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- and ß-Dependent Gene Expression in the Brain1
Center for Behavioral Neuroscience (H.B.P., P.L.W., L.J.Y.) and Departments of Anthropology (P.L.W.) and Psychiatry and Behavioral Sciences (L.J.Y.), Emory University, Atlanta, Georgia 30329
Address all correspondence and requests for reprints to: Dr. Heather B. Patisaul, Center for Behavioral Neuroscience, Emory University, 954 Gatewood Road NE, Atlanta, Georgia 30329.
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Abstract |
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and ß (ER and
ERß). Because isoflavone dietary supplements are becoming
increasingly popular and are frequently advertised as natural
alternatives to estrogen replacement therapy, we have examined the
effects of one of these supplements on estrogen-dependent behavior and
ER- and ERß-dependent gene expression in the brain. In the adult
female rat brain, 17ß-estradiol treatment decreased ERß messenger
RNA signal in the paraventricular nucleus by 41%, but supplement
treatment resulted in a 27% increase. The regulation of ERß in the
paraventricular nucleus is probably via an ERß-dependent mechanism.
Similarly, in the ventromedial nucleus of the hypothalamus, supplement
treatment diminished the estrogen-dependent up-regulation of oxytocin
receptor by 10.5%. The regulation of oxytocin receptor expression in
the ventromedial nucleus of the hypothalamus is via an ER-dependent
mechanism. Supplement treatment also resulted in a significant decrease
in receptive behavior in estrogen- and progesterone-primed females. The
observed disruption of sexual receptivity by the isoflavone supplement
is probably due to antiestrogenic effects observed in the brain. These
results suggest that isoflavone phytoestrogens are antiestrogenic on
both ER- and ERß-dependent gene expression in the brain and
estrogen-dependent behavior. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A variety of phytoestrogens have been shown to bind to both isoforms of
the estrogen receptor (ER and ERß) in vitro (9, 10) and activate ER-dependent gene transcription. This evidence
combined with previous observations that these compounds can increase
uterine weight in vivo (11, 12) has led to the
classification of phytoestrogens as weakly estrogenic. However, new
evidence obtained in vivo has suggested that at least one
phytoestrogen, coumestrol, may actually function as an antiestrogen in
the brain (13).
Although the potentially beneficial effects of a high phytoestrogen diet have been well documented (14, 15, 16), very little is known about the potential impact of these compounds on the brain and estrogen-dependent behavior. Because commercially prepared phytoestrogen supplements are becoming more widely available, and their consumption is increasing in popularity, this study seeks to examine the effects of one of them on multiple endpoints including estrogen-dependent gene transcription in the brain and the resulting impact on female sex behavior.
Lordosis is a reflexive posture made by female rodents in response to
male mounting. Females will only display this posture when sexually
receptive. Ovariectomized (OVX) females can be reliably induced into
behavioral estrus by administering 17ß-estradiol, followed 48 h
later by progesterone (for a detailed review, see Ref.
17). The absence of female sexual behavior in ER
knockout mice (ERKO mice) demonstrates that ER is critically
important for the regulation of female sexual behavior
(18, 19, 20). By contrast, ERßKO mice show far less dramatic
behavioral deficits (21). Thus, if the supplement is
antiestrogenic, particularly through ER, it should attenuate the
lordosis response in females primed with progesterone in the absence of
estrogen. Similarly, if it is estrogenic, it should enhance the
lordosis response in females primed with both 17ß-estradiol and
progesterone.
To distinguish through which estrogen receptor the supplement is
acting, we have chosen to look at oxytocin receptor (OTR) expression in
the ventromedial nucleus of the hypothalamus (VMN) because estrogen is
known to significantly up-regulate OTR via an ER-dependent mechanism
(22). If the isoflavone supplement is estrogenic via
ER, it should up-regulate OTR in a manner similar to that of
estrogen. We have also chosen ERß messenger RNA (mRNA) expression in
the paraventricular nucleus of the hypothalamus (PVN) because estrogen
down-regulates ERß mRNA (13). This down-regulation of
ERß transcription is probably an ERß-dependent mechanism because
the PVN is devoid of ER (23, 24). Again, if the
isoflavone supplement is estrogenic, it should down-regulate ERß
mRNA in a manner similar to that of estrogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1.4 g). The label directs consumers to take two tablets (76 mg
total isoflavones) daily. The presence of both genistein and daidzein
was confirmed using HPLC analysis.
