BIOLOGY OF REPRODUCTION 81, 690–699 (2009)
Published online before print 17 June 2009.
Neonatal Bisphenol-A Exposure Alters Rat Reproductive Development
and Ovarian Morphology Without Impairing Activation of Gonadotropin-
Releasing Hormone Neurons1
Heather B. Adewale,3Wendy N. Jefferson,4Retha R. Newbold,4and Heather B. Patisaul2,3
Department of Biology,3North Carolina State University, Raleigh, North Carolina
Developmental Endocrinology Section,4Laboratory of Molecular Toxicology, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina
Developmental exposure to endocrine-disrupting compounds
is hypothesized to adversely affect female reproductive physi-
ology by interfering with the organization of the hypothalamic-
pituitary-gonadal axis. Here, we compared the effects of
neonatal exposure to two environmentally relevant doses of
the plastics component bisphenol-A (BPA; 50 lg/kg and 50 mg/
kg) with the ESR1 (formerly known as ERalpha)-selective agonist
4,40,400-(4-propyl-[1H]pyrazole-1,3,5-triyl)trisphenol (PPT; 1 mg/
kg) on the development of the female rat hypothalamus and
ovary. An oil vehicle and estradiol benzoate (EB; 25 lg) were
used as negative and positive controls. Exposure to EB, PPT, or
the low dose of BPA advanced pubertal onset. A total of 67% of
females exposed to the high BPA dose were acyclic by 15 wk
after vaginal opening compared with 14% of those exposed to
the low BPA dose, all of the EB- and PPT-treated females, and
none of the control animals. Ovaries from the EB-treated females
were undersized and showed no evidence of folliculogenesis,
whereas ovaries from the PPT-treated females were character-
ized by large antral-like follicles, which did not appear to
support ovulation. Severity of deficits within the BPA-treated
groups increased with dose and included large antral-like
follicles and lower numbers of corpora lutea. Sexual receptivity,
examined after ovariectomy and hormone replacement, was
normal in all groups except those neonatally exposed to EB. FOS
induction in hypothalamic gonadotropic (GnRH) neurons after
hormone priming was impaired in the EB- and PPT-treated
groups but neither of the BPA-treated groups. Our data suggest
that BPA disrupts ovarian development but not the ability of
GnRH neurons to respond to steroid-positive feedback.
AVPV, corpora lutea, development, disruption, endocrine
disruptors, ERa, ESR1, estradiol, estradiol receptor, estrogen,
estrogen receptor, GnRH, gonad, hypothalamus, lordosis,
neuroendocrinology, ovary, PPT, puberty
Bisphenol-A (BPA) initially entered commercial develop-
ment in the 1930s as a synthetic estrogen  but is now used
primarily in the production of polycarbonate plastic products,
epoxy resins, and the linings of soda and soup cans. The U.S.
Centers for Disease Control recently estimated that nearly all
Americans have detectable levels of BPA in their bodies and
that children have higher levels than adults [2, 3]. Infants in
neonatal intensive care units may have notably high exposure
to BPA, presumably from its use in medical devices .
Newborns can also be exposed through lactational transfer .
Fetal exposure is also highly likely because relatively high
levels in umbilical cord blood and fetal plasma indicate that
BPA fails to bind to the estrogen-sequestering protein alpha-
fetoprotein and can cross the placenta [6, 7]. In multiple
species, exposure to BPA during the prenatal or postnatal
period has been shown to have an impact on female
reproductive physiology, including the timing of pubertal
onset and the induction of an early, persistent estrus [8–10].
Presumably, BPA produces its effects by interfering with one
or both of the primary forms of the estrogen receptor (ER;
ESR1 or ESR2, formerly known as ERa and ERb) within the
hypothalamic-pituitary-gonadal (HPG) axis, but it is not well
understood through which ER BPA primarily acts in vivo. In
the present study, we took a comparative approach and
examined the effects of neonatal exposure to BPA or an
ESR1-selective agonist on the structure and function of the
adult hypothalamus and ovary in female rats. We have shown
previously that exposure to the ESR1-specific agonist 4,40,400-
(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) but not the
ESR2-specific agonist diarylpropionitrile (DPN) during the
neonatal period can defeminize the female rat hypothalamus
and impair steroid-positive feedback . Here, we sought to
determine whether neonatal exposure to BPA could similarly
impact hypothalamic organization, a result which would then
implicate a mechanistic role for ESR1 in endocrine disruption
by BPA in the female hypothalamus.
Within the HPG axis, reproductive maturation and function
are coordinated by the release of gonadotropin-releasing
hormone (GnRH) [12, 13]. The neural components of the
HPG axis that regulate GnRH secretion are sexually differen-
tiated by endogenous gonadal hormones, primarily estradiol,
through a series of gestational, prenatal, and perinatal critical
periods . The GnRH release is regulated through feedback
effects of gonadal steroids. In both males and females, GnRH
secretion is suppressed by steroid-negative feedback, the signal
for which is thought to arise from the arcuate nucleus of the
hypothalamus [15, 16]. In females, however, GnRH release is
augmented once per cycle by estrogens. This positive feedback
potentiates the surge in GnRH and, subsequently, luteinizing
hormone (LH) that precedes ovulation . In rats, this process
is now thought to be mediated within the anterior ventral
periventricular nucleus (AVPV) of the hypothalamus [15, 16,
1Supported by National Institute of Environmental Health Sciences grant
R01 ES016001 to H.B.P.
