AMER. ZOOL., 16:57-65 (1976).
Comparative Biochemistry of the Pineal Glands of Birds and Mammals
SUE BINKLEY
Department of Biology, Temple University,
Philadelphia. Pennsylvania 19122
SYNOPSIS. Melatonin is synthesized from serotonin in the pineal gland by N-acetyltransferase and hydroxyindole-O-methyltransferase (HIOMT). The activities of these two enzymes
have been compared in the pineal glands of rats, hamsters, chickens, and sparrows. Three
different patterns of pineal biochemistry have been observed in the four species. Evidence
for the regulation of pineal N-acetyltransferase activity by a noradrenergic system exists in
rats but not in chickens, hamsters, and sparrows.
The physiology and biochemistry of the ectomy on gonads (reviewed in Wight,
pineal glands of a number of species appear 1971; Reiter, 1972). No comparable effect
to be related in one important respect: of pinealectomy has been shown on the
They involve daily or circadian rhythms re- gonads of male sparrows (Donham and
lated to periods of environmental light and Wilson, l970a,b; Menaker et al, 1970).
dark. In this report, I shall present comIt is now common knowledge that the
parative biochemical data for four different pineal gland secretes melatonin (Lerner et
organisms: rats, hamsters, chickens, and al, 1958; Pelham et al, 1972). In rats,
sparrows. The information in the literature serotonin is converted to N-acetylserotonin
will be supplemented by data from recent by the enzyme N-acetyltransferase.
experimental studies.
N-acetylserotonin is converted to melatoSome functional aspects of the pineal nin by the enzyme hydroxy-O-methyltransgland have been revealed by pinealectomy. ferase, abbreviated as HIOMT (Weissbach
In sparrows, pinealectomy results in a per- etal, 1969; Axelrod and Weissbach, 1960).
manent loss of the circadian rhythms of N-acetyltransferase activity and melatonin
locomotor activity and of body temperature and serotonin levels in the gland have been
normally seen in a constant dark condition shown to exhibit clear-cut daily rhythms in
(Gaston and Menaker, 1968; McMillan, higher vertebrates (Lynch, 1971; Klein and
1972; Binkley, Kluth and Menaker, 1971). Weller, 1970; Quay, 1963). Activity of
This effect has thus far been reported only HIOMT has been found by some authors
for the two species of Passerine birds that to exhibit daily rhythms (Axelrod et al,
have been studied {Passer domesticus and1965; Pelham and Ralph, 1972); other auZonotrichia albicollis). In hamsters, pineal-thors, however, have not found rhythmic
ectomy prevents the degeneration of the activity of HIOMT (Binkley et al, 1973).
gonads that ordinarily occurs when the The activity of HIOMT changes in rats,
hamsters are kept in photoperiods of less chickens, and sparrows subjected to prothan 12 hours (Reiter, 1970; Gaston and longed (several-day) periods of constant
Menaker, 1967). The gonads of rats re- light (LL) or constant dark (DD), or lightspond similarly to photoperiod and pineal- dark cycles with differing light-dark ratios
ectomy (Reiter, 1968). Chickens sometimes (Axelrod et al. 1965; Lauber et al. 1968;
show an age-dependent effect of pineal- Barfussand Ellis, 1971).
To fully interpret the meaning of the
I thank Kathy Hall, Sylvia Goddes, Jules Fleischner, results of the experiments reported herein,
S. E. MacBride, C. L. Ralph, D. C. Klein, and the it is necessary to consider the regulation of
Temple University Computer Activity Center for their
assistance. The research was supported by grants to S. pineal N-acetyltransferase activity in some
Binkley: NSF GB-43215, NSF Institutional Supple- detail. Figure 1 summarizes data previously
ment Award, and Temple Grant-in-Aid.
