Maternal programming of sexual attractivity in female Long Evans rats.

Abstract

In mammals, maternal care influences the developing offspring across multiple domains. In Long Evans rats, for example, the quality of maternal care received as a pup influences later cognitive function, neuroendocrine responses to stress and behavioral measures of emotionality. Data from humans, non-human primates, and rodents also suggest that early life events may similarly perturb measures of sexual reproduction, with possible consequences for reproductive fitness. The current study examined whether or not male conspecifics differentially prefer females, as adult mating partners, that were reared under varying maternal conditions (assessed via the quantity of licking and grooming received; LG). Additionally, the impact of maternal care on adult female sexual motivation and behavior were quantified to determine if these behavioral characteristics are associated with any preference observed. In a mate preference task, male rats chose, almost exclusively, to mount, copulate and ejaculate with female rats reared under Low LG conditions. Under non-paced mating conditions, female Low LG rats display significantly more paracopulatory and copulatory behaviors compared to High LG rats. Due to its critical role in female paracopulatory behavior, progesterone receptor immunoreactivity (PR-ir) in the ventromedial nucleus of the hypothalamus (VMH) was also assessed in both groups of female rats. Estradiol induced PR-ir in the VMH was significantly higher in Low LG relative to High LG rats. Together, these data suggests that early life parental care may developmentally program aspects of behavior and physiology that subsequently influence sexual attractivity and behavior in adult females.

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Psychoneuroendocrinology (2011), doi:10.1016/j.psyneuen.2011.02.016
Maternal programming of sexual attractivity in female
Long Evans rats
Samuel A. Sakhaia,*, Lance J. Kriegsfelda,b, Darlene D. Francisb,c
aDepartment of Psychology, 3210 Tolman Hall MC 1650, University of California at Berkeley, Berkeley, CA 94720, USA
bHelen Wills Neuroscience Institute, 3210 Tolman Hall MC 1650, University of California at Berkeley, Berkeley, CA 94720, USA
cSchool of Public Health, 3210 Tolman Hall MC 1650, University of California at Berkeley, Berkeley, CA 94720, USA
Received 18 October 2010; received in revised form 28 February 2011; accepted 28 February 2011
1. Introduction
Early-life environments and experiences, both pre and
post natal, shape the development of numerous biological
processes in humans and other mammals (Fish et al., 2004;
Boyce et al., 2006; Evans and Schamberg, 2009; Lyons et al.,
2010). Diverse disciplines such as social epidemiology and
behavioral neuroscience have documented that challenging
and/or adverse experiences that occur early in life can
influence developmental processes to impact an individual
throughout its lifetime. These effects include changes in
reproductive behavior and corresponding biology, both of
which have been documented in insects, reptiles, amphi-
Psychoneuroendocrinology (2011) xxx, xxx—xxx
KEYWORDS
Maternal programming;
Early life;
Female sexual behavior;
Progesterone receptor;
Attractivity;
VMH;
Parental care
Summary
domains. In Long Evans rats, for example, the quality of maternal care received as a pup
influences later cognitive function, neuroendocrine responses to stress and behavioral measures
of emotionality. Data from humans, non-human primates, and rodents also suggest that early life
events may similarly perturb measures of sexual reproduction, with possible consequences for
reproductive fitness. The current study examined whether or not male conspecifics differentially
prefer females, as adult mating partners, that were reared under varying maternal conditions
(assessed via the quantity of licking and grooming received; LG). Additionally, the impact of
maternal care on adult female sexual motivation and behavior were quantified to determine if
these behavioral characteristics are associated with any preference observed. In a mate
preference task, male rats chose, almost exclusively, to mount, copulate and ejaculate with
female rats reared under Low LG conditions. Under non-paced mating conditions, female Low LG
rats display significantly more paracopulatory and copulatory behaviors compared to High LG
rats. Due to its critical role in female paracopulatory behavior, progesterone receptor immuno-
reactivity (PR-ir) in the ventromedial nucleus of the hypothalamus (VMH) was also assessed in
both groups of female rats. Estradiol induced PR-ir in the VMH was significantly higher in Low LG
relative to High LG rats. Together, these data suggests that early life parental care may
developmentally program aspects of behavior and physiology that subsequently influence sexual
attractivity and behavior in adult females.