Analysis of isoflavonoid content of the supplement
The quantities of genistein and daidzein in the supplement were
determined by HPLC after extraction and hydrolysis by the method of
Franke et al. (1). A sample (0.1 g) of the
finely ground supplement was suspended in 5 ml 77% ethanol with 2
M HCl by sonicating for 30 min and hydrolyzed at
80 C for 2 h. Ethanol lost during hydrolysis was replaced to bring
the volume to 2 ml, and the mixture was clarified by centrifuging at
2000 rpm for 10 min. Aliquots (40 and 200 µl) were reconstituted to
200 µl mobile phase, and 20 µl were injected into the HPLC
system.
Chromatography was carried out using a Hypersil ODS column (5 µm, 25 cm x 4.6 mm; Aldrich, Milwaukee, WI). Phytoestrogens were analyzed using a reverse phase isocratic technique for the detection of isoflavonoids (25, 26). The mobile phase was 60% methanol in 0.1 M ammonium acetate buffer, pH 4.6, with a flow rate of 1 ml/min for 16 min at 30 C. Analytes were monitored using a dual channel photodiode array detector at 255 and 280 nm. Peaks were scanned between 0–600 nm, and the resultant absorption spectra were compared with the spectra of authentic standards to confirm the identity of peaks corresponding to the retention times of authentic standards. Retention times for isoflavonoids were as follows: daidzein, 6.1; phloretin, 7.2; equol, 6.8; genistein, 7.4; coumestrol, 9.6; and formonetin, 11.4 min. Retention times were reproducible to within a 1% coefficient of variation over a 3-month period. Detection limits obtained from authentic standards were 2–3 ng. Isoflavonoid concentrations were calculated from standard curves of the peak area responses for authentic isoflavonoid standards. Calibration curves showed high linearity (0.97–0.99) over a concentration range of 2–300 ng. Within- and between-assay coefficients of variation were less than 10%.
Animal care
Animal care, maintenance, and surgery were conducted in
accordance with the applicable portions of the Animal Welfare Act and
the U.S. DHHS Guide for the Care and Use of Laboratory Animals. Adult
male (n = 8) and female (n = 73) Long Evans rats
(Charles River Laboratories, Inc., Raleigh, NC) were
housed in a 12-h light, 12-h dark cycle at 23 C and 50% humidity.
Females were housed in groups of two; males were individually housed.
Animals used for the behavioral study were kept on a reverse light
cycle.
Because standard laboratory chows contain significant amounts of phytoestrogens given their high soy content (27, 28), a semipurified, phytoestrogen-free test diet (American Institute of Nutrition formulation 76A, Purina Test Diets, Richmond, IN) was used for the preparation of the treatment diets. All animals were maintained on this diet for at least 4 days before beginning each experiment.
Behavioral testing
After a 1-week acclimation to the vivarium, all females (n
= 32) were OVX under ketamine anesthesia. Within a few weeks after
surgery, all females were induced into estrus with estrogen (10 µg in
0.1 ml sesame oil) and progesterone (500 µg in 0.1 ml sesame oil) and
tested with males to confirm each male’s vigor and each female’s
sexual responsiveness to ovarian hormones. Each female was tested
twice, and each male (retired breeders, Charles River Laboratories, Inc.) was tested up to six times. Males that did
not reliably engage in sexual behavior after repeated exposures
(reliability = mating in at least 80% of pairings) were not used
in the study (n = 5 vigorous males). Testing began 1 week after
the completion of this initial priming, and all females were fed the
AIN-76A diet for 5 days before testing.
The females were divided into four treatment groups (n = 8/group): isoflavone-free diet and no estrogen, isoflavone-free diet and estrogen, isoflavone diet and no estrogen, and isoflavone diet and estrogen. All animals were given progesterone. Estradiol benzoate (10 µg in 0.1 ml sesame oil) was sc injected 48 h before testing, and progesterone (500 µg in 0.1 ml sesame oil) was sc injected 4–5 h before testing. Animals not receiving estrogen were injected with sesame oil (0.1 ml) only. The isoflavone diet was prepared by mixing the ground supplement into the semipurified diet as described above at a 0.35% concentration [13 parts/million (ppm) genistein and 33 ppm daidzein]. This dose was selected because we wanted to use a physiologically, rather than pharmacologically, relevant dose. A recent study has demonstrated that this dose of genistein should produce plasma levels in Sprague Dawley rats that fall between plasma levels seen in humans eating a traditional Western diet (29) and levels in humans consuming a traditional Asian diet (16) or phytoestrogen supplements (30). Testing took place in a double-sided arena linked by a tunnel small enough for the female, but not the male, to pass through. Each side of the arena was identical in size and shape to the home cage of each animal and was thoroughly cleaned between trials. The females were given 5 min to adapt to the arena before introduction of the male. Testing sessions were 30 min in length, and all interactions were videotaped under red light and scored from the tape. Receptivity was assessed using the lordosis quotient (LQ) as calculated by the number of lordosis responses in 10 min divided by the number of mount attempts made in the same 10 min multiplied by 100. To eliminate variability between subjects due to mount latency, the beginning of the 10-min scoring period was defined as 5 sec before the first mount attempt by the male.
