European Journal of Neuroscience, Vol. 23, pp. 1860–1868, 2006 doi:10.1111/j.1460-9568.2006.04703.

Estrogen receptor-b gene disruption potentiates estrogen-

inducible aggression but not sexual behaviour in male mice

Masayoshi Nomura,1 Sandra Andersson,2 Kenneth S. Korach,3 Jan-Åke Gustafsson,2 Donald W. Pfaff1 and Sonoko
Ogawa1
1
Laboratory of Neurobiology and Behaviour, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
2
Department of Medical Nutrition, Karolinska Institute, S-141–86 Hudding, Sweden
3
The Laboratory Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research
Triangle Park, North Carolina 27709, USA

Keywords: androgen receptor, knockout mouse, estrogen receptor a, sex difference, testosterone

Abstract
Aggressive behaviour of gonadally intact male mice is increased by estrogen receptor (ER)-b gene disruption, whereas sexual
behaviour remains unchanged. The elevated aggression levels following ER-b gene disruption is pronounced during repeated
aggression tests in young animals and the first aggression test in adults. In the present study, the roles of ER-b activation in the
regulation of aggressive and sexual behaviour were investigated in gonadectomized ER-b knockout (bERKO) and wild-type (WT)
male mice treated with various doses of estrogen. Overall, estradiol benzoate (EB) treatment induced higher levels of aggression in
bERKO mice than in WT mice. In WT mice, the levels of aggression induced by EB were highest in the lowest-dose (2.5 lg ⁄ day)
group and gradually decreased in higher-dosage groups. On the other hand, equally high levels of aggressive behaviour were
induced by all three doses of EB in bERKO mice. A marked genotype difference in dose responses is inferred, such that the ER-a-
mediated facilitatory action of estrogen is more pronounced at lower and physiological doses and the ER-b-mediated inhibitory action
is only unveiled at higher doses of estrogen. In contrast to aggression, the levels of sexual behaviour induced by EB were not different
between bERKO and WT at either dose of EB (2.5 and 12.5 lg ⁄ day) examined. These findings support the notion that ER-b
activation may exert an attenuating action on male aggression induced by estrogen through ER-a-mediated brain mechanisms,
whereas its effect on male sexual behaviour is relatively small.

Introduction
It is well established that testosterone is a major hormone facilitating
aggressive and sexual behaviour in male mice. Testosterone exerts its
neural function not only by acting through androgen receptors (AR), in
its original form or as the 5a-reduced form (dihydrotestosterone), but
also through estrogen receptors (ER) after aromatization to estradiol
(E2). There is accumulating evidence that not only AR-dependent
mechanisms but also ER-dependent mechanisms play crucial roles in the
regulation of aggressive and sexual behaviour in male mice directly in
adulthood as well as through organizational action during perinatal
development (Simon & Whalen, 1986; Simon & Whalen, 1987; Ogawa
et al., 1997; Wersinger et al., 1997). Two types of ERs, ER-a and the
more recently identified ER-b, have been identified in the central
nervous system (Shughrue et al., 1997; Laflamme et al., 1998; Shughrue
& Merchenthaler, 2001; Mitra et al., 2003). ER-a and -b are similar
estrogen-binding proteins which act as ligand-dependent transcription
factors. Neuroanatomical, transfection and behavioural studies collec-
tively suggest that ER-a and ER-b play different roles in the regulation
of neuroendocrine functions and behaviour (Laflamme et al., 1998;
Couse & Korach, 1999; Shapiro et al., 2000; Shughrue et al., 2000;
Vasudevan et al., 2001; Ogawa et al., 2002; Gustafsson, 2003).
Correspondence: Dr Sonoko Ogawa, as above.
E-mail: ogawa@mail.rockefeller.edu
Received 2 October 2005, revised 6 January 2006, accepted 18 January 2006
In a series of behavioural studies using single (aERKO and
bERKO) or double (abERKO) ER-knockout mice, we confirmed the
involvement of ERs in male aggressive behaviour and found that the
two types of ERs differentially regulate them. Aggressive behaviour
was greatly reduced in both aERKO and abERKO male mice, whereas
they were increased in bERKO male mice (Ogawa et al., 1997; Ogawa
et al., 1998; Ogawa et al., 1999; Ogawa et al., 2000; Scordalakes &
Rissman, 2003), particularly in young (5–12 weeks) age groups
(Nomura et al., 2002a) compared to their respective wild-type (WT)
control mice. The opposite effects ER-a and ER-b gene disruption on
aggressive behaviour led us to hypothesize that activation of ER-a by
E2 may be necessary for the induction of aggressive behaviour whereas
activation of ER-b by E2 may inhibit aggression in male mice
depending on age and social situation. In gonadally intact mice,
however, we could not rule out the possibility that elevated levels of
aggression in bERKO mice were simply because AR-dependent neural
systems of aggression were compensatory in a potentiated fashion.
Furthermore, it is not known whether ER-b activation directly inhibits
aggression induced by activation of ER-a by E2, as an aromatization
product of testosterone, or ER-b activation inhibits aggression induced
by activation of AR by androgens. In the present study therefore we
tested whether ER-b activation may actively participate in modulating
aggressive behaviour induced by ER-a-mediated actions of E2 by
examining aggressive behaviour in response to various doses of
estrogen in gonadectomized (GDX) male mice.

