Pineal melatonin synthesis and release are not altered throughout the estrous cycle in female rats.

Abstract

Melatonin times reproduction with seasons in many photoperiodic mammalian species. Whether sexual hormones reflect on melatonin synthesis is still debated. The aim of this work was to study, using a large panel of technical approaches, whether the daily profile of pineal melatonin synthesis and release varies with the estrous cycle in the female rat. The mRNA levels and enzyme activities of the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase were similar at the four stages of the rat estrous cycle. The endogenous release of melatonin, followed by transpineal microdialysis during six consecutive days in cycling female rats, displayed no significant variation during this interval. Taken together, the present results demonstrate that there is no regular fluctuation in the pineal metabolism leading to melatonin synthesis and release throughout the estrous cycle in female rats.

Full text

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Pineal melatonin synthesis and release are not altered throughout
the estrous cycle in female rats
Introduction
The pineal gland, via the rhythmic secretion of melatonin, is
essential for transducing seasonal changes in day length
(photoperiod) into physiological responses, such as sea-
sonal reproduction [1]. Interestingly, it has been proposed
that the gonadal steroids may have a negative feedback
effect on the pineal gland, especially on the melatonin-
synthesizing enzymes arylalkylamine N-acetyltransferase
(AANAT) and hydroxyindole-O-methyltransferase (HI-
OMT) [2]. In rats, however, various studies reported
contradictory results concerning the possible alteration in
melatonin synthesis and release throughout the estrous
cycle. Quay [3] showed a trend in reduction of melatonin
during the diestrus. Ozaki et al. [4] reported a sudden
reduction of melatonin during proestrus, whereas Johnson
et al. [5] observed a reduction during the estrous phase of
the cycle. Additionally, other studies with rats reported
estrous cycle-dependent variations in HIOMT activity with
the highest and lowest activities being, respectively, at
diestrus and estrus [6], diestrus and proestrus [7] or estrus
and proestrus [8]. No significant difference in AANAT
activity was found associated with the estrous stages [9, 10].
In other species (hamster, ewe and monkey), pineal or
plasma melatonin level do not appear to vary as a function
of the reproductive state [11–13].
This brief survey of the literature shows that there is no
clear evidence of estrous cycle-dependent variation in the
daily rhythm of pineal melatonin in female rats. The
purpose of this study was to use new methods including a
combination of microdialysis, radioenzymatic and in situ
hybridization techniques in an attempt to solve this
controversy. Using pineal microdialysis it is possible to
follow the endogenous release of melatonin in the same
animal throughout its estrous cycle, overcoming any
possible interference of inter-individual variations. Sec-
ondly, AANAT and HIOMT activities and melatonin
content were assayed at night in the same pineal of female
rats at different stages of the estrous cycle. Finally, pineal
AANAT and HIOMT mRNAs transcript levels were
measured at the various stages of the estrous cycle using
in situ hybridization.
Materials and methods
Animals
Adult female Wistar rats weighing 240–270 g were kept on
a 12-hr light/12-hr dark cycle in a temperature-controlled
room (21 ± 2?C), with water and food ad libitum for at
least 2 wk before the experiments. Vaginal smears were
taken daily in the morning (or 6 hr after lights off, for the
Abstract: Melatonin times reproduction with seasons in many photoperiodic
mammalian species. Whether sexual hormones reflect on melatonin synthesis
is still debated. The aim of this work was to study, using a large panel of
technical approaches, whether the daily profile of pineal melatonin synthesis
and release varies with the estrous cycle in the female rat. The mRNA levels
and enzyme activities of the melatonin synthesizing enzymes, arylalkylamine
N-acetyltransferase and hydroxyindole-O-methyltransferase were similar at
the four stages of the rat estrous cycle. The endogenous release of melatonin,
followed by transpineal microdialysis during six consecutive days in cycling
female rats, displayed no significant variation during this interval. Taken
together, the present results demonstrate that there is no regular fluctuation
in the pineal metabolism leading to melatonin synthesis and release
throughout the estrous cycle in female rats.