Brain collection and analysis
Adult female Long Evans rats (Charles River Laboratories, Inc.; n = 41) were housed in groups no larger than three on
a 12-h light, 12-h dark cycle at 23 C. After a 1-week acclimation to
the vivarium, all animals were OVX under ketamine anesthesia and placed
on the AIN-76A diet. Ultimately, the animals were divided into four
groups: no estrogen and isoflavone-free diet (n = 11), estrogen
and isoflavone-free diet (n = 10), isoflavone diet and no estrogen
(n = 10), and isoflavone diet and estrogen (n = 10). Estrogen
was administered by SILASTIC brand capsule (Dow Corning Corp., Midland, MI), and the isoflavone diet was prepared by
mixing the ground supplement into the semipurified diet as described
above at a 0.35% concentration (13 ppm genistein and 33 ppm daidzein).
One week after OVX, each group was placed on its designated treatment
diet. Two days later, all animals were sc implanted with either an
empty or a 17ß-estradiol (Sigma, St. Louis, MO)-filled
SILASTIC brand capsule (9 mm in length; id, 2 mm; od, 3.2 mm) under
ketamine anesthesia. Implants were chosen over injections or oral
administration because implants release a steady dose of estrogen over
the entire course of treatment and better simulate endogenous estrogen
secretion. Similarly sized tubing has previously been shown to deliver
physiological levels of 17ß-estradiol in rats (31). Four
days after implantation all animals were killed by
CO2 asphyxiation for a total of 6 days on the
treatment diet and 4 days on the hormone treatment. Uteri were
collected and weighed at the time of death. Blood was collected and
spun down within 20 min of collection to isolate the plasma. All brains
were immediately frozen on dry ice at the time of death. The plasma and
brains were kept at -80 C until use. The estrogen content of each
plasma sample was quantified by RIA using a modification of the
Pantex Direct 125I Estradiol kit
(Pantex, Santa Monica, CA). As described previously
(32, 33) the assay has a sensitivity of 9 pg/ml. The
brains were cut on a cryostat into sections 20 µm thick at 80-µm
intervals and thaw-mounted on SuperFrost Plus microscope slides
(Fisher, Pittsburgh, PA). Serial sections were taken from
the lateral septum to the caudal end of the VMN (corresponding to
bregma -0.3 to -3.0 mm).
In situ hybridization
ERß in situ hybridization was performed using a set
of two 48-bp, 35S-labeled oligonucleotide probes
(GTG AGG GAC ATC ATC ATG GAG GCC TCG GTG AAG GGC ATG CTG GGA CGG and
GAG CTC CAC AAA GCC AGG GAT TTT CTT AGC CCA GCC AAT CAT GTG CAC). These
oligonucleotides are complimentary to nucleotides 714–762 and 784–832
of rat ERß mRNA (GenBank accession no. 2801690) and are 60% and 77%
homologous to ER, respectively. This sequence encodes the hinge
region of the ERß receptor. The oligonucleotides were labeled using
terminal transferase in a reaction containing
[35S]ATP (NEN Life Science Products, Boston, MA) at a concentration of 35 pmol 10 mCi/ml
[35S]ATP/7.5 pmol total oligonucleotides. The
probe was diluted to a final concentration of 3.75 pmol of each
oligonucleotide/3 ml hybridization solution (520,000 cpm/slide).
In situ hybridization was performed using a well established protocol in our laboratory as described previously (34). After in situ hybidization, the rinsed and dried sections were exposed to Kodak BioMax MR film (Eastman Kodak Co., Rochester, NY) with 14C-labeled autoradiographic standards (Amersham Pharmacia Biotech, Arlington Heights, IL) for 4 weeks to produce autoradiograms for quantitative analysis.
OTR autoradiography
OTR autoradiography was performed using
125I-d(CH2)5[Tyr(Me)2,Tyr-
NH29]OVT (NEN Life Science Products) exactly as previously described
(22). After air-drying, the slides
were exposed to BioMax MR film (Kodak) for 48 h.
