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Reductions in Hamster Serum Thyroxine Levels by Melatonin Are Not Altered by Changes in Serum Testosterone
General and Comparative Endocrinology 123, 121–126 (2001) doi:10.1006/gcen.2001.7665, available online at http://www.idealibrary.com on
Reductions in Hamster Serum Thyroxine Levels by Melatonin Are Not Altered by Changes in Serum Testosterone Thomas H. Champney Department of Human Anatomy and Medical Neurobiology, College of Medicine, Texas A&M University Health Science Center, College Station, Texas 77843-1114 Accepted April 16, 2001
mechanism as castration-induced reductions.
Daily melatonin injections reduce reproductive and thyroid hormones in male Syrian hamsters. The interrelationship between the decline in these hormones is not known. To explore this relationship, male Syrian hamsters were divided into four groups: castrated, implanted with testosterone (5-mm silastic implants), both treatments, or neither treatment. One-half of each group of hamsters (n ⴝ 7 or 8) were injected with melatonin (25 mg) daily at 1730 h. The other half of each group received daily vehicle injections. Ten weeks later, the hamsters were anesthetized and decapitated. Testes weights, serum testosterone, and serum thyroxine levels were measured. As expected, testes and serum testosterone levels were uniformly low in all of the melatonin-treated hamsters. All of the melatonin-treated groups also had lower than normal thyroxine values irrespective of gonadal treatment. Interestingly, in the non-melatonin-treated hamsters, serum thyroxine values were decreased in the castrated group and increased in the testosterone-implanted group. These results suggest that castration can reduce serum thyroxine levels in male Syrian hamsters and that replacement of testosterone restores these levels to normal. Notably, the declines in thyroxine levels produced by daily melatonin injections were not restored by testosterone implants in castrated or intact hamsters. Therefore, melatonin-induced reductions in thyroxine are not mediated by concurrent reductions in serum testosterone levels. It appears that melatonin-induced reductions in serum thyroxine levels do not use the same 0016-6480/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
©
2001
Academic Press
Key Words: melatonin; thyroxine; testosterone; hamster; testes.
INTRODUCTION Endogenous pineal melatonin production occurs during the dark phase of the photoperiod, providing a signal to the rest of the organism that mirrors the photoperiod (Bartness et al., 1993; Reiter, 1987). Therefore, during the winter months, a longer period of melatonin production occurs. This period length can be mimicked by provision of daily late-afternoon melatonin injections to long-photoperiod-exposed animals. When Syrian hamsters are exposed to long photoperiods and given daily late-afternoon melatonin injections, they display numerous physiologic changes that parallel changes observed during short-photoperiod exposure (Bartness et al., 1993; Stetson and Watson-Whitmyre, 1984). The best known response is a decline in reproductive physiology that is demonstrated by a drastic reduction in testes size (from over 3000 mg to less than 500 mg). This decline in testes size produces a parallel decline in serum testosterone levels. The reproductive effects, however, are not the only physiologic changes that occur after daily afternoon 121
122
melatonin injections to hamsters; a reduction in thyroid physiology is also observed. The changes in thyroid physiology in hamsters are usually demonstrated by a reduction in serum thyroid hormones or free thyroxine index (Champney, 1988, 1989; Creighton and Rudeen, 1989; Petterborg and Rudeen, 1989; Reiter et al., 1988; Singh and Turner, 1972; Vaughan et al., 1982a, 1984; Vriend, 1984; Vriend and Reiter, 1977; Vriend et al., 1979, 1987). Changes in hamster thyroid morphology and serum thyroid stimulating hormone levels have also been observed (Vaughan et al., 1982b; Vriend, 1985; Vriend and Thliveris, 1985). In other species, the effects of melatonin on thyroid physiology are species specific with increases, decreases, or no changes depending on the species examined and the time frame of melatonin administration (Ashley et al., 1999; Esquifino et al., 1997; Gower et al., 1996; Krotewicz and Lewinski, 1994a,b; Levine et al., 1995; Ozturk et al., 2000; Rasmussen et al., 1999; Wright et al., 1996, 2000). Previous research in rats has found that changes in thyroid activity can be produced by alterations in gonadal hormone levels, specifically that castration reduces thyroid hormone levels and testosterone replacement restores or increases thyroid hormone levels (Chen and Walfish, 1979; Christianson et al., 1981; Paloyan et al., 1982; Steger et al., 1989). Therefore, it could be surmised that the effects of melatonin on serum testosterone levels in the hamster are responsible for the observed thyroid effects. The present study examined this interaction by investigating the impact of reduced serum testosterone levels (and its replacement) on serum thyroxine levels in control and melatonin-treated hamsters.
