LD TV,”
n
8.0 5.5 5.2 6.6 5.9 5.9 4.8 5.7
9 9 9 10 10 10
Slope
-0.00117~ -0.00190
-0.00225 -0.00213 -0.00233 -0.00223
Tm
10.9 5.5 5.8 5.5 6.1 5.4
THYROID
FUNCTION
the daily release of radioiodide from the thyroid gland relative to 24-hr release when they remained euthermic (Table 2) (P < 0.001; r(30) = 331). Animals which became torpid exhibited a release of 3 to 7% of the radioiodide present in the gland on the previous day compared to a release rate of 10 to 25% when they did not become torpid. This latter result is similar to the
3.37
IN I’. /eucopu.s
daily release rate of 10 to 21% obtained from Animal A (Table 2) which never became torpid. The 24hr radioiodide release was positively correlated with the m inimum daily body temperature (r = 0.795; P < 0.01; 12= 32) and negatively correlated with the duration of daily torpor (r = -0.762; P < 0.01; n = 32). No correspondence was found between the rate of entry (r =
TABLE 2 ENTRY RATES, AROUSAL RATE, PERCENTAGE lzsI RELEASED FROM THE THYROID PER 24 HR, DURATION OF TORPOR, AND MINIMUM DAILY BODY TEMPERATURE IN SIX INDIVIDUAL P. leucopus MONITORED FOR DAILY TORWR WITH BODY-TEMPERATURE TRANSMITTERS IMPLANTED IN THE ABDOMINAL CAVITY
Minimum T,, (“)
Day
Duration of torpor (hr)
Percentage Pz5 released from gland/24 hr
0.0 0.0 0.0 0.0 0.0 0.0 0.0
21.0 16.0 16.0 18.0 12.0
1.5 4.5 3.5
5.0 5.0
0.0
0.0 6.5 5.0 4.5
0.0
0.0
12.0
0.5 3.5
Animal A 34.0 34.0 35.0 32.0 31.0 33.0 32.0
Entry rate (Vmin)
Arousal rate (“imin)
-
-
-
10.0 11.0
Animal B 29.0 20.0 24.5 32.5
Animal C 18.5 18.0 18.5 37.0
0.05 0.08 0.08
0.12 0.16
18.0
-
6.0 3.0
0.13 0.15 0.15
0.18 0.19 0.15
0.11
-
-
14.0
0.05
3.0 13.0 12.0
0.10
0.12 0.14
0.07
0.13
4.5 3.5
6.0 25.0 24.0 25.0 4.0 7.0
0.09 0.08
0.14 0.17
0.0
25.0
5.0
5.0
0.0
25.0
-
0.5 6.5 5.0 3.5
10.0
0.12 0.14 0.12 0.08
Animal D 29.0 25.0 30.5 31.0
0.0 0.0
Animal E 26.0 33.0 35.0 33.5 21.5 25.0
4.5
0.0 0.0 0.0
Animal F 35.0 21.0 35.0 29.5 19.0 19.5 22.0
6.0 6.0 4.0
-
-
-
-
0.17
0.19
0. IO 0.18 0.18 0.14
138
DONALD
-0.301; P > 0.05; n = 16) or arousal (r = -0.172; P > 0.05; n = 16) from torpor and the 24-hr radioiodide release rate.
H.