2Correspondence: Heather B. Patisaul, Department of Biology, North
Carolina State University, Raleigh, NC 27695. FAX: 919 515 5327;
Received: 17 April 2009.
First decision: 7 May 2009.
Accepted: 9 June 2009.
? 2009 by the Society for the Study of Reproduction, Inc.
eISSN: 1259-7268 http://www.biolreprod.org
Downloaded from www.biolreprod.org.
In rodents, sexual differentiation of this feedback circuit
takes place primarily during the neonatal period and has been
shown to be particularly sensitive to disruption by hormones or
hormone-like compounds [11, 19, 20]. It is well established
that the administration of steroid hormones, including testos-
terone or estradiol benzoate (EB), during the neonatal critical
period can defeminize the female rodent brain, whereas
castration can effectively prevent defeminization of the male
rodent brain [21–23]. Thus, in males castrated as neonates, the
potential for estrogen to evoke a GnRH surge is preserved
whereas, conversely, in females neonatally exposed to
estrogens, this capacity is diminished or lost. Exposure to sex
steroids or endocrine disrupting compounds (EDCs) during the
neonatal critical period can also advance puberty and alter
ovarian morphology [15, 24–26]. The present experiments
targeted this neonatal critical window, which corresponds to
approximately the third trimester of gestation in humans .
In a prior study, we demonstrated that neonatal administration
of the phytoestrogens genistein (GEN) and equol (EQ) can
advance puberty and induce an early, persistent estrus .
This disruption of female reproductive physiology was
accompanied by impaired GnRH activation (as measured by
the colocalization of GnRH and FOS immunoreactivity),
suggesting that perturbation of the hormone-dependent orga-
nization of steroid-positive feedback circuits within the
hypothalamus was a potential mechanism for the effects on
pubertal timing and ovarian cyclicity. Animals neonatally
exposed to the ESR1-specific agonist PPT but not the ESR2-
specific agonist DPN also displayed an early, persistent estrus
and inhibited FOS induction in GnRH neurons after hormone
priming, demonstrating that ESR1 plays a critical role in the
defeminization of the hypothalamus by estrogens. By exten-
sion, ESR1 might play a mechanistic role in the disruption of
hypothalamic organization by EDCs, such as BPA. Reduced
colocalization of GnRH and FOS immunoreactivity after
neonatal exposure to BPA would potentially indicate that
BPA can interact with ESR1 in the hypothalamus during
development, a finding that would provide key information
about the possible mechanism(s) by which BPA affects the
organization of the female reproductive system.
Developmental exposure to estrogenic EDCs can induce
additional effects elsewhere in the HPG axis, such as the ovary
[10, 28, 29]. Previous studies have shown that perinatal
exposure to GEN can lead to altered ovarian differentiation
characterized by multioocyte follicles and reduced fertility in
mice . In contrast, relatively little is known about how
exposure to BPA at doses considered relevant for human risk
assessment and over the neonatal critical period alone can affect
ovarian development and function. Therefore, the present study
had two main goals: 1) to determine whether advanced puberty
and impaired estrus function in female rats after neonatal
exposure to one of two relatively low doses of BPA were
associated with impaired sexual receptivity and FOS induction
in hypothalamic GnRH neurons and 2) to characterize ovarian
morphology in the adult after neonatal BPA exposure.
Because ERs are expressed in all three HPG components, not
just the hypothalamus, disruption by BPA could potentially
occur anywhere within it, including the pituitary, the ovary, the
brain, or any combination of the three. It is not well understood
whether ovarian deficits after neonatal exposure to endocrine
disruptors result from direct effects in the ovary itself, disrupted
organization of the hypothalamus, both of these mechanisms, or
some other potential mechanism, perhaps at the level of the
pituitary. Although the present experiments were not designed
to explore this critical knowledge gap directly, we again
compared the effects of PPT and BPA as a first step in the
exploration of this key issue. A recent study by Nakamura and
colleagues reported that ovaries collected from mice neonatally
exposed to PPT, but not the ESR2 agonist DPN, at 13 wk of age
lack corpora lutea (CLs), a condition that is accompanied by
persistent estrus . This finding suggeststhat ESR1 may have
an important mechanistic role in endocrine disruption of ovarian
function. We have also reported persistent estrus in female rats
after neonatal exposure to PPT  but did not explore ovarian
morphology in that prior study. Thus, in the present study, we
sought to determine whether neonatal exposure to BPA or PPT
induces similar ovarian malformations.
Multiple laboratories have now demonstrated that BPA can
impair female reproductive function at doses equivalent to or
below those that regulatory bodies have deemed to be ‘‘safe’’
for human exposure, but the question of whether or not there
are significant ‘‘low-dose effects’’ of BPA remains controver-
sial. At the time the present studies were undertaken, the lowest
observed adverse effect level (LOAEL) for BPA established by
the U.S. Environmental Protection Agency (EPA) was 50 mg/
kg body weight (bw) per day, and the EPA reference dose (the
dose considered ‘‘safe’’ for human exposure) was 50 lg/kg bw
per day. For our experiments, we used the EPA LOAEL as our
high dose and the EPA reference dose as our low dose. The
synthetic estrogen EB, at a dose sufficient to masculinize the
hypothalamus, advance pubertal onset, and prevent the onset of
regular estrous cycles, was used as a positive control [31–33].
