Paternal influence on female behavior: the role of Peg3 in exploration, olfaction, and neuroendocrine regulation of maternal behavior of female mice.

Abstract

Genomic imprinting represents a mechanism through which parent-of-origin effects on offspring development may be mediated. However, investigation of the influence of imprinted genes on behavior has been limited. Here the authors investigate the role of the maternally imprinted/paternally expressed gene, Peg3, in several aspects of behavior using both 129Sv- and B6-Peg3 mutant female mice. Virgin Peg3 females on both genetic backgrounds were less exploratory and had higher rates of defecation with strain-dependent effects on activity levels and olfactory discrimination. Reproductive success, pup retrieval, and postnatal maternal care of pups were reduced in these females whereas indices of maternal aggression were higher among B6 Peg3-KO females. Differences in maternal care were apparent in females caring for biological or cross-fostered offspring and deficits in pup retrieval apparent beyond the immediate postpartum period. Oxytocin receptor binding in the MPOA and LS was reduced in Peg3-KO females. Thus, