ORIGINAL PAPER

Jpn J Reprod Endocrinol ⓒ2003 by
Japan Society of Reproductive Endocrinology

Behavioral Evaluation of Sociosexual Dysfunction in
Aromatase Knockout Mice
Kazuo YAMADA1), Yasuhiko KONDO2), Katsumi TODA3) and Yasuo SAKUMA2)
1) Department of Psychology, Mejiro University, Tokyo 161-8539, Japan
2) Department of Physiology, Nippon Medical School, Tokyo 113-8602, Japan
3) Department of Molecular Genetics, Kochi Medical School, Kochi 783-8505, Japan

Abstract. Estrogen has been known to play a critical role in regulation of male sexual behavior. Recent studies
demonstrated severe impairment of male mouse sexual and aggressive behavior by targeted disruption of aromatasegene, Cyp19. In the present study, as basis for a loss of sexual behavior, we compared odor difference between wild
and aromatase-knockout (ArKO) mice using probe estrous females in sexual preference paradigm. The probe females reacted equally to body smell and soiled bedding of both ArKO and normal mice, suggesting androgen regulation of pheromones and/or attractive odorants emitted by sexually vigorous male mice. Emotionality of those mice
was also measured in open field and elevated plus maze. ArKO mice had significantly low activity in open field, but
not in elevated plus maze, compared to wild-type mice. We suggest that absence of aggressive behavior is based on
their high emotionality (low activity in unfamiliar area).

Key words: Estrogen, Emotion, Pheromone, Knock-out, Sexual differentiation

Introduction

in male animals throughout development.
Recently, transgenic techniques have been well devel-

There has been accumulated ample evidence showing

oped for studying neuroendocrine mechanisms of sexual

critical roles of estrogen in brain differentiation of sexual

differentiation. Deletion of estrogen receptor gene, both

dimorphism in physiology and behavior. Early exposure

ERs α and β, demonstrates the importance of estrogento either estrogen or aromatizable androgen in ontogeny

in development of reproductive systems in mice. Female

produces masculine phenotype such as male copulatory

mice lacking ER α showed a complete loss of female sex-

[1, 2] and aggressive behaviors [3, 4], whereas absence ofual behavior [13], and males lacking ER α showed partial

these hormones in the critical period induces female phe-disruption of male sexual behavior [14-16]. Although ER

notype, lordosis behavior [5] and ovarian functions [6, 7].

β had no effect on both female and male sexual behavior

Estrogen also plays an important role in expression of[17], double knockout of ERs α and β eliminated male

copulatory behavior in adult males. Prevention of metabo-mouse sexual behavior [18]. Presumably, ERs α and β

lism from androgen to estrogen by aromatase inhibitors

may interact each other to regulate male and female sexu-

disrupts mount behavior in male rats [8], hamsters [9]al behavior [19].

and mice [10], whereas treatment of estrogen activates

Another line of studies uses aromatase knockout

mount behavior in male and female rats [11, 12]. Thus, es-(ArKO) mice that cannot produce estrogen [20-22].

trogen exerts both the organizational and priming effects

These ligand-deficiency mice showed severe impairmentof copulatory behavior in the male and infertility in the female [21-23]. They also showed a loss of intermale ag-

Accepted: August 22, 2003
Correspondence: Yasuhiko KONDO, Department of Physiology, Nippon Medical School, Sendagi 1, Bunkyo-ku, Tokyo
113-8602, Japan
TEL: +81-3-3822-2131 (ext. 5356)
FAX: +81-3-5685-3055
E-mail: ykondo@nms.ac.jp

gression [24, 25] and sex partner preference [26, 27].
In the present study, we investigated how the malemice lacking aromatase (disruption of Cyp19) fail to display sexual behavior and aggression. It is possible that alack of estrogen in peripheral organs changes the produc-Japanese Journal of Reproductive Endocrinology (2003) 8 : 61-66

61Kazuo YAMADA et al.

tion of pheromone and/or other odorants, causing changes in their attractiveness and female approaches. Then,we examined differences of their body smell between
ArKO and wild-type mice using partner/odor preference
paradigm. In addition, central estrogen might change
their emotional responsiveness on which aggressive behavior is based. Then, we also examined their emotionality measured in open field and elevated plus maze.

