Journal of Pineal Research 6:367-373 (1989)

Effects of Daily Afternoon Melatonin

Administration on Body Weight and Thyroid
Hormones in Female Hamsters
Larry J. Petterborg and P. Kevin Rudeen zyx
Department of Anatomy, School of Medicine, University of Missouri, Columbia

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Female hamsters were treated each afternoon for 8 weeks with subcutaneous
injections of 25 kg of either melatonin or vehicle solution. Animals were sacrificed in
either the morning or afternoon of diestrus and proestrus, along with their respective
melatonin-induced acyclic pairs. Melatonin-treated hamsters had significantly greater
mean body weights than did the vehicle-treated hamsters (P < 0.05). Terminal mean
serum T4 levels and free thyroxine index (FT4I) were significantly reduced (P <
0.05) in melatonin-treated hamsters compared with values obtained from animals
subjected to the vehicle alone. In addition, T4 levels, FT41, T3 levels, and FT3I were
significantly influenced by day and time of sacrifice independently of the melatonin
treatment. T3 uptake was significantly lower on the morning of proestrus in
melatonin-treated animals. These results demonstrate that chronic afternoon melato-
nin administration in female hamsters results in the loss of estrous cyclicity, a

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significant gain in body weight, and the reduction of T4 levels and T3 uptake. Changes

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in serum T3 levels are a function of the time of sample collection and are not
influenced by melatonin treatment. In addition, these data indicate that thyroid
function in general changes during the estrous cycle in these animals.

Key words: estrous cycle, T3 levels, T 4 levels, free thyroxine index

INTRODUCTION
The pineal hormone melatonin provides a photoperiodically dependent
signal utilized by many different mammalian species to coordinate and regulate
physiological processes such as reproduction and metabolism [ Reiter, 1987;
Vriend, 1981; Wade and Bartness, 19841. In female Syrian hamsters, daily
afternoon melatonin injections lead to a loss of vaginal cyclicity and reduced

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gonadotropin and thyroid hormone levels [Vriend et al., 1982; Stetson and
Hamilton, 198 1 1. Melatonin, either experimentally administered or manipulated
by the daily light-dark cycle, most likely acts at the level of the neuroendocrine

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Received April 11, 1988; accepted October 7, 1988.

Address reprint request to Dr. L.J. Petterborg, Department of Anatomy, University of Missouri
School of Medicine, Columbia, M O 65212.

0 1989 A,bn R. Liss, Inc.

368 zyxwv
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Petterborg and Rudeen

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hypothalamus to influence the regulation of the anterior pituitary gland through
the various releasing factors and hormones [Vriend, 1981, Petterborg, 1986;
Steger et al., 19851. Although the site(s) and mechanism(s) of action of
melatonin on the gonadal and metabolic axes may be similar, they are not
necessarily the same. Wade and Bartness [ 19841 have demonstrated that the
administration of small doses of melatonin (2.5 pg/day) cause an increase in
body weight and fat content while not affecting reproductive function at all.
Previous studies concerning the effects of melatonin on thyroid hormones
in the female Syrian hamster have tended to group the experimental animals
solely on the basis of the melatonin regimen while overlooking time of day and
day of the estrous cycle as possible confounding variables. This approach could
obscure potentially interesting diurnal and cyclic variations in hormone levels.
To obtain a more complete description of the effects of melatonin on serum
thyroid hormone levels in female hamsters, the present study compared serum
thyroxine (T4), tri-iodothyronine (T3), free thyroxin index (FT4I), free T3
index (FT3I), and T3 uptake in groups of animals sacrificed in the morning or
afternoon of diestrus or proestrus. Hamsters rendered acyclic because of
melatonin treatment were killed at the same times on the same day as their
cyclic pairs, which received the vehicle treatment.

MATERIALS AND METHODS

Young, 60-70 gm female Syrian hamsters (Lak:LVG SYR strain; Charles

River, Wilmington, MA) were housed in clear plastic cages in a light- and
temperature-controlled (25 +- 2°C) room within the Department of Laboratory
Animal Medicine at the University of Missouri-Columbia. A lighting cycle of 14
hr of light and 10 hr of darkness (LD 14:10, lights on at 0400 hr) was maintained
throughout the experiment. The animals had free access to food and water.
Beginning soon after the arrival of the animals and continuing for the duration
of the study, hamsters were examined daily for vaginal discharge as a means of
monitoring estrous cycles [ Orsini, 19611. The day preceding the vaginal
discharge was designated as proestrus, and the day following the discharge was
denoted diestrus. All hamsters used in the experiment exhibited at least two
consecutive 4 day cycles prior to the initiation of daily melatonin injections. In
addition, the animals were weighed every week and the weights recorded.
Animals were paired according to the day of their respective cycle and
maintained within this matched pairing throughout the experiment. This group
pairing allowed for each hamster in one group of animals to receive a daily
subcutaneous (sc) injection of ethanolic-saline vehicle, while each animal in the
matched group received 25 pg melatonin (sc) each afternoon at approximately
1600 hr for 8 weeks. At the termination of the study, groups of vehicle-treated
animals were sacrificed by decapitation in the morning (0900-1000 hr) or
afternoon (1500-1600 hr) on either diestrus or proestrus along with their
acyclic melatonin-treated pairs. The resulting serum samples were stored frozen
( - 20°C) until assayed for thyroid hormones.
Serum T4 and T3 levels and T3 uptake (%T3U) were determined by
radioimmunoassay utilizing kits purchased from Diagnostic Products, Co. (Los
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Melatonin and Thyroid Hormones 369