# 2011 Elsevier Ltd. All rights reserved.
In mammals, maternal care influences the developing offspring across multiple
* Correspondingauthor.Tel.:+15106428661;fax:+15106425293.
E-mail address: ssakhai@berkeley.edu (S.A. Sakhai).
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/psyneuen
0306-4530/$ — see front matter # 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2011.02.016

Page 2

bians, fish, birds, rodents, and primates (Danforth, 1991;
Bass, 1992; Tollrian, 1995; Denver, 1997; Rhen and Crews,
2002). In humans, childhood sexual abuse is strongly asso-
ciated with early pubertal maturation (Wierson et al., 1993;
Turneret al.,1999; Zabinetal.,2005).In rats,lower levelsof
parental investment markedly change reproductive pheno-
types, including precocial puberty (Moore, 1984; Uriarte
et al., 2007; Cameron et al., 2008a). Despite these well-
established effects of early experience on adult physiology
and behavior, the means by which these early-experiences
are biologically embedded and persist long term is not well
understood. The present experiments investigate the impact
of variations in early maternal care on physiological and
behavioral measures of sexual attractivity and behavior in
female laboratory rats.
Life History Theory postulates that humans have evolved
to be responsive to early childhood environments that sub-
sequently bias children to adopt different behavioral and
reproductive strategies (Belsky et al., 1991; Coall and
Chisholm, 2003; Crews, 2003; Boyce and Ellis, 2005). Envir-
onments may directly influence child development or, alter-
natively, changes may occur through perturbations in
parental care and investment that arise in response to vary-
ing ecological demands. For example, girls raised with
increased exposure to paternal absence are at significantly
elevated risk for early sexual activity and adolescent preg-
nancy (Surbey, 1990; Ellis et al., 2003). Early sexual activity
has been shown to have several deleterious effects that
include:(i)having moreand oldersexualpartnersthroughout
the teen years, (ii) increased risk of sexually transmitted
disease and (iii) increased risk of adolescent pregnancy
(Coker et al., 1994; Sandfort et al., 2008). Thus, the accel-
eration and maturation of the hypothalamo-pituitary-gona-
dal (HPG) sex axis in young girls has profound consequences
that persist throughout the life course.
Research employing animal models has focused on the role
of early maternal care on the regulation and programming of
sexual development in offspring, allowing cause-effect rela-
tionships between early care quality and adult phenotypes to
be established. In rats, for example, developing offspring are
very sensitive to the quality of parental care received during
theearlypostnatalperiod.Maternallickingandgrooming(LG)
of offspring during the first week of life has been shown to
developmentally program the hypothalamo-pituitary-adrenal
(HPA) axis of Long Evans rats, later impacting adult stress-
reactivity profiles (Francis et al., 1999b). Specifically, off-
spring that experience Low maternal LG as pups are more
stress reactive, and less exploratory than their High LG coun-
terparts as adults (Francis et al., 1999b; Fish et al., 2004;
Weaver et al., 2004). Recent findings (Moore, 1995; Cameron
etal.,2005;Uriarteetal.,2007)suggestthatasimilarprocess
occursfordevelopmentalprogrammingoftheHPGaxisinrats,
subsequently influencing adult sexual behaviors.
Disruptions to early postpartum maternal care in rats can
perturb neuroendocrine and behavioral processes in the off-
spring that influence sexual activity and reproduction. For
example,femaleratssubjectedtohandlingaspupsexhibitless
copulatory behavior and increased anovulatory estrous cycles
compared to non-handled controls when assessed later in life
(Gomesetal., 2005, 2006;Rainekietal., 2008).Similartothe
influence of maternal care on the developing HPA axis,
Cameronetal.(2008a,b,c)reportsthatvariationsinmaternal
care received during the early postnatal period can also sig-
nificantlyinfluenceaspectsoffemalesexualbehaviorslaterin
life.OffspringofLowLGfemalesarereportedtoreachpuberty
at an earlier age relative to High LG females. Low LG females
exhibit a higher lordosis rating, a lower (but not significant)
inter-intromission interval and have a higher incidence of
pregnancy compared to High LG animals. With the exception
of pregnancy rate and pubertal onset, these are measures of
copulatory behaviors, behaviors that result in the successful
transfer of male gametes to the female (Blaustein, 2008).
