Disruption of adult expression of sexually selected
traits by developmental exposure to bisphenol A
Eldin Jašarevic ´a,b,c, Paizlee T. Sielib,d, Erin E. Twellmanc, Thomas H. Welsh, Jr.e, Todd R. Schachtmana,f,
R. Michael Robertsb,g,h,1, David C. Gearya,f,1, and Cheryl S. Rosenfeldb,c,1
aInterdisciplinary Neuroscience Program,bBond Life Sciences Center, and Departments ofcBiological Sciences,dBiomedical Sciences,fPsychological Sciences,
gBiochemistry, andhAnimal Sciences, University of Missouri, Columbia, MO 65211; andeDepartment of Animal Science, Texas A&M University, College Station,
Contributed by R. Michael Roberts, May 18, 2011 (sent for review March 31, 2011)
Exposure to endocrine disrupting compounds (EDCs), such as
bisphenol A (BPA), may cause adverse health effects in wildlife
and humans, but controversy remains as to what traits are most
sensitive to EDCs and might serve as barometers of exposure.
Expression of sexually selected traits that have evolved through
intrasexual competition for mates and intersexual choice of mating
partner are more dependent on developmental and physical con-
dition of an animal than naturally selected traits and thus might
to EDCs. We have used the deer mouse (Peromyscus maniculatus)
as a model to test this hypothesis. Adult male–male competition
for mates in this species is supported by enhanced spatial naviga-
tional and exploratory abilities, which enable males to search for
prospective, widely dispersed females. Male deer mice exposed to
BPA or ethinyl estradiol (EE) through maternal diet showed no
changes in external phenotype, sensory development, or adult
circulating concentrations of testosterone and corticosterone, but
spatial learning abilities and exploratory behaviors were severely
compromised compared with control males. Because these traits
are not sexually selected in females, BPA exposure predictably
had no effect, although EE-exposed females demonstrated en-
hanced spatial navigational abilities. Both BPA-exposed andcontrol
females preferred control males to BPA-exposed males. Our dem-
onstration that developmental exposure to BPA compromises
cognitive abilities and behaviors essential for males to reproduce
successfully has broad implications for other species, including our
own. Thus, sexually selected traits might provide useful biomarkers
toassess riskof environmental contaminationin animal andhuman
mate choice|sexual selection|spatial abilities|cognition|sex differences
(EDCs) has posed a major threat to wildlife since the large-
scale production of these industrial chemicals (1). Numerous stud-
ies have documented disturbances of sex-typical development,
reproductive tract pathologies, and abnormal adult behaviors
through environmental contact with EDCs, including bisphenol
A (BPA) (2–7). However, scant information is available regard-
ing exposure to EDCs during development within the context of
sexual selection (8, 9). Expression of sexually selected traits is
critical to reproductive fitness and may be particularly vulnerable
to EDCs because these traits show greater phenotypic variation
than naturally selected traits, owing in part to dependence on
more genetic loci and overall body condition (10, 11). Moreover,
optimal expression of these traits in adulthood requires a com-
plex orchestration of developmental exposure to estrogens and
androgens, processes that can be compromised by EDC expo-
sure (3, 4). We predicted that traits that evolved through intra-
sexual competition for mates and influence intersexual choice
of mating partner would be particularly sensitive to EDCs (3, 12,
13) and might serve as useful barometers for detecting such
chemicals in the environment.
evelopmental exposure to endocrine-disrupting compounds
To capture the variation in sexually selected traits found in wild
populations, we used polygynous deer mice (Peromyscus man-
iculatus) to assess the effects of developmental exposure to BPA
and ethinyl estradiol (EE), a synthetic estrogen, on male com-
petitive behaviors and males’ attractiveness to females. Males of
this species compete by expanding their territorial range during
prospective mates widely dispersed throughout the ecology. This
enhanced adult male spatial ability and exploratory behavior
requires not only seasonal increases in testosterone but prenatal
exposure to this hormone (14), with the latter requirement ren-
dering these traits potentially vulnerable to developmental EDC
exposure. Once a prospective mate is found, females choose
whether to mate with the male. Female choice is largely mediated
by olfactory cues and behaviorally indicated by time engaged in
nose-to-nose contact with and inspecting the male (15–17). On
the basis of the sexually selected traits particular to this species,
we assessed the effect of developmental exposure to BPA or EE
on spatial navigational ability and exploratory behavior in male
and female deer mice and female preference through a mate
Litter Data. Average litter size did not differ across maternal diet
(2.95 ± 0.28, 2.76 ± 0.31, and 2.85 ± 0.20 for control, EE, and
BPA groups, respectively) (Table S1). However, dams fed the
EE diet had relatively fewer male offspring (41% male) than
females receiving the control (62% male) and BPA-supple-
mented (65% male) diets (Table S1). Binomial tests, assessing
whether the differences differed from the expected 50% sex
ratio, were significant for the control (P = 0.009) and EE (P =
0.001) groups, and there was a trend for the dams on the BPA
diet (P = 0.053). The more critical finding, however, was that the
sex ratios for dams on the control and BPA-supplemented diets
did not differ from each other (P = 0.83).