Ana-Lucia Skorupa1, Marie-Laure
Garidou2, Be ´atrice Bothorel2,
Michel Saboureau2, Paul Pe ´vet2,
Jose ´ Cipolla Neto1and Vale ´rie
Simonneaux2
1Department of Physiology and Biophysics,
Institute of Biomedical Sciences, University of
Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil;2Laboratoire
de Neurobiologie des Rythmes,
CNRS/Universite ´ Louis Pasteur, Strasbourg,
France
Key words: arylalkylamine
N-acetyltransferase, estrous cycle,
hydroxyindole-O-methyltransferase,
melatonin, pineal microdialysis
Address reprint requests to Vale ´rie
Simonneaux, Laboratoire de Neurobiologie
des Rythmes, UMR 7518, CNRS/Universite ´
Louis Pasteur, 67000 Strasbourg, France.
E-mail: simonneaux@neurochem.u-strasbg.fr
Received July 17, 2002;
accepted September 5, 2002.
J. Pineal Res. 2003; 34:53–59
Copyright ? Blackwell Munksgaard, 2003
Journal of Pineal Research
ISSN 0742-3098
53

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pineal microdialysis experiment) to monitor estrous cycles.
Only animals exhibiting at least two consistent 4-day
estrous cycles were used in the following studies. All
experiments with animals were performed in accordance
with ‘Principles of Laboratory Animal Care’ (NIH publi-
cation no. 86-23, revised 1985) as well as in accordance with
the French national laws.
Experimental design
Experiment 1
Eleven animals were used to follow the daily profile of the
endogenous pineal melatonin release as a function of the
estrous cycle, using transpineal microdialysis. The animals
were housed in a reversed 12-hr light/12-hr dark cycle
(lights off from 10:00 to 22:00 hr). The dark onset (10:00
hr) corresponded to Zeitgeber time ZT-12. The animals
were kept under these conditions for 3 wk before surgery
(see below). The experiment lasted 6 days during which
the pineal gland was continuously perfused with a
Ringer’s solution and the rats were checked for the
estrous cycle stages.
Experiment 2
Forty female rats were used to measure nocturnal pineal
melatonin content, AANAT and HIOMT mRNAs and
activities at the four stages of estrous cycle. The animals
were housed for 3 wk (checked daily for their estrous stage)
before the experiment. They were sacrificed in the dark (dim
red light) at ZT-19 (7 hr after lights off). Vaginal smears
were taken 1 hr before being killed and the rats were
distributed into four groups according to the estrous stage
(proestrus, P; estrus, E; metestrus, M, and diestrus, D). In
half of these animals (five per estrous stage) the pineal gland
was removed to assay melatonin and to measure AANAT
and HIOMT enzymatic activities. In the remaining animals
(five per estrous stage) the brain was removed with the
pineal attached, to perform in situ hybridization.
Surgery and dialysis
Animals were anaesthetized with Equithesin i.p. (0.4 mL/
100 g body weight). The implantation of the microdialysis
probe was performed as already described [14]. The
microdialysis probe (0.22 mm i.d., 0.27 mm o.d., 10,000
molecular weight cutoff) was prepared from saponified
cellulose ester membrane (Cordis Dow Medical Interna-
tional, Oosterwolde, the Netherlands). The surface of the
probe was coated with silicone glue (CAF3, Rho ˆ ne Pou-
lenc, France) except for a 2-mm wide zone. The microdi-
alysis probe bearing a tungsten wire with a sharpened point
was fastened horizontally in a transverse position in a
holder mounted a stereotaxic apparatus (David Kopf
Instruments, Roucaire, Les Ulis, France). One hole was
drilled on each side of the temporal bone, 1.6 mm ventral to
the skull and 0.7 mm posterior to lambda according to the
atlas of Paxinos and Watson [15], and the probe was
pushed laterally through the pineal gland. Both inlet and
outlet tubes were fixed on the skull in a vertical position
with dental cement. The rats were allowed to recover from
the surgery for at least 4 days in individual cages. During
experiments, the inlet of the probe was connected to a
microinjection pump (Pump 22, Harvard Biosciences, Les
Ullis, France) via a fluid swivel (375/22, Instech Laborat-
ories, Plymouth Meeting, PA, USA) with polyethylene
tubing. The probe was perfused with Ringer’s solution at a
flow rate of 3 lL/min. The composition of the Ringer’s
solution was 147 mmol/L of NaCl, 4 mmol/L of KCl,
1.2 mmol/L of CaCl2and 1.0 mmol/L of MgCl2. The outlet
connection consisted of microbore PEEK tubing (0.13 mm
i.d., 0.51 mm o.d.) connected to a 1.5 mL polypropylene
microvial. Collected samples were stored at )20?C until
assayed by radioimmunoassay (RIA). Samples were collec-
ted hourly from ZT-11 to ZT-1 and every 2 hr from ZT-1 to
ZT-11, during 6 days. At the end of the experiment, rats
were decapitated and the brains, dissected with the pineal
glands, were frozen at )20?C. Cryostat sections (25 lm) of
the brain/pineal were stained with cresyl violet in order to
determine the location of the microdialysis probe.