Whereas paracopulatory behaviors, behaviors performed by
female rats to elicit mounting behavior from males, are sub-
ject to early-life regulation have been explored using a paced
matingparadigm(Cameronetal.,2008b).Inthepresentseries
ofstudies,wesoughttodetermineiffemalesexualbehaviors,
specifically, paracopulatory behaviors potentially lead to dif-
ferences in male preference based on maternal history.
The aim of the following study was to investigate the role
of maternal care in the developmental regulation of physio-
logical and behavioral measures implicated in female para-
copulatory behavior. Specifically, we hypothesized that
sexually experienced male rats, when allowed to choose
between High or Low LG females in estrus will prefer to
copulate preferentially with Low females. We hypothesized
that Low female rats will exhibit increased paracopulatory
and copulatory behaviors during estrus relative to High LG
female rats, behaviorally rendering them more attractive to
males when compared to High LG females. Attractivity, a
term coined by Frank Beach (Beach, 1976), is defined by the
stimulus value of the female in evoking male sexual behavior.
As progesterone treatment potentiates the effects of estra-
diol on female sexual behaviors (specifically paracopulatory
behaviors) we also assessed whether or not estradiol-induced
progesterone receptor immunoreactivity (PR-ir)in the ven-
tromedial nucleus of the hypothalamus (VMH) paralleled
behavioral differences observed.
2. Methods and materials
2.1. Animals and housing
Male Long Evans rats used in the study were purchased
from Charles River Breeding Laboratories (Wilmington,
MA) andpairhoused
(27.8 cm ? 17.5 cm ? 13.0 cm). Female rats (n = 48) used in
the study were born in our colony which was generated using
LongEvansratsoriginallypurchasedfromCharlesRiver.Forall
animals, temperature was kept constant at 20 ? 2 8C and
relative humidity was maintained 50 ? 5%. Rats were main-
tained on a 12-h light—dark cycle (lights on 0700 h to 1900 h)
and allowed access to food (Purina Rat Chow, Purina Mills, St.
Louis, Missouri) and tap water ad libitum. Female offspring
underwent behavioral testing between PND 85—90. Housing
and care of the rats were carried out in accordance with the
standards and practices of the UC Berkeley Animal Care and
Use Committee.
in polypropylenecages
2.2. Observations of maternal behavior
Female rats were bred and permitted to give birth. Day of
birth was marked as postnatal day (PND) 0. Maternal obser-
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2 S.A. Sakhai et al.

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vations were performed beginning on PND 1 and continued
until PND 8 (Liu et al., 1997; Francis et al., 1999a; Cham-
pagne et al., 2003a). Each litter was observed for 5 h a day at
the following times: 0600—0800 h, 1200—1300 h and 1800—
2000 h. During each observation session, litters were
observed and behaviors recorded every 2 min (in sum, each
litter was observed 150 times per day for eight days). Beha-
viors recorded included: mother on/off the nest and mater-
nal licking behaviors directed at self or at pups. A maternal
care distribution curve was generated by calculating the
frequency with which pup-directed maternal licking was
observed. Maternal licking was expressed as a percentage
ofthetotal numberofobservationsperformedforeachlitter.
The mean (?SD) frequency of maternal licking was calcu-
lated for the cohort and High or Low maternal designations
were made relative to this mean. High and Low licking litters
were assessed as those falling one SD above or below the
mean, respectively. Animals were weaned on PND 22, and
pair housed with same sex littermates.
2.3. Male mate preference task
All testing was performed during the beginning of the dark
phaseofthelight—darkcycle(between1900 hand0200 h)and
digitally recorded. All animals were examined in a counter-
balanced manner with an equal number of animals from both
maternal conditions being tested at each time point. The
testing apparatus consisted of three transparent plastic Plex-
iglas chambers (21 ? 15 ? 10 inches) connected by two cylin-
ders (800? 400dia.) in a linear manner. The outer chambers
housed High and Low LG tethered female rats in estrus (n = 11
pairs of High/Low females) that were behaviorally screened
prior to the task. Females were screened by pairing with an
established stud male. Females who did not exhibit lordosis
uponmalestimulationwereexcludedfromtesting.Themiddle
chamber was neutral. A vasectomized male rat was placed in
the neutral chamber. Female rats were given 10 min to accli-
mate,whilemalesweregiven5 minpriortotestingwithaccess
to females prohibited. They were otherwise left undisturbed.