MATERIALS AND METHODS Male Syrian hamsters (80 –100 g) were purchased from Harlan Sprague–Dawley (Madison, WI). They were housed 2 per cage in a temperature-controlled (23°) room with long photoperiod (14L:10D h, lights on at 0500 h). Food and water were available continuously. The hamsters were subdivided into two large groups: one group (32 hamsters) received daily melatonin injections (25 g in 0.1 ml of 1:10 ethanolic saline
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Thomas H. Champney
subcutaneously) and the other group (31 hamsters) received vehicle (0.1 ml of 1:10 ethanolic saline subcutaneously). Both groups received their injections at 1730 h each day. Each of the groups was subdivided into four subgroups: (1) implanted with testosterone (subcutaneous 5-mm silastic capsule (1.5 mm ID, 2.5 mm OD) replaced 6 weeks later), (2) castrated (testes removal under methoxyflurane (metofane) anesthesia), (3) both treatments (implanted ⫹ castrated), and (4) left untreated (control). All subgroups contained 8 hamsters except for the vehicle/control group which had 7 hamsters. For the 10-week duration of the experiment, the hamsters were injected daily at 1730 h, were weighed weekly, and had their testosterone implants replaced at the beginning of the 6-week time point. At the end of the experiment, the hamsters were killed over a 2 day period with one-half of each subgroup killed on each day. One animal from each subgroup was killed sequentially to minimize the impact of time of day on tissue collection. The hamsters were killed by decapitation after metofane anesthesia. Body weights, testes weights, and trunk blood were collected. The blood was chilled (4°) and centrifuged for 30 min at 2000g, and the resulting serum was transferred to glass vials and frozen (⫺20°). Serum testosterone, thyroxine (T4), and triiodothyronine (T3) uptake levels were determined by radioimmunoassays (Diagnostic Systems Laboratories, Webster, TX) that were validated for use in the hamster (Champney, 1988). For the testosterone assay, the least detectable level was 0.006 ng/ml with an intraassay variation of 2.24%. For the thyroxine assay, the least detectable value was 0.199 g/dl and the intraassay variation was 4.55%. For the T3 uptake assay, the least detectable percentage uptake was 9% with an intraassay variation of 1.52%. Free thyroxine index (FTI) was calculated by multiplication of each serum thyroxine value by each T3 uptake value and division by 35 (a standard reference value) (FTI ⫽ T4 ⫻ T3 uptake/35) (Diagnostic Systems Laboratories; DSL15100 package insert). Statistical significance was determined by a threeway analysis of variance (melatonin vs castration vs implant) with the least squares method used as a post hoc test to determine significance between groups.
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Melatonin Reduces Serum Thyroxine Levels
TABLE 1 Body Weight, Testes Weight, and Serum T3 Uptake in Male Syrian Hamsters That Received Vehicle or Melatonin (25 g) at 1730 h for 10 Weeks Vehicle
Body weight (g) Testes weight (g) T3 uptake (%)
Melatonin
Con
Imp
Cas
I⫹C
Con
Imp
Cas
I⫹C
142 ⫾ 4 3.87 ⫾ 0.17 60.3 ⫾ 0.5
144 ⫾ 5 3.44 ⫾ 0.14 58.5 ⫾ 0.8
159 ⫹ ⫾ 5 — 57.5 ⫹ ⫾ 0.9
160 ⫹ ⫾ 4 — 58.0 ⫹ ⫾ 0.3
149 ⫾ 6 0.49* ⫾ 0.04 61.1 ⫾ 0.6
144 ⫾ 6 0.33* ⫾ 0.09 58.5 ⫾ 0.7
150 ⫾ 6 — 58.6 ⫾ 0.7
145 ⫾ 3 — 59.7 ⫾ 0.4
Note. The hamsters were also implanted with testosterone (Imp, 5-mm silastic capsule), were castrated (Cas), received both treatments (I⫹C), or received neither treatment (Con). Values are means ⫾ standard errors with 7 or 8 hamsters per group. ⫹ P ⬍ 0.05 vs vehicle/control. * P ⬍ 0.001 vs vehicle/control.