Melatonin radioimmunoassay
Melatonin was measured in pineal glands and pineal
microdialysates by RIA [14, 16]. Single pineal glands were
sonicated in 110 lL sodium phosphate buffer (0.05 m,
pH 7.9), centrifuged and 25 lL of the supernatant was
assayed for melatonin whereas the remaining supernatant
was used for enzymatic activity and protein assays. Mela-
tonin was assayed using a specific antiserum (R19540,
INRA, Nouzilly, France) at a final dilution of 1:200,000
and [125I]-2-iodomelatonin as a tracer. Sheep anti-rabbit
antiserum (INRA, Nouzilly, France) was used to separate
the bound and free tracer. Protein content was measured in
20 lL tissue homogenate following the protocol of Lowry
with BSA as standard [17]. Melatonin concentration in the
pineal gland was expressed as pg/lg of protein.
Melatonin concentrations in dialysates were determined
directly without extraction in duplicate 25 lL samples. The
assay was validated for pineal dialysates as already reported
by Barassin et al. [14]. The limit of sensitivity of the assay
was 0.5 pg/tube. Melatonin in the pineal microdialysate was
expressed as pg/25 lL microdialysate.
Pineal AANAT and HIOMT activity radioenzymatic
assay
Arylalkylamine N-acetyltransferase activity was assayed by
measuring the amount of14C–N-acetyltryptamine formed
from14C–acetyl CoA (14C–ACoA) and tryptamin. HIOMT
activity was assayed by measuring the amount of melatonin
formed from S-adenosyl-l-[14C–methionine] and N-acetyl-
serotonin (NAS). Immediately after the sacrifice of the
animals, individual pineal glands were sonicated in 110 lL
sodium phosphate buffer (0.05 m, pH 7.9), and AANAT
and HIOMT activities were determined as previously
described [18, 19] in the same pineal homogenate.
For AANAT activity, 30 lL of the tissue homogenate
were incubated for 20 min at 37?C with 80 mm tryptamine
and 32 mm
Nuclear (NEN)-Dupont, Le Blanc Mesnil, France] in a
14C–ACoA [44.1 mCi/mmol, New England
Skorupa et al.
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final volume of 60 lL (pH 6.8), then the reaction was
stopped by the addition of 1 mL chloroform. Newly
synthesized N-acetyltryptamine was measured after extrac-
tion in 1 mL of water-saturated chloroform and counting of
the radioactivity after evaporation of the organic solvent.
For HIOMT activity, 25 lL of the tissue homogenate
were incubated for 30 min at 37?C with 1 mm NAS and
43.8 lm S-adenosyl-l-[14C–methionine] (59.3 mCi/mmol;
NEN-Dupont) in a final volume of 100 lL (pH 7.9), then
the reaction was stopped by the addition of 200 lL sodium
borate buffer (12.5 mM; pH 10). Newly synthesized
melatonin was measured after extraction in 1 mL water-
saturated chloroform and counting of the radioactivity
after evaporation of the organic solvent. Enzymatic activ-
ities were expressed as picomoles of reaction product
formed per hr/lg of protein.
In situ hybridization of AANAT and HIOMT mRNAs
Coronal sections of frozen brains (20 lm thick) were thaw-
mounted onto gelatin-coated slides. For each animal, the
pineal AANAT and HIOMT mRNA levels were quantified
by quantitative in situ hybridization performed on two
separate sets of sections according to a protocol previously
described (HIOMT: [20], AANAT: [16]).
The sense and antisense riboprobes were synthesized
from the linear pBluescript-CMV phagemid and pBlue-
script plasmid containing the cDNA encoding AANAT
(1311 bp, [21]) or HIOMT (1542 bp, [22]) with either T7 or
T3 RNA polymerase (MAXIscript transcription kit, Am-
bion, Montrouge, France; a[35S]-UTP, 46.3 TBq/mmol,
NEN-Dupont). The AANAT and HIOMT probes were
hydrolyzed by an alkaline treatment to generate 200-bp-
long fragments. The AANAT and HIOMT mRNA analysis
was performed according to the same protocol as follows.