Aftertheshortacclimationperiod,malesweregiven15 minto
explore, inspect, investigate and ‘‘select’’ a female. Male
behaviors scored included the following: (i) time spent in
proximity to High or Low female rat, (ii) frequency of mounts,
(iii) intromission frequency, (iv) inter-mount interval, (v)
inter-intromission interval and (vi) ejaculations.
2.4. Female sexual behaviors
Similar to the male mate preference task, High and Low LG
adult female rats in estrus (behaviorally confirmed) were
tethered to the outer-chambers of the testing apparatus and
all behaviors were digitally recorded. Females were accli-
mated to the tethers for at least 10 min before the onset of
the task, and were tethered behind the forelimbs using an 800
nylon harness. The location of the female rats was counter-
balanced, with High and Low females being equally repre-
sentedintheleftandrightchambersamongtrials.During the
15 min test, the following proceptive and receptive beha-
viors were scored: (i) frequency of ear-wiggling, (ii) fre-
quency of hopping/darting and (iii) lordosis quotient
(lordosis responses/mounts and intromissions).
2.5. Male olfactory preference task
To assess if male mate selection was due to olfactory cues
emitted rather than female behaviors, a simple olfactory
preference task was performed. Using the three-chambered
apparatus described above, soiled bedding from High and
Low LG estrus female rats was placed in the outer test
chambers. Males were placed in the neutral chamber,
allowed to acclimate for 5 min and then were given equal
access to the two outer chambers. The placement of the
bedding was counterbalanced in the outer chambers for
each trial to minimize lateralization effects. Total time
spent in the High LG, Low LG and neutral chambers was
recorded.
2.6. Hormone treatment and perfusion
Upon completion of behavioral testing, 20 female rats
(n = 10 High and 10 Low) were bilaterally ovariectomized
and allowed to recover for seven days. After recovery,
femaleswereinjected(s.c.)with10 mgofestradiolbenzoate
(EB) in 0.1 ml sesame oil vehicle followed 48 h later by a
500 mg (s.c.) injection of progesterone in 0.1 ml vehicle.
This hormone treatment regimen is sufficient to facilitate
the expression of sexual behavior in female rats as well as
induce progesterone receptor immunoreactivity in brain
regions critical to sexual behavior (Boling and Blandau,
1939; Blaustein and Olster, 1989). Four hours after the final
hormone treatment, rats were screened for behavioral
estrus and subsequently administered a lethal dose of
sodium pentobarbital (50 mg/ml). Rats were perfused using
0.9% saline followed by 4% fresh paraformaldehyde in 0.1 M
PBS. Brains were removed, post-fixed in 4% paraformalde-
hyde in 0.1 M PBS for 3 h and subsequently placed in 30%
sucrose/0.1 MPBSsolution overnightat4 8Cuntilsaturated.
Forty mm sections were then cut on a cryostat and saturated
in anti-freeze cryoprotectant at ?20 8C until immunohisto-
chemistry (IHC) wasperformed to label progesterone recep-
tors (PR).
2.7. Progesterone receptor
immunocytochemistry and image analysis
For the PR IHC, sections were first washed (6?) in 0.01 M PBS
for 10 min each wash. Sections were then incubated in 0.5%
hydrogen peroxide (10 min) in order to reduce endogenous
peroxidase activity. The hydrogen peroxide rinse was fol-
lowed by three (10 min) PBS washes and incubated in 20%
normal goat serum with 0.3% PBT in order to reduce non-
specific staining. Sections were then incubated overnight in a
rabbit polyclonal primary antiserum generated against the
DNA binding domain of human progestin receptor (1:500,
A0098, DAKO, Carpinteria, CA) in 0.3% PBT for 72 h. Residual
primary antibody was removed by three (10 min) washes in
0.3% PBT.Sections were then incubated in a biotinylated goat
anti-rabbit secondary antibody (1:200, T0411, Vector, Bur-
lingame, CA) for 1 h, followed by three (10 min) PBTwashes.
Next, sections were incubated in avidin—biotin complex
(Vectastain Elite, Vector Laboratories, Burlingame, CA) for
1 h followed by three (10 min) PBS washes. Sections were
exposed to diaminobenzine (DAB, SK-4100, Vector Labora-
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Maternal programming of sexual attractivity3

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tories, Burlingame, CA) for 5 min. Sections were immedi-
ately washed in three (10 min) washes in PBS to stop the
reaction. Sections were mounted onto gelatin-coated
slides and cover slips were applied using Permount mount-
ing medium (ProSciTech, Australia). PR-immunoreactive
cells in the VMH were assessed using three matched sec-
tions of the VMH (Plate 56, 59, 60, Paxinos and Watson, 6th
ed.) for each animal. Sections were analyzed by capturing
images under 20? magnification with a Zeiss Axio Imager
M1 with an Axio Cam Mrm TV2/cc 0.63x Camera. Cells were
counted using NIH Image J to label cells with a pixel
density darker than 130 (where 0 = black and 255 white).