Animals were exposed to either vehicle, EB, PPT, low-dose
BPA (50 lg/kg bw), or high-dose BPA (50 mg/kg bw) in the
first 4 days of life via subcutaneous injection. Injection has
recently been demonstrated to produce equivalent or lower
plasma BPA levels in neonatal rodents than oral administration,
thus making injection an appropriate route of exposure when
considering potential effects in humans [34, 35]. After neonatal
exposure, females were then monitored for changes in
reproductive physiology, sexual receptivity, GnRH activation,
and ovarian morphology. We hypothesized that BPA would
adversely affect all endpoints examined in a dose-dependent
MATERIALS AND METHODS
Animals and Neonatal Treatment
Animal care, maintenance, and surgery were conducted in accordance with
the applicable portions of the Animal Welfare Act and the U.S. Department of
Health and Human Services ‘‘Guide for the Care and use of Laboratory
Animals’’ and were approved by the North Carolina State University (NCSU)
Institutional Animal Care and Use Committee. Female pups were obtained
from cross-fostered litters born to timed pregnant Long Evans rats (n ¼ 10;
Charles River Laboratories). Cross-fostering was done to minimize potential
litter effects. Each litter contained a mixture of animals (12 maximum), only
two of which were genetically related to each other. All dams were individually
housed in a humidity- and temperature-controlled room with a 12-h light cycle
(lights on from 0700 to 1900 h) at 238C and 50% average relative humidity at
the Biological Resource Facility at NCSU and were maintained on a
semipurified, phytoestrogen-free diet ad libitum for the duration of the
experiment (AIN-93G; Test Diet, Richmond, IN).
Beginning on the day of birth, the female pups were cross-fostered and
subcutaneously (sc) injected with vehicle (0.05 ml), EB (25 lg; Sigma, St.
Louis, MO), 50 lg/kg bw BPA (low-dose BPA; Sigma), 50 mg/kg bw
bisphenol-A (high-dose BPA), or the ESR1 agonist PPT (1 mg/kg bw; Tocris
Biosciences, Ellisville, MS). PPT is a selective agonist for ESR1, with a 400-
fold preference for ESR1 and minimal binding to ESR2 . The EB was used
as a positive control and administered at a dose previously established to
masculinize the hypothalamus and eliminate GnRH activation in response to
hormone priming [31–33]. All compounds were dissolved in ethanol and then
sesame oil at a ratio of 10% EtOH and 90% oil, as we have done previously
[11, 37]. The vehicle was also prepared with this ratio. We have found this
vehicle to cause less skin irritation than the alternative vehicle, DMSO. The
animals (n ¼ 10–12 per group) received injections daily from the day of birth,
defined as Postnatal Day 0 (PND 0), through PND 3 (four injections total).
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All pups were weaned into same-sex littermate pairs on PND 22, ear
tagged, and maintained on a reverse light schedule (lights off from 1000 to
2200 h) for the remainder of the experiment. Upon weaning, a cohort of
animals (n ¼ 8–9 per group) were checked daily for day of vaginal opening
(DOV). The primary mechanism for pubertal onset remains unclear [38, 39],
but in rats, DOV is a hallmark of puberty and was therefore used as the
physiological indicator of puberty. Monitoring of the estrous cycle by vaginal
lavage  commenced approximately 2 wk after vaginal opening and
occurred every 24 h for 4 days (longer if needed) every other week, for 13 wk.
We opted to sample every other week to reduce handling stress and minimize
the potential for inducing a pseudopregnancy. All animals observed to have
stopped cycling (defined by the failure to sequentially progress through
proestrus, estrus, and diestrus) were resampled (for 3–5 days) 2 wk later to
confirm that the failure to cycle was not due to a pseudopregnancy.
Females were ovariectomized (OVX) under isoflurane anesthesia (com-
pleted during 3 wk beginning on Postnatal Day 148) and allowed 2 wk to
recover to provide adequate time for endogenous hormone levels to diminish.
At the time of OVX, ovaries were removed and fixed in 10% formalin for 24 h
at 48C, then postfixed in 70% ethanol and stored at 48C until processing.
Testing for Sexual Receptivity
To induce sexual receptivity and test for lordosis behavior, the OVX
females were injected sc with 10 lg of EB at 0900 h, followed 48 h later by an
sc injection of 500 lg of progesterone (same vehicle as above) and paired with
vigorous males as described previously [41–43]. Lordosis quotient (LQ) was
calculated by dividing the number of lordosis responses in each trial (10 min)
by the number of mount attempts, then multiplying the result by 100. In two
pairings, the male made no attempt to mount the female. These trials were not
included in the analysis.
Ovaries were coded at collection and were subsequently processed at the
National Institute of Environmental Health Sciences (NIEHS). For each animal,
both ovaries were paraffin embedded side by side and sliced into 6-lm
sections. Sections were slide mounted (Superfrost Plus; Fisher, Pittsburgh, PA),
and two slides per animal were deparaffinized, stained with hematoxylin-eosin,
coverslipped, and qualitatively examined for histological abnormalities at
NIEHS. The slides and the remaining uncut tissue were then returned to NCSU
for the quantification of CLs. Corpus luteum counts are a reliable indicator of
successful ovulation . Although the morphological appearance of CLs
changes across the cycle, the overall number does not , eliminating the need
to control for cycle stage at OVX. For each animal, CLs were counted on two
slides (each containing one section from each ovary) and averaged to yield a
final value for statistical analysis.
