Effects of pinealectomy and melatonin administration on thyroid follicles of blind syrian hamsters.код для вставкиСкачать
THE ANATOMICAL RECORD 211:29-33 (1985) Effects of Pinealectomy and Melatonin Administration on Thyroid Follicles of Blind Syrian Hamsters JERRY VRIEND AND JAMES A. THLIVERIS Department of Anatomy, University of Manitoba School of Medicine, Winnipeg, Manitoba, Canada R3E OW3 ABSTRACT Since previous studies have shown that a n active pineal gland exerts a n inhibitory effect on circulating levels of thyroxin in the Syrian hamster, a study was conducted to determine whether the histology and ultrastructure of the thyroid gland supported the conclusions drawn from the hormone data. The ultrastructure of thyroid glands of blinded male Syrian hamsters was compared to that of intact controls kept under a 14W10D photoperiod, to that of blinded hamsters also pinealectomized, and to that of blinded hamsters receiving 80 pg/ml of melatonin in the drinking water. Serum thyroxin (T4) and serum thyrotropin (TSH) concentrations were determined by radioimmunoassay. After 10 weeks serum thyroxin concentrations were less than 50% of controls and concentrations were significantly reduced. EM examination revealed that blinded hamsters had a n increased number of follicular cells with flattened epithelium and nondilated endoplasmic reticulum compared to intact controls. In blinded hamsters that were pinealectomized or treated with melatonin in the drinking water, the ultrastructure of the thyroid was not different from controls and serum thyroxin concentrations were restored to near normal. These ultrastructural data support the conclusion that the pineal gland is required to obtain inhibition of the pituitary-thyroid axis in blinded hamsters and that melatonin has a counter-inhibitory effect when administered via the drinking water. Reduced concentrations of circulating thyroxin and TSH are among the results of light restriction in the Syrian hamster (Vriend and Reiter, 1977; Vriend et al., 1982). Placing hamsters in short photoperiod or blinding them by orbital enucleation results in depression of circulating thyroxin levels as early as 3 weeks after initiation of treatment (Vriend et al., 1979). The role of the pineal gland in this phenomenon was demonstrated by data showing that if blinded hamsters or hamsters kept under short photoperiod were pinealectomized they had circulating T4 levels that were not different from controls (Vriend et al., 1977: Vaughan et al., 1982). This effect of the pineal gland, like its effects on gonads, appears to be mediated by melatonin. The effect of melatonin depends on the mode of administration (Reiter, 1980; Vriend et al., 1982).Acute injections of microgram amounts of melatonin given toward the end of the light cycle inhibit T4 concentrations and result in gonadal involution. Continuously available melatonin in subcutaneous depots or in the drinking water, on the other hand, prevents the effects of light restriction (Vriend et al., 1982; Gibbs and Vriend, 1983; Vriend and Gibbs, 1984). Although the effects of photoperiod, pineal, and melatonin on thyroid hormones are now well documented, little is known concerning the effects of the pineal or melatonin on the structure of the thyroid gland. Thepur- 0 1985 ALAN R. LISS, INC pose of the present study was to investigate the effects of light restriction on the histology and ultrastructure of the thyroid glands of Syrian hamsters and to determine whether the effects of light restriction were influenced by pinealectomy and melatonin in the drinking water. An additional goal of the study was to determine whether light microscopic and ultrastructural data supported the conclusions drawn from the hormone data. MATERIALS AND METHODS Sixteen young adult male hamsters (90-110 gm)were used for this investigation. The animals were housed four per cage in a room automatically illuminated for 14 hours daily (L:D 14:lO). Lights were on from 6:30 A.M. to 8:30 P.M. Food and water water were continuously available. The animals were divided into the following groups: 1) control hamsters, 2) hamsters that were blinded by bilateral orbital enucleation, 3) hamsters that were blinded and pinealectomized, 4) hamsters that were