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

In male sexual behaviour, on the other hand, simple mounting was
altered greatly only in the mice lacking both ER genes, but not in
single-knockout mice (Ogawa et al., 1997; Ogawa et al., 1998; Ogawa
et al., 1999; Ogawa et al., 2000). These findings suggest that, for the
induction of male sexual behaviour, androgenic stimulation of AR
alone may not be sufficient and at least one ER needs to be intact.
Although in bERKO mice all components of sexual behaviour were
intact (Ogawa et al., 1999; Ogawa et al., 2000), it is not determined
yet whether the levels of sexual behaviour may still remain intact in
bERKO mice even when only ER-a, but not AR, are activated.
Therefore, in the present study we also examined the levels of male
sexual behaviour in estrogen-treated gonadectomized bERKO and WT
male mice.
Materials and methods
Mice
One hundred and forty-five male bERKO mice and their WT
littermates were used. They were obtained from the breeding colony
maintained at the Rockefeller University by mating heterozygous
mice. Original breeding pairs (mixed background of C57BL ⁄ 6J and
129) were obtained from the National Institute of Environmental
Health Sciences. Genotyping of tail DNA was accomplished by
using PCR with primers from intron 2 (5¢-GTGATGAGCT-
GAGGTGGTGCTT-3¢), the 3¢ end of the Neo (5¢-GCAGCCTCTG
TTCCACATACA-3¢) and exon 3 (5¢-CATCCTTCACAGGACC-
AGACAC-3¢); a 1435-bp band (intron 2 and exon 3 primers) is
amplified for homozygous wild-type (+ ⁄ +) mice, a 1479-bp band
(intron 2 and Neo primers) for homozygous mutant mice, and both
bands for heterozygous (+ ⁄ –) mice. All mice were gonadectomized
under isoflurane inhalation anaesthesia at the age of 9–15 weeks and
individually housed in plastic cages (30 · 20 · 12 cm) throughout
the study. They were maintained on a 12–12 h light–dark cycle (light
off at noon) at constant temperature (22
C) throughout the period of
the studies. Food and water were available ad libitum. All
procedures were approved by the IACUC at the Rockefeller
University.
Aggressive behaviour tests
Eighty-two mice were tested for aggression once on the 9th day after
GDX (preimplant test) and assigned to one of four different treatment
groups (see below) matched in the levels of total aggression during the
preimplant test. Eight days after the preimplant test, animals were
implanted under isoflurane inhalation anaesthesia with an estradiol
benzoate (EB) pellet (Innovative Research of America, USA) between
subcutaneous and muscle layers around the shoulder at doses of 2.5
(WT, n ¼ 9; bERKO, n ¼ 13), 5.0 (WT, n ¼ 9; bERKO, n ¼ 12) or
12.5 lg ⁄ day (WT, n ¼ 10; bERKO, n ¼ 11), or a placebo pellet
(WT, n ¼ 7; bERKO, n ¼ 11). All pellets were on a 21-day release
schedule and the average amount released per day was calculated
based on the total amount in the pellets. Mice were then tested for
aggressive behaviour five more times (first to fifth postimplant tests)
every 3–4 days.
Aggressive behaviour was assessed in a resident–intruder paradigm.
Each mouse was tested in his home cage (as a resident) against a
group-housed (four or five mice per cage) weight-matched olfactory
bulbectomized male mouse (heterozygous mouse from the bERKO
breeding colony) for 15 min. Expression of aggression in mice is
mainly regulated by olfactory cues; therefore, olfactory bulbectomized
intruders rarely show aggression. However, as their gonads are intact,
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 1860–1868
ER-b and male aggressive and sexual behaviour 1861
they can elicit aggressive behaviour from resident mice. By testing
against olfactory bulbectomized intruder mice, aggressive behaviour
of resident animals, which were not influenced by any experience of
defeat, were therefore measured (Denenberg et al., 1973). Each
resident mouse was tested against a different intruder mouse during six
aggression tests. All aggressive behaviour tests were performed under
red light during the dark phase of the light–dark cycle starting 2 h after
lights were turned off. The aggressive behaviour tests were videotaped
for further analysis by an observer who did not know the genotypes or
the treatment groups of the mice.
Definition and measurements of aggressive behaviour
Chasing, boxing, tail rattling, biting, lunging and offensive lateral
attack (often accompanied by biting and wrestling), previously shown
to be typical for intermale (male vs. male) aggression, were defined as
aggressive behavioural acts (Ogawa et al., 1996). An aggression bout
was defined as a continuous series of behavioural interactions that
included at least one aggressive behavioural act. Three seconds was
