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Behavioural Brain Research 187 (2008) 284–288

Research report

Environmental prenatal stress alters sexual dimorphism

of maternal behavior in rats
Carmen Pérez-Laso a , Santiago Segovia a , José Luis R. Martı́n a ,
Esperanza Ortega b , Francisco Gómez c , M. Cruz R. Del Cerro a,∗
a Departamento de Psicobiologı́a, Universidad Nacional de Educación a Distancia, c/Juan del Rosal, 10, 28040 Madrid, Spain
bDepartamento de Bioquı́mica y Biologı́a Molecular, Universidad de Granada, Avda. de Madrid, s/n, 18012 Granada, Spain
c Centro de Salud Joaquı́n Rodrigo, Área 11, IMSALUD, Madrid, Spain

Received 12 January 2007; received in revised form 6 July 2007; accepted 18 September 2007
Available online 29 September 2007

Abstract
The prenatal external environment can affect fetuses, altering the maternal behavior that they express when mature. In the present study,
environmental prenatal stress (EPS) was applied to pregnant rats in their final week of gestation, and when their female offspring reached maturity,
the long latency effect of the stress on those offspring was ascertained on their induced maternal behavior (MB), accessory olfactory bulb (AOB)
morphology and plasma levels of ACTH and corticosterone (Cpd B). EPS reduced: the percentage of these virgins that showed induced MB,
their retrieval of foster pups, the time spent crouching, and the quality of nest building; it also increased the incidence of their cannibalism of
foster pups. The EPS-treated females presented a male-like pattern of induced MB. They showed increased plasma levels of ACTH and Cpd B
and increased numbers of mitral cells in the AOB. These findings provide evidence that stress applied to the pregnant rat produces long-lasting
behavioral, neuroanatomical and hormonal alterations in the female offspring that can be observed when they reach maturity.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Prenatal stress; Maternal behavior; Brain development; Corticosterone; Accessory olfactory bulb

1. Introduction The effects of prenatal stress are mediated by maternal stress

It is well known that exposure to stress during pregnancy
(EPS) produces physiological and behavioral alterations in the
offspring [28], including reproduction and maternal behavior.
Fertility and fecundity of female offspring rats was reduced
after EPS [12]. After EPS, when they reached maturity, virgin
females manifested a longer, male-like, latency to show induced
MB [14]. Moreover, EPS significantly reduced the percentage of
female rats that became maternal. EPS decreased c-fos activity
in the mitral cells of the matured female AOB [17]. The AOB
is an olfactory structure of the sexually dimorphic vomeronasal
pathway [10,22,24] and is involved in the neural control of MB
[3,7,8,13,18].
Abbreviations: ACTH, adrenocorticotrope hormone; AOB, accessory olfac-
tory bulb; Cpd B, corticosterone; E2 , estradiol; EPS, environmental prenatal
stress; HPA, hypothalamic–pituitary–adrenal axis; MB, maternal behavior;
MBR, maternal behavior register; RIA, radio immunoassay; T, testosterone.
∗ Corresponding author. Tel.: +34 91 3986291; fax: +34 913986287.
E-mail address: mcrdelcerro@psi.uned.es (M.C.R. Del Cerro).
hormones that reach the fetal brain and impair the development
of the response of the hypothalamic–pituitary–adrenal (HPA)
axis to stress at maturity [1]. Furthermore, acute EPS during the
late gestational period affects the fetal HPA axis, significantly
increasing fetal plasma ACTH [14]. These findings provide evi-
dence that the fetal HPA axis is able to respond to EPS and
that it may play a role in mediating the behavioral and neural
alterations observed in the offspring.
The aim of the present study was to ascertain whether the
effects of EPS on the induced MB of female offspring when
they reach maturity are correlated with HPA axis dysfunction
and morphological alterations in the AOB.
2. Materials and methods
2.1. Animals
Forty-four rats of the Wistar Strain (Iffacredo, Barcelona, Spain), born from
two groups of mothers (control and stressed during gestation), were assigned to
one of three groups: control females (C-F, n = 16), control males (C-M, n = 14)