125I-Labeled autoradiographic standards
(Amersham Pharmacia Biotech) were included in the cassette
for quantification.
Data analysis
Female sexual behavior. All behavior was scored from the
videotape by a single investigator blind to the treatment groups, then
validated by a second investigator. The number of mounts by the male,
the number of lordotic responses by the female, and the time spent by
the female in both sides of the arena were scored for each 10-min test
session and averaged across each of the four treatment groups. Lordosis
was defined as complete dorsoflexion of the spine in response to a
mount by the male as previously described in detail (35).
A mount was defined as placement of both front limbs on the
hindquarters of the female with or without intermission or ejaculation.
All data, including male mounting, was analyzed by one-way ANOVA using
SYSTAT (SPSS, Inc., Chicago, IL), and group
differences were identified using Fisher’s least significant
difference post-hoc test.
Estrogen-dependent gene expression. ERß in situ hybridization and OTR film autoradiograms were analyzed on a Macintosh computer using the public domain NIH Image program. Brain regions from three adjacent sections per subject were measured bilaterally from anatomically matched sections, and care was taken to ensure that the area of the regions selected for measurement did not differ by more than 10% between sections and subjects. For the ERß autoradiograms, optical densities were converted to nanocuries per g tissue equivalents, and for the OTR autoradiograms, optical densities were converted to disintegrations per min/mg tissue equivalents using 14C- and 35S-labeled standards (Amersham Pharmacia Biotech). All data were analyzed by one-way ANOVA using SYSTAT, and group differences were identified using Fisher’s least significant difference post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Daidzein and genistein were
identified by comparison of retention times and absorption spectra to
authentic standards. An additional peak was tentatively identified as
glycitein by comparison of its spectra to published data
(36). One gram of the supplement was estimated to contain
3.7 mg genistein and 9.4 mg daidzein, levels very similar to those
stated by the manufacturer (3.57 mg genistein and 12.85 mg daidzein). A
standard was not available for quantification of glycitein content.
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). Supplement-treated
animals (LQ = 7.8%) were similar to the controls, but supplement
intake significantly reduced lordosis behavior in estrogen-treated
animals (LQ = 72.8%; P < 0.006).
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). The
time spent by the female in the arena containing the male was the same
across all groups, implying that the difference seen in male mounting
attempts is not complicated by differences in the amount of time that
males had access to the females (data not shown).
ERß mRNA expression
Hybridization with the antisense oligonucleotide probe for ERß
mRNA resulted in strong signals for ERß mRNA in the PVN but no
detectable levels in the VMN (Fig. 3).
This is consistent with previous reports of ERß distribution in the
brain (23, 24, 37, 38, 39), which identified ERß to
magnocellular neurons. Quantitative analysis of the signal on the film
autoradiograms showed that treatment with 17ß-estradiol resulted in a
41% decrease in ERß mRNA expression in the PVN (Fig. 4; P < 0.001) compared
with that in the control group. In contrast, ingestion of a 0.35%
isoflavone-supplemented diet resulted in a 27% increase
(P < 0.02) compared with the control group. Treatment
with both 17ß-estradiol and the isoflavone supplement resulted in a
43% decrease in ERß mRNA signal compared with the control group
(P < 0.001) which is not significantly different from
the group treated with 17ß-estradiol alone.
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). Quantitative analysis of the signal
on the film autoradiograms revealed that treatment with 17ß-estradiol
resulted in a 195% increase in OTR expression in the VMN compared with
the controls (Fig. 4; P < 0.001), whereas treatment
with the 0.35% isoflavone-supplemented diet resulted in no significant
change in OTR expression compared with the controls. However, treatment
with both the isoflavone diet and 17ß-estradiol resulted in a 166%
increase in OTR expression in the VMN, which is significantly higher
than that in the controls (P < 0.001), but
significantly lower than the increase in the animals treated with
17ß-estradiol alone (Fig. 4; P < 0.02).
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Treatment with the supplement did not significantly increase uterine weight (mean, 0.19 ± 0.011 g) compared with the controls (mean, 0.18 ± 0.009 g) or diminish the uterotropic effects of estrogen (mean, 0.62 ± 0.02 g) when given in combination (mean, 0.57 ± 0.026 g). This is consistent with the results of an immature rat uterotropic bioassay, which showed that a 0.35% supplemented diet did not significantly increase uterine weight in juvenile females compared with the controls (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ERß-dependent gene expression in the brain. The behavioral data
suggest that the supplement is acting as an antiestrogen.