the maximum amount of time that could elapse between aggressive
behavioural acts to be considered part of the same aggressive bout; if
intervals between the occurrences of two aggressive behavioural acts
exceeded 3 s, the two behavioural acts were scored as two separate
aggressive bouts.
All aggressive bouts observed during the 15-min aggression tests
were combined and defined as total aggressive bouts. In addition,
aggressive behaviour bouts with offensive lateral attacks (the most
vigorous aggressive behaviour acts) were counted separately from
those without offensive attacks. For each experimental male, cumu-
lative durations of total aggressive bouts, offensive attack bouts and
nonattack bouts were recorded.
Sexual behaviour tests
An additional 63 mice were tested for sexual behaviour on the 14th
and 21st day (first and second preimplant tests, respectively) after
GDX and assigned to one of three different treatment groups (see
below) matched in the levels of total sexual behavioural acts during
the preimplant tests. Three days after the second preimplant test,
animals were implanted with an EB pellet at a dose of 2.5 (WT,
n ¼ 11; bERKO, n ¼ 9) or 12.5 lg ⁄ day (WT, n ¼ 9; bERKO,
n ¼ 13), or a placebo pellet (WT, n ¼ 11; bERKO, n ¼ 10). All
pellets were on a 21-day release schedule and the average amount
released per day was calculated based on the total amount in the
pellets. Mice were then tested for sexual behaviour four more times
(first to fourth postimplant tests), one test every 5 days. Male sexual
behaviour was measured during a 30-min behavioural test with a
female mouse in the male’s home cage during the dark phase (2–6 h
after lights off) of the light–dark cycle under red light. All stimulus
females (heterozygous mice from the bERKO breeding colony) were
ovariectomized and primed with 10 lg of EB (48 h prior to the tests)
and 500 lg of progesterone (4–7 h prior to the tests) to ensure high
sexual receptivity.
Definition and measurements of sexual behaviour
Male sexual behaviour, i.e. mount, intromission and ejaculation,
were defined as previously described (McGill, 1970). For each male,
the number and latency (1800 s was used for mice that did not show
the behaviour) of mounts, intromissions and ejaculations were
recorded.
1862 M. Nomura et al.
Measurement of serum estradiol
After decapitation trunk blood was collected for measurements of
serum estradiol one day after the completion of behaviour tests (19th
and 21st day after pellet implant for mice tested for aggression and
sexual behaviour, respectively). Serum levels of estradiol were
determined using Coat-A-Count Total Estradiol Kit (DPC, USA).
Data analysis and statistics
All data are presented as mean ± SEM. Data on cumulative duration of
total aggressive bouts, offensive attack bouts and nonattack bouts were
log-transformed before further statistical analyses to ensure homogen-
eity of the variance. Behavioural data were first analysed using a two-
way anova for repeated measurements for the main effects of treatment
(for aggressive behavioural data) or genotype (for sexual behavioural
data), test day as a repeated measure, and their interactions to examine
the time course of behavioural changes before and after hormonal
treatment. Behavioural data combined for multiple test days and blood
estradiol data were analysed using a two-way factorial anova for the
main effects of genotype, treatment and their interaction followed by
post hoc pair-wise comparisons. Fisher’s exact probability test was used
for post hoc pair-wise comparisons at a ¼ 0.05.
Results
Aggressive behaviour
Changes in aggressive behaviour during pre- and postimplant tests
Changes in cumulative duration of total aggressive bouts during six
aggression tests were first examined in each genotype by combining
data from three EB treatment groups and comparing them with those
of the placebo control group.
In the preimplant tests, which were performed 9 days after GDX,
some mice of both genotypes were still aggressive (Fig. 1A and B).
Mean ± SEM cumulative durations of total aggression in WT and
bERKO mice were 7.9 ± 4.3 and 15.1 ± 7.4 s, respectively, and there
was no genotype difference. In bERKO mice, as seen in the placebo
group, aggressive behaviour had subsided by the first postimplant test
(21 days after GDX and 4 days after pellet implant) and completely
disappeared by the second postimplant test. On the other hand, WT
mice of the placebo group were aggressive up to the third postimplant
test (28 days after GDX and 11 days after pellet implant) and became
completely nonaggressive thereafter. However, no significant interac-