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2007.09.029
 Author's personal copy
C. Pérez-Laso et al. / Behavioural Brain Research 187 (2008) 284–288
and environmental prenatally stressed females (EPS-F, n = 14). Subjects of the
EPS group were randomly selected between the pups of a group of pregnant
rats that were exposed to three daily stress sessions of 45 min each during the
last week of gestation. We used the paradigm of Ward [25], which involves
exposure of the pregnant rat to restraint, light (2500 lux) and heat (31 ± 1 ◦ C).
Subjects of the control group were selected from the pups of mothers that were
left undisturbed throughout their entire pregnancy.
On the day of birth, pups were counted and sexed. Sex ratio was 4–5 males
vs. 5–7 females. Group size was adjusted to five males and five females per
dam. EPS females were assigned to stressed mothers and control pups to control
mothers. Birth weight did not differ among groups and was measured every
fourth day until weaning. EPS female pups showed a significant lower weight
in comparison with control females (p < .0008) on the fourth day after birth, but
was recovered on the next day of weight measure and no weight differences
among the three groups were found until weaning. Rats were also weighed on
the day of sacrifice (103–109 days of age) at which time normal sex differences
were found (p < .0001).
2.2. Maternal behavior induction test
At 97 days of age, the above mentioned groups C-F (n = 16), C-M (n = 14)
and EPS-F (n = 14) were tested for induction of maternal behavior, which was
carried out under dim red light. Testing commenced each day at 10 a.m. Every
day at that time and during 12 consecutive days, a fresh litter of four nourished
pups (3–6 days of age) was placed opposite the test rat in its home cage. MB
patterns (nest-building quality, pup retrieving, licking of the pups’ anogenital
area and infanticide rate) were recorded during the next 10 min. The quality of the
nest was rated according to the scale developed in this laboratory [6]. An animal
was considered to have reached “sensitization” criterion (i.e., became maternal)
when it performed retrieval of the entire litter during the 10-min observation
period on 2 consecutive days.
Pups were left with the virgin females and naive males during a 24 h period,
after which they were removed and returned to their biological dams. Each day,
four new recently fed pups were presented to each subject and MB observa-
tions again commenced. These pups, either male or female, from donor dams,
were used as stimuli only three times during the experiment. Behavioral obser-
vation data were recorded using the “MBR” computer program designed in our
laboratory [5].
2.3. Hormone measurement
Animals were sacrificed as soon as they reached sensitization criterion or at
the end of the experiment on day 12. Blood samples, for hormone determina-
tion, were collected, exclusively, between 16:00 and 17:00 h, to avoid HPA axis
activity fluctuations [15].
Trunk blood from each group (n = 14 for the three groups) was obtained
from the left ventricle after anesthesia and stored in pre-cooled heparinized
vials. The samples were centrifuged at 2000 rpm for 10 min at 2 ◦ C and the
plasma was collected and placed in marked, pre-cooled sample tubes that were
kept at −20 ◦ C until assays were performed. Plasma Cpd B was measured with
RIA kit (ICN Biomedical Inc. Costa Mesa, California, USA) and plasma ACTH
was measured with an immunoradiometric kit (CIS Biointernational, Gif-Sur-
Yvette Cedex, France). All the samples were assayed in duplicate concurrently.
The intraassay coefficients of variations were 4.4 and 3.4%, respectively.
2.4. Estimation of AOB mitral cell number by stereology
Between days 103 and 109 of age, animals were sacrificed using deep anes-