RESULTS At the end of the experiment, daily melatonin injections increased body weight (F ⫽ 4.09, P ⫽ 0.048) (Table 1). Castration also increased body weight (F ⫽ 5.66, P ⫽ 0.021). In addition, there was a significant interaction between melatonin and castration on body weight (F ⫽ 5.48, P ⫽ 0.023). Body weights in the vehicle/castrated and the vehicle/implanted ⫹ castrated hamsters were significantly increased over those in the vehicle/control hamsters (P ⬍ 0.05) (Table 1). Testes weights in all of the intact, melatonintreated hamsters were significantly lower than those in the intact, vehicle-injected hamsters (P ⬍ 0.001) (Table 1). As expected, there were significant differences in serum testosterone levels in melatonin-treated hamsters (F ⫽ 59.44, P ⫽ 0.0001), in castrated hamsters (F ⫽ 112.52, P ⫽ 0.0001), and in testosterone-implanted hamsters (F ⫽ 61.13, P ⫽ 0.0001) (Fig. 1). In addition, there were significant interactions between melatonin and castration (F ⫽ 79.46, P ⫽ 0.0001), between melatonin and testosterone implants (F ⫽ 13.15, P ⫽ 0.001), and among all three factors (F ⫽ 6.08, P ⫽ 0.017). Serum testosterone values in all of the melatonin-treated hamsters and in the vehicle/ castrated and vehicle/implanted ⫹ castrated hamsters were significantly lower than those in the vehicle/ control animals (P ⬍ 0.01) (Fig. 1). Significant difference were observed in serum thyroxine levels in the melatonin-treated hamsters (F ⫽ 74.35, P ⫽ 0.0001) and in the testosterone-implanted hamsters (F ⫽ 13.81, P ⫽ 0.001) (Fig. 2). Interactions
between melatonin and castration (F ⫽ 9.11, P ⫽ 0.004) and between melatonin and testosterone implants (F ⫽ 7.16, P ⫽ 0.01) were also significant. Serum thyroxine values were significantly reduced in all of the melatonin-treated hamsters and in the vehi-
FIG. 1. Serum testosterone levels in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
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Thomas H. Champney
trated group, whereas it was increased in the vehicle/ implanted group compared to that in the vehicle/ control hamsters (P ⬍ 0.01) (Fig. 3).
DISCUSSION This study found that castration of vehicle/control hamsters reduced serum thyroxine levels and that this reduction was reversed by testosterone replacement (Figs. 2 and 3). This finding suggests that testosterone has an impact on thyroid physiology in male hamsters similar to that found in other species (Chen and Walfish, 1979; Steger et al., 1989). This is further supported by the lack of major changes in the thyroid binding proteins as determined by the T3 uptake assay (Table 1). Previous research shows little or no effect of gonadal hormone manipulations on thyroid binding
FIG. 2. Serum thyroxine levels in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
cle/castrated group, whereas they were increased in the vehicle/implant group compared to those in the vehicle/control hamsters (P ⬍ 0.05) (Fig. 2). Castration significantly reduced T3 uptake (F ⫽ 6.28, P ⫽ 0.015) (Table 1). There was also a significant interaction between castration and testosterone implants on T3 uptake (F ⫽ 10.92, P ⫽ 0.002). T3 uptake in the vehicle/castrated and the vehicle/implanted ⫹ castrated hamsters was significantly decreased compared to that in the vehicle/control hamsters (P ⬍ 0.05) (Table 1). The free thyroxine index was significantly different in the melatonin-treated hamsters (F ⫽ 76.09, P ⫽ 0.0001) and in the testosterone-implanted hamsters (F ⫽ 13.23, P ⫽ 0.001) (Fig. 3). Significant interactions between melatonin and castration (F ⫽ 11.64, P ⫽ 0.001) and between melatonin and testosterone implants (F ⫽ 7.36, P ⫽ 0.009) were also found in the free thyroxine index. The FTI was lower in all of the melatonin-treated hamsters and in the vehicle/cas-
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
FIG. 3. Serum free thyroxine index in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
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Melatonin Reduces Serum Thyroxine Levels
proteins (Emerson et al., 1990; Valenti et al., 1982) and the current data are consistent with these findings. This study also found that, irrespective of reproductive state, daily melatonin injections reduced serum thyroxine levels and the free thyroxine index (Figs. 2 and 3). This indicates that melatonin-mediated reductions in serum thyroxine levels are not due to concurrent declines in serum testosterone levels. These results suggest that melatonin’s effects on the thyroid system are independent of melatonin’s effects on the reproductive system. Interestingly, since testosterone replacement did not correct the melatonin-induced decline in thyroxine levels, it appears that there are at least two mechanisms that can modify thyroid hormone levels: a melatonin-mediated mechanism and a testosterone-mediated mechanism. The current hypothesis for melatonin-induced changes in serum thyroxine levels suggests that melatonin acts at the hypothalamic level, altering the downstream hypothalamic–pituitary– endocrine organ axis (Hoover et al., 1992; Miguez and Aldegunde, 1996; Vriend, 1989). These authors suggest that melatonin alters neurotransmitter levels in the hypothalamus, which modify the current hypothalamic set point, putting in motion the observed endocrine effects. The current results do not directly support or refute this hypothesis. They do, however, provide some indirect support for the hypothesis by indicating that the effects of melatonin on the thyroid system are independent of its effects on the reproductive system. It is not known whether testosterone regulation of thyroid function acts by a similar hypothalamic mechanism or at the thyroid directly, but the present results suggest that two separate mechanisms exist for thyroid hormone regulation—a melatonin-mediated response and a testosterone-mediated response. In summary, daily afternoon melatonin injections reduced serum thyroxine content and free thyroxine index in male hamsters irrespective of the hamster’s serum testosterone levels. This indicates that melatonin-mediated declines in thyroid hormone levels are not regulated by the concurrent decline in serum testosterone content. There does, however, appear to be a testosterone effect on thyroid hormone levels in hamsters with castration reducing thyroxine levels and testosterone replacement restoring these levels to normal.