Materials and Methods
１）Animals
Animal care and experiments adhere to the Guidelines
for Care and Use of Laboratory Animals of the Institutionof Laboratory Animal Resources, National Research Council (1996). All animal were maintained in controlled temperature and illumination (lights on 23:00-11:00). Foodand water were given ad libitum. ArKO male mice weregenerated by targeted disruption of Cyp19 gene as previously described [28, 29]. Heterozygous males and femalesof C57BL/6 strain were bred to generate wild-type (+/+),

Fig. 1. Schematic representation of the apparatus used in test forbody smell in ArKO mice. A probe female was located between ArKO and control male mice, and time spent by thefemale to stay in the vicinity of each stimulus male wasmeasured.

heterozygous (+/-) and homozygous-null (ArKO) offspring. Nine ArKO, 6 +/- and 5 +/+ males (8 weeks old at

had air-inlets (mesh-covered holes, 3 cm in diameter), andbeginning) were used for the following behavioral experi-

the center compartment had an air-outlet (10 cm in diame-ments.

ter) at the back. A motor-driven blower, attached to the

As probe females, another 9 C57BL/6 females (8 weeks

air-outlet via a flexible duct, introduced air from the left

at beginning) were ovariectomized under ether anesthe-and right compartments into female areas (approximately

sia, and injected subcutaneously 48 hours before each be-

0.2m3/min).

havioral test with 5 µg estradiol benzoate (EB) followed
by 0.5 mg progesterone (P) 4 hours before the test.
２）Tests for Body Smell of ArKO Mice

During acclimation, each of paired experimental males
was located in the side compartments, respectively, and aprobe female was placed in center of the apparatus. The
blower attached in opposite direction to make air flow out

Partner preference paradigm was used for testing theirfrom female areas into the male compartments (no pre-body smells. Probe females were located between ArKO

sentation of odors). The acclimation period lasted for 10

males and +/- males or between ArKO and +/+ males. The

min.

acrylic cage (30 cm x 50 cm x 30 cm) divided into 3 areas;

After the acclimation, the probe female was relocated toright, center and left areas, by the acrylic walls with 5 cm

the center area and the direction of blower was returned

wide pathway (see Fig. 1). The apparatus was bedded

to proper position. The time spent by the female in eachwith white paper tips (Alpha-dri, Shepherd Specialty Pa-

area was recorded for 10 min. After every test, the appara-pers, Inc., MI). The back ends of right and left areas had

tus was cleaned and sprayed with 70% ethnol. Fresh paper

small compartments separated by duplicate opaque panels

beddings were put into the apparatus ready for the next

for experimental males. Each panel, with a 2 cm hole intest.

diameter, bored at different position, were assembled into

A week after the tests, the same tests with different

a partition, which allowed the passage of air but prevented

combinations of paired males were carried out as the sec-visual and physical interactions. The male compartments

ond tests.

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Japanese Journal of Reproductive Endocrinology Vol. 8 2003

Behavioral Traits of Aromatase-KO Mice

３）Tests for Excretion-Odors of ArKO Mice
Using their soiled bedding in home cage as stimuli,
odor preference test was conducted in an acrylic observation box (30 cm × 50 cm × 40cm). Probe females were located between soiled beddings of ArKO males and clean
beddings, between soiled beddings of +/- males and clean
beddings, and between soiled beddings of ArKO males
and of +/- males. The beddings were collected from eachmale’s home cage immediately before each test. The beddings on a grass Petri dish (12 cm in diameter) wereplaced in the observation box apart from each other. The

Fig. 2. Mean time spent by probe females in each area in bodysmell test. The probe females spent almost same time close
to ArKO and +/+ male mice (left) and to ArKO and +/- malemice (right). Vertical bars represent SEM.

time spent for contacting each dish was measured during
5 min.