Brain Collection and Immunohistochemistry
Nineteen days after the behavioral tests were completed, all animals were
again sequentially administered EB and progesterone and were killed by
transcardial perfusion with 4% paraformaldehyde 6–8 h after the progesterone
injection, as we have done in past experiments . It is well established that
this is the point at which GnRH activity, after sequential steroid hormone
administration, is maximal [45, 46]. Brains were removed, postfixed,
cryoprotected, and stored at ?808C (14), then sliced into 35-lm coronal
sections and divided into four series of free-floating alternating sections, one of
which was used for the present study. For each animal, one set of coronal
sections comprising the organum vasculosum of the lamina terminalis (OVLT)
through the caudal border of the AVPV were immunolabeled for GnRH and
FOS using immunohistochemistry methods described in detail elsewhere [11,
47]. The GnRH and FOS were detected using a cocktail of primary antibodies
directed against GnRH (raised in rabbit; 1:20000; generously gifted by Dr.
Robert Benoit, McGill University Health Center, Montreal, QC, Canada) and
FOS (raised in goat; 1:250; SC-52–6; Santa Cruz Biotechnology), followed by
the secondary antibodies Alexa-Fluor donkey anti-rabbit 488 and Alexa-Fluor
donkey anti-goat 568, each at 1:200. After secondary antibody incubation,
sections were rinsed, mounted onto slides (Superfrost Plus; Fisher, Pittsburgh,
PA), and coverslipped using a glycerol-based mountant (50% glycerol in 4 M
Quantification of GnRH and FOS Immunoreactivity
The GnRH/FOS double-immunofluorescent label was visualized and
quantified as described in our prior publications [11, 48]. Briefly, GnRH and
FOS immunostaining was observed to be consistent and distributed both
laterally and dorsally to the third ventricle throughout the OVLT, as has been
described previously [49, 50]. Thus, two to three midlevel sections per animal
were selected for analysis. Only those sections showing consistent GnRH and
FOS immunostaining were used. They were photographed with a Retiga 1800
monochrome camera attached to a Leica 5000DM microscope fitted with 203
and 403 objective lenses and filter cubes for Cy3 and fluorescein
isothiocyanate. Anatomical identification was made using a brain atlas .
The images of each label (GnRH and FOS) were then merged using the MCID
Elite Image Analysis (Interfocus Imaging Ltd., Cambridge, England) software
package. Cells immunostained for GnRH only and cells immunolabeled for
both GnRH and FOS were then hand counted by an individual blind to the
treatment groups and were verified by a second independent observer.
The DOV, LQ, CL number, and percentage of GnRH and FOS colabeled
cells were compared across treatment groups by one-way ANOVA with
treatment as a factor and were followed up with Fisher least significant
difference posthoc tests for individual comparisons (SYSTAT; Systat Software
Inc., Chicago, IL). As anticipated [49, 50], the number of GnRH neurons
counted did not significantly differ between groups (data not shown). In all
cases, comparisons were two tailed, and the significance level was set at P ?
Age at Vaginal Opening
There was a significant effect of treatment on DOV (F[5,50]
¼18.34; P ? 0.001). Compared with the oil-treated controls (n
¼9), DOV was significantly advanced by neonatal exposure to
EB (n ¼ 11; P ? 0.001), PPT (n ¼ 8; P ? 0.04), and the low
dose of BPA (50 lg/kg bw; n¼8; P ? 0.01; Fig. 1). The high
dose of BPA (50 mg/kg bw; n ¼ 14), however, had no
significant effect on DOV.
Estrous Cycle Data
Beginning approximately 2 wk after DOV, regularity of the
estrous cycle was assessed every 24 h for 4 days (longer if
needed) every other week, for 13 wk by vaginal lavage in a
cohort of animals. Regular 4-day estrous cycles commenced in
all treatment groups except the group neonatally exposed to EB
(Fig. 2). As expected [31–33], all of the EB-treated females (n
¼8) displayed persistent estrus or diestrus and failed to enter a
PND 3) administration of EB and the low (50 lg/kg bw) dose of BPA but
not the high (50 mg/kg bw) dose of BPA or the ESR1-selective agonist PPT
compared with the control animals (means 6 SEM; *P ? 0.04).
The DOV was significantly advanced by neonatal (PND 0 to
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normal estrous cycle. By 7 wk after DOV, all (n¼8) neonatally
treated PPT females had entered a state of persistent estrus or
diestrus. By 15 wk after DOV, only 33% of the females
exposed to the high dose of BPA (n ¼ 9) were still cycling,
compared with 86% of the females exposed to the low dose of
BPA (n ¼ 8).
Sexual receptivity was assessed after ovariectomy and
hormone replacement. There was a significant main effect of
treatment on LQ (F[4,35] ¼ 17.046; P ? 0.001; Fig. 3);
however, this effect was exclusively due to the abolition of
sexual behavior in the group neonatally treated with EB (n¼9;
P ? 0.001). There was a trend for suppressed LQ in the group
neonatally exposed to low-dose BPA, but this effect did not
reach statistical significance (n ¼ 7; P ¼ 0.157). Both the PPT
and high-dose BPA exposures had no appreciable effect on LQ.
These observations are consistent with what we have observed
in past studies .
Both a qualitative and quantitative analysis of ovarian
morphology was conducted (Table 1). Qualitative histological
comparison between groups revealed that only the negative
control group showed consistently healthy ovaries character-
ized by all stages of follicular development and the presence of
numerous healthy CLs (Fig. 4). All other groups displayed
some degree of abnormalities, including hemorrhagic follicles,
large antral-like follicles that did not seem to support ovulation,
multioocyte follicles, and ovarian cysts. The type and severity
of abnormalities present differed between treatment groups
(Table 1). Females neonatally exposed to EB as our positive
control group (Fig. 4) displayed the most severe effects,
qualitatively characterized by undersized ovaries, no CLs, and
no signs of folliculogenesis. Females neonatally exposed to the
ESR1-selective agonist PPT also displayed abnormal ovarian
development, but not in the same way that the EB females did.
