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Effects of pinealectomy and melatonin administration on thyroid follicles of blind syrian hamsters.

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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
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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.
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