published on N-acetyltransferase activity
57
58
SUE BINKLEY
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LIGHT CYCLE
LDI2M2
TIME (hours)
FIG. 1. Summary of daily rhythm in pineal N-acetyltransferase activity in the chicken. The solid line
( • — • ) is the daily rhythm in N-acetyltransferase activity in an LD12A2 light cycle represented by the
horizontal bar under the figure, (a) is the change that is
seen if chicks are subjected to a 4 hour dark period
during their normal light-time (A — A); (b) is the
change that is seen if chicks are subjected to a 4 hour
dark period during their expected dark-time ( A — • ) ;
(c) is the change that is seen if chickens are subjected to
light unexpectedly during their normal dark-time
(•—o). Closed symbols mean birds were killed using
the dark-phase procedure; open symbols mean birds
were killed using the light-time procedure. The results can be summarized as follows: (i) The enzyme
activity has a daily rhythm; (ii) During part of the
light-time the birds are refractory to dark, that is, dark
will not increase the enzyme activity; (iii) Light imposed during the normal dark-time causes a precipitous decrease in enzyme activity. Data are from Binkley et al., 1973, 1975.
in chickens. A daily rhythm in pineal
N-acetyltransferase activity was present.
Enzyme activity was high in the dark and
low in the light. In a light-dark LDI2A2
regime, chickens were experimentally exposed to unexpected periods of light and
dark to ascertain whether the enzyme activity was directly affected by the presence of
light and dark or whether there were refractory periods. The following results
were obtained: (i) During the normal lighttime, four hours of dark do not cause an
increase in pineal N-acetyltransferase activity (Fig. la); (ii) During the expected darktime, dark increases pineal N-acetyltransferase activity (Fig. lb); during the normal
dark-time, light causes a rapid decrease in
pineal N-acetyltransferase activity when it
was already high (Fig. lc). These results
are consistent with the hypothesis that the
dark-time rise in N-acetyltransferase activity is under control of an entrained endogenous circadian system that has a permissive period during the normal darktime. Evidence that there is such an endogenous circadian rhythm in N-acetyltransferase activity is derived from an unpublished study of the N-acetyltransferase
rhythm in DD in chicks (Binkley and Geller,
in preparation). The rhythm persists and
the period length is close to 24 hours; the
N-acetyltransferase activity decreases in anticipation of the expected onset of lights
(i.e., enzyme activity decreases without oc-
PINEAL GLAND: COMPARATIVE BIOCHEMISTRY
currence of a light stimulus as well as with
one).
Rats and chickens are distinctly different
in their pineal biochemistry. Eyes are
necessary for the rapid response to light in
rats, but nonvisual light perception can
mediate the response in chickens (Klein
and Weller, 1972; Binkley etal, 1975). Furthermore, the regulation of pineal
N-acetyltransferase activity in the rat involves the superior cervical ganglion and a
noradrenergic transmitter system (Klein et
al., 1970; 1971). This is not the case in
chickens (MacBride et al., 1973; Binkley et
al., 1975). In addition to these differences
in N-acetyltransferase activity regulation,
the response of HIOMT activity to constant
light or dark is opposite for rats and chickens (Axelrod et al., 1965; Lauber et al.,
1968).
In the work reported here, the following
key experiments were done to compare the
biochemistry of the pineal glands of rats,
hamsters, chickens, and sparrows: (1)
N-acetyltransferase activity and HIOMT
activity were compared in light vs. dark in
light-dark cycles to ascertain whether a
daily rhythm exists; (2) N-acetyltransferase
activity was measured 3 hours after a dose
of isoproterenol in vivo or 6 hours after
treatment with norepinephrine in vitro to
ascertain whether an increase in enzyme
activity occurred that would implicate an
adrenergic regulatory system as observed
for rats (norepinephrine in vitro, Klein et
al., 1970; isoproterenol in vivo, Deguchi
and Axelrod, 1972).
Furthermore, N-acetyltransferase and
HIOMT activities were measured in
hamsters that had been subjected to long
and short photoperiods in order to change
their gonadal state to determine whether
the enzyme activities were correlated with
photoperiodically induced changes in reproduction.
59
from Charles River. Chickens (Gallus domesticus. White Leghorn males and females)
were obtained as day-old chicks from
George Shaw in Philadelphia. Sparrows
(Passer domesticus) were trapped as juveniles
or adults in the vicinity of Philadelphia.
All animals were housed indoors in
rooms or environmental boxes with
fluorescent overhead lights (Sylvania F30
T12 Lifeline intensity — 4600 lux measured with an Eppley thermopile and
Keithley Instrument No. 153 null detector
microvolt meter) controlled by automatic
timers. Food (mixed grain for sparrows,
chick starter for chickens, and Purina lab
chow for rats and hamsters) and water were
provided ad libitum.