Assessments of Sensory and Neuromuscular Function. Initial obser-
vations indicated offspring exposed to BPA and EE through
maternal dietduring prenatal development untilweaning(day 25)
demonstrated no visible abnormalities in phenotype, including
coat color and weight. Males and females in all of the groups
exhibited intact sensory systems, including olfaction, neuromus-
cular strength, response to sound, and vision (Table S2).
Author contributions: E.J., T.R.S., R.M.R., D.C.G., and C.S.R. designed research; E.J., P.T.S.,
E.E.T., and T.H.W. performed research; D.C.G. analyzed data; and E.J., T.R.S., R.M.R., D.C.G.,
and C.S.R. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, Gearyd@
missouri.edu, or email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1107958108 PNAS Early Edition
| 1 of 6
Spatial Learning and Memory. At sexual maturity (approximately
day 60), spatial learning was assessed with the Barnes maze, a
circular, dry-land maze that assesses the animal’s ability to use
visual cues to navigate out of the maze (Fig. S1). Behavior of
each mouse was recorded and analyzed with automated tracking
software (EthoVision XT; Noldus), allowing a composite track-
ing image to be assembled for every trial. After 2 d of habituation
(5 min each), testing was performed on 7 successive days, with
two trials per day. Fig. 1 shows the time required to reach the
correct exit (latency) for males (Fig. 1A) and females (Fig. 1C)
across training. After 2 d, control males exhibited shorter la-
tencies to exit the maze than BPA- or EE-exposed males (P <
0.0001 for each) and females in all diet groups (P < 0.004). Males
exposed to BPA or EE did not differ from each other on any day
(P > 0.0834) (Fig. 1A). Except for day 2, females exposed to EE
reached the exit hole sooner than control females (P < 0.0051).
Frequency of entering the wrong exit hole for males (Fig. 1B)
and females (Fig. 1D) was also affected by maternal diet. Control
males and EE-exposed females did not differ in number of
committed errors (P = 0.1767), but both groups committed
fewer errors than males exposed to either BPA or EE (P <
0.0001 for each comparison) and control females (P < 0.0001).
Automated tracking of movement in the Barnes maze allowed
three types of search strategy to be identified: random (with no
particular pattern), serial (in which the mice patrolled the pe-
rimeter), and spatial (in which the most direct approach to the
exit hole was used, presumably based on learned use of visual
cues) (Fig. 2A; further details supplied in SI Results). The
dominant strategy on day 1 was random across the two trials,
with the exception of EE-exposed females (Fig. 2B). Strategy
differences between groups of males emerged within several days
of training (Fig. 2B). On average, control males acquired the
spatial-oriented strategy by day 3 of training, whereas the ma-
jority of males exposed to BPA or EE failed to shift to the
spatial-oriented strategy by the end of training (P < 0.0002).
Although behaviors for the BPA and EE male groups became
less random, few demonstrated use of the more efficient spatial-
oriented strategy used by control males (Movies S1–S3). EE-
exposed females rapidly acquired the spatial-oriented strategy
typical of control males (Movie S4) and, after day 2, outper-
formed control and BPA-exposed females (P < 0.0046 and P <
0.0084, respectively). Moreover, EE-exposed females did not
differ from control males after day 1 (P > 0.20).
These combined results reveal that spatial learning in males is
compromised by developmental exposure to either BPA or EE in
the maternal diet. Conversely, EE but not BPA exposure seemed
to render female spatial behavior more like that of control males
with respect to error frequency and spatial strategy; they also had
shorter latencies than control females. When total distance
traveled was used as a covariate, it did not alter the significance
of the latency data (SI Results, Spatial Learning and Memory).