thesia (tribromoethanol, 250 mg/kg i.p.), followed by 4% paraformaldehyde via
transcardiac perfusion. Brains were then removed and stored in paraformalde-
hyde for 2 days, after which they were placed in a 30% sucrose solution for 3–5
days. The olfactory bulbs were frozen and sectioned in the sagittal plane at 40 ␮.
All sections were stained with a 0.1% solution of Cresyl violet (Sigma, Madrid,
Spain) titrated with glacial acetic acid to pH 4.
In each group, six animals were randomly selected in which the total num-
ber of mitral cells of the AOB was estimated using the optical fractionator [11].
This method combines a (multistage) sampling procedure as a tool for counting
285
particles (in this case, mitral cells). Every second section was used. The final
thickness of sections, once stained and dried, using the microcator (depth mea-
surement device) was approximately 25 ␮ for the three groups. Beginning at a
random starting position, visual fields were sampled by step-wise movements of
80 ␮ in the X-axis and 120 ␮ in the Y-axis. Neurons were counted in every frame
when their nucleus came into focus, using the optical dissector with a height of
12 ␮, which means that counting started at a depth of 6 ␮ and ended at 18 ␮.
We, thus, counted neurons, exclusively, in the middle zone of the sections, thus
avoiding over-counting.
2.5. Statistical analysis
Since morphological data showed homogeneity of variance, one-tailed
ANOVA and post hoc t-test were applied. However, the data from behavioral
and hormonal tests did not show homogeneity of variance, and consequently for
those data, non-parametric Kruskal–Wallis and Mann–Whitney U-tests were
performed.
3. Results
Environmental prenatal stress altered the expression of MB
in the female offspring. Results of the maternal behavior induc-
tion test are shown in Fig. 1. In the C-F group, the percentage
of female offspring becoming maternal (reaching sensitization),
when adult, was 42.9% vs. 7.1% found in the EPS-F group
(p < .0001) and 0% observed in the C-M group (p < .0001)
(Fig. 1A). This difference is also expressed as the cumula-
tive percentage of females showing retrieval of any pup, but
not the complete litter: 50% in the C-F vs. 21.42 and 6.25%
in the EPS-F and C-M groups, respectively (Fig. 1B). Prena-
tally stressed females showed significantly more infanticidal
behavior than controls: 35.70% vs. 13.30% in the C-M group
(p < .0001) and none in the C-F group (p < .0001) (Fig. 1C).
As can be seen in Table 1, nest quality and crouching behavior
were also affected by EPS treatment. EPS-F and C-M groups
showed lower nest-building scores than the C-F group (p < .001
and p < .0001, respectively), while EPS-F did not differ from C-
M (p > .05). EPS-F and C-M showed less crouching time than
F-C (p < .002 and p < .005, respectively), but there was no signifi-
cant difference between EPS-F and C-M (p > .05). No significant
differences were observed in licking behavior.
Table 2 shows the plasma levels of ACTH and Cpd B observed
in the control groups and EPS females. There were no significant
differences in levels of these two hormones between C-F and C-
M. However, there was a significant increase of ACTH in EPS-F
in comparison with the levels found in C-F (p < .03), although
Table 1
Sex differences between control groups and the effect of EPS on nest-building
quality and time spent crouching over the pups
Groups Nest score (scale 0–4) Crouching time (s/session)
median + s.i.r. median + s.i.r.
C-F 2.41 + 0.35 0.79 + 9.89
EPS-F 1.58 + 0.31a 0.00 + 0.00b
C-M 1.37 + 0.44b 0.00 + 0.00b
Data show median and upper interquartile range. Differences compared with
C-F.
a p < .001.
b p < .0001.
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286 C. Pérez-Laso et al. / Behavioural Brain Research 187 (2008) 284–288