ACKNOWLEDGMENTS The technical help of Gregg Allen and Dr. Wei-Jung A. Chen is appreciated. These data were previously presented at the 82nd Annual Endocrine Society meeting (June 2000) (No. 1185).
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126 tropin; Dependence of effects on the used doses. Neuroendocrinol. Lett. 16, 263–268. Krotewicz, M., and Lewinski, A. (1994b). Effects of pinealectomy and of late afternoon injections of pineal indole substances on thyroid hormone secretion in male Wistar rats. Biomed. Lett. 50, 101–107. Levine, M., Duffy, L., Moore, D. C., and Matej, L. A. (1995). Acclimation of a non-indigenous sub-Arctic population: Seasonal variation in thyroid function in interior Alaska. Comp. Biochem. Physiol. 111A, 209 –214. Miguez, J. M., and Aldegunde, M. (1996). Changes in the hypothalamic serotonergic function may mediate the endocrine effects of melatonin. Rev. Esp. Fisiol. 52, 239 –245. Ozturk, G., Coskun, S., Erbas, D., and Hasanoglu, E. (2000). The effect of melatonin on liver superoxide dismutase activity, serum nitrate and thyroid hormone levels. Jpn. J. Physiol. 50, 149 –153. Paloyan, E., Hofmann, C., Prinz, R. A., Oslapas, R., Shah, K. H., Ku, W. W., Ernst, K., Smith, M., and Lawrence, A. M. (1982). Castration induces a marked reduction in the incidence of thyroid cancers. Surgery 92, 839 – 848. Petterborg, L. J., and Rudeen, P. K. (1989). Effects of daily afternoon melatonin administration on body weight and thyroid hormones in female hamsters. J. Pineal Res. 6, 367–373. Rasmussen, D. D., Boldt, B. M., Wilkinson, C. W., Yellon, S. M., and Matsumoto, A. M. (1999). Daily melatonin adminstration at middle age suppresses male rat visceral fat, plasma leptin, and plasma insulin to youthful levels. Endocrinology 140, 1009 –1012. Reiter, R. J. (1987). The melatonin message: Duration vs coincidence hypotheses. Life Sci. 40, 2119 –2131. Reiter, R. J., Li, K., Gonzalez-Brito, A., Tannenbaum, M. G., Vaughan, M. K., Vaughan, G. M., and Villanua, M. A. (1988). Elevated environmental temperature alters the responses of the reproductive and thyroid axes of female Syrian hamsters to afternoon melatonin injections. J. Pineal Res. 5, 301–315. Singh, D. V., and Turner, C. W. (1972). Effect of melatonin upon thyroid hormone secretion rate in female hamsters and male rats. Acta Endocrinol. 69, 35– 40. Steger, R. W., Chandrashekar, V., Bartke, A., Wortsman, J., Newton, S. C., Smallridge, R. C., and Dietrich, J. (1989). Effects of elevated gonadotropin levels on thyroid function in the male rat. Life Sci. 45, 85–90. Stetson, M. H., and Watson-Whitmyre, M. (1984). Physiology of the pineal and its hormone melatonin in annual reproduction in rodents. In “The Pineal Gland” (R. J. Reiter, Ed.), pp. 109 –153. Raven Press, New York.