Exploratory and Anxiety-Like Behavior.After completing the spatial
learning and memory testing in the Barnes maze, exploratory
behavior in the same group of mice was assessed by using the
elevated-plus maze (EPM), which consisted of two open and two
closed arms projecting from a central platform raised 1 m from
the floor. Each mouse was placed on the center platform and
allowed to explore the maze for 5 min. Each trial was scored for
total time spent in the arms, and the proportion of total time
spent in open arms was calculated. In addition, experiments
measured time spent on the central platform and the number of
closed and open arm entries and exits. Control males spent more
time in the open arms, an indicator of exploratory behavior, than
EE- or BPA-exposed males (P = 0.014 and P < 0.0001, re-
spectively) and control females (P = 0.0039) (Fig. 3 A and C).
in the Barnes maze. (A and C) Latency (i.e., time required to escape the maze) across days of training for males (A) and females (C) (mean ± SEM). (B and D)
Number of escape errors across days of training for males (B) and females (D) (mean ± SEM, *P < 0.01) (see SI Results for detailed comparisons).
Effects of early developmental exposure to BPA and EE on spatial learning and memory of adult male and female deer mice (Peromyscus maniculatus)
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| www.pnas.org/cgi/doi/10.1073/pnas.1107958108 Jašarevic ´ et al.
However, male exploratory behavior was more strongly com-
promised by BPA than EE. Control males also entered open
arms more frequently than either EE- or BPA-exposed males
(P = 0.0044 and P = 0.0024, respectively) and control females
(P = 0.0006), again emphasizing the potential demasculinizing
effect of BPA and EE on male deer mice behaviors. In particular,
BPA males spent a large proportion of their time in the closed
arms of the maze (Fig. 3 B and C). Whether such sheltering in the
closed arms reflects a lowered exploratory behavior, increased
anxiety, or both, is unclear. Finally, males exposed to BPA spent
more time immobile than control and EE-exposed males (means,
158 s, 135 s, and 124 s, respectively; P = 0.0591) (Fig. 3D).
Mate Choice Experiment. After a 2-wk resting period following
EPM testing, female mate preference for control vs. BPA-ex-
posed males was compared (Fig. S2). Control and BPA-exposed
females spent more time engaged in nose-to-nose contact with
control males than with males exposed to BPA (29.5 ± 5.86 s vs.
14.3 ± 5.76 s; P = 0.0114) (Fig. 4). Inspection time, however, did
not vary with maternal diet of the females (P = 0.2727). We
conclude that females have a reduced sexual preference for
males exposed to BPA, although it remains to be determined
whether this effect is due to behavioral cues, reduced chemical
signaling through pheromones, or both (Movie S5). Male choice
of female partners was not assessed because male choice is
rare among species with polygynous mating systems, including
Adult Serum Testosterone and Corticosterone Concentrations in Male
Deer Mice. Finally, because spatial abilities and exploratory be-
havior in male deer mice are likely to be influenced by adult
concentrations of circulating testosterone and corticosterone in
the peripheral blood (19), we measured the concentration of
these hormones in serum of adult males from the three diet
groups. No differences in serum testosterone (P > 0.3180) or
corticosterone (P > 0.2540) concentrations were observed be-
tween the control, EE, and BPA males (Fig. S3). Thus, the be-
havioral disturbances manifested in adult males caused by early
exposure to BPA seem not to be linked to either changes in adult
testosterone or corticosterone serum concentrations.
Together, the results demonstrate that developmental exposure
to BPA and the synthetic estrogen EE severely disrupts spatial
learning of male P. maniculatus and simultaneously decreases
their exploratory behavior, without altering adult concentrations
of circulating testosterone and corticosterone (Fig. S3). In a mate
choice experiment, females also seemed to detect the compro-
mised quality of males exposed during early development to BPA
and discriminated against them. Depressed male sexual vigor has
also been observed in Japanese quail (Corturnix coturnix japonica)
exposed in ovo to EE (20). In the latter study, there were also no
accompanying changes in circulating testosterone concentrations
to account for the abnormal behavior. The sex ratio bias in dams
on the EE-supplemented diet might partially explain some of the
effects of EE on male spatial cognition: males of this species born
to female-biased litters have been previously demonstrated to
exhibit some deficits in spatial abilities and a lower tendency to
disperse during the mating season (14). However, these results do
not explain the spatial deficits exhibited by BPA-exposed males,
If anything, this sex ratio bias in the BPA group would have been
anticipated to improve male spatial learning.