tion between genotype and test was observed in the placebo-treated
group. WT mice from the EB-treated group showed steady levels of
aggression during five postimplant tests. In WT mice, there were no
overall significant differences between placebo- and EB-treated groups
in the cumulative duration of total aggression (Fig. 1A). In marked
contrast, there were overall significant effects of EB treatment on the
induction of aggression in terms of cumulative duration of total
aggressive bouts (F1,45 ¼ 7.60, P < 0.01) in bERKO mice (Fig. 1B).
bERKO mice from EB-treated group were highly aggressive
throughout the postimplant tests. Particularly, they were significantly
more aggressive than the placebo control mice during the second, third
and fourth postimplant tests (F1,45 ¼ 4.49, P < 0.05; F1,45 ¼ 7.12,
P < 0.05; and F1,45 ¼ 5.58, P < 0.05, respectively; Fig. 1B). A small
interaction between genotype and test was also observed in EB
treatment group (F1,62 ¼ 2.11, P ¼ 0.079).
Effects of different doses of EB on the levels of aggression
Genotype differences in the effects of different doses of EB on
aggressive behaviour were analysed for the data from the second to
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
fourth postimplant tests, when EB had the maximum effects on
aggression in bERKO mice. The levels of aggression were examined
in terms of (i) the number of tests with the behaviour (ranging from 0
to 3) and (ii) the mean cumulative duration during the three
postimplant tests, for each of three aggressive behavioural bouts, i.e.
total aggressive bouts, offensive attack bouts and nonattack bouts.
Total aggressive bouts
During the second to fourth postimplant tests, there were overall
significant genotype differences in EB-treated groups in both the
number of tests with aggression (F1,58 ¼ 5.28, P < 0.05) and the
cumulative duration of total aggressive bouts (F1,58 ¼ 4.04, P < 0.05;
Fig. 2A), whereas the interaction between genotype and treatment in
the number of tests with aggression did not reach statistical
significance (F1,78 ¼ 2.80, P ¼ 0.102). Overall, EB-treated bERKO
mice were more aggressive than WT mice. These genotype differences
were most pronounced in the group treated with 5.0 lg ⁄ day of EB. At
this dose, bERKO mice were significantly more aggressive than WT
mice in terms of both the number of tests with the behaviour
(F1,19 ¼ 5.48, P < 0.05) and cumulative duration of bouts
(F1,19 ¼ 4.53, P < 0.05). WT mice showed relatively high levels of
aggression only in the group treated with 2.5 lg ⁄ day of EB. At the
higher doses, EB failed to induce aggressive behaviour beyond the
placebo levels. In contrast to WT mice, in bERKO mice all three doses
of EB induced aggression. Particularly, bERKO mice from groups
treated with 2.5 and 5.0 lg ⁄ day of EB showed significantly higher
levels of aggression than the placebo group (Fig. 2A).
Offensive attack bouts
EB-treated bERKO mice showed a significantly higher number of
tests with offensive attack bouts than WT mice (F1,58 ¼ 4.31,
P < 0.05). EB-treated bERKO mice also tended to show longer
duration of offensive attack bouts than WT mice although this
difference did not reach statistically significant levels (F1,58 ¼ 3.47,
P ¼ 0.067; Fig. 2B). WT and bERKO mice showed similar levels of
offensive attacks in the group treated with 2.5 lg ⁄ day of EB, whereas
bERKO mice showed higher levels of aggression than WT mice in the
rest of the groups treated with EB (Fig. 2B). It should be noted that
WT mice from the placebo group, although showing nonattack
aggression, did not show any offensive attacks (Fig. 2B).
Non-attack bouts
Genotype differences and EB dose effects in nonattack bouts were
similar to those of total aggression, although there were no overall
statistically significant genotype differences in either the number of
tests or the cumulative duration (number of tests: F1,58 ¼ 3.20,
P ¼ 0.079; duration: F1,58 ¼ 3.82, P ¼ 0.055; Fig. 2C). EB treatment
tended to be more effective in bERKO mice than in WT mice at all
doses except 2.5 lg ⁄ day of EB.
Sexual behaviour
Changes in sexual behaviour during pre- and postimplant test
Unlike aggressive behaviour, regardless of genotype GDX eliminated
sexual behaviour and the number of mounts with intromission in the
placebo groups remained low throughout the four postimplant tests.
Therefore, we examined genotype differences in the time course of
restoration of sexual behaviour by EB treatment. Data from two EB-
treatment groups (2.5 and 12.5 lg ⁄ day) were combined and compared
between WT and bERKO mice.
European Journal of Neuroscience, 23, 1860–1868
 ER-b and male aggressive and sexual behaviour 1863