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VEHICLE IETOH-SALINE1
180 -
c _

0
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-0 *
MELATONIN
MELATONIN II Z
ZSS uugg//D
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0
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= 120
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100

80
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100

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20 - 20 bQ
20 bQ

0
0 1 2 3 4 5 6 7 8
WEEKS OF TREATMENT
Fig. 1. Effect of daily afternoon melatonin (25 kg) or ethanolic (ETOH)-saline (vehicle)
injections for 8 weeks o n body weight ('P < 0.05) and estrous cyclicity in female Syrian hamsters.
The vertical axis o n the left indicates weekly mean body weight, whereas the vertical axis on the
right indicates the percentage (% ) of the animals in each treatment group that were cycling during
that week.

Angeles, CA). The free thyroxine index (FT4I) and free T3 index (FT3I) was
derived from the product of %T3U and total T4 or total T3, respectively.
The weekly body weight data were compared between the two treatment
groups by way of Student's t test. The hormone data were analyzed by
employing a three-factor multivariate analysis of variance (MANOVA), which
analyses all five dependent variables simultaneously. When significant interac-
tions were found, subsequent MANOVAs were conducted for each level of the
factors involved in the interaction term. Then, univariate analyses of variance
(ANOVAs) were conducted to see which of the dependent variables were
responsible for the overall MANOVA result.

RESULTS
Daily afternoon melatonin injections resulted in a significant acceleration
in the normal rate of weight gain in female Syrian hamsters maintained under
long laboratory photoperiods (Fig. 1). Animals that received the afternoon
melatonin injections showed a significant increase in body weight by 4 weeks of
treatment. This effect preceded any observable disruption of regular estrous
cycles, since 80% of the melatonin-treated animals remained cyclic after 5
weeks of treatment.
The results of the thyroid hormone determinations are presented in Table
1. The three-factor (melatonin or vehicle treatment, time of day, and day of
cycle) analysis revealed that the highest order interaction term was not
statistically significant (F [ 5,491 = 0.28). The only significant two-way interac-
tion was day by time (F [5,49] = 13.34; P < 0.0001). To interpret this effect,
a two-way MANOVA was conducted on day and treatment for each level of time
and a two-way MANOVA was conducted on time and treatment for each day.
Since all of these subsequent MANOVAs were statistically significant, the
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370 Petterborg and Rudeen

Treatment Time of No.

Diestrus
SAL
MEL
Sacrifice

AM
AM
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TABLE 1. Effect of Daily Afternoon Melatonin (MEL) or V e h i c l e (SAL.) I n j e c t i o n for 8 Weeks
on Thyroid H o r m o n e s in F e m a l e Syrian Hamsters

9
8
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( Cl.g/dl)

5.93? 0.44
2.64? 0.24
T3
(ng/dl)

59.72 ? 4.03
56.11 + 5.37
% T3U FT4I FT31

50.81 2 0.88 3.01 k 0.22 30.18 2 1.81

51.64 ? 0.60 1.37 k 0.12 28.82? 2.53
SAL PM 8 *
4.47 0.52 44.03? 5.50 49.45 2 0.79 2.19 t 0.24 21.50 + 2.57
MEL PM 8 2.15 ? 0.28 42.62 + 5.12 49.45 2 0.40 1.07 ? 0.14 21.04 2 2.49
Proestrus
SAL AM 7 5.72? 0.47 37.59 k 2.58 47.37 * 0.47 2.70 f 0.20 *
17.47 1.22
MEL AM 7 *
3.41 0.52 41.19 + 5.44 45.39 * 0.33 1.54 2 0.23 18.64 2 2.41
SA L PM 7 4.98 t 0.46 33.79 2 5.81 51.74 ? 0.85 2.58 2 0.25 17.46 2 1.18
MEL PM 7 3.46? 0.44 44.59 ? 3.54 50.30 * 0.79 1.73 k 0.20 22.53 f 2.05

ANOVA results were examined to determine which of the five dependent

variables ("4, T3, % T3U, FT41, and FT3I) were responsible for the significant
MANOVAs (Table 2). Serum T4 levels were significantly depressed in the
melatonin-treated hamsters when compared with vehicle-treated animals. Thy-
roxine levels were found to be significantly higher in the morning and on
proestrus than in the afternoon and on diestrus. Serum T3 only differed
between diestrus and proestrus at the morning time point. Melatonin adminis-
tration resulted in a marked reduction in the FT41, whereas the FT3I was not
affected by the treatment but was influenced by the time of day and day of the
cycle. The % T 3 U was significantly greater in cyclic vehicle-treated control
hamsters on proestrus than in melatonin-treated animals. Interestingly, in
general, the % T 3 U was lower during the morning of proestrus compared with
afternoon levels and just the opposute during diestrus.