RHODES
daily torpor in P. leucopus relative to animals exposed to a long photoperiod. (2) Not all cold-exposed mice exhibited altered thyroid function, which concurs with the DISCUSSION finding that daily torpor appears to be charExposure of many small mammals to acteristic of only certain individual P. ambient temperatures outside the limits of leucopus (Hill, 1975; Lynch et al., 1978). (3) their thermal neutral zone induces an in- Mice which were known to undergo bouts crease in radioiodide release from the of daily torpor on the basis of bodythyroid gland during cold exposure and a temperature records exhibited pronounced decrease during heat exposure (Collins and decreases in the release of radioiodide from Weiner, 1%8; Chaffee and Roberts, 1971). the thyroid gland. Thus, although body However, in this study chronic temperature temperatures of mice chronically exposed and photoperiod treatment did not influ- to low ambient temperature were not meaence the rate of radioiodide release from the sured, it is suggested that the observed reductions in thyroid activity were correlated thyroid gland in the majority of Peromyscus leucopus examined. Together with the con- with the occurrence of daily torpor in the stant proportions of thyroid iodoamino experimental animals. acids observed throughout all temperaThe finding that thyroid activity is subture - photoperiod treatments, these lines of stantially depressed in mice known to have evidence suggest that thyroid function does undergone daily torpor suggests two posnot play a critical role in the thermal adap- sibilities with respect to the regulation of tation of this species to long-term temper- thyroid activity: (1) thyroid hormone reature exposure. lease is being “turned off’ prior to and in However, a significant reduction in the preparation for entrance into daily torpor, secretion of radioiodide from the thyroid or (2) thyroid hormone release is decreased gland was observed in 12% of the P. due to the metabolic effect of a reduction in leucopus included in this study. This de- body temperature during the several hours crease in thyroid activity was observed of daily torpor. Since thyroidal radioiodide only in mice exposed to ambient tempera- release was depressed only on days when tures of 15” or less which contrasts with the animals became torpid but did not differ established reports of elevated thyroid from values on days during which the mice function in cold-exposed mammals. remained euthermic, a reduction in body Further, since only a few mice exhibited temperature is the most likely cause of the reduced thyroid activity when exposed to altered thyroid activity. This conclusion is cold, this would not seem to be a consistent supported by the findings that thyroidal adaptive response to low ambient temper- radioiodide release is significantly correature. lated with the minimum body temperature One possible explanation for the ob- reached during bouts of torpor, and negaserved decline in thyroid function is that tively correlated with the amount of time some P. leucopus maintained at low am- spent in torpor (Table 2). Since thyroid hormones are known to bient temperature were undergoing daily torpor. Three observations indicate that stimulate the metabolic rate, it might be this may have been the case. (1) The major- expected that hormone release would deity of mice exhibiting reduced thyroid se- crease preceding entry into torpor or, altercretion were maintained on a short photo- natively, increase during arousal from period. Lynch et al. (1978) have recently torpor. No significant correlation exists demonstrated that a short-day lighting re- between either entry or arousal rate of gime significantly increases the incidence of body-temperature change and the radio-
THYROID
FUNCTION
iodide release, further evidence that changes in thyroid activity associated with the occurrence of daily torpor are the result of a reduction in body temperature and not an active inhibition of thyroid activity. It seems unlikely, however, that the observed depression in radioiodide release from the thyroid gland in conjunction with torpor was due to the effect of a low body temperature on thyroid metabolism alone. In comparison with radioiodide release rates of euthermic mice, radioiodide release rates were reduced by 50% or more on days during which animals underwent bouts of daily torpor. Since torpor seldom exceeded 6 hr in duration, body temperatures remained high for at least 75% of the day. If we assume that hormone release fi-om the thyroid gland may be reduced to a minimum level, or even zero, at the low body temperatures associated with torpor, then radioiodide release should approximate 75% of that observed on days during which the mice did not undergo torpor. Since release rates of radioiodide were always less than the predicted 75%, thyroid function must have been suppressed for longer periods of time than the 6 hr of daily torpor. This suggests that low body temperature may affect thyroid function so as to depress activity subsequent to a bout of torpor, or alter TSH secretion during daily torpor. Tashima (1965) concluded that the observed decline in thyroid activity after the onset of hibernation in the golden hamster resulted from not only a direct effect of temperature on the metabolism of the thyroid gland, but also a reduction in TSH secretion from the anterior pituitary at the low body temperatures of hibernation. Thus, it is possible that the lengthened radioiodide half-lives observed in P. feucopus were the result of a decrease in both TSH secretion and the metabolism of the thyroid gland at the low body temperatures of daily torpor. In summary, thyroid activity in P. leucopus is not directly affected by photo-
IN P. leucopus
139
period or chronic temperature exposure, but the occurrence of daily torpor in this species does result in a substantial decrease in thyroid activity. Further studies are required to determine the mechanism by which this reduction in thyroid activity occurs. ACKNOWLEDGMENTS I thank Dr. M. Richmond for valuable suggestions concerning this manuscript. The research was funded in part by NSF Grant PCM77-21799 to Jack W. Hudson and a departmental research grant to D.H.R.