In our experiments, maternal diet exposure to BPA was 50
mg BPA/kg feed weight, which is consistent with prior studies
that have demonstrated that such a dose can induce epigenetic
(21) and behavioral changes (22) in laboratory rodents. It was
designed to be well below the diet-administered maximum non-
toxic dose for rodents (200 mg/kg body weight per day), within the
presumptive no observed effect level for mice (23), and likely to
provide circulating serum concentrations close to those observed
for humans (24). We have recently demonstrated that dietary
exposure of laboratory mice to 100 mg deuterated [dimethyl-d6]-
of 18.8 ± 4.4 ng/mL within 6 h after initiating the diets (24). On
the basis of the linear response curve observed with BPA (25–28),
was anticipated to yield serum concentrations in these animals
within the range of human exposure (27, 29).
Although our initial assumption was that maternal consump-
tion of BPA had a direct, endocrine-disrupting effect on pup
brain development, it is also possible that maternal exposure to
BPA led to decreased maternal investment in her pups, as has
been observed in mice and rats (30, 31). Such reduced maternal
care by dams receiving the BPA- and EE-supplemented diet
could, in turn, account for the extended effects on offspring
social and cognitive competencies, as has been proposed else-
where (32). Further studies are therefore planned to examine
search strategy of adult male and female deer mice (Peromyscus man-
iculatus) in the Barnes maze. (A) Examples of composite images from single
animals tracked from entry to escape, illustrating different spatial strategies
used to exit the maze. (B) Distribution of different spatial strategies
according to sex, diet exposure, and day of training. During the initial
training period (day 1), most animals navigated by using a random strategy
(black), followed by a serial search strategy (yellow). The most efficient
spatial search strategy (green) emerged when the animals began to use di-
rectional and positional intramaze cues. By day 3 of training, control males
used more efficient strategies than control females and EE- and BPA-exposed
males, who in turn did not differ on any day (P < 0.0002). EE-exposed females
used more efficient strategies than control and BPA-exposed females on all
days except day 2 (see SI Results for further details).
Effects of early developmental exposure to BPA and EE on spatial
Jašarevic ´ et al. PNAS Early Edition
| 3 of 6
maternal care by BPA- and EE-exposed female deer mice and to
test whether such effects on maternal behaviors influence the
subsequent spatial learning abilities of male offspring.
A feature of polygynous rodent species, in which males com-
pete by expanding home range and searching for mates, is in-
creased hippocampal cell density, volume, and spine density
during the breeding season (14, 33–35). BPA exposure can also
result in a variety of biochemical and structural changes in the
hippocampus of laboratory rodents (36–38). Although these
effects of BPA, if direct and not an indirect outcome of poor
maternal care, might be due to its ability to act as an analog of
estradiol and engage and inappropriately alter the expression of
genes for estrogen receptors (Esr1 and Esr2) in the fetal brain
(39, 40). It is also possible that the spatial navigational deficits in
exposed males are an outcome of suppression of Leydig cell
testosterone production at the time that androgens from the
testes normally masculinize the developing brain through local
conversion to estradiol by aromatase (41, 42). Another possibility
is that BPA effects are more immediate and occur through
membrane-initiated events that disrupt specific subset of neurons
(43). Whatever the basis of the learning defects, the foundation
of BPA pathologies in adult offspring seems likely to be epige-
netic in origin (21, 44–48).
On the other hand, even though the proximate mechanisms
mediating BPA effects on spatial learning and exploratory be-
haviors in P. maniculatus remain to be clarified, the reproductive
consequences are quite apparent. The disruption of male spatial
cognition and the supporting brain systems would severely com-
promise the ability of the male deer mice to find mates in natural
settings, and even if they did locate females, such animals would
seem to be less likely to be chosen as mates than males that had
not been exposed to BPA. Moreover, these abnormalities in traits
associated with the likelihood of male reproductive success cannot
be explained by altered adult testosterone or corticosterone con-
centrations of affected males.
Spatial abilities among female deer mice were not affected by
developmental exposure to BPA, most probably because such
traits have not been elaborated by sexual selection and were,
therefore, less sensitive to disruption than the same traits in
males. These traits are not sexually selected in females because
home range expansion and increased exploration is unlikely to
confer reproductive benefit to females and could be costly in
termsofpredationrisk.Inotherwords,therelative insensitivity of
some traits to BPA exposure, as demonstrated here for female
spatial abilities in P. maniculatus, can be readily understood
within the purview of sexual selection. Sex differences in ex-
pression of such traits provide a theoretical context within which
to understand EDC exposure for a broad range of species. Subset
examples include the decrease in sexually dimorphic physical size
of alligators born in contaminated streams of Lake Apopka (49),
the reduced plumage coloration around the facial region of male
American kestrels (Falco sparverius) exposed to polychlorinated
whose great grandmothers had been treated with the fungicide
vinclozolin (9). The latter study also provides an example of the
transgenerational effects of vinclozolin exposure on male quality
and female choice.