Assays. N-acetyltransferase activity was
measured using xh of each pineal gland as
described elsewhere (Klein and Weller,
1970; Binkley et al., 1973; Klein, 1972).
HIOMT activity was measured on Ms of a
pineal gland using either of two assays
(Klein, 1972; Pelham and Ralph, 1972;
Binkley et al., 1973). Two assays were used
since chicken pineal glands were more active using the modified assay, and the modified assay was not sensitive enough to detect rat HIOMT activity.
Protocol—Light-dark cycles. Animals were
kept in artificially programmed light-dark
cycles and were killed 2 hours before the
scheduled lights-off to obtain a light-time
point and 4 hours after the scheduled
lights-off to obtain a dark-time point. The
light cycles were shifted with respect to
solar time so that lights-off occurred at
1200 (noon) to facilitate experimentation
during the normal work day. All animals
were kept on the artificial cycles for at least
2 weeks prior to killing.
Protocol-Isoproterenol experiment (in vivo).
Animals were injected subcutaneously with
L-isoproterenol (10 mg/kg dissolved in 0.9%
saline) during the light-time of their lightdark cycles and sacrificed 3 hours later.
Control animals were injected with saline.
MATERIALS AND METHODS
Protocol—Norepinephrine experiment (in
Animals. Adult male rats (Rattus nor- vitro). Pineal glands were removed from
vegicus, outbred albino CrhCOBS animals sacrificed during the light-time of
CD(SD)BR) and adult male and female their light-dark cycles. The glands were
hamsters [Mesocricetus auratus, Lakeview organ cultured using the method described
outbred Lak: LVG(SYR)] were obtained by Klein (1972) with small modifications: A
60
SUE BINKLEY
plastic organ culture dish (Falcon 3010) was
used and the glands were supported with
millipore filters (HAWP 02500) in place of
a glass dish with a wire screen. After half an
hour of culturing, L-norepinephrine
monohydrate bitartrate was added to the
cultures so that the final concentration of
norepinephrine would be 10~ 4 M (i.e., 5 /xl
of norepinephrine dissolved in 0.01 N HC1
was added to the media while 5 /xl of 0.01 N
HC1 was added to the control media). After
6 hours of culturing, the glands were frozen and assayed as above.
for hamsters. Also noteworthy are the
quantitative differences in amounts of
dark-time N-acetyltransferase activity (note
variation in y-ordinates of Figure 2): on a
per milligram of pineal basis, hamsters had
only 14% of the activity of rats while chickens had 4.6 times and sparrows 2.8 times
the activity of rats.
Noradrenergic regulation of N-acetyltransferase activity (Fig. 2). Norepinephrine in
vitro and isoproterenol in vivo increased
N-acetyltransferase activity only in rats.
The chemicals did not increase N-acetylProtocol-Hamster gonad experiment. In the transferase activity in the pineal glands of
hamster gonad experiment, lighting the other three species.
Photoperiodic effect on N-acetyltransferase
schedules of LD14A0, LD10A4, LD16.8,
and LD8:16 were used to achieve photo- and H10MT activities (Fig. 4). T h e two paralperiods of different lengths. After expo- lel experiments using different light cycles
sure to the cycles for varying periods of were qualitatively similar. The testis size
time (specified in the figure captions), was substantially smaller on short photohamsters were sacrificed by decapitation periods than on long ones. There was a
and then pineal glands and testes were slight positive relationship between
quickly dissected from the hamsters. The HIOMT activity and testis weights, but the
testes were preserved in formalin and were changes in HIOMT activity were not nearly
so marked as in the testicular weights.
subsequently blotted dry and weighed.
HIOMT activity. There were quantitative
Protocol-Sacrifice. All animals were killed
by decapitation followed by quick dissec- differences in HIOMT activities in that the
tion of the pineal glands. This procedure birds had markedly higher values on a
for the pineal gland took 30 seconds on the pineal weight basis than the mammals (Taaverage for chicks and slightly longer for ble 1). Differences between light and dark
the other species. "Dark" killed animals values were not marked in any species. The
were briefly exposed to a G.E. 7.5-watt red dark/light ratio was 2 for rats, 0.9 for
photographic safelamp during decapita- hamsters, 1.2 for chickens, and 0.9 for
tion followed by dissection of the pineal sparrows. Hamsters had 76% of the darktime HIOMT activity of rats; chickens had
glands under a white incandescent light.