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Thomas H. Champney
Valenti, G., Banchini, A., Zavaroni, D., Vescovi, P. P., Ponticelli, P., and Hafez, E. S. (1982). Pituitary–thyroid axis after bilateral orchiectomy in men. Arch. Androl. 9, 171–174. Vaughan, G. M., Vaughan, M. K., Seraile, L. G., and Reiter, R. J. (1982a). Thyroid hormones in male hamsters with activated pineals or melatonin treatement Prog. Clin. Biol. Res. 92, 187–196. Vaughan, M. K., Powanda, M. C., Brainard, G. C., Johnson, L. Y., and Reiter, R. J. (1982b). Effects of blinding or afternoon melatonin injections on plasma cholesterol, triglycerides, glucose, TSH and thyroid hormone levels in male and female Syrian hamsters. Prog. Clin. Biol. Res. 92, 177–186. Vaughan, M. K., Richardson, B. A., Petterborg, L. J., Holtorf, A. P., Vaughan, G. M., Champney, T. H., and Reiter, R. J. (1984). Effects of injections and/or chronic implants of melatonin and 5-methoxytryptamine on plasma thyroid hormones in male and female Syrian hamsters. Neuroendocrinology 39, 361–366. Vriend, J. (1983). Evidence for pineal gland modulation of the neuroendocrine–thyroid axis. Neuroendocrinology 36, 68 –78. Vriend, J. (1984). Influence of the pineal gland and circadian rhythms in circulating levels of thyroid hormones of male hamsters. J. Pineal Res. 1, 15–22. Vriend, J. (1985). Effects of melatonin and thyroxine replacement on thyrotropin, luteinizing hormone, and prolactin in male hypothyroid hamsters. Endocrinology 117, 2402–2407. Vriend, J. (1989). Endocrine effects of blinding in male Syrian hamsters are associated with increased hypothalamic 5-hydroxyindoleacetic acid/serotonin ratios. J. Pineal Res. 7, 401– 409. Vriend, J., and Reiter, R. J. (1977). Free thyroxine index in normal, melatonin-treated and blind hamsters. Horm. Metab Res. 9, 231– 234. Vriend, J., Reiter, R. J., and Anderson, G. R. (1979). Effects of the pineal and melatonin on thyroid activity of male golden hamsters. Gen. Comp Endocrinol. 38, 189 –195. Vriend, J., and Thliveris, J. A. (1985). Effects of pinealectomy and melatonin administration on thyroid follicles of blind Syrian hamsters. Anat. Rec. 211, 29 –33. Vriend, J., Borer, K. T., and Thliveris, J. A. (1987). Melatonin: Its antagonism of thyroxine’s antisomatotrophic activity in male Syrian hamsters. Growth 51, 35– 43. Wright, M. L., Pikula, A., Cykowski, L. J., and 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. Wright, M. L., Cuthbert, K. L., Donohue, M. J., Solano, S. D., and Proctor, K. L. (2000). Direct influence of melatonin on the thyroid and comparison with prolactin. J. Exp. Zool. 286, 625– 631.
Reductions in Hamster Serum Thyroxine Levels by Melatonin Are Not Altered by Changes in Serum Testosterone Thomas H. Champney Department of Human Anatomy and Medical Neurobiology, College of Medicine, Texas A&M University Health Science Center, College Station, Texas 77843-1114 Accepted April 16, 2001
mechanism as castration-induced reductions.
Daily melatonin injections reduce reproductive and thyroid hormones in male Syrian hamsters. The interrelationship between the decline in these hormones is not known. To explore this relationship, male Syrian hamsters were divided into four groups: castrated, implanted with testosterone (5-mm silastic implants), both treatments, or neither treatment. One-half of each group of hamsters (n ⴝ 7 or 8) were injected with melatonin (25 mg) daily at 1730 h. The other half of each group received daily vehicle injections. Ten weeks later, the hamsters were anesthetized and decapitated. Testes weights, serum testosterone, and serum thyroxine levels were measured. As expected, testes and serum testosterone levels were uniformly low in all of the melatonin-treated hamsters. All of the melatonin-treated groups also had lower than normal thyroxine values irrespective of gonadal treatment. Interestingly, in the non-melatonin-treated hamsters, serum thyroxine values were decreased in the castrated group and increased in the testosterone-implanted group. These results suggest that castration can reduce serum thyroxine levels in male Syrian hamsters and that replacement of testosterone restores these levels to normal. Notably, the declines in thyroxine levels produced by daily melatonin injections were not restored by testosterone implants in castrated or intact hamsters. Therefore, melatonin-induced reductions in thyroxine are not mediated by concurrent reductions in serum testosterone levels. It appears that melatonin-induced reductions in serum thyroxine levels do not use the same 0016-6480/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
©
2001
Academic Press
Key Words: melatonin; thyroxine; testosterone; hamster; testes.