In contrast to the effects of BPA, developmental exposure of
female deer mice to the synthetic estrogen EE, itself a potent
environmental contaminant with known adverse effects on a variety
opmental exposure to BPA and
EE on exploratory behavior in the
EPM. Diet and sex differences in
time spent in (A) open arms; (B)
closed arms; (C) open arms in
proportion to total time in open
and closed arms; and (D) time
spent immobile. Same letters in-
dicate no difference, whereas dif-
ferent letters indicate significant
differences (see SI Results for de-
Effects of early devel-
exposed females exhibited longer duration of nose-to-nose contact (mean ±
SEM) with control males than with BPA-exposed males. *P < 0.05.
Female choice of control vs. BPA-exposed males. Control and BPA-
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| www.pnas.org/cgi/doi/10.1073/pnas.1107958108Jašarevic ´ et al.
of species (39, 50–52), masculinized their spatial abilities and
exploratory behaviors and led to a phenotype more similar to that
of control males than control females. Laboratory mice (Mus
musculus domesticus) and rats (Rattus norvegicus) exposed during
prenatal through postnatal development to EE similarly demon-
a similar regimen of BPA exposure did not induce comparable
responses (53, 54). These contrasting data obtained after EE
and BPA exposure suggest that the endocrine-disrupting effects
of the latter cannot be entirely attributed to its estrogenic actions.
In conclusion, we have shown that in a polygynous deer mouse
species, sexually selected traits that drive intrasexual competition
for mates and influence intersexual choice of mating partner are
particularly sensitive to BPA. Such a paradigm might be extended
to other species and provide a roadmap as to what sex-specific
traits might be most vulnerable to such chemicals. In the case of
P. maniculatus, male spatial navigational ability is disrupted by
early BPA exposure, and the mate choice experiment revealed
that females are sensitive to the compromised condition of such
malesandprefermales thatwere notexposedtoBPA.Inthewild,
BPA exposure might thus reduce the chances of these males to
reproduce successfully. To the extent that sexually selected traits
are particularly vulnerable to disruption by BPA and other EDCs,
they might constitute particularly useful biomarkers to assess
for environmental contamination (13, 55), provide a means to
resolve the accumulating, apparently contradictory accounts of
BPA effects appearing in the EDC literature, and allow future
studies of human risk assessments to be targeted in ways that are
not currently considered.
Materials and Methods
Animals. Thirty outbred female and 30 male deer mice (Peromyscus man-
iculatus bairdii) were purchased from the Peromyscus Genetic Stock Center
(University of South Carolina, Columbia, SC). All experiments were approved
by University of Missouri Animal Care and Use Committee and performed in
accordance with National Institutes of Health Animal Care and Use Guide-
lines. Virgin females, 8–12 wk of age, were randomly assigned to receive one
of three diets: (i) a low phytoestrogen AIN 93G diet supplemented with 7%
corn oil (control); (ii) AIN93G supplemented with 50 mg of BPA/kg feed
weight (BPA); or (iii) AIN93G diet supplemented with 0.1 parts per billion
feed weight of EE, as a positive control (56). Diets were administered 2 wk
before mating, and dams remained on the diet through pregnancy and
lactation, because early brain development extends into the postnatal pe-
riod (57). The total number of F1 offspring analyzed was 57 males (n = 20,
18, 19, for control, EE, and BPA diets, respectively) to capture male variability
and 32 females (n = 13, 9, 10, for control, EE, and BPA diets, respectively) to
determine whether sexually selected traits demonstrate differential sus-
ceptibility to EDCs.
After weaning, all offspring were placed on control diet and housed with
same-sex siblings until sexual maturity (age ≈60 d). Mice remained singly
housed throughout behavioral testing and were culled at ≈90 d of age. In
contrast to common laboratory rodent species, P. maniculatus does not re-
spond well to repeated handling (58), and thus animals were only handled
during weekly cage changes and behavioral testing. To minimize back-
ground exposure to BPA beyond treatment regimen (59), deer mice were
housed in white polypropylene cages (32 × 18 × 24 cm), maintained at
standardized environmental conditions, at 25 ± 2 °C and 50% ± 10% hu-
midity, with ad libitum access to water from glass bottles and food specific
to each treatment group. All animals were maintained on a long daylight
cycle (16 h light/8 h dark) to induce sexual maturity in males and females.