Results４）Open Field Test

１）Test for Body Smell

Open field tests were daily carried out for 3 consecutive

During the acclimation period, the mean time (sec)days in a white polyvinyl chloride circular chamber (90spent by probe females in each area was not significantly

cm in diameter with 50 cm walls). The floor was marked

different in both paired stimulus males (ArKO and WT,

by 8 radial lines and a 50 cm circle, which divided the are-123.33 ± 18.62 and 171.28 ± 34.19; ArKO and HET, 147.98

na into the center and rim. The apparatus was illuminated± 38.58 and 106.12 ± 18.29). In the test period, the meansby overhead fluorescent lighting.

of time spent in each area is shown in Fig. 2. Although theExperimental males were placed in the center of theprobe females tended to spend longer time in the vicinity

arena. The number of line crossings, rearings, and fecesof stimulus experimental males, compared to that during

were measured for 3 min.

the acclimation period, they showed no preference at all.

５）Elevated Plus Maze Test
One week after open field tests, all males were subjected to elevated plus maze test. The elevated plus maze con-

They spent almost same time close to ArKO and +/+ malemice and to ArKO and +/- male mice (t < 1, ns).
２）Test for Soiled beddings

sisted of a black and white acrylic plus shaped maze ele-

Fig. 3 shows the results of tests for soiled beddings ofvated 60 cm from floor. Two opposite arms, 5 × 30 cm,

experimental male mice. The probe females explored sig-

were black, and another two arms of the same dimensions

nificantly longer the soiled beddings collected from homewere white. The black arms were enclosed by 15 cm highcages of ArKO males (t(8) = 5.78, p < .01) or +/- males

walls (closed arms), and white arms had no wall (open(t(8) = 4.76, p < .01), compared to clean fresh beddings.

arms). The proportion of time spent onto open arms was

They spent time for exploring either soiled beddings

measured for 10 min as an index of emotionality. Experi-

longer than 3 times of clean beddings.

mental males received one trial per day on each consecutive 3 days.
６）Statistics
All data were first analyzed by two-way ANOVA with repeated measures. When comparing a pair among thegroups in each test, statistical significance was evaluatedby t test.

When presented a pair of soiled beddings collectedfrom ArKO and +/- males, the probe females tended to explore the soiled beddings of ArKO males longer thanthose of +/- males, but the difference did not yielded statistical significance because of the large variances.
３）Open Field Behavior
In +/+ male mice, highest locomotion activity was observed on Day 1, and the activity gradually decreased

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Kazuo YAMADA et al.

Fig. 3. Mean time spent by probe females for exploring each bedding. The probe females explored significantly longer the soiled bedding collected from ArKO (left) or +/- male s cages (center), compared
to clean bedding. No difference was found in test between ArKO and +/- males (right). Verticalbars represent SEM. **p < .01.

Fig. 4. Mean number of line-crossings in open field test over 3 consecutive days. Locomotion activity of ArKO males was significantly lower than that of +/+ males on Day 1. Verticalbars represent SEM. **p < .01.

Fig. 5. Mean proportion of time stayed in open arms in elevatedplus maze test over 3 consecutive days. No difference wasfound among ArKO, +/+ and +/- males throughout 3 days.
Vertical bars represent SEM.

through the 3 test days (Fig. 4). The tendency was almost

most of the test period throughout 3 days.

same in +/- males, but variance was much larger thanthose of +/+ males. In contrast to those males, locomotion
activity of ArKO males was lower from Day 1 and no

Discussion

change through the 3 test days (Fig. 4). On Day 1, theThe first half of the present study clearly showed thatmean number of line crossings was significantly lower inreceptive females do not differently react to ArKO and

ArKO males than that of +/+ males (t(12) = 4.01, p < .01).

normal male mice. The probe females equally accessed to

The mean numbers of rearings and defecations did notodors of either ArKO mice or +/+, +/- mice. In addition,differ significantly among all genotypes throughout 3 days

the females were also interested in soiled beddings of(data not shown).

ArKO males, when compared to clean one.