INTRODUCTION Endogenous pineal melatonin production occurs during the dark phase of the photoperiod, providing a signal to the rest of the organism that mirrors the photoperiod (Bartness et al., 1993; Reiter, 1987). Therefore, during the winter months, a longer period of melatonin production occurs. This period length can be mimicked by provision of daily late-afternoon melatonin injections to long-photoperiod-exposed animals. When Syrian hamsters are exposed to long photoperiods and given daily late-afternoon melatonin injections, they display numerous physiologic changes that parallel changes observed during short-photoperiod exposure (Bartness et al., 1993; Stetson and Watson-Whitmyre, 1984). The best known response is a decline in reproductive physiology that is demonstrated by a drastic reduction in testes size (from over 3000 mg to less than 500 mg). This decline in testes size produces a parallel decline in serum testosterone levels. The reproductive effects, however, are not the only physiologic changes that occur after daily afternoon 121
122
melatonin injections to hamsters; a reduction in thyroid physiology is also observed. The changes in thyroid physiology in hamsters are usually demonstrated by a reduction in serum thyroid hormones or free thyroxine index (Champney, 1988, 1989; Creighton and Rudeen, 1989; Petterborg and Rudeen, 1989; Reiter et al., 1988; Singh and Turner, 1972; Vaughan et al., 1982a, 1984; Vriend, 1984; Vriend and Reiter, 1977; Vriend et al., 1979, 1987). Changes in hamster thyroid morphology and serum thyroid stimulating hormone levels have also been observed (Vaughan et al., 1982b; Vriend, 1985; Vriend and Thliveris, 1985). In other species, the effects of melatonin on thyroid physiology are species specific with increases, decreases, or no changes depending on the species examined and the time frame of melatonin administration (Ashley et al., 1999; Esquifino et al., 1997; Gower et al., 1996; Krotewicz and Lewinski, 1994a,b; Levine et al., 1995; Ozturk et al., 2000; Rasmussen et al., 1999; Wright et al., 1996, 2000). Previous research in rats has found that changes in thyroid activity can be produced by alterations in gonadal hormone levels, specifically that castration reduces thyroid hormone levels and testosterone replacement restores or increases thyroid hormone levels (Chen and Walfish, 1979; Christianson et al., 1981; Paloyan et al., 1982; Steger et al., 1989). Therefore, it could be surmised that the effects of melatonin on serum testosterone levels in the hamster are responsible for the observed thyroid effects. The present study examined this interaction by investigating the impact of reduced serum testosterone levels (and its replacement) on serum thyroxine levels in control and melatonin-treated hamsters.
MATERIALS AND METHODS Male Syrian hamsters (80 –100 g) were purchased from Harlan Sprague–Dawley (Madison, WI). They were housed 2 per cage in a temperature-controlled (23°) room with long photoperiod (14L:10D h, lights on at 0500 h). Food and water were available continuously. The hamsters were subdivided into two large groups: one group (32 hamsters) received daily melatonin injections (25 g in 0.1 ml of 1:10 ethanolic saline
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Thomas H. Champney
subcutaneously) and the other group (31 hamsters) received vehicle (0.1 ml of 1:10 ethanolic saline subcutaneously). Both groups received their injections at 1730 h each day. Each of the groups was subdivided into four subgroups: (1) implanted with testosterone (subcutaneous 5-mm silastic capsule (1.5 mm ID, 2.5 mm OD) replaced 6 weeks later), (2) castrated (testes removal under methoxyflurane (metofane) anesthesia), (3) both treatments (implanted ⫹ castrated), and (4) left untreated (control). All subgroups contained 8 hamsters except for the vehicle/control group which had 7 hamsters. For the 10-week duration of the experiment, the hamsters were injected daily at 1730 h, were weighed weekly, and had their testosterone implants replaced at the beginning of the 6-week time point. At the end of the experiment, the hamsters were killed over a 2 day period with one-half of each subgroup killed on each day. One animal from each subgroup was killed sequentially to minimize the impact of time of day on tissue collection. The hamsters were killed by decapitation after metofane anesthesia. Body weights, testes weights, and trunk blood were collected. The blood was chilled (4°) and centrifuged for 30 min at 2000g, and the resulting serum was transferred to glass vials and frozen (⫺20°). Serum testosterone, thyroxine (T4), and triiodothyronine (T3) uptake levels were determined by radioimmunoassays (Diagnostic Systems Laboratories, Webster, TX) that were validated for use in the hamster (Champney, 1988). For the testosterone assay, the least detectable level was 0.006 ng/ml with an intraassay variation of 2.24%. For the thyroxine assay, the least detectable value was 0.199 g/dl and the intraassay variation was 4.55%. For the T3 uptake assay, the least detectable percentage uptake was 9% with an intraassay variation of 1.52%. Free thyroxine index (FTI) was calculated by multiplication of each serum thyroxine value by each T3 uptake value and division by 35 (a standard reference value) (FTI ⫽ T4 ⫻ T3 uptake/35) (Diagnostic Systems Laboratories; DSL15100 package insert). Statistical significance was determined by a threeway analysis of variance (melatonin vs castration vs implant) with the least squares method used as a post hoc test to determine significance between groups.