Ovaries from PPT-treated females (Fig. 4) were also undersized
and lacked CLs but were characterized by numerous large
antral-like follicles that did not appear to support ovulation.
Some contained oocytes (Fig. 4, yellow arrows), but most did
not. Females neonatally exposed to the high dose of BPA (50
mg/kg) displayed abnormal folliculogenesis and follicle
degeneration, with a few follicles containing multinucleated
cells. Ovaries from females exposed to the low dose of BPA
(50 lg/kg) displayed all stages of follicular development;
however, a few animals had hemorrhagic follicles. Ovaries
from both BPA-treated groups also contained a number of large
antral-like follicles that were similar in appearance to those
seen in the PPT-treated animals and were structurally
consistent with ovarian cysts. All of the PPT-treated animals
and most of the high-dose BPA-treated animals were acyclic at
the time of ovariectomy, and CLs were not typically observed
in these animals. Thus, it is unlikely that these follicles
progressed to ovulation. Because the number of antral follicles
changes across the estrus cycle and we did not stage our
animals at OVX, we did not compare the mean number of
antral-like follicles across treatment groups.
group neonatally exposed to EB. The percentage of females within each
treatment group displaying a regular estrous cycle diminished over time in
all groups except for the control group. By 15 wk after vaginal opening,
only 33% of animals exposed to the low (50 lg/kg bw) dose of BPA
neonatally still displayed a regular estrous cycle compared with 86% of
the animals exposed to the high (50 mg/kg bw) dose of BPA.
Regular estrous cycles commenced in all groups except the
significantly affected by neonatal exposure to any of the compounds
except for EB (means 6 SEM; *P ? 0.001).
Female sexual receptivity after hormone priming was not
TABLE 1. Summary of ovarian morphology.
All stages of
Appears likely to
EB (25 lg)
PPT (1 mg/kg)
Abnormal: small follicles
Abnormal: many larger antral and cystic
follicles, degenerating eggs
Somewhat abnormal: hemorrhagic
follicles in some
Abnormal: degenerated, multinucleated,
and some hemorrhagic tissue present
BPA (50 lg/kg) 100 (7/7)9.75 Slightly undersizedYes Yes
BPA (50 mg/kg) 36 (4/11)2.1 NormalNo No
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Quantitative analysis of ovulatory capacity was made by
counting CLs (Table 1). Corpora lutea appear after the release
of an egg from the ovary and are a reliable and quantitative
indicator of successful ovulation, regardless of cycle stage [24,
43, 52]. There was a significant treatment effect on the number
of CLs present (F[4,35]¼22.484; P ? 0.001). Compared with
the control females, a significant decrease in CL number was
observed in the females neonatally exposed to EB (n¼8; P ,
0.001) or PPT (n ¼ 7; P , 0.001), neither of which displayed
any CLs. The number of CLs was also significantly lower in
the high-dose BPA-treated females (n ¼ 10; P , 0.001), with
only 3 of 10 containing any CLs. The mean number of CLs
also trended lower in the low-dose-treated BPA females
compared with the control group, but this effect did not reach
statistical significance (n ¼ 8; P , 0.36). This was largely
because whereas most animals in the low-dose BPA group had
fewer CLs than the vehicle-treated control animals, two
animals had nearly twice as many. Both of these animals were
still cyclic, but not staged, at the time of ovariectomy.
Colocalization of GnRH and FOS in the OVLT
Hormone treatment was repeated approximately 2 wk after
sexual testing was completed, and animals were killed 6 h after
the progesterone injection. This is the point at which FOS
expression in GnRH neurons (an indicator of GnRH activation)
is known to be maximal [45, 46]. The GnRH activation was
assessed by quantifying the percentage of GnRH and FOS
colabeled OVLT neurons. There was a significant effect of
treatment (F[4,29] ¼ 17.331; P ? 0.001). Hormone adminis-
tration successfully induced FOS immunoreactivity (-ir) in
93% of GnRH neurons of the oil-treated control females (n¼7;
Fig. 5). In contrast, 6% of GnRH neurons in EB-treated
females (n ¼ 5; P , 0.001), and only 22% in PPT-treated
females (n ¼ 7; P , 0.001), were immunoreactive for FOS.
This PPT effect is consistent with what we have reported in
past studies . FOS labeling within GnRH neurons was not
significantly reduced in either of the BPA groups compared
with the controls, suggesting that the sex-specific organization
of the hypothalamus may not have been disrupted by neonatal
BPA exposure at the doses used.
The accelerated pubertal timing, premature anestrus, and
ovarian malformations induced by neonatal exposure to BPA
could result from disrupted organization anywhere within the
HPG axis, including the hypothalamus, ovary, and/or pituitary
gland. The exposure was specifically timed to target the
hypothalamus. Our results do not support the hypothesis that
BPA can defeminize the hypothalamus, a process in which, as
we have shown previously , ESR1 appears to play a
ly different across treatment groups. Fe-
males neonatally exposed to EB had very
small ovaries with no clear signs of active
oogenesis or ovulation. Ovaries from fe-
males neonatally exposed to PPT were
characterized by numerous large antral-like
follicles, only some of which contained
oocytes (yellow arrows). Similar structures
were observed in both of the BPA-treated
groups but not to the degree seen in females
neonatally exposed to PPT. Generally, ova-
ries from the high-dose (50 mg/kg bw) BPA
treatment group more closely resembled
those from the PPT treatment group than the
EB treatment group. Bar ¼ 500 lm.