blinded and had melatonin added to their drinking water at a concentration of 80 pg/ml. Pinealectomies were performed in hamsters anesthetized with pentobarbital. Bilateral orbital enucleation Received May 14,1984;accepted July 31,1984 30 J. VRIEND AND J.A. THLIVERIS was performed under ether anesthesia 2 days after pinealectomy; at this time melatonin was added to the drinking water in the required concentration. A fresh solution was prepared twice weekly. The water bottle containing melatonin was covered with aluminum foil to prevent light-induced oxidation of the melatonin. All animals were kept for 10 weeks after which they were sacrificed. Following decapitation, trunk blood samples were collected and serum stored frozen for later hormone analysis. Gonads were dissected out at this time and their weights recorded. T4 concentrations were determined by radioimmunoassay using reagents and protocol of Nuclear Medical Laboratories (Dallas, Texas). TSH concentrations were determined by RIA using NIAMDD reagents. The data were analyzed by analysis of variance and t test for multiple means. For ultrastructural study, one lobe of the thyroid glands of control and experimental animals was removed, cut into small pieces, and fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4)for 2 hours at 4°C. After an overnight rinse in 0.1 M phosphate buffer (pH 7.4)containing 0.2 M sucrose, the tissues were postfixed in 1%osimum tetroxide in 0.1 M phosphate buffer (pH 7.4) for 2 hours at 4"C, dehydrated in ascending concentrations of ethanol, and embedded in epon 812. Thick 1 p ) sections were cut and stained with toluidine blue and examined for routine orientation. Thin sections were stained with uranyl acetate and lead citrate and viewed and photographed in a Philips EM 201 electron microscope. For light microscopic study, the remaining lobe of the thyroid glands were fixed in Bouin's solution and stained with hemotoxylin and eosin. RESULTS The T4 and TSH data of this study are shown in Figure 1. Blinding resulted in a significant decrease (P < 0.01) in T4 to 38% of controls. This depression was prevented by pinealectomy. Addition of melatonin to the drinking water for 10 weeks resulted in partial restoration to normal. Similar results were obtained for serum TSH concentrations; blinding resulted in a significant (P < 0.01) reduction which was prevented by pinealectomy. Testicular weights were reduced to 13%of controls by blinding (P < 0.001). Pinealectomy restored testicular weights to 90% of controls and melatonin adminis- -5 -4 E 6- 2 v) 0 - -3 2 -2 ? *- - 5 v) c W a E 2 v) 0 W 2- -1 ' 0- Control Blind BI-Px -0 Bl-kt Fig. 1. Effects of blinding, pinealectomy, and melatonin administration on serum T4 and TSH concentrations. *P <0.01 compared to controls. Bars indicate means + standard errors. tration to 84%of controls, confirming previous reports (Gibbs and Vriend, 1983; Vriend and Gibbs, 1984). Light microscopy of thyroid glands revealed that the follicles of thyroid glands of control hamsters were comprised of cuboidal epithelium (Fig. 2). In blinded hamsters most of the follicles also appeared to be cuboidal, but there was an increased number of follicles with squamous epithelium, particularly near the center of the gland (Fig. 3). Ultrastructural data confirmed that blinded hamsters had an increased number of follicular cells with flattened epithelium (Figs. 4 and 5). These cells lacked the dilated endoplasmic reticulum found in cuboidal epithelium. Histology and ultrastructure of the thyroid glands of blinded, pinealectomized hamsters and of thyroid glands of blinded hamsters receiving melatonin in the drinking water were not different from controls. DISCUSSION The results of this study confirm that light restriction of hamsters by bilateral orbital enucleation results in a substantial decrease in circulating levels of thyroxin (Vriend and Reiter, 1977).The decrease observed in this study was somewhat greater than that observed in prior studies, presumably because the experiment was allowed to continue through 10 weeks, somewhat longer than in prior studies. The reversal of the depression by pinealectomy also confirmed previous reports (Vriend et al., 1977: Vaughan et al., 1982). The decreased