Pineal weights. Since N-acetyltransferase 1200 times the activity; and sparrows had
activity is rapidly lost in handling, separate 68 times the activity of rats.
series of animals were killed to obtain
pineal gland weights for size comparisons.
DISCUSSION
The glands were weighed in the wet state.
Daily rhythms in pineal N-acetyltransBody weights were obtained for these aniferase activity are found in rats (Klein and
mals prior to sacrifice.
Further experimental details are present Weller, 1970), chickens (Binkley et al.,
1973), quail (Backstrom et al., 1972) and
in the legends of the figures and tables.
sparrows. The hamster is the first species to
be studied in which a distinct daily rhythm
RESULTS
in the enzyme activity has not been found.
Daily rhythm in pineal N-acetyltransferase ac- The data presented here do not preclude
tivity (Figs. 1,2,3). Daily rhythms were pres- the possibility that the hamster may have a
ent in the rat, chicken, and sparrow. On a daily N-acetyltransferase activity rhythm in
pineal weight basis, the dark/light ratio of other light cycles than those tested, or that
N-acetyltransferase activity was 21 for rats, the peak activity was simply missed because
27 for chickens, 21 for sparrows, and 1.2 of the differences in the shape of the oscilla-
PINEAL GLAND: COMPARATIVE BIOCHEMISTRY
RAT
8.5
14.2
15.8
HAMSTER
CHICKEN
SPARROW
FIG. 2. Effects on pineal N-acetyltransferase activity of
three treatments in four species: (1) effect of
norepinephrine (10~4M) on enzyme activity in pineal
glands in organ culture; (2) effect of injection of 10
mg/kg (s.c.) L-isoproterenoI in vivo; dark versus lighttime values in a light-dark cycle. Numbers below each
pair of bars are the ratio of the N-acetyltransferase
activity values of the treated to the control group and
of dark to light. Note that the ordinates are not the
same for all species which reflects a quantitative difference in activity. All values are averages for groups of
four animals ± one standard error. Enzyme activity
was detectable for all groups, i.e., well above experimental blanks. (LD data for chickens are from Binkley
etal., 1973)
tion between the hamster and the other
species. Of the species tested (rats, chickens, hamsters, and sparrows), only for rats
was there any implication of adrenergic
mechanisms in the regulation of pineal
61
N-acetyltransferase activity. This is despite
the fact that noradrenergic terminals
and/or connections with the superior cervical ganglia have been implicated in some
way for the regulation of the pineal glands
of chickens and hamsters as well as rats
(MacBride, 1973; Reiter and Hester, 1966;
Klein et al., 1971). Possible dose-response
or enzyme assay optima differences among
the species are not excluded at this time.
Three patterns of pineal N-acetyltransferase are thus seen in the four species: (1)
Rats have a daily rhythm and a betaadrenergic system for regulation of the
rhythm; (2) Sparrows and chickens exhibit
the rhythm but not the beta-adrenergic
regulation; and (3) Hamsters have neither a
marked rhythm nor beta-adrenergic regulation of N-acetyltransferase. Noteworthy
among these patterns and the results above,
is the fact that only in rats is N-acetyltransferase activity clearly regulated by a betaadrenergic system and the superior cervical
ganglion. This fact must be borne in mind
when studying the system in other species
and for application to studies of humans.
In the photoperiodism experiment, the
melatonin-producing enzyme activities do
not correlate well with gonadal state in the
hamster so as to implicate melatonin as the
anti-gonadal pineal factor. Evidence is now
accumulating that melatonin is not the
major pineal anti-gonadal factor (Benson et
al, 1971; Vaughane<a/., 1974; Kexteretal,
1974) in contrast with reports indicating
that it might be the factor (Yochim and
Wallen, 1974; Wurtman, Axelrod and
Kelly, 1968). The experiment reported
herein, however, does not exclude the possibility that melatonin might be an antigonadal factor.