Assessments of Sensory and Neuromuscular Function. On postnatal day 25,
offspring were assessed to ensure that neuromuscular, sensory, and other
functional systems were intact, as described previously (60, 61). Olfaction was
tested by placing a small piece of food (cookie crumbs, 20 mg) under the
bedding in a clean mouse cage that was visually, but not physically, divided
into nine quadrants. The time required to find the food (latency) was
recorded (maximum of 10 min). Neuromuscular strength was tested by
placing mice individually on a wire lid 15 cm above their home cage. The lid
was gently turned upside down, and the latency to falling into their cage
was recorded (maximum of 60 s). To test acoustic startle, the experimenter
clapped his or her hands ≈0.65 m from the individually caged mouse and
recorded whether the animal became startled by the noise. Vision was
tested by holding the deer mouse ≈20 cm above a wire cage and slowly
lowering it. Normal vision was indicated by an arched back posture and
reaching for the lid.
Spatial Learning and Memory. At 60 d of age, spatial navigational ability of
F1 male and female deer mice was assessed with a modified black poly-
propylene Barnes maze for use with Peromyscus (62–64) (Fig. S1). Offspring
were randomly assigned an escape hole number. Exit holes were alternated
90° to eliminate odor cues for consecutively tested mice, whereas the escape
hole location and visual cues remained constant for any individual deer
mouse across all test trials. All testing occurred late in the light phase, and
animals were returned to the vivarium immediately after testing. Two days
before testing, mice were provided two habituation trials to acclimate the
mice to the maze design, followed by 7 d of two-trial evaluations per day for
300 s each, with a 30-min intertrial interval. At the beginning of each testing
day, animals were transferred from the vivarium to testing room 30 min
before behavioral assessments to reduce any confounding stress. A trial
consisted of carefully placing the mouse in the center of the maze in an
opaque starting box toallowthe tracking software todetect thecenter body-
point of the mouse. The starting box was lifted and a trial initiated once the
mouse began to move in the maze. If the mouse failed to enter the escape
box within 300 s, the observer gently guided the animal to the escape hole.
Stimulatory light measured ≈1,200 lx (vivarium room lighting measured 420
lx). If a deer mouse did not enter the exit hole within 30 s, a recording of
a barn owl (Tyto alba) screech was played to motivate predator avoidance
(65). The maze platform was cleaned after every trial with 70% ethanol so-
lution. Additional information is provided in SI Materials and Methods.
Exploratory and Anxiety-Like Behavior. Two weeks after the Barnes Maze
testing, exploratory and anxiety-related behaviors were assessed by using the
EPM (66). Additional information is provided in SI Materials and Methods.
Mate Choice Experiment. Control and BPA-exposed females were tested for
their preference of control vs. BPA-exposed males, similar to a previous study
(Fig. S2) (9). Additional information is provided in SI Materials and Methods.
Adult Male Serum Testosterone and Corticosterone Concentrations. One week
after completing mate-choice tests, males were killed, and cardiac serum
from 12 males per group was used to quantify circulating testosterone and
corticosterone concentrations by RIA (67). Males were 90 ± 7 d of age. Blood
was always collected between 700 and 900 hours, Central Standard Time.
Additional information is provided in SI Materials and Methods.
Statistical Analyses. Sensory and EPM scores were submitted to a 2 (sex) × 3
(diet) ANOVA or analysis of covariance (ANCOVA); Barnes maze latencies
were analyzed with a 2 (sex) × 3 (diet) × 7 (day) repeated-measures ANCOVA,
with trial (nested within day) as covariate (see SI Materials and Methods
for additional analyses). Hierarchical linear models (PROC MIXED, SAS, 2004),
whereby males and females were treated as separate random variables,
were used to assess female preference for control or BPA-exposed males.
Additional information is provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. Dennis Miller for his advice on the
sensory battery tests; Dr. Rex Hess for his suggestions on the manuscript;
Mr. Wayne Shoemaker, Bond Life Sciences Center, for constructing the
behavior testing apparatuses; Lisa Caldwell and Denise Warzak for technical
assistance; and Joseph Reddy and Scott Williams for their assistance with
husbandry and care of the deer mice. This work was supported by a Mizzou
Advantage grant (to D.C.G. and C.S.R.) and National Institutes of Health
Challenge Grant RC1 ES018195 (to C.S.R.).
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