serum TSH and its reversal by pinealectomy in the present study suggest that the decreased T4 is due to decreased secretion of TSH. Short photoperiod has been reported to reduce plasma levels of TSH and T4 in female hamsters (Vriend et al., 1982). The histological and ultrastructural studies also suggest reduced thyroid secretion in blinded hamsters. Light microscopic study showed an increased number of follicles with flattened epithelium and reduced colloid near the center of the gland. Previous investigators have noted that the central follicles are more active than peripheral follicles in rodents (Nadler, 1953; Nadler et al., 1954). There appeared to be no difference in the large, peripheral follicles of control and blinded hamsters. These follicles are apparently used as long-term storage sites (Nadler et al., 1954).Ultrastructural study also provided evidence for an increase in number of follicular cells with flattened epithelium; these cells lacked the dilated endoplasmic reticulum found in cuboidal follicular cells. Since the light and electron microscopic data suggested reduced thyroid activity they were in agreement with the T4 and TSH data. The data showing that pinealectomy reversed the effects of blinding support the view that a pineal gland activated by light restriction has an inhibitory effect on release of TSH by the pituitary. Light and electron microscopic study lent support to this view, since the histology and ultrastructure of blinded, pinealectomized hamsters was not different from that of controls. The present study showed that adding melatonin to the drinking water was effective in restoring serum T4, serum TSH,and gonadal weights of blinded hamsters to near normal. No difference in ultrastructure of control and blinded hamsters receiving melatonin in the drinking water could be detected. The present study does not support the conclusion of Panda and Turner (1968) that PINEAL INHIBITION OF THYROID Fig. 2. Light micrograph of thyroid of control hamster. Noter cuboidal epithelium (arrows). x460. Fig. 3. Light micrograph of thyroid of blinded hamster. Note flattened epithelium (arrows). x460. 31 32 J. VRIEND AND J.A. THLIVERIS Fig. 4. Electron micrograph of thyroid of contrnl hamster. Nnt,e dilated profiles of endoplasmic reticulum (arrows). Co, colloid; FC, follicular cells, ~ 5 , 6 0 0 . Fig. 5. Electron micrograph of thyroid of blinded hamster. Note flattened epithelial cells and nondilated endoplasmic reticulum (arrows).Co, colloid; FC, follicular cells, x 5,600. PINEAL INHIBITION OF THYROID melatonin has a goitrogenic effect. The present results, on the other hand, lead to the conclusion that the effects of the pineal and melatonin on the thyroid occur via a CNS site. Effects of pinealectomy on thyroid structure and function of rats and mice have been reported. Mild hypertrophy of the thyroid of mice and rats have been found (Houssay et al., 1966; Losada, 1977). Relkin (1972) observed that prepubertal rats kept in constant darkness for several days had significantly lower plasma TSH and plasma protein-bound iodine than rats kept in standard diurnal lighting. The role of the pineal in this phenomenon was suggested by data showing that pinealectomy prevented the effects of constant darkness. More recent data were provided by Niles et al. (19791, who found highly significant increases in plasma TSH of pinealectomized rats kept in a short photoperiod (1L/ 23D) room compared to SKam-operated-rats kept under conditions*These studies thus provided evidence that the Pineal gland had an inhibitory action on secretion of TSH of rats and mice. The interpretation that the pineal influences secretion of thyrotropin releasing hormone (TRH) in the hypothalamus melkin, 1978; Vriend, 1978) was supported by recent data showing significant effects of blinding and pinealectomy on hypothalamic TRH content in Syrian hamsters (Vriend and Wilber, 1983). Houssay et al. (1966) found that administration of melatonin prevented the hypertrophy obtained in mice after pinealectomy. In rats melatonin administration has generally resulted in inhibitory effects on thyroid function (Baschieri et al., 1963; Reiter et al., 1965; DeProspo et al., 1968);one exception is the report of Gordon et