All the prehybridization steps were carried out at room
temperature. Sections were incubated in 4% paraformal-
dehyde/1 · phosphate-buffered saline (PBS) for 15 min.
They were washed successively into 1 · PBS and 2 ·
sodium saline citrate (SSC) for 2 min each. Sections were
then acetylated with 0.5% acetic anhydrate/0.1 m trietha-
nolamine (pH 7.4) for 10 min and rinsed in 2 · SSC and
1 · PBS for 2 min each. They were then incubated for 30
min in 0.1 m glycine/0.1 m Tris (pH 7.0) and rinsed in
2 · SSC and 1 · PBS before being dehydrated in graded
ethanol solutions (70, 90, 95, and 100%, 1 min each) and
dried at room temperature.
For hybridization, probes were dissolved in a hybridiza-
tion solution containing 2 · SSC/20% Dextran sulfate/
50% deionizedformamide/10
Denhardt’s solution. Dehydrated brain sections were over-
laid with 80 or 50 pm of the antisense or sense AANAT or
HIOMT probes, respectively, and incubated overnight at
54?C in a moist chamber.
Posthybridization treatment consisted of washing the
sections for 10 min at room temperature in 2 · SSC before
being incubated for 30 min at 37?C with 0.02 Kunitz unit/
mL ribonuclease type X-A (from bovine pancreas, Sigma,
Saint Quentin Fallavier, France) in 0.5 m NaCl/10 mm Tris
(pH 7.4)/10 mm EDTA. The sections were then washed
three times (5 min each) in 2 · SSC at room temperature
mm
dithiothreitol/1 ·
before dehydration in graded ethanol solutions (70, 90, 95
and 100%, 1 min each) and air dried.
The slides were then exposed to autoradiographic films
(Hyperfilm MP, Amersham, Les Ulis, France) for 48 hr at
room temperature. Quantitative analysis of the autoradio-
grams was performed using the BIOCOM computerized
analysis program RAG200. Specific hybridization was
determined by densitometry as the difference between total
(antisense) and non-specific (sense) hybridization.
Statistical analysis
For microdialysis experiment, each individual melatonin
profile was characterized by its onset time (IT50), its offset
time (DT50) and the peak amplitude (Yampl). These values
were determined by fitting a logistic peak with the following
equation:
Y ¼ Y0þ
Yampl
ð1 þ e2:91ðIT50?xÞÞð1 þ e2:77ðx?DT50ÞÞ
where Y was the nth data point, x the time point of the nth
point, Y0 basal level during daytime, and Yampl the
amplitude of the nocturnal peak. IT50 was defined as the
time point at which 50% of the increase in melatonin level
was reached and DT50 as the time at which 50% of the
decrease occurred. The non-linear regression analysis was
performed with SigmaPlot software (SPSS ASC GmbH,
Erkrath, Germany) and fitted through datapoints of each
experimental day of each animal [14]. The results from the
individual regressions gave a mean Y0close to zero, thus in
further analyses, only the three parameters IT50, DT50 and
Yamplwere considered to characterize the pattern of the
melatonin peaks. To compare the variations in melatonin
peak among the four estrous cycles an analysis of variance–
covariance was carried out considering the variances and
residues of variance given by the regressions characterizing
the melatonin peak. We only considered the first occurrence
of one of the four stages observed in each animal.
In the experiment 2, results on melatonin content,
AANAT and HIOMT activity and mRNA expression are
expressed as mean ± S.E.M. and were computed using
GraphPad Prism data analysis and graph package (version
3.01, GraphPad Software Inc., San Diego, CA, USA).
Data, distributed according to the estrous cycle phase of
each animal just before the sacrifice, were compared using
the one-way ANOVA procedure.
The level of probability was fixed at 0.05 for statistical
significance.
Results
In experiment 1, all the 11 female rats had the probe
correctly implanted within the pineal gland but only three
of them recovered in displaying a regular 4-day estrous
cycle. Therefore the individual profile of melatonin pro-
duction has been followed during 5–6 consecutive days in
three cycling female rats with raw values of melatonin
release presented as a function of ZT for each animal
(Fig. 1). No regular pattern of variation in the melatonin
profile is observed among the four stages of the estrous
cycle in any of the female rats. The statistical analyses
Estrous cycle-independent synthesis of melatonin
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carried on the parameters characterizing the melatonin
peak (onset, amplitude, offset) revealed no significant
difference related to the estrous stage (F9
P ¼ 0.6).