Fig. 1. Effect of EB treatment on aggression in terms of the cumulative duration of total aggressive behaviour bouts in (A) WT and (B) bERKO mice.
Gonadectomized mice were tested once before (Pre) and five times after (Post 1–Post 5) pellet implant with the intervals indicated at the bottom of the figure. Pre-
implant test data include all mice, which were assigned to one of four treatment groups (three different doses of EB and placebo). For the postimplant tests, data from
all three EB-treatment groups were combined and compared with those of the placebo control group in each genotype. The data were log-transformed and presented
as mean ± SEM. *P < 0.05, **P < 0.01 vs. respective placebo group. WT n ¼ 7, bERKO n ¼ 11 for the placebo-treated groups and WT n ¼ 28, bERKO n ¼ 36
for the EB-treated groups.

During two preimplant tests, there were no overall significant
genotype differences in the number of mounts with intromission
(Fig. 3A) or the latency to first mount (Fig. 3B). Both WT and bERKO
mice showed low levels of sexual behaviour in the first preimplant
tests and even less in the second preimplant tests. EB treatment
restored sexual behaviour in both genotypes of mice, but with a
different time course after EB pellet implant. During postimplant tests,
there were statistically significant interactions between genotype and
test in the number of mounts with intromission (F3,171 ¼ 3.42,
P < 0.05; Fig. 3A) but not in the latency to the first mount
(F3,171 ¼ 0.48, P ¼ 0.483; Fig. 3B). In WT mice, the levels of
sexual behaviour were initially low but increased gradually in
subsequent tests. bERKO mice, on the other hand, showed steadily
high levels of sexual behaviour throughout all four postimplant tests,
including the first two tests. No mice showed ejaculation throughout
pre- and postimplant sexual behavioural tests.

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 1860–1868
1864 M. Nomura et al.

Fig. 2. Effects of different doses of EB on aggression in terms of (A) total aggression, (B) offensive attack and (C) nonattack aggression during the second to
fourth postimplant tests. In each mouse, the number of tests with the behaviour and the mean cumulative duration during these three tests were calculated for each of
three aggressive behavioural bouts. In EB-treated mice, there were overall significant genotype differences in total aggression (both the number of tests and the mean
cumulative duration) and offensive attack (the number of tests) but not in the mean cumulative duration of offensive attack and nonattack aggression (both the
number of tests and the mean cumulative duration). The data for the cumulative duration were log-transformed. All data are presented as mean ± SEM. *P < 0.05 vs.
WT, aP < 0.05 vs. respective placebo group.