Where cells were clustered and difficult to individually
discriminate, images of the masked region were captured
at 40? magnification and analyzed in the same manner
using Image J. The numbers of immunoreactive cells were
quantified by observers blind to the experimental condi-
tions. A total of six hemi sections were assessed per animal
(three matched sections/animal), averaged for a total
count and expressed as the mean ? SEM. Sections were
countedby two independent
ensure reliable and valid quantification of immunoreactive
cells.
investigators to
2.8. Data analysis
All behavioral measures were analyzed by a one-way analysis
of variance (ANOVA) or a Student t-test. Prior to analysis, a
D’Agostino—Pearson omnibus test for normality was con-
ducted. If behaviors failed normality (p < 0.05), a Mann—
Whitney t-test was used in order to account for non-Gaussian
distributions. The following data was determined to be non-
parametric: intromission frequency, inter-intromission inter-
val, and male olfactory preference. All other data was
parametric. Densitometric measurements of PR-ir were
determined by Student’s t-test. Results were considered
statistically significant when p < 0.05.
3. Results
3.1. Maternal observations
Dams naturally differed in frequency of licking/grooming and
arched-back nursing over the first six—eight days postpar-
tum. A frequency distribution of maternal licking across all
litters was created as previously described (Francis et al.,
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High Low Neutral Chamber
0
50
100
150
200
250
300
350
400
450
**
A
Proximity to Female (sec)
HighLow
0.0
2.5
5.0
7.5
10.0
*
B
Mount Frequency
HighLow
0.0
2.5
5.0
7.5
10.0
12.5
*
C
Intromission Frequency
HighLow
0
300
600
900
1200
**
D
Inter-Mount Interval
High Low
0
300
600
900
1200E
Inter-Intromission Interval
High Low
0
2
4
6
8
10
F
*
Total Ejaculations
Across Trails
Figure 1
represent females reared in High maternal litters and the open bars represents females reared in Low maternal litters. The grey bar
representstheneutral chamberwherenofemaleswerepresent.MalesspentsignificantlymoretimeinvestigatingLowfemales relative
to Highs (n = 11). (B) Mean (?SEM) mount frequency with High and Low female rats. (C) Mean (?SEM) intromission frequency with High
and Lowfemale rats. (D) Mean (?SEM) inter-mount interval withHigh and Low female rats. (E) Mean (?SEM) inter-intromission interval
with High and Low female rats. (F) Total number of ejaculations across all trials (n = 11) in both High and Low female rats. All observed
ejaculations (n = 3) occurred with Low LG females. *p < 0.05; **p < 0.01.
Male mate preference. (A) Mean (?SEM) time spent by male rats in proximity to High or Low LG females. The filled bars
4 S.A. Sakhai et al.

Page 5

1999b). A maternal care score was generated by calculating
the frequency of maternal licking observed relative to the
total number of observations performed over the entire
observation period. Percent licking in this cohort ranged
from 4.00 to 12.33% with a mean licking score of 6.84% across
all litters. Female rats reared in litters that fell ?1 SD away
from the mean were used in the remainder of the study. In
sum,fiveHighandsevenLowLGlitters weregeneratedoutof
a total of 30 litters.
3.2. Male mate preference task
Male rat sexual preference for female rats reared under a
High or Low LG maternal conditions was tested. Males spent
significantly more time in proximity to Low LG females
compared to High LG females (p < 0.01). Indeed, males
spent nearly twice the amount of time with Low LG females
relative to High LG females (6 min 52 s vs. 3 min 28 s, respec-
tively) (Fig. 1A). Males also exhibited greater mounting
(p < 0.05) and intromission frequencies (p < 0.05) with
Low LG females compared to High LG females (Fig. 1B and
C) as well as exhibiting a shorter inter-mount interval
(p < 0.01; Fig. 1D). Whereas male behavior did not differ
in inter-intromission intervals (p > 0.05; Fig. 1E), a general
trend of shorter latency between intromissions exists
towards Low LG females. Furthermore, across all trials
(n = 11/group) ejaculations occurred exclusively with Low
females (n = 3 total ejaculations) (p < 0.05; Fig. 1F).