In experiment 2, melatonin content (Fig. 2A), AANAT
(Fig. 2B) and HIOMT (Fig. 2C) activities, and protein level
were measured in the same pineal gland of female rats, at
191¼ 0:818,
either of the four estrous stages, sacrificed 7 hr after dark
onset (when the melatonin peak amplitude is at its
maximum). No significant difference in nocturnal mela-
tonin content or enzyme activity is found as a function of
thestage oftheestrous
D ¼ 25.43 ± 7.15 pg/lg protein, P ¼ 20.78 ± 5.84 pg/lg
protein, E ¼ 21.61 ± 9.37 pg/lg protein, M ¼ 19.11 ±
3.82 pg/lg protein, ANOVA, P ¼ 0.53; AANAT values:
D ¼ 84.44 ± 24.07 pmol/hr lg protein, P ¼ 93.30 ±
22.78 pmol/hr lg protein, E ¼ 82.07 ± 30.01 pmol/hr lg
protein, M ¼ 67.85 ± 14.84 pmol/hr lg protein, ANOVA,
P ¼ 0.64; HIOMT values: D ¼ 1.12 ± 0.20 nmol/hr lg
protein, P ¼ 1.05 ± 0.16 nmol/hr lg protein, E ¼ 1.03 ±
cycle (melatoninvalues:
Fig. 1. Daily pattern of melatonin release measured in pineal
microdialysates for five to six consecutive days in three cycling
femalerats. Femalerats(F, H, I)werekept undera12-hrlight/12-hr
dark cycle with lights off at 10:00 am and were checked everyday
for estrous stage (P, E, M, D). Pineal microdialysates were sampled
every 1 hr (night) or 2 hr (day), and assayed for melatonin.
Fig. 2. (A) Melatonin content, (B) arylalkylamine N acetyltrans-
ferase (AANAT) activity and (C) hydroxyindole-O-methyltrans-
ferase (HIOMT) activity in the pineal gland of female rats at each
of the four stages of the estrous cycle. Animals were sacrificed 7 hr
after lights off. Data are given as mean ± S.E.M. of n ¼ 5 ani-
mals.
Skorupa et al.
56

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0.28 nmol/hr lg protein, M ¼ 0.89 ± 0.13 nmol/hr lg
protein, ANOVA, P ¼ 0.39; mean ± S.E.M. of five ani-
mals per stage).
The expression of AANAT (Fig. 3A) and HIOMT (Fig.
3B) mRNAs was measured in the same pineal gland of
female rats, at either of the four estrous stages, sacrificed 7
hr after dark onset. No significant difference has been
found in the amount of either AANAT mRNA (D ¼
2308.94 ± 124.06 dpm, P ¼ 2601.26 ± 187.38 dpm, E ¼
3048.40 ± 384.47 dpm, M ¼ 2530.54 ± 185.55 dpm;
ANOVA, P ¼ 0.23; mean ± S.E.M. of five animals per
stage) or HIOMT mRNA (D ¼ 1881.40 ± 126.65 dpm,
P ¼ 1902.49 ± 143.10 dpm, E ¼ 1867.96 ± 226.94 dpm,
M ¼ 2159.37 ± 51.45 dpm; ANOVA, P ¼ 0.40; mean ±
S.E.M. of five animals per stage) throughout the estrous
cycle.
Discussion
This paper shows that there is no significant variation in the
daily pattern of pineal melatonin release throughout the
estrous cycle in female rats. This result is strengthened by
the demonstration that gene expression and activity of the
two melatonin-synthesizing enzymes AANAT and HIOMT
did not vary as a function of the estrous stage.
These data are in agreement with those obtained for ewe
and hamster melatonin contents, but they are in contrast
with some results obtained for rats. The data showing a
strong reduction of melatonin during proestrus [4] were
obtained from urine samples. Another study, monitoring
urinary 6-sulphatoxymelatonin excretion, showed a signifi-
cant variation during the estrous cycle, with higher values
on proestrus [23]. An explanation for these results could be
an estrous cycle variation in the hepatic ability for
metabolizing melatonin or, even more likely, a greater or
smaller ability of the kidney to excrete metabolic melatonin
products according to the estrous stages. Quay [3] showed a
small reduction in melatonin during the diestrus whereas
another study [5] reported a reduction of pineal melatonin
during proestrus. It seems possible that the inconsistencies
of finding reproducible variations in melatonin secretion
throughout the rat estrous cycle is because of large inter-
individual differences.