DISCUSSION

The results of the present study indicate that when melatonin is given late
in the light phase of the photoperiod, it acts to reduce thyroxine levels (Table
1). An increase in body weight was also observed and occurred prior to any
noticeable disruption of regular estrous cycles (Fig. 1). In a similar experiment,
Wade and Bartness [ 19841 demonstrated that an increase in body weight caused
by afternoon melatonin injections was accomplished without a concomitant
increase in food consumption. This result could be explained by suggesting that
the effect of melatonin was to lower serum T4 levels, leading to a decrease in
the metabolic rate, while the hamsters maintained a normal level of food intake.
The net result would be the storage of excess calories as body fat. A subsequent
report by McElroy and Wade [ 19871 showed that an increase in body weight by
female hamsters kept in a short photoperiod was associated with an increase in
food intake. This apparent discrepancy between the effects of exogenously
administered melatonin and short photoperiod requires further investigation.
The results of the present study confirm the reports of others [Vriend et al.,
1982;Vaughan et al., 1982, 19841that serum T4 levels are significantly reduced
by melatonin administration and extend these observations to include the effect
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Melatonin and Thyroid Hormones

TABLE 2. Significant Differences (P < 0.05) in Thyroid

Hormone Levels as Presented in Table 1
Day of
sacrifice
Dependent
Variable Comparison
371

Proestrus T4 SAL > MEL

FT41 SAL > MEL
T3U SAL > MEI.
PM > AM
Diestrus T4 SAL > MEL
AM > P M
T3 AM > P M
FT4I SAL > MEI.
AM > PM
FT31 AM > PM
T3U AM > PM
Time of sacrifice
AM T4 SAL > MEI.
T3 DIE > P R O
FT41 SAL > MEL
FT31 DIE > P R O
T3U DIE > P R O
PM T4 SAL > MEL
P R O > DIE
FT41 SAL > MEL
P R O > DIE
T3U P R O > DIE

Data were analyzed by t tests following analysis of variance

(ANOVA). SAL, vehicle; MEL, melatonin; PRO, proestrus; DIE,
diestrus.

of the estrous cycle on thyroid hormones in the Syrian hamster (Table 1).
Vriend et al. [ 19821 have reported a depression in serum T4, T3, and TSH levels
and lowered free hormone indexes (FT4I and FT3I) in female hamsters
following melatonin injections for 8 weeks. Neither the age nor body weights of
the animals were reported. Separate observations by Vaughan and coworkers
[1982, 19841 provide data concerning the effects of age and melatonin on
thyroid hormones. In one study comparing melatonin treatment for 8 weeks in
young ( 4 month) and old ( 15 month) female hamsters, it was found that T4,
FT41, and FT3I were lowered by melatonin only in the old animals [Vaughan et
al., 1982). In the other study, plasma T4 but not T3 was significantly reduced in
“adult” female hamsters injected for 9 weeks with 25 pg of melatonin given
every afternoon [Vaughan et al., 19841. In the present study, it was found that
serum T4 levels were lowered by melatonin injections while T3 levels
fluctuated with the estrous cycle, unaffected by the administration of melatonin.
The current investigation demonstrated that at least serum T4 and FT4I
are reduced in acyclic female hamsters given melatonin when compared with
matched cyclic controls during both diestrus and proestrus. In the previous
studies, there were no attempts to discriminate differences in the serum levels
of thyroid hormones during the estrous cycle when compared with matched
melatonin-induced acyclic hamsters. The present study demonstrates that the
372 Petterborg and Rudeen zyxwv
day of the cycle and the time of the day need to be taken into consideration in
studies of the effects of melatonin o n the thyroid axis in female hamsters.
Changes in the pattern of circulating thyroid hormone levels during the
estrous cycle in the female Syrian hamster have not been well characterized.
Pituitary and ovarian reproductive hormones have been shown to fluctuate
greatly between days of the cycle [Bast and Greenwald, 1974; Saidapur and
Greenwald, 19781. Since a significant reduction in serum T3 occurred on
proestrus relative to diestrus in this study (Table l), a possible relationship
between regular variations in thyroid hormone levels and the estrous cycle
warrants further investigation. A thorough examination of such a relationship
may provide a possible explanation for observed variations in body weight that
occurs during the estrous cycle in hamsters [Schneider et al., 19861. These data
indicate that chronic melatonin treatment leads to the loss of estrous cyclicity,
an increased gain of body weight, and a reduction in serum T4 levels, while T3
levels appear to be determined by the day and the time of day even in the
absence of any overt sign of reproductive cyclicity.

ACKNOWLEDGMENTS

This work was supported by NIH grant Nos. HD17658 (L.J.P.) and
M O O 1 0 7 (P.K.R.). The authors thank the Office of Biostatistics, School of

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Medicine, University of Missouri, for their assistance in the analysis of the data.

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