al. (1980). In hamsters melatonin may be either antithyrotrophic or prothyrotrophic depending on the mode of administration (Vriend and Reiter, 1977: Vriend and Gibbs, 1984). These effects of melatonin are similar to the antigonadal and progonadal effects of melatonin, which also depend on the mode of administration of melatonion (Gibbs and Vriend, 1983; Carter and Goldman, 1983a,b). The effects of melatonin on the thyroid seems to share a CNS site of action with the effects of melatonin on the Dituitarv-honadal axis. 33 Experientia, 19:15-17. Carter, D.C., and B.D. Goldman (1983) Antigonadal effects of timed melatonin infusion in pinealectomized male Djungarian hamsters (Phodopus sungorus sungorus): Duration is the critical parameter. Endocrinology, 113:1261-1267. Carter, D.C., and B.D. Goldman (1983) Progonadal role of the pineal in the Djungarian hamster (Phodopus sungorus sungorus): Mediation by melatonin. Endocrinology, 1131268-1273. DeProspo, N.D., L.J. DeMartino, and E.T. McGuinness, (1968) Melatonin's effect on 13'1 uptake by the thyroid glands in normal and ovariectomized rats. Life Sci., 7:183-188. Gibbs, F.P., and J. Vriend, (1983) Counterantigonadotropic effect of melatonin administered via the drinking water. Endocrinology, 113:1447-1451. Gordon, J., J.E. Morley, and J.E. Hershman (1980) Melatonin and the thyroid. Hormone Metab. Res., 12:71-73. Houssay, A.B., J.H. Pazo, and C.E. Epper (1966) Effects of the pineal gland upon the hair cycles in mice. J. Invest. Dermatol., 47:230234. Losada, J., (1977) Effects of experimental pinealectomy. Ann. Anat., 26t133-153. Nadler, N.J. (1953) The quantitative estimation of radioactive isotope by radioautography. Am. J. Roentgenol., 703314-823. Nadler, N.J., C,p, L&lond, and R. Bogoroch, (1954). m e rate of iodine metabolism by the thyroid follicles are a function of its size. Endocrinology, 54:154-172. Niles, L.P., G. Brown, and L.J. Grota, (1979) Role of the pineal gland in diurnal endocrine secretion and rhythm regulation. Neuroendocrinology, 29: 14-2 1. Panda, J.N., and C.W. Turner, (1968) The role of melatonin in the regulation of thyrotrophin secretion. Acta Endocrinol., 57:363-373. Reiter, R.J. (1980) The pineal and its hormones. Endocrine Rev., It109131. Reiter, R.J., R.A. Hoffman, and R.J. Hester (1965) Inhibition of 1311 uptake by the thyroid glands of male rats treated with melatonin and pineal extracts. Am. Zool., 5:727-728. Relkin, R. (1972) Effects of pinealectomy, constant light and darkness on thyrotropin levels in the pituitary and plasma of the rat. Neuroendocrinology, IOt46-52. Relkin, R. (1978) Use of melatonin and synthetic TRH to determine site of pineal inhibition of TSH secretion. Neuroendocrinology, 2 5 3 10-3 18. Vaughan, G.M., M.K. Vaughan, L.G. Seraille, and R.J. Reiter, (1982) Thyroid hormones in male hamsters with activated pineals or melatonin treatment. Prog. Clin. Biol. Res., 92.187-196. Vriend, J. (1978) Testing the TRH hypothesis of pineal function. Med. Hypotheses, 4:376-387. Vriend, J., and F.P. Gibbs, l(1984) Coincidence of counter-antigonadal and counter-antithyroid action of melatonin administration via the drinking water in male golden hamsters. Life Sci., 34:617-623. Vriend, J., and R.J. Reiter, (1977) Free thyroxin index in normal, melatonin treated 2nd blind hamsters. Hormone Metab. Res., 9:231-234. Vriend., J.., R.J. Reiter. and G.R. Anderson (1979) Effects of the nineal and melatonin on 'thyroid activity of male golden hamsters: Gen. ACKNOWLEDGMENTS Comp. Endocrinol., 38: 189-195. Vriend, J., B.A. Richardson, M.K. Vaughan, L.Y. Johnson, and R.J. This l?3E%3Xhwas supported by the Research Reiter, (1982) Effects of melatonin on thyroid physiology of female Council of Canada. hamsters. Neuroendocrinology, 35:79-85. Vriend, J., J.W. Sackman, and R.J. Reiter, (1977) Effects of blinding, LITERATURE CITED pinealectomy and superior cervical ganglionectomy on free thyroxin index of male golden hamsters. Acta Endocrinol., 86t758Baschieri, L., F. DeLuca, L. Cramarosa, C. Demartino, A. Oliverio, and 762, M. Negri, (1963) Modification Of thyroid activity by melatonin. Vriend, J.,and J.F. Wilber, (1983) Influence of the pineal gland on hdypothalamic content of TRH in the Syrian hamster. Hormone Res., 27:108-113.