To overcome these putative inter-individual variations
we used the transpineal microdialysis technique which
allowed for the monitoring of endogenous melatonin
release for several consecutive days in the pineal gland of
a single animal. We have previously validated this tech-
nique and demonstrated that for each animal, the melato-
nin profile is stable up to 5 consecutive days with very low
intra-individual variability [14].
The endogenous melatonin secretion from the pineal
gland of three cycling female rats was followed for 5–6
consecutive days. No consistent or regular variation of the
nycthemeral melatonin profile was detected that could be
strictly associated to any of the four stages of the estrous
cycle. Occasionally, one melatonin peak appeared of higher
amplitude but with no significant relation with the estrous
cycle (during the diestrus in rat H, during the metestrus in
rat F, during estrus in rat I). These inter-individual
variations could explain the inconsistent results found in
the literature. Therefore, the absence of a significant
variation in the melatonin pattern in relation with the
estrous stage of three female rats exclude any regular or
coherent variation of pineal melatonin synthesis depending
on the hormonal physiologic environment because of the
estrous cycle.
To strengthen these observations, gene expression and
enzyme activity of AANAT and HIOMT were examined
during the night at each of the estrous stages. Enzyme
activities were already examined according to the repro-
ductive status in mammals, but, to our knowledge, this
study is the first to analyze the effect of sexual hormones on
pineal enzyme gene expression. Nocturnal pineal AANAT
mRNA and activity displayed no significant variation
associated with the four estrous stages. This observation
is in agreement with data in the literature reporting no
difference in AANAT activity throughout the estrous cycle
[9, 10]. Similar to AANAT, nocturnal HIOMT mRNA
level and activity displayed no significant variation during
the estrous cycle. This is in contrast to data reported in the
literature [7, 8, 10] although these three studies reported
contradictory results. Therefore, it is not surprising that, in
fact, we found no significant difference in HIOMT gene
expression and enzyme activity in relation to the estrous
cycle.
Our results, however, do not exclude an effect of gonadal
steroids per se on pineal metabolism. The pineal gland
Fig. 3. (A) arylalkylamine N-acetyltransferase (AANAT) mRNA
and (B) hydroxyindole-O-methyltransferase (HIOMT) mRNA
levels measured by in situ hybrodization in the pineal gland of
female rats at each of the four stages of the estrous cycle. Animals
were sacrificed 7 hr after lights off. Data are given as mean ±
S.E.M. of n ¼ 5 animals.
Estrous cycle-independent synthesis of melatonin
57

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accumulates specifically estradiol and testosterone [24, 25]
and contains nuclear binding sites for estradiol, testoster-
one [26–28]. Furthermore, testosterone exhibits stimulatory
effects and castration reduces cAMP concentrations [29],
AANAT activity [30] and melatonin secretion [31]. In
female rats, estradiol displays an inhibitory effect on the
a1b1-AR-induced cAMP and Ca2+levels, AANAT activ-
ity and melatonin production while ovariectomy leads to a
significant increase in the cAMP/AANAT/melatonin path-
way [32–37]. Similarly, an increase in nocturnal melatonin
secretion (associated with an increase in AANAT not
HIOMT activity in rat pineal) is observed during meno-
pause in relation to the existence of a low estrogen
environment in rat [37] and human [38].
In summary, this is the first time that a potential effect of
estrouscycleonpinealmetabolismhasbeenstudiedinsucha
thorough way looking at all steps of melatonin synthesis
(enzyme gene expression, enzyme activities, melatonin con-
tent and melatonin release). This study clearly demonstrates
thatthemelatoninsynthesispathwayisnotalteredduringthe
estrous cycle in female rats, which is similar to the results
reported for hamster [12], sheep [11] and monkey [13].
Acknowledgments
Authors are grateful to Berthe Vivien-Roels, Christiane
Calgari, Ste ´ phane Barassin and Me ´ lanie Franco for tech-
nical assistance and to Andre ´ Malan for statistical analyses
of the microdialysis data. This research was supported by
FAPESP grant 97/090904-5, the FAPESP-INSERM agree-
ment, FAPESP fellowships to ALS (98/-01761-3) and EMJr
(98/03927-6) and the foundation Simone et Cino del Duca.
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