Fig. 1. Effect of EPS on the induced maternal behavior (median and upper interquartile range) and on the AOB mitral cell number (means ± S.E.M.). (A) Percentage
of animals expressing sensitization; (B) cumulative percentage of animals showing retrieval; (C) percentage of animals that cannibalized; (D) AOB mitral cells.
Differences compared with C-F * p < .0001; compared with EPS-F + p < .0001.

Table 2 AOB, one of the sexually dimorphic brain nuclei involved in the
Effects of EPS on plasma hormone levels control of MB [7,8], and the HPA endocrine axis [19].
Groups ACTH (pg/ml) % Cpd. B (pg/ml) % Prenatal stress was reported to increase the latency to express
C-F 51.23 ± 7.04 387.27 ± 80.80
full MB in virgin female Sprague–Dawley rats when adult [14]
EPS-F 82.26 ± 10.81 ↑60.56a 730.67 ± 36.11b ↑88.67 and to reduce the percentage of virgin female rats that became
C-M 62.69 ± 15.00 400.89 ± 51.42 maternal [17]. The present findings confirmed that the expres-
Number of determinations: 14 per group and % column indicate the difference in
sion of MB was also markedly altered by EPS in virgin female
percentage compared with the control female group. Data show mean ± S.E.M. rats of the Wistar strain. This treatment significantly reduced
Differences compared with C-M. the number of females that became fully maternal, the time
a p < .03.
spent crouching over pups and the quality of their nest build-
b p < .0001.
ing, while it increased their infanticide behavior. An additional
differences between the groups was that the percentage of the
not with C-M levels. There was a significant increase of Cpd B control females showing pup retrieval increased progressively
in EPS-F vs. C-F (p < .0001) and C-M (p < .0001), but no sex from day 8 until day 12 (the last test day), whereas the percent-
differences were found. age of EPS females showing this response remained unchanged
ANOVA of the number of AOB mitral cells indi- over the same period. With the exception of cannibalism, in
cated that there was a significant difference among groups which EPS-F exceeded C-M, EPS-F and C-M groups did not
(F(2,15) = 26.51; p < .0001). As seen in Fig. 1D, C-M showed differ significantly from each other in the rest of behavioral
significantly more AOB mitral cells than C-F (p < .0001). More- patterns recorded. Based upon the present findings, we con-
over, this sex differences was abolished by EPS, EPS-F showing clude that female rats, exposed to EPS as fetuses, display a
significantly more AOB mitral cells than C-F (p < .0001), but did male-like (i.e., attenuated) pattern of induced MB when they
not differ significantly from the C-M group. are mature.
Based upon the present findings EPS seems to have affected
4. Discussion the HPA endocrine axis function increasing ACTH and Cpd B
levels in adult EPS females. These results are consistent with
The present study provides evidence that EPS during the last the hypercorticism reported by Koehl et al. [15]. However, we
week of pregnancy exerts long-lasting effects on the expression did not find sex differences between C-F and C-M groups in
of induced maternal behavior in the female offspring when they Cpd B levels. This may be due to our procedure of collecting
are adult. Moreover, the changes observed in the induced mater- blood samples at 16:00 while light offset was at 20:00. Results
nal behavior are related to alterations in the development of the supported by findings of Koehl et al. [15], showing no sex dif-
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C. Pérez-Laso et al. / Behavioural Brain Research 187 (2008) 284–288
ferences between control animals in the same time period. As
mentioned above, the effects of EPS are mediated by the HPA
axis [1,19]. These endocrine conditions observed in EPS female
rats resemble those reported in women diagnosed with congen-
ital adrenal hyperplasia. Those women were reported to show
less interest in infants than their sisters unaffected by the pathol-
ogy [2]. The present study provides evidence that EPS disrupts
female maternal behavior and suggests that ACTH and Cpd
B may play a role in the alterations of MB, which is a sex-
ually dimorphic behavior. Without specific testing of the role
of ACTH and Cpd B in MB, this is just a hormonal correlate.
Oxytocin [29] and opioid peptides [26,27] have also been pro-
posed as mediating the effects of environmental stress of the
pregnant dam on the subsequent sexually dimorphic behavior of
her pups.
The present results indicate that EPS increases the num-
ber of AOB mitral cells in the females to the level observed
in males. It has been shown that stress elevates Cpd B levels
in the pregnant dams and their fetuses [19] and that Cpd B,
administered to rat pups during the first postnatal week, sup-
presses granule cell death in the dentate gyrus [9]. Thus, the
increase of Cpd B levels observed after EPS may have attenuated
AOB mitral cell death and it could be the process responsible
for the male-like numbers of these cells in the EPS females.
This increase of AOB mitral cells to a male-like level, which is
associated with a disruption of MB in EPS virgin female rats,
also occurs after early postnatal administration of the GABAA
antagonist, picrotoxin [23]. Picrotoxin elevates Cpd B levels in
nonstressed rats [16]. The present findings support the hypoth-
esis that the functional significance of sexual dimorphism in the
pattern of number of neurons (male > female), is related to the
inhibition of feminine reproductive behavior patterns in male
rats [22].
Induced MB may be considered a model of maternal adoption
and care expressed to foster infants. In addition to the effects on
the fetuses, the environmental stress of the dam during her preg-
nancy has been shown to affect her own maternal behavior, as
we have shown in a previous study [20]. Moreover, these effects
are persistent in the adult female offspring from stressed dams
towards their own pups [4,21]. The eventual adult behavioral
disruptions expressed by the pups that were subjected to envi-
ronmental stress as fetuses, is most likely a function not only
of their own disrupted endocrine and neuronal systems, but also
a function of their dam’s disrupted maternal behavior that they
experienced while pups.
5. Conclusion
Environmental stress applied to pregnant rats altered the
induced MB of their female offspring when, as virgins, they
reached maturity. These virgin females showed a male-like pat-
tern of care for foster pups and their plasma levels of ACTH and
Cpd B were increased by EPS. Moreover, EPS masculinized the
number of AOB mitral cells. The present findings indicate that
EPS and, probably, altered maternal cares are a potent neuro-
functional teratogenic agent that exerts a long-lasting effect on
brain morphology, HPA axis and maternal behavior.
287
Acknowledgements
We thank L. Carrillo, L. Troca, G. Moreno for the technical
assistance and A. Marcos for his editorial help. This work was
funded by DGICYTs grants PB-96-00107/C03-02 and DGESIC
BSO 2000-019 (M.C.R.C).
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