Ovarian morphology was distinct-
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significant mechanistic role. Instead, GnRH neurons seem to
retain the capacity to respond (by producing FOS) to hormone
priming by estrogen and progesterone. These results suggest
that steroid-positive feedback was not defeminized by BPA but
do not rule out the possibility, however, that other signaling
mechanisms on GnRH neurons could have been altered.
Effects through ESR1 (by PPT or other endocrine
disruptors) on GnRH are most likely indirect, because ESR1
is not known to be present in GnRH neurons. Instead,
disruption likely occurs within ESR1-containing neurons in
the AVPV, the arcuate nucleus (ARC), or elsewhere, which
send projections to GnRH neurons . This may include the
recently discovered population of neurons in the AVPV that
express the peptide kisspeptin and send afferents to GnRH
neurons [15, 16]. The vast majority of kisspeptin neurons in
this region coexpress ESR1, and the administration of
kisspeptin can induce early puberty in adolescent animals
and generate a GnRH surge in ovariectomized animals [53, 54].
A recent study by Navarro et al.  found reductions in
hypothalamic Kiss1 mRNA levels after neonatal BPA exposure
at doses higher than those used here. We subsequently
determined that the density of hypothalamic kisspeptin-
immunoreactive fibers is reduced by the high, but not the
low, dose of BPA used in the present study, and that the effect
was more pronounced in the ARC than the AVPV .
Collectively, these observations support the hypothesis that
signaling pathways on GnRH neurons may be altered by
developmental exposure to BPA and that steroid-negative
(rather than positive) feedback may be impaired.
Even if GnRH release following estrogen administration is
normal, the subsequent release of LH from the anterior
pituitary may not be. A new study by Fernandez and colleagues
 targeted the critical period for pituitary differentiation
(PND 13) in female rats. Levels of GnRH-induced LH released
from pituitary cells in vivo and in vitro were lower than
controls in BPA-treated animals. Additionally, BPA-treated
animals showed increased GnRH pulse frequency, hypothe-
sized to cause desensitization of the pituitary, thus leading to
blunted LH secretion [56, 57]. Prepubertal exposure to BPA
has also been shown to decrease LH pulse frequency and
amplitude in gonadally intact ewes [58, 59]. Similarly,
perinatal but not adult exposure to 1.2 mg/kg bw BPA
decreased plasma LH levels in adult OVX mice, an observation
that also indicates possible interference with hypothalamic
organization and emphasizes that exposure sensitivity is
heightened during critical developmental periods. Bisphenol-
A has also been shown to reduce ERK activation, which may in
turn affect GnRH receptor expression . Reduced GnRH
receptor expression in the pituitary could be a mechanism by
which LH release is reduced while steroid-induced GnRH
activation remains intact. Collectively, these prior results
combined with the abnormalities seen in the present study
could also be indicative of disrupted steroid-negative feedback.
Although it is still unclear by what mechanism it might
occur, our results clearly demonstrate that ovarian cyclicity is
compromised by a very brief, neonatal BPA exposure at doses
at and above the current EPA reference dose for human
exposure. Prior studies examining whether BPA can affect
ovarian function have used equivalent or higher doses than
those used here, and they have shown that the window of
vulnerability may extend beyond the neonatal period [56, 61,
62]. For example, exposures to 10 mg/kg BPA on PNDs 15–
18, 100 lg/kg BPA across PNDs 1–5, or 50 mg/kg BPA on
Gestational Days 6–21 have all been shown to result in ovarian
and vaginal malformations in mice [63–65], including the
absence of CLs, cystic ovaries, endometrial hyperplasia, and
lack of ESR1 expression in the vagina. Higher doses of BPA (1
or 4 mg per pup) have also been reported to decrease ovarian
area occupied by CLs and multiple cystic follicles in rats .
Within that study, however, a dose of 250 lg per pup was not
found to alter ovarian morphology, a finding which is
inconsistent with our own, but the ovaries were collected
approximately 6 wk after vaginal opening (earlier than was
done in the present study), so it is possible that abnormalities
following low-dose exposure could have manifested later.
Although the low dose of BPA accelerated pubertal onset in the
present study, most of these animals maintained a regular
estrous cycle for 15 wk after DOV, whereas animals exposed to
the high dose did not. Our results indicate that lower doses of
neonatal BPA exposure may result in reduced ovulatory
capacity, although the onset of irregular cycles may take
somewhat longer to manifest. This type of effect has also been
observed after neonatal low-dose exposure to the phytoestro-
gen GEN . Therefore, it is important to observe animals for
several weeks beyond pubertal onset when seeking to
determine whether an EDC can affect ovarian function. The
effect of low-dose BPA on ovarian cyclicity may be more
robust if exposure comprises both the gestational and neonatal
periods, a condition that is more consistent with human
exposure, which is low and lifelong .