Regarding the possible role of the pineal
gland in antigonadal function, the experiment reported herein is subject to further
reservations. It is possible that the enzyme
activities should be measured while the
gonads are in a state of flux in response to a
lighting change instead of in animals whose
gonads have attained a relatively stable
large or small state. Barfuss and Ellis (1971)
found that sparrow HIOMT activity varied
in an annual cycle that correlated negatively with testis growth. Parenthetically,
62
SUE BINKLEY
SPARROWS
LDI2H2
24
12
TIME (hr)
FIG. 3. Daily rhythm in sparrow pineal N-acetyltransferase activity. Data from two separate experiments
have been combined to make this curve (closed symbols, experiment 1; open symbols, experiment 2).
Each point represents the average N-acetyltransferase
activity from 4 birds ± one standard error. The birds
were kept in the LD12:12 cycle (indicated by the horizontal bar) for at least two weeks prior to sacrifice; the
sparrows were adults of undetermined age and both
sexes.
since the sparrow pineal gland has not been
found to control photoperiodically induced
changes in testis size (Donham and Wilson,
l970a,b; Menaker et al., 1970) the implication is that even where HIOMT activity varies seasonally, the pineal gland does not
control seasonal reproductive changes.
The photoperiodic effect on HIOMT activity seen in the hamsters in this investigation
is not striking. The data contrast with
findings for HIOMT in rats and in
hamsters where blinded animals have
higher HIOMT activity and smaller testes
than animals which were not surgically
blinded (Reiter et al.. 1969; Axelrod et al.,
1965; Eichler and Moore, 1971). The difference may be attributable to the use of
long vs. short photoperiods in the experiment reported here whereas the other investigators used blinding, constant light,
TABLE 1. Pineal HIOMT activity in light versus da rk for four species."
HIOMT activity
(pmoles/pineal gland/hr)
Species
Rat
Hamster
Chicken"
Sparrow
a
Assay
type
R
R
C
R
Light
25
27
121000±9800
810
LD cycle
Pineal weight
(nig)
Body weight
Dark
52
23
14200O±127OO
756
10:14
14:10
12:12
12:12
1.1 ±0.20
0.64±0.14
2.5±0.10
0.33±0.05
178 ±10
141 ± 5
695 ±25
22.1± 0.3
(g)
R represents the Klein type assay for HIOMT, C means the Pelham type assay was used. Data are from
pooled samples or are averages of activity for four animals ± one standard error. Body and pineal weights were
obtained in separate experiments using male rats, male hamsters, male chickens (8-weeks old), and sparrows of
both sexes. Data for chickens are from Binkley et al., 1973.
b
statistically significant P < .05.
PINEAL GLAND: COMPARATIVE BIOCHEMISTRY
: 400-1
1I
300-
"200-
N-acetyltransferase
activity
I 1000-
..
80-|
?
60H
HIOMT
activity
I
o.
20H
0J
2-
I
I-
Testis
weights
0LD8I6
LDI66
FIG. 4. Pineal enzyme activity and testis weights in
long and short photoperiods. The bar graphs represent data from an experiment in which LD8A6 and
LD16.8 were the experimental lightcycles. The data to
the side of the bars are from an experiment in which
LD14-A0 and LD10A4 were the light cycles. In the
latter experiment, the enzyme activities were measured in both light (open symbols) and dark (closed
symbols). In the first experiment (bar graph), 90-100g
hamsters were put into the controlled environmental
chambers and kept there for 96 days; lights-off occurred at 1300; killing time was 2300. In the second experiment (points to the right of bar graph) adult
hamsters were placed in LD10.14 for 61 days before
they were killed; killing times were 1000 and 1600,
lights-off occurred at 1200. The points represent
means from four or five animals ± one standard error
except forthe HIOMT values(14: lOand 10:14)which
are means of duplicate assays on pooled samples.
and constant dark in their experiments.
HIOMT activity is substantially greater
in sparrows and chickens than in hamsters
and rats. This difference may be partly
explained by the fact that there appear to
be different properties of the enzyme in
birds and mammals (Axelrod and Lauber,
1968). No distinct daily .rhythms in
HIOMT activity were seen in any of the
species in this study, but the two-point experiments reported here do not absolutely
rule out the existence of a daily rhythm in
HIOMT activity.