3.3. Female sexual behaviors
Low LG females exhibited a significantly higher lordosis
quotient (number of lordosis responses/number of mounts;
p < 0.05) compared to High females (Fig. 2A). High and Low
LGfemalesalsodifferedsignificantlyinparacopulatorybeha-
viors. Low females engaged in more hopping and darting
(p < 0.01) compared to High LG females, whereas ear wig-
gling was not significantly different (p > 0.05) (Fig. 2B and
C). Collectively, Low females appear to exhibit more para-
copulatory and copulatory behaviors compared to High
females.
3.4. Male olfactory preference task
When placed in the neutral arena of a three-chambered
testing apparatus and allowed to explore olfactory cues
(soiled bedding) from High and Low LG females, males did
not demonstrate a preference for odors from females from
either maternal condition (p > 0.05). Males did, however,
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HighLow
0.00
0.25
0.50
0.75
1.00
*
A
Lordosis Qoutient
(mean ± SEM)
HighLow
0.0
2.5
5.0
7.5
10.0
12.5B
**
Frequency of Hops/Darts
(mean ± SEM)
HighLow
0
10
20
30
C
Frequency of Ear Wiggles
(mean ± SEM)
Figure 2
(?SEM)frequency ofhoppinganddartinginHighandLowLGfemalesexposedtoanovelmale.(C)Mean(?SEM)earwigglefrequency of
High and Low females exposed to a novel male. *p < 0.05; **p < 0.01.
Female behavior. (A) Mean (?SEM) lordosis quotient for High and Low LG females exposed to a novel male rat. (B) Mean
High Low Neutral
0
100
200
300
400
**
**
Time spent in
each chamber (sec)
Figure 3
inoneofthreechambersthatcontaineddifferentolfactorycues.
Males spent roughly equal time investigating olfactory cues from
High and Low females and significantly less time in the neutral
chamber. **p < 0.01.
Male olfactory preference. Mean (?SEM) time spent
Maternal programming of sexual attractivity5

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spend significantly more time in the chambers containing
female olfactory cues relative to the neutral center chamber
(p < 0.0001; Fig. 3).
3.5. Progesterone receptor immunoreactivity
Estradiol-induced PR was quantified in the VMH for both Low
and High LG females. Consistent with the behavioral data,
the number of cells expressing PR-ir in the VMH was signifi-
cantly higher in estrogen-primed Low LG females compared
to High LG females (p < 0.05; Fig. 4).
4. Discussion
The findings presented suggest that the quality of maternal
care received early in life may differentially program phy-
siological and behavioral measures implicated in rat female
sexual function in adulthood. Specifically, female rats raised
in a litter that received low levels of maternal care, when
tested later as adults, demonstrated higher levels of para-
copulatory and copulatory behaviors when compared to
femalesraisedinalitterthatreceivedhighlevelsofmaternal
care. Interestingly, females reared under conditions of vary-
ing maternal care were also differentially ‘attractive’ to
novel male rats. Males, when allowed to select between
High or Low LG female rats in estrus, chose primarily to
mount, intromit, and ejaculate with Low LG females. This
was not due to differences in pheromonal/odor cues emitted
by the Low LG females as males spent the same amount of
time exploring olfactory cues from High and Low LG animals.
Differences between High and Low LG female rats were not
limited to behavior, but also extended to neurophysiology. In
female rats, the ventromedial nucleus of the hypothalamus
(VMH) has been studied extensively for its role in sexual
behavior. Interestingly, Low LG females, as adults, had
greater estrogen-induced PR-ir in the VMH compared to High
LG females. In accordance with the literature, these results
confirm that early life maternal care may influence female
sexual behavior; these findings further demonstrate that
differences in female sexual behavior can drive male mate
preferencethroughbehavioralandnotpheromonalsignaling.