It has been hypothesized that neonatal exposure to EDCs
enhances ovulation rate when animals are young, which then
results in compromised fertility later in life and induces a
premature reproductive senescence . The intriguing
observation that two females exposed to the low (50 lg/kg
bw) dose of BPA in the present study had nearly twice as many
and FOS (green) in the OVLT. Double-
labeled cells are indicated by the white
arrows, and single-labeled GnRH neurons
are indicated by the blue arrows. The
number of GnRH-immunopositive cells did
not significantly differ among groups. Orig-
inal magnification 320 (A) and 363 (inset);
bar ¼ 40 lm. B) The percentage of GnRH
cells that were colabeled with FOS was
significantly lower in animals neonatally
exposed to PPT or EB but not significantly
affected by neonatal exposure to either the
low (50 lg/kg bw) or high (50 mg/kg bw)
doses of BPA (means 6 SEM; P ? 0.001; *P
? 0.001). 3v, third ventricle.
A) Immunolabeling of GnRH (red)
HYPOTHALAMUS NOT DEFEMINIZED BY BISPHENOL-A
Downloaded from www.biolreprod.org.
CLs as control females whereas the rest had fewer is consistent
with this hypothesis and requires further investigation. It is
possible that among these animals, CL counts were different
simply because they were at different points in their estrous
cycle when the ovaries were collected. A recent study,
however, found that although the morphologic features of
CLs change across the cycle, the number of CLs does not .
Our results also indicate that lower rather than higher doses
of BPA have the greatest potential to accelerate pubertal onset,
a finding that is counterintuitive but consistent with what has
been seen in other studies. An even lower dose (20 lg/kg)
administered over Gestational Days 11–17 has also been
shown to advance vaginal opening , demonstrating that
perturbations of the hormonal milieu in other critical
developmental windows besides the neonatal period have the
potential to advance puberty. Conversely, higher doses of BPA
typically fail to affect pubertal timing, or they delay rather than
advance it . The mechanism by which lower, but not
higher, doses of BPA can advance puberty remains poorly
understood. It has been hypothesized previously that BPA, like
hormones, has a U-shaped or ‘‘nonmonotonic’’ dose-response
curve [8, 68–70]. Our results are consistent with that
hypothesis. The mechanism(s) by which this might occur have
not been satisfactorily elucidated but could include stimulation
of hormone receptors at low doses but downregulation of
hormone receptors at higher doses. (For a critical discussion of
nonmonotonic dose curves, see Vandenberg et al. .) It is
important to note that although the effect of neonatal exposure
to the low dose of BPA on DOV was significant, it was not as
robust as neonatal exposure to EB. Neonatal exposure to the
ESR1 agonist PPT was also not sufficient to advance pubertal
onset to the same degree as EB. This could suggest that strong
agonism of both ESR1 and ESR2 is necessary to evoke a
It is also possible that BPA alters pubertal timing by an ER-
independent mechanism. For example, bw at the time of
vaginal opening may influence this endpoint, and could
therefore be a confounding factor. Delayed pubertal onset
after exposure to 500 mg/kg BPA via maternal dietary
exposure during gestation and nursing was most pronounced
in the lightest animals . Other studies in rats and sheep
have also found an effect of BPA on body weight at pubertal
onset [59, 61]. Therefore, it could be that vaginal opening in
our high-dose (50 mg/kg) animals was later than in our low-
dose (50 lg/kg) animals because of a difference in bw.
Unfortunately, this possibility was not taken into account;
however, we did not note any obvious differences in bw at the
time of vaginal opening. Regardless, our data suggest that
exposure to BPA during a developmental window that
corresponds to late gestation in humans, at levels relevant to
human exposure, has the potential to accelerate pubertal onset.
The specific mechanisms by which this occurs remain to be
Malformations in the ovary after exposure to EDCs,
including BPA, during development have been reported
previously, but the mechanism(s) by which they might occur
has not been well established . For this aspect of the present
study, the ESR1-selective agonist PPT was employed for
comparative purposes. All groups except the control group
showed some degree of abnormalities, the characteristics of
which were distinctly different between groups. Malformations
within the BPA females were dose dependent, with ovaries
from the high-dose group showing the most significant adverse
effects. Generally, the BPA ovaries more closely resembled
those from the PPT than the EB group which, as expected from
prior reports , were notably undersized (a qualitative
assessment) and showed no sign of folliculogenesis. Unlike the
EB group, ovaries from animals in the PPT and high-dose BPA
groups were characterized by numerous antral-like cavities,
most of which contained either a degenerating or no oocyte. In
many cases, these cavities resembled cystic follicles, but some
appeared to be large atretic follicles, particularly in the PPT
animals (Fig. 4). This observation is consistent with a previous
study that also described the presence of large, antral-like
cavities in the ovaries of mice exposed to 250 lg/kg bw BPA
in utero , and to a more recent study  that observed
similar structures in cultured follicles directly exposed to BPA.
It should be noted, however, that a different mechanism of
action is likely involved in the cell culture study, because the
ovarian malformations arose from acute rather than develop-
mental exposure. Finally, none of the PPT females had CLs, an
observation that is consistent with what has been reported
previously in mice  and suggests that the PPT animals are
anovulatory and fail to progress beyond the follicular phase. It
appears that agonism of ESR1 during the rodent neonatal
period can curtail the capacity of oogenesis to progress into the
Although the ovaries from the animals neonatally treated
with PPT and the high dose of BPA share some similar
characteristics, they do not necessarily indicate a mechanistic
role for ESR1 in the emergence of the BPA-induced effects.