Effects of different doses of EB on the levels of sexual behaviour mounts with intromission (Fig. 4A) and (ii) the mean latency to the
The effects of different doses of EB on the levels of sexual behaviour first mount (Fig. 4B) during four tests. There were significant effects
during four postimplant tests were further analysed. The levels of of EB treatment on the number of mounts with intromission
sexual behaviour were examined in terms of (i) the mean number of (F2,57 ¼ 8.22, P < 0.01) and the latency to the first mount

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 1860–1868
 ER-b and male aggressive and sexual behaviour 1865

Fig. 3. Genotype differences in the time course of restoration of sexual behaviour by EB treatment, in terms of (A) the number of mounts with intromission and
(B) the latency to the first mount. Gonadectomized mice were tested twice before (Pre 1 and Pre 2) and four times after (Post 1–Post 4) pellet implant with the
intervals indicated at the bottom of the figure. Data from two EB treatment groups (2.5 and 12.5 lg ⁄ day) were combined and compared between WT and bERKO
mice. During the postimplant tests, there were significant interactions between genotype and test in the number of mounts with intromission. The levels of sexual
behaviour in WT mice were initially low but increased gradually in subsequent tests, whereas those of bERKO mice were steady throughout the postimplant tests.
The data are presented as mean ± SEM. WT n ¼ 20, bERKO n ¼ 22. *P < 0.05 vs. WT,  P < 0.1 vs. WT.

(F2,57 ¼ 24.36, P < 0.01), but not those of genotype or genotype ·
treatment. Two doses of EB, 2.5 and 12.5 lg ⁄ day, equally restored
sexual behaviour.
Serum estradiol levels
Serum E2 levels were measured in 145 samples. Although blood
samples were collected on different days after pellet implant, i.e. 19th
day for aggression test groups and 21st day for sexual behavioural test
groups, there were no significant differences between these two groups
of mice in any treatment groups. Therefore, the data from these two
behavioural testing groups were combined. There were no significant
genotype differences in the levels of serum E2 in each EB-treated
group. The serum levels were nondetectable in the group treated with
placebo, 54.0 ± 11.9 (WT, n ¼ 20) and 58.7 ± 12.6 pg ⁄ mL (bERKO,
n ¼ 22) in the group treated with 2.5 lg ⁄ day of EB, 428.4 ± 116.4
(WT, n ¼ 9) and 247 ± 77.0 pg ⁄ mL (bERKO, n ¼ 12) in the group
treated with 5.0 lg ⁄ day of EB, and 762.4 ± 76.9 (WT, n ¼ 19) and