4.1. Neuroendocrine underpinnings of female
sexual behavior
Our results suggest that male preferences for Low LG females
is guided, in part, by the pattern of female sexual behavior
(Figs. 1A—F and 2A—C) rather than changes in pheromonal
cues emitted by females (Fig. 3). During proestrus, female
copulatory and paracopulatory behaviors are dependent on
estrogen and progesterone stimulation of the neural circuits
driving sexual behavior (Boling and Blandau, 1939; Blaustein
and Erskine, 2002). The present findings suggest that differ-
ences in maternal care manifest in adulthood as changes in
the activity of this hormone-dependent circuitry (Fig. 4A)
and consequent changes in paracopulatory and copulatory
behaviors that drive male partner preference.
Activation of VMH progestin receptors by estrogens
influences many neuroendocrine processes including pre-
ovulatory gonadotropin secretion and female sexual beha-
vior (Rubin and Barfield, 1983; Olster and Blaustein, 1988).
One mechanism by which maternal care may be influencing
the immunoreactivity of PR in the VMH is by influencing the
pathway leading to PR induction by estradiol. Several co-
regulators of PR induction involved in female sexual beha-
vior may be regulated by differential maternal care in the
VMH. Potential targets include steroid receptor co-activa-
tor 1 (SRC-1), SRC-2, and cAMP binding protein, all of
which have been shown to play a modulatory role in PR
and estrogen receptor (ER) induction involved in female
sexual behavior (Molenda et al., 2002; Molenda-Figueira
et al., 2006). Similarly, estrogen receptor beta (ERb), like
estrogen receptor alpha (ERa), can influence the expres-
sion of female sexual behavior by conferring estrogen
sensitivity and PR induction (Helena et al., 2009).
Whereas, ERb has not been shown to influence PR-ir in
the VMH, this receptor has been shown to act in the locus
coeruleus to mediate sexual behavior.
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Please cite this article in press as: Sakhai, S.A., et al., Maternal programming of sexual attractivity in female Long Evans rats.
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Figure 4
(VMH) of female rats. (A) Low-power photomicrograph depicting representative immunostaining for PR in High and Low females (left,
upperpanel)andplate56ofPaxinosandWatson(2007),depictinglocationoftheVMH(left,lowerpanel).(B)Themean(?SEM)number
of cells expressing PR immunoreactivity in High and Low LG female rats (n = 7/group). *p < 0.05.
Effects of early maternal rearing on estrogen-induced progesterone receptor expression in the ventromedial hypothalamus
6 S.A. Sakhai et al.

Page 7

The previously mentioned transcription factors may pro-
vide proximate mechanisms for the role of maternal care on
PR-ir and sexual behavior. However, maternal care has also
been explicitly implicated in altering the neuroendocrine
circuits involved in regulating gonadotropin-releasing hor-
mone (GnRH), a key peptide involved in regulating the HPG
axis. This is believed to be mediated by epigenetic regulation
ofERa inthe medialpreoptic area (MPOA) and increasing ERa
immunoreactivityin theanteroventral
nucleus (AVPV) of the female LE rat (Champagne et al.,
2003b, 2006; Cameron et al., 2008c). Previous studies have
shown that Low LG females exhibit higher ERa expression in
both the MPOA and AVPV and can increase the likelihood of
female paracopulatory behaviors (Champagne et al., 2003b,
2006; Cameron et al., 2008b). The MPOA is rich in GnRH cell
bodies and input to this nucleus from estrogen-responsive
cells in the AVPV can influence GnRH and downstream lutei-
nizing hormone (LH) and estradiol. As estradiol enables PR-ir
in the VMH, feedback signaling may increase PR-ir in the VMH
and related paracopulatory behaviors shown in the present
work. In accordance with our findings, Cameron et al.
(2008a,b,c) report that Low LG females exhibit greater GnRH
immunoreactivity, higher estradiol and progesterone profiles
during proestrus, and a significantly higher amplitude LH
surge after ovariectomy and estrogen replacement. Thus,
Low LG animals have greater LH, estradiol and progesterone
release, likely due, at least in part, to greater ERaexpression
in the AVPV. In such a fashion, natural differences in copu-
latory and paracopulatory behaviors may arise that can
influence male preference.