Moreover, a mechanistic role for ESR2 in the disruption of
ovarian function by BPA cannot be ruled out. Recent studies
have reported that ESR2-deficient mice exposed gestationally
to 400 ng/day BPA do not display the same abnormalities as
BPA-exposed wild-type controls , leading the authors to
conclude that BPA exerts its effects in the ovary via ESR2 [76,
77]. It is important to be mindful, however, that effects
resulting from the elimination of ESR2 function across the
lifespan may differ from those observed after selectively
agonizing or silencing ESR2 during discrete time periods. We
and others have shown that postnatal exposure to ESR2-
specific agonists can also result in the premature loss of a
regular estrous cycle, although not necessarily at the same rate
as animals exposed to ESR1-specific agonists [11, 78].
Therefore, the loss of ovarian cyclicity appears to be possible
after postnatal agonism of either of the two major ER subtypes.
ESR1 is primarily expressed in the interstitial and thecal cells
as early as PND 1, whereas ESR2, expressed in the granulosa
cells, is not detectable on PND 1 but is present by PND 5 .
Thus, the BPA exposure in our study was sufficiently long
enough for either ER subtype to play a mechanistic role. It is
also possible that BPA is acting as an ESR 1 or ESR2
antagonist. Selective antagonists for ESR1 and ESR2 are only
now becoming available, so it may soon be possible to directly
test how ER-specific antagonism affects ovarian structure and
function as part of future studies. These and other types of
experiments are needed delineate the relative roles each ER
subtype play in the organization and function of the HPG axis,
including the ovary.
It is also possible that BPA action on ovarian development
and HPG organization may not be limited to ERs. Bisphenol-A
is thought to bind to the thyroid hormone receptor, androgen
receptor, estrogen-related receptor, and the aryl hydrocarbon
receptor [71, 80–82], and it may have direct or indirect actions
on the ovary through these alternate pathways. For example,
estrogen-related receptor gamma (ESRRG) and estrogen-
related receptor alpha (ESRRA) expression have been found
in human ovarian cells, with increased expression in ovarian
cancer cells [83, 84]. Bisphenol-A can also bind to a
membrane-bound form of the ER and a transmembrane ER
called G protein-coupled receptor 30 (GPER, formerly called
ADEWALE ET AL.
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GPR30) [85–87]. Emerging evidence now indicates that
epigenetic modifications, such as DNA methylation of
transposable elements and cis-acting, imprinting regulatory
elements, may also be a potential mechanism by which BPA
and other endocrine disruptors affect reproductive function [88,
89]. The potential for BPA and other endocrine disruptors to
modify the epigenome and the health consequences of those
disruptions remain largely unexplored.
Interestingly, when hormone primed after ovariectomy, all
of the animals except those treated with EB displayed normal
sexual receptivity, regardless of whether or not they had lost
their estrous cycle, demonstrating that the capacity to display
sexual behavior may persist despite impaired reproductive
physiology. This observation is consistent with what we have
seen before following neonatal exposure to GEN, EQ, PPT, or
DPN [11, 43]. Enhanced lordosis has been reported previously
after gestational exposure to 40 lg/kg per day BPA  or a
low (5 mg/kg) dose of the chlorinated pesticide chlordecone, a
compound which is also classified as an estrogenic EDC .
Our observations are also consistent with a body of literature
produced by Gorski , Dohler , and Yanase and Gorski
 showing that lordosis behavior can persist after postnatal
hormone manipulation, depending on dose and timing of
administration. We have yet to determine whether or not these
females would display normal sexual behavior if tested with
ovaries in place and without hormone priming.
Here, we have shown that neonatal exposure to BPA at or
below the LOAEL set by the EPA adversely affects pubertal
timing and ovarian function. Our results are consistent with the
conclusions of the National Toxicology Program , which
stated that there is ‘‘some concern for adverse effects of
developmental toxicity for fetuses, infants and children.’’ The
National Toxicology Program also concluded that there is
‘‘minimal’’ concern for effects on puberty in females, largely
because the mouse literature on this effect is inconsistent, and
not enough information has been obtained from rat studies.
Therefore, our study adds to the literature demonstrating an
effect on sexual maturation in females. Bisphenol-A is known
to cross the placenta and pass to infants by lactational transfer
[5, 95, 96]. Blood and amniotic fluid samples obtained from
pregnant women reveal that amniotic levels of BPA (8.3–8.7
ng/ml) can be even higher than fetal serum levels (1–2 ng/ml)
[97–99]. Whether or not the levels of BPA obtained through
maternal exposure in humans are sufficient to elicit reproduc-
tive deficits remains to be determined. However, as contam-
ination levels and routes of exposure to both naturally
occurring and synthetic EDCs differ across populations,
determining exactly how they interfere with normal reproduc-
tive development warrants further exploration. Our finding that
the capacity for GnRH neurons to produce FOS in response to
hormone priming is apparently intact in female rats neonatally
exposed to BPA suggests that defeminization of the hypothal-
amus may not be a mechanism by which developmental
exposure to BPA can affect DOV, estrous cyclicity, and
ovarian morphology. Further studies are needed to better
characterize the specific mechanisms through which BPA and
similar EDCs disrupt the organization of the HPG axis and
impair female reproduction.
The authors thank Barbara (BJ) Welker and Linda Hester for their
assistance with animal husbandry, as well as Jillian Mickens and Karina
Todd for their help with the tissue slicing and labeling. We are also
grateful to the pathologists at National Institute of Environmental Health
Services for helping us with ovarian histology, and Dr. Robert Benoit
(McGill University Health Center, Montreal, QC, Canada) for supplying us
with the GnRH antibody. We appreciate John Vandenbergh for his
constructive comments on the experimental design and Charles Patisaul
for his critical reading of this manuscript.
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HYPOTHALAMUS NOT DEFEMINIZED BY BISPHENOL-A
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