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 1860–1868
1866 M. Nomura et al.
Fig. 4. Effects of different doses of EB (2.5 and 12.5 lg ⁄ day) on sexual behaviour in terms of (A) the number of mounts with intromission and (B) the latency to
the first mount. Two doses of EB, 2.5 and 12.5 lg ⁄ day, equally restored sexual behaviour in both genotypes of mice. Data are presented as mean ± SEM.
**P < 0.01 vs. respective placebo group.
919.7 ± 223.8 pg ⁄ mL (bERKO, n ¼ 24) in the group treated with
12.5 lg ⁄ day of EB.
Discussion
Aggressive behaviour
The present study revealed a clear genotype difference between WT
and bERKO male mice in estrogen-inducible aggression in terms of
both time course and dose dependency. Overall, EB-treated bERKO
mice showed significantly higher levels of aggressive behaviour
during the five postimplant tests, particularly second to fourth tests,
than did the placebo-treated mice. On the other hand, in WT mice
relatively low levels of aggression were seen in EB-treated mice
throughout the postimplant tests and difference from the placebo
group was only apparent during the last two tests. In addition, there
was a small interaction between genotype and treatment in the levels
of aggression during postimplanted tests. These genotype differences
in the effects of EB treatment reflect the difference in responses to
different doses of EB. Detailed analyses during second to fourth
postimplant tests revealed that, in WT mice, only the treatment with
2.5 lg ⁄ day of EB was effective for the induction of aggressive
behaviour beyond the placebo levels. In contrast, the treatments with
all doses of EB were effective in bERKO mice although the levels of
aggression tended to be higher in the groups treated with two lower
doses, 2.5 and 5 lg ⁄ day, than the higher 12.5 lg ⁄ day dose group.
These results indicate that estrogenic stimulation by itself is sufficient
to induce aggression in gonadectomized male mice. Our findings that
the overall effects of EB were more pronounced in bERKO mice than
in WT mice suggest that facilitatory behavioural effects of estrogen are
mediated by ER-a and that additional activation of ER-b plays an
inhibitory role in estrogen-inducible aggression in male mice.
Furthermore, a marked genotype difference in dose responses suggests
that the ER-a-mediated facilitatory action of estrogen may be more
pronounced at lower and physiological doses and the ER-b-mediated
inhibitory action may only be unveiled at higher doses of estrogen. It
should be noted that the possibility that activation of ER-b by
moderate to high doses of E2 causes lethargy in WT mice cannot be
completely ruled out, although we did not notice any genotype
differences in general activity during aggression tests in the mice
treated with these doses of EB.
The notion that ER-b activation mediates inhibitory effects of
gonadal steroids on aggressive behaviour in male mice is consistent
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
with our previous studies in gonadally intact mice. In our earlier
studies in gonadally intact adult males, we found that aggressive
behaviour was higher in bERKO than WT mice in the first aggression
tests but not in subsequent tests, in which the levels of aggression in
WT mice steadily increased whereas those in bERKO mice were
unchanged (Ogawa et al., 1999). Potentiation of aggressive behaviour
by ER-b gene disruption was more obvious in younger age bERKO
mice (5–12 weeks), which showed significantly higher levels of
aggression than WT mice during all three repeated tests (Nomura
et al., 2002a). In these gonadally intact mice, however, elevated levels
of aggression were not simply attributed to the lack of ER-b-mediated
inhibitory action in bERKO mice. Rather, the AR-mediated neural
system of aggression might be altered in bERKO mice due to the lack
of ER-b. Although the possibility that this may additionally contribute
to the elevated levels of aggression in bERKO mice is not completely
excluded, the present study supports the idea that the very lack of
ER-b can increase the levels of aggression in male mice. As the levels
of intermale aggression are greatly reduced or even completely
abolished in terms of the most vigorous offensive attacks in aERKO
mice (Ogawa et al., 1997; Ogawa et al., 1998; Ogawa et al., 2000;
Scordalakes et al., 2003), ER-a is necessary for the induction of male
aggression. Taken together, we can conclude that activation of ER-a
facilitates and activation of ER-b inhibits aggression. It should be
noted that this is the first demonstration that two types of estrogen
receptors regulate one specific behavioural endopoint in opposite
directions, although it has been shown that ER-a and ER-b seem to
operate in such an antagonistic ‘yin–yang’ mode in other physiolo-
gical contexts (Gustafsson, 2003; Lindberg et al., 2003; Matthews &
Gustafsson, 2003; Weihua et al., 2003). It still remains to be
determined whether activation of ER-b may also have opposite effects
to AR-activation on aggression: whether aggressive behaviour induced
by activation of AR can be reduced by activation of ER-b.
Even though the effects may not be completely opposite to those of
ER-a, there are other cases in which the final outcome of behaviour
may be modulated by ER-b activation (Ogawa et al., 2002). For
instance, expression of female sexual behaviour measured as lordosis
quotient was not affected by ER-b gene disruption on the day of the
behavioural estrus, when females are most receptive (Ogawa et al.,
1999). However, bERKO females showed significantly higher recep-
tivity than WT females on the day after behavioural estrus. These
findings suggest that ER-b may be responsible for fine-tuning of
expression of receptivity, particularly the turning-off phase during the
estrous cycle. As some ER-a- and ER-b-specific agonists are currently
European Journal of Neuroscience, 23, 1860–1868