High and Low LG animals expressed similar levels of
copulatory and paracopulatory behaviors following ovariect-
omy and hormonal replacement (data not shown, also
reported in Cameron, 2008). This finding suggests that early
life maternal care may have an organizing effect on the
developing neuroendocrine systems responsible for female
sexual behavior whereas thedownstreamtargets ofthesesex
steroids remains intact. It should be noted, however, that the
arcuate nucleus, a neural locus largely implicated in regulat-
inganterior pituitary hormonalrelease and GnRH,exhibits no
differences in PR-ir between groups, indicating that the
impact of differential maternal care is selective for proges-
terone sensitive targets that are specifically involved in
female sexual behavior. The means by which early life
experience leads to these neuroendocrine changes repre-
sents an important area for further inquiry.
paraventricular
4.2. Strengths and limitations
The results from this study are in agreement with those
investigating the effects of neonatal handling on offspring
sexual behavior as well as reports using cross-fostering para-
digms to study similar phenomena (Moore, 1984; Padoin
et al., 2001; Gomes et al., 2005, 2006; Uriarte et al.,
2007; Cameron et al., 2008b). However, several caveats
should be considered in interpreting the present findings:
i. ecological validity in the method of testing female sexual
behavior in a constrained environment, ii. vaginal cervical
stimulation (VCS) received by females during the male pre-
ference task may potentiate any effects observed, and iii.
female auditory cues, which may influence male preference
behaviors (in conjunction with paracopulatory behaviors),
were not recorded.
i. The current findings, unlike previous studies, document
differencesinHigh/LowLGfemaleparacopulatorybeha-
viors in addition to the previously noted differences in
copulatory behavior. Female sexual behaviors are most
commonly studied through the use of a paced mating
procedure. This is traditionally assessed using a pacing
chamber, in which the female regulates the timing and
number of interactions with male conspecifics by
approaching and withdrawing. Females pace interac-
tions with males in order facilitate fertility and fecundi-
ty (Frye and Erskine, 1990). The current experiments did
not utilize a female paced mating paradigm, but rather
used a protocol in which female rats were tethered. This
limited the control exerted by the female over the
‘sexual repertoire’ but allowed for the assessment of
male mate selection between two female rats. Whereas
this task has allowed us to gain some insight into female
sexual behavior and male mate selection that could not
be assessed using conventional methods, it does con-
strain the ecological validity of our findings.
ii. Another caveat lies in the manner in which male rats
approached, mounted, intromitted, and ejaculated with
High or Low LG females. Female copulatory behaviors
areregulated,inpart,byVCSinconjunctionwith steroid
hormones. VCS, including male intromissions and ejacu-
lations can, over time, facilitate the expression of lor-
dosis in the absence of estradiol or progesterone
(Blaustein et al., 2009). Furthermore, male copulatory
stimulation and non-intromissive copulatory behavior
can facilitate female copulatory and paracopulatory
behaviors (Blaustein et al., 2009). Consequently, the
order in which male conspecifics ‘‘chose’’ females in
our task may have affected female behavior. This point
did not likely impact the present findings, as our data
indicate that males initially ‘‘chose’’ High and Low LG
females with an equal probability.
iii. Finally, whereas olfactory and visual cues were assessed
to determine if these cues influence female attractivity,
auditory cues emitted by the female were not investi-
gated. It is possible that Low LG rats attract male
conspecifics through this mechanism (White and Bar-
field, 1989).
4.3. Conclusion
The present findings reveal that early life experiences in
female rats influence multiple aspects of later sexual
functioning. The quality of maternal care received early
in life influences later paracopulatory behaviors, copula-
tory behaviors and progesterone receptor immunoreactiv-
ity in relevant neural loci. By extension, these differences
in sexual function result in male rats choosing, almost
exclusively, to partner with female rats reared under
Low LG maternal conditions. While it remains to be deter-
mined if research on sexual behavior in rodents has pre-
dictive validity for aspects of human sexual function it is
compelling to consider the possibility that similar pro-
cesses of developmental programming are at play in
humans. The findings from the current study suggest that
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Maternal programming of sexual attractivity7

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social interventions targeted at ameliorating conflict and
strife in the home and improving parental support
could have a powerful impact on the sexual maturity
and function of young girls at the level of behavior and
physiology.
Role of the funding source
Funding for this study was provided by NIMH Grant R24
MH081797 (to D.D.F) and NIH Grant HD050470 (to L.J.K.);
the NIH had no role in the study design; in the collection,
analysis, and interpretation of data; in the writing of this
article;orinthedecisiontosubmitthisarticleforpublication.
Conflict of interest
All authors declare no conflict of interest.
Acknowledgments
We would like to thank Dr. Daniela Kaufer and Elizabeth Kirby
for guidance in manuscript preparation.
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