REFERENCES Barry, W. (1976). Environmental effects on food hoarding in deer mice (Peromyscus). J. Mammal. 57, 731-746. Bauman, T. R., Clayton, F. W., and Turner, C. W. (1965). The L-thyroxine secretion rate, Ltriiodothyronine equivalent, and the biological halflife of L-thyroxine- ‘3’1 in the raccoon (Procyon lotor). Gen. Comp. Endocrinol. 5, 261-266. Chaff=, R. R. J., and Roberts, J. C. (1971). Temperature acclimation in birds and mammals. Annu. Rev. Physiol. 33, 155-202. Collins, K. J., and Weiner, J. W. (1968). Endocrinological aspects of exposure to high environmental temperatures. Physiol. Rev. 48, 785-839. Heroux, 0. (1%9). Catecholamines, corticosteroids, and thyroid hormones in nonshivering thermogenesis under different environmental conditions. In “Physiology and Pathology of Adaptation Mechanisms” (E. Bajusz, ed.), pp. 347-365. Pergamon, New York. Heroux, 0.. and Brauer, R. (1965). Critical studies on determination of thyroid secretion rate in cold adapted animals. J. Appl. Physiol. 20, 597-606. Heroux, O., Depocas, F., and Hart, J. S. (1959). Comparison between seasonal and thermal acclimation in white rats. I. Metabolic and insulative changes. Canad. J. Biochem. Physiol. 37, 474-478. Hill, R. W. (1975). Daily torpor in Peromyscus leucopus on an adequate diet. Comp. Biochem. Physiol. 51, 413-423. Hudson, J. W. (1973). Torpidity in mammals. In “Comparative Physiology and Thermo-regulalion,” Vol. II (G. Causey Whittow, ed.), pp. 98- 158. Academic Press, New York. Hudson, J. W., and Deavers, D. (1976). Thyroid function and basal metabolism in the ground squirrels Ammospermophilus leucurus and Spermophi1u.r spp. Physial. Zool. 49, 425-444. Hudson, J. W., and Wang, L. C. (1969). Thyroid function in desert ground squirrels. In “Physiologi-
140
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cal Systems in Semiarid Environments” (C. C. Hoff and M. S. Riedesel, eds.), pp. 17-33. Univ. of New Mexico Press, Albuquerque. Inoue, E., and Taurog, A. (1968). Acute and chronic effects of iodide on thyroid radioiodine metabolism in iodine deficient rats. Endocrinology 83, 279-290. Knigge, K. (1957). Influence of cold exposure upon the endocrine glands of the hamster, with an apparent dichotomy between morphological and functional response of the thyroid. Anat. Rec. 127, 157-163. Lynch, G. R. (1970). Effect of photoperiod and cold acclimation on nonshivering thermogenesis in Peromyscus leucopus. Amer. Zool. 10, 308.
H. RHODES Lynch, G. R., White, S. E., Gnmdel, R., and Berger, M. S. (1978). Effects of photoperiod, melatonin administration and thyroid block on spontaneous daily torpor and temperature regulation in the white-footed mouse, Peromyscus leucopus. J. Comp. Physiol.
125, 157-163.
Tashima, L. S. (1%5). The effects of cold exposure and hibernation on thyroidal activity of Mesocricetus
auratus.
Gen.
Comp.
Endocrinol.
5,
267-277. Wenberg, G. M., and Holland, J. C. (1973). The circannual variations of thyroid activity in the woodchuck, Marmota monax. Comp. Biochem. Physiol.
44, 775-780.