available, a complementary study using these agonists would solidify
behavioural alterations observed in genetically modified mice.
There are a number of hypotheses of potential brain mechanisms
underlying modulatory roles of ER-b activation on aggressive
behaviour. It is known that ER-b protein and mRNA are expressed
more abundantly than ER-a in brain areas such as the paraventricular
nucleus, bed nucleus of stria terminalis, medial amygdala and
midbrain dorsal raphe nuclei (Shughrue et al., 1997; Shughrue &
Merchenthaler, 2001; Mitra et al., 2003; Nomura et al., 2003).
Neuropeptides, e.g. oxytocin and vasopressin, as well as neurotrans-
mitters, e.g. serotonin, known to be synthesized in these brain areas,
are also implicated in the regulation of aggressive behaviour. Both
oxytocin (Richard & Zingg, 1990; Mohr & Schmitz, 1991) and
arginine vasopressin (Shapiro et al., 2000) genes contain an ER-
responsive element and recent in vitro studies in ER-b transfected cells
have demonstrated that ER-b activation by estradiol regulates the
oxytocin and arginine vasopressin promoter activity (Shapiro et al.,
2000; Loven et al., 2001). It has been shown that both oxytocin- and
vasopressin-synthesizing neurons in the paraventricular nucleus
coexpress ER-b (Simonian & Herbison, 1997; Alves et al., 1998;
Hrabovszky et al., 1998; Laflamme et al., 1998) and estrogenic up-
regulation of oxytocin and down-regulation of vasopressin in this
brain area are either abolished or attenuated in bERKO male mice
(Nomura et al., 2002b). A number of behavioural studies have
collectively demonstrated that oxytocin facilitates affiliative behaviour
(Insel, 1992; Nelson & Panksepp, 1998; Young et al., 2001) and
inhibits aggression (Harmon et al., 2002; Lubin et al., 2003), and
vasopressin facilitates aggression (Ferris & Potegal, 1988; Wersinger
et al., 2002). Therefore, it is possible that the elevated levels of
aggression in bERKO male mice may be partially due to an alteration
of estrogenic regulation on syntheses of these neuropeptides.
Another possible mechanism is ER-b-mediated regulation of brain
serotonergic systems. It is now well established that ER-b is the
predominant form of ER localized in the dorsal raphe nuclei (Lu et al.,
1999; Gundlah et al., 2001; Lu et al., 2001). Our preliminary studies
revealed that > 90% of ER-b-immunopositive cells in the dorsal raphe
nuclei coexpress tryptophan hydroxylase (TPH), the rate-limiting
enzyme of serotonin synthesis, whereas only 20% of ER-a positive
cells express TPH. Although the exact outcome of colocalization of
ER-b and TPH still needs to be determined, it is assumed that a lack of
ER-b in TPH-positive cells may profoundly affect estrogenic regu-
lation of the brain serotonin system. As serotonin is the most well
characterized neurotransmitter for its inhibitory effect on aggression
(Miczek et al., 2001; Nelson & Chiavegatto, 2001), it is hypothesized
that alteration of ER-b-mediated regulation of serotonin systems may
also be involved in the modulatory role of ER-b on aggression.
Sexual behaviour
Unlike its effects on aggression, the effects of estrogen treatment on
the restoration of sexual behaviour were not different between WT and
bERKO mice, except a small, but statistically significant, difference in
the time course of behavioural recovery after EB pellet implant. Also,
two doses of EB, the lower (2.5 lg ⁄ day) and the higher
(12.5 lg ⁄ day), induced equal amounts of male sexual behaviour in
both WT and bERKO mice. This is also in striking contrast to possible
EB dose-dependent differential roles of the two types of ER in
aggressive behaviour. These results collectively demonstrate that the
lack of ER-b does not affect the expression of male sexual behaviour.
Furthermore, these results suggest that the levels of sexual behaviour
remain intact in bERKO mice even when only ER-a, but not AR, is
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 1860–1868
ER-b and male aggressive and sexual behaviour 1867
activated. This does not necessarily mean, however, that ER-b is not
involved in the control of male sexual behaviour. A recent study in
pubertal age bERKO male mice reported that they showed reduced
levels of sexual behaviour (Temple et al., 2003). In addition, our
previous studies in gonadally intact adult male mice revealed that male
sexual behavioural components such as ultrasonic vocalization and
mounting behaviour were intact not only in bERKO (Ogawa et al.,
1999) but also in aERKO mice, although aERKO mice showed
reduced levels of intromission and complete abolition of ejaculation
(Ogawa et al., 1997, 1998, 2000; Wersinger et al., 1997). On the other
hand, all components of male sexual behaviour were completely
abolished in double knockout mice (Ogawa et al., 2000). These
findings suggest that ER-b plays a role, at least partially, in the
expression of sexual behaviour in male mice.
Acknowledgements
The authors are thankful to Ms L. Durbak and Ms M. Markey for their technical
assistance. Support for this work was provided by NIMH 62147 to S.O. M.N.
was a recipient of postdoctoral fellowships for research abroad from the Japan
Society for Promotion of Science.
Abbreviations
AR, androgen receptor; E2, estradiol; EB, estradiol benzoate; ER, estrogen
receptors; ERKO, ER-knockout; GDX, gonadectomized ⁄ gonadectomy; WT,
wild-type.
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