Administration of the supplement in combination with progesterone in
the absence of estrogen failed to increase female receptivity.
Similarly, ingestion of the isoflavone supplement in combination with
both estrogen and progesterone significantly decreased female receptive
behavior. This observed decrease in the lordosis quotient of
hormone-primed females fed the isoflavone supplement may be the
consequence of the antiestrogenic actions of the isoflavone supplement
on ER- and ERß-dependent gene expression in the hypothalamus.
Studies using ERKO mice have conclusively demonstrated that ER is
required for the normal expression of both male and female sexual
behavior (18, 19, 20). Oxytocin is also known to be critical
for the facilitation of sexual behavior (41, 42), and in
the PVN, ERß is colocalized with oxytocin (37, 38).
Thus, phytoestrogenic disruption of estrogen-dependent OTR
up-regulation in the VMN coupled with antiestrogenic effects on ERß
mRNA expression in the PVN could explain the reduced sexual receptivity
of hormone-primed female rats on an isoflavone-supplemented diet.
In vitro assays have shown that phytoestrogens are capable
of binding to ER and activating estrogen-dependent gene
transcription (43), and that at least one phytoestrogen,
resveratrol, has antagonist activity through ER (10).
It has also been shown that the up-regulation of OTR by estrogen in the
VMN is mediated exclusively by ER (22). The failure of
the isoflavone supplement to alter OTR expression in the absence of
estrogen implies that it has no estrogenic activity through ER in
this region at the dose used in this experiment. However, the
isoflavone supplement significantly decreased the induction of OTR by
estrogen in the VMN. This demonstrates that the phytoestrogens
contained in the supplement interfere with the ability of estrogen to
up-regulate OTR.
Similarly, in situ hybridization (24, 38) and
immunocytochemical (23, 37), studies have failed to detect
ER in the PVN. Therefore, it is likely that the estrogen-dependent
down-regulation of ERß mRNA in this region is an autoregulatory
effect mediated by ERß rather than heterologous regulation by ER.
The up-regulation of ERß mRNA in the PVN by the isoflavone supplement
is probably also via this mechanism and suggests that it has
antiestrogenic effects on ERß. The antiestrogenic effects on
estrogen-dependent gene expression seen in the hypothalamus are
consistent with at least one previous study showing that the individual
phytoestrogen, coumestrol, also had the opposite effect of estrogen on
ERß mRNA expression in the PVN (13).
These results demonstrating that isoflavones act as antiestrogens on
ER and ERß in the brain are in contrast with results obtained
in vitro showing that a variety of phytoestrogens, including
genistein and daidzein, activate estrogen-dependent gene transcription
(9, 43). The difference between the effects found
in this transfection reporter gene assay and our own in vivo
findings may be due to the metabolism of the supplement after
consumption or to interactions of the ligand-receptor complex with
accessory binding proteins not present in the transfection assay tissue
culture, but present in the PVN or VMN.
Although most studies of the estrogenic action of phytoestrogen action have been conducted using only one individual phytoestrogen, it is virtually impossible for consumers to obtain the purified compounds. Diets high in phytoestrogens, like traditional Asian diets and vegetarian diets, are high in a vast multitude of phytoestrogens, and all of the commercially prepared supplements, powders, and even soy-based infant formulas contain at least the two most common phytoestrogens, genistein and daidzein, in addition to several others (1, 2, 44). Multiple studies have shown that human plasma, urine, and breast milk from a wide variety of cultures (45, 46, 47, 48) contain many different phytoestrogens, making it necessary and imperative to study the combined effects of these compounds.
The mental health benefits of estrogen in women are well known and often used to argue for the use of ERT in postmenopausal women. Specifically, ERT may protect against a decline in memory (49, 50, 51) and alleviate depression (52, 53). Because more and more women are looking to phytoestrogen-rich foods and supplements as a natural alternative to ERT, the impact of phytoestrogen supplementation on estrogen-dependent end points in the brain needs to be examined in greater detail before they can be advocated as an effective alternative to traditional ERT.
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Acknowledgments |
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Footnotes |
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Received November 21, 2000.
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References |
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and ß. Endocrinology 141:3657–3667 is essential for induction of oxytocin receptor
by estrogen. NeuroReport 9:933–936[Medline]
and ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
(ER) and ß
(ERß) mRNA in the rat pituitary, gonad, and reproductive tract.
Steroids 63:498–504[CrossRef][Medline]
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, P < 0.06
(vs. E-S-).