63
Chemical rhythms in the pineal gland
have been studied in other avian species.
Quail have been studied and found to have
daily changes in their pineal enzyme activities: 1.8 to 2.4 fold for N-acetyltransferase activity (Backstrom et ai, 1972), 1.1
to 1.3 fold for HIOMT activity (Backstrom
etal, 1972; Alexanderrfa/., 1970a,b), 5 fold
for melatonin (Lynch, 1971). In quail,
melatonin changes shift if the photoperiod
is reversed (Ralph etal., 1967), and quail do
not require the superior cervical ganglion
for gonad responses though they have
photoperiodic control of oviposition. In
this species the ganglion is believed to be
connected to the pineal gland by innervation (Ralph etal., 1970). Daily changes have
also have been reported for pigeons (i.e.,
serotonin levels and HIOMT activity,
Quay, 1966) and for African weaver birds
(melatonin content, Ralph et al., 1967).
In comparative studies of the type reported here, it is hoped that the biochemistry can be correlated in some meaningful way with the physiology, environmental adaptations, or the evolution of the
different species.
Physiologically, there is as yet no clear
correlation between those species in which
the pineal gland is important for photoperiodically regulated reproductive function and those species in which biochemical
rhythms occur. Nor is the control of endogenous circadian locomotor activity correlated with the presence or absence of
rhythmicity in the pineal enzyme activities
as yet. There does seem to be more evidence for the observation made at the beginning of this report, that circadian
rhythms and light/dark effects are common
aspects of pineal function.
In their behavioral relationships to environmental illumination, rats and
hamsters are nocturnal; chickens and sparrows are diurnal. A priori one would expect
opposite biochemical responses to environmental lighting in the diurnal versus
nocturnal groups. This expectation is not
borne out when one examines the results
for pineal N-acetyltransferase activity. In
all the species where N-acetyltransferase
activity is high, it is high during dark, regardless of whether the species is behavior-
64
SUE BINKLEY
ally nocturnal (rats) or diurnal (sparrows,
chickens, and quail).
From an evolutionary standpoint, there
seems to be a divergence between mammals
and birds in terms of the control system for
pineal enzyme activities. Chickens have
nonvisual light perception (Binkley et al.,
1975; MacBride et al., 1973; Lauber et al,
1968), do not require the superior cervical
ganglion for light-dark responses (Lauber
et al, 1968; MacBride et al, 1973), and do
not appear to use the beta-adrenergic system for N-acetyltransferase activity regulation. The rat does require its eyes (Klein
and Weller, 1970 and 1972), the superior
cervical innervation (Klein et al, 1971), and
a beta-adrenergic system to control pineal
enzyme activities (Klein et al, 1970). That
the hamster does not fit clearly into either
scheme is indicative of a further evolutionary divergence within the mammalian
group.
The experimental results together with
observations from the literature support
the idea that more than two systems are
utilized for environmental regulation of
the pineal gland enzyme activity. Why the
animals have so diverged, and what exactly
are the functions of the enzyme activities
and melatonin, are questions yet to be
answered.
NOTE ADDED IN PROOF: Since this
manuscript was prepared, it has been shown
by Rudeen and his colleagues (Rudeen,
P. K., R. J. Reiter.and M. K. Vaughan. 1975.
Pineal serotonin-N-acetyltransferase activity
in four mammalian species. Neuroscience
Letters 1:225-229) that hamsters do exhibit
a daily rhythm of enzyme activity. Data
taken in LD16:8 and presented in the form
of a curve revealed a single point of peak
activity at 8 hours after lights-out. This peak
activity value was three times the values obtained at other points (taken at 4 hour intervals).
In experiments reported in the present
paper (Figs. 2, 4) points were obtained at 4
and 10 hours after lights-out and at 2 and 12
hours after lights-on. The data at these
points agree with the data published by
Rudeen et al. at the comparable time points.
The finding by Rudeen et al., that hamsters
have an N-acetyltransferase activity rhythm
(though only 3-fold in comparison with 16to 17-fold in rats, chickens, and sparrows),
may permit eventual correlation of biochemical rhythms in pineal melatonin synthesis with photoperiodic regulation of reproduction in hamsters.
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