４）Elevated Plus Maze Activity

Chemosensory signals play extremely important roles
in sociosexual behavior in mice, as well as other mamma-

The results of elevated plus maze test, which are indi-lian species [30, 31]. The signals mainly excreted withcated by proportion of time stayed in open arms, areurine are received via two distinctive systems, olfactory

shown in Fig. 5. No difference was found among ArKO,

epithelium and vomeronasal organ, and travel into the+/+ and +/- males. They all stayed in the closed arms forlimbic and hypothalamic regions, regulating sociosexual64

Japanese Journal of Reproductive Endocrinology Vol. 8 2003

Behavioral Traits of Aromatase-KO Micebehaviors. Recently, a complete loss of sex discrimination

ing ArKO males were more anxious than wild or heterozy-and intermale aggression was reported in mice lacking

gous males [27].

TRP2, a putative ion channel of the transient receptor po-

On the other hand, there was no clear difference be-tential family that is required for sensory activation of

tween ArKO males and +/+ or +/- males in elevated plus

vomeronasal neurons and exclusively expressed in thosemaze. ArKO males seem to spend rather longer time inneurons [32]. Thus, chemosensory signals broadly affectopen arms, but with large variances and no statistical dif-social behavior including copulatory, maternal and ag-

ference, than other controls. Although both open field andgressive behaviors.

elevated plus maze tests has been believed to measure

There is also evidence revealing that secretion of thoseemotionality or anxiety in rodents, these 2 tests may havepheromonal and attractive chemicals is regulated by go-different sensitivity or measure somewhat different aspectnadal hormones [33]. Attractiveness of male odors to fe-of emotionality. Indeed, there is some contradiction inmales depends directly on the level of circulating testos-studies of mice lacking ER α, increased in elevated plus

terone in voles. Gonadectomy decreases attractiveness ofmaze [36] and decreased in open field [14]. As ArKO andodors to opposite sex conspecifics in both males and fe-

ER-knockout are not the same, our present results andmales, while replacement of testosterone in males or es-

previous study of ArKO [27] suggest that estrogen rather

trogen in females restores it [34]. ArKO males have no es-

reduce emotionality in mice.

trogen and elevated level of testosterone in blood [20, 22].

The present study showed no difference in body smell

However, our probe females that were highly receptive by

and high emotionality in ArKO male mice. The high emo-

treatment with estrogen and progesterone react indiffer-tionality (nearly equal to timidity) might be related to low

ently to odors of both ArKO and control male mice. Thisaggression in ArKO mice [37]. Presumably ArKO micesuggests that secretion of male pheromones or attractive

may have much more other characteristics in behavior.

odorants is regulated by androgen but not estrogen con-Further studies are required to understand ArKO mice,verted by aromatase.

and of course, roles of estrogen and aromatase.

Our soiled bedding tests indicate that (1) odors fromboth ArKO males and other controls are attractive, notaversive, because longer time spent for exploration of it
compared to clean beddings, and (2) attractive odorants
was contained in excretions. Electrophysiological recording in behaving mice indicated activation in some neurons
in the accessory olfactory bulb, secondary sensory neurons in vomeronasal system, when their nose contacted tohead or face area of stimulus conspecifics [35]. However,substances in urine may be primarily important for sexual
attractiveness.
In conclusion, we can reject the hypothesis that copulatory deficit in ArKO males is due to loss of sexual pheromone or attractive odorants to females emitted by malemice. The impairment of copulatory activity in ArKO mice
may be purely caused by absence of estrogen action inneural circuits responsible to male sexual behavior.
As to second part of the present study, emotionality of
ArKO males, locomotion activity of ArkO males was lower
than that of +/+ males in open-field test, suggesting that
disruption of aromatase gene resulted in increased emotionality. This result agrees with the previous study show-

Acknowledgement
The authors thank Mss. Natsue Shimizu, Mai Ito,
Chisano Sakai, and Kanako Moriya for helping behavioral
testing, and also thank Mr. Kai Xiao for helping experiment and animal care.

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