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TABLE 1 Body Weight, Testes Weight, and Serum T3 Uptake in Male Syrian Hamsters That Received Vehicle or Melatonin (25 g) at 1730 h for 10 Weeks Vehicle
Body weight (g) Testes weight (g) T3 uptake (%)
Melatonin
Con
Imp
Cas
I⫹C
Con
Imp
Cas
I⫹C
142 ⫾ 4 3.87 ⫾ 0.17 60.3 ⫾ 0.5
144 ⫾ 5 3.44 ⫾ 0.14 58.5 ⫾ 0.8
159 ⫹ ⫾ 5 — 57.5 ⫹ ⫾ 0.9
160 ⫹ ⫾ 4 — 58.0 ⫹ ⫾ 0.3
149 ⫾ 6 0.49* ⫾ 0.04 61.1 ⫾ 0.6
144 ⫾ 6 0.33* ⫾ 0.09 58.5 ⫾ 0.7
150 ⫾ 6 — 58.6 ⫾ 0.7
145 ⫾ 3 — 59.7 ⫾ 0.4
Note. The hamsters were also implanted with testosterone (Imp, 5-mm silastic capsule), were castrated (Cas), received both treatments (I⫹C), or received neither treatment (Con). Values are means ⫾ standard errors with 7 or 8 hamsters per group. ⫹ P ⬍ 0.05 vs vehicle/control. * P ⬍ 0.001 vs vehicle/control.
RESULTS At the end of the experiment, daily melatonin injections increased body weight (F ⫽ 4.09, P ⫽ 0.048) (Table 1). Castration also increased body weight (F ⫽ 5.66, P ⫽ 0.021). In addition, there was a significant interaction between melatonin and castration on body weight (F ⫽ 5.48, P ⫽ 0.023). Body weights in the vehicle/castrated and the vehicle/implanted ⫹ castrated hamsters were significantly increased over those in the vehicle/control hamsters (P ⬍ 0.05) (Table 1). Testes weights in all of the intact, melatonintreated hamsters were significantly lower than those in the intact, vehicle-injected hamsters (P ⬍ 0.001) (Table 1). As expected, there were significant differences in serum testosterone levels in melatonin-treated hamsters (F ⫽ 59.44, P ⫽ 0.0001), in castrated hamsters (F ⫽ 112.52, P ⫽ 0.0001), and in testosterone-implanted hamsters (F ⫽ 61.13, P ⫽ 0.0001) (Fig. 1). In addition, there were significant interactions between melatonin and castration (F ⫽ 79.46, P ⫽ 0.0001), between melatonin and testosterone implants (F ⫽ 13.15, P ⫽ 0.001), and among all three factors (F ⫽ 6.08, P ⫽ 0.017). Serum testosterone values in all of the melatonin-treated hamsters and in the vehicle/ castrated and vehicle/implanted ⫹ castrated hamsters were significantly lower than those in the vehicle/ control animals (P ⬍ 0.01) (Fig. 1). Significant difference were observed in serum thyroxine levels in the melatonin-treated hamsters (F ⫽ 74.35, P ⫽ 0.0001) and in the testosterone-implanted hamsters (F ⫽ 13.81, P ⫽ 0.001) (Fig. 2). Interactions
between melatonin and castration (F ⫽ 9.11, P ⫽ 0.004) and between melatonin and testosterone implants (F ⫽ 7.16, P ⫽ 0.01) were also significant. Serum thyroxine values were significantly reduced in all of the melatonin-treated hamsters and in the vehi-
FIG. 1. Serum testosterone levels in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
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Thomas H. Champney
trated group, whereas it was increased in the vehicle/ implanted group compared to that in the vehicle/ control hamsters (P ⬍ 0.01) (Fig. 3).
DISCUSSION This study found that castration of vehicle/control hamsters reduced serum thyroxine levels and that this reduction was reversed by testosterone replacement (Figs. 2 and 3). This finding suggests that testosterone has an impact on thyroid physiology in male hamsters similar to that found in other species (Chen and Walfish, 1979; Steger et al., 1989). This is further supported by the lack of major changes in the thyroid binding proteins as determined by the T3 uptake assay (Table 1). Previous research shows little or no effect of gonadal hormone manipulations on thyroid binding
FIG. 2. Serum thyroxine levels in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
cle/castrated group, whereas they were increased in the vehicle/implant group compared to those in the vehicle/control hamsters (P ⬍ 0.05) (Fig. 2). Castration significantly reduced T3 uptake (F ⫽ 6.28, P ⫽ 0.015) (Table 1). There was also a significant interaction between castration and testosterone implants on T3 uptake (F ⫽ 10.92, P ⫽ 0.002). T3 uptake in the vehicle/castrated and the vehicle/implanted ⫹ castrated hamsters was significantly decreased compared to that in the vehicle/control hamsters (P ⬍ 0.05) (Table 1). The free thyroxine index was significantly different in the melatonin-treated hamsters (F ⫽ 76.09, P ⫽ 0.0001) and in the testosterone-implanted hamsters (F ⫽ 13.23, P ⫽ 0.001) (Fig. 3). Significant interactions between melatonin and castration (F ⫽ 11.64, P ⫽ 0.001) and between melatonin and testosterone implants (F ⫽ 7.36, P ⫽ 0.009) were also found in the free thyroxine index. The FTI was lower in all of the melatonin-treated hamsters and in the vehicle/cas-
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FIG. 3. Serum free thyroxine index in male Syrian hamsters that received vehicle or melatonin (25 g) at 1730 h for 10 weeks. The hamsters were also implanted with testosterone (5-mm silastic capsule), were castrated, received both treatments, or received neither treatment. Values are means ⫾ standard errors with 7 or 8 hamsters per group.
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proteins (Emerson et al., 1990; Valenti et al., 1982) and the current data are consistent with these findings. This study also found that, irrespective of reproductive state, daily melatonin injections reduced serum thyroxine levels and the free thyroxine index (Figs. 2 and 3). This indicates that melatonin-mediated reductions in serum thyroxine levels are not due to concurrent declines in serum testosterone levels. These results suggest that melatonin’s effects on the thyroid system are independent of melatonin’s effects on the reproductive system. Interestingly, since testosterone replacement did not correct the melatonin-induced decline in thyroxine levels, it appears that there are at least two mechanisms that can modify thyroid hormone levels: a melatonin-mediated mechanism and a testosterone-mediated mechanism. The current hypothesis for melatonin-induced changes in serum thyroxine levels suggests that melatonin acts at the hypothalamic level, altering the downstream hypothalamic–pituitary– endocrine organ axis (Hoover et al., 1992; Miguez and Aldegunde, 1996; Vriend, 1989). These authors suggest that melatonin alters neurotransmitter levels in the hypothalamus, which modify the current hypothalamic set point, putting in motion the observed endocrine effects. The current results do not directly support or refute this hypothesis. They do, however, provide some indirect support for the hypothesis by indicating that the effects of melatonin on the thyroid system are independent of its effects on the reproductive system. It is not known whether testosterone regulation of thyroid function acts by a similar hypothalamic mechanism or at the thyroid directly, but the present results suggest that two separate mechanisms exist for thyroid hormone regulation—a melatonin-mediated response and a testosterone-mediated response. In summary, daily afternoon melatonin injections reduced serum thyroxine content and free thyroxine index in male hamsters irrespective of the hamster’s serum testosterone levels. This indicates that melatonin-mediated declines in thyroid hormone levels are not regulated by the concurrent decline in serum testosterone content. There does, however, appear to be a testosterone effect on thyroid hormone levels in hamsters with castration reducing thyroxine levels and testosterone replacement restoring these levels to normal.
ACKNOWLEDGMENTS The technical help of Gregg Allen and Dr. Wei-Jung A. Chen is appreciated. These data were previously presented at the 82nd Annual Endocrine Society meeting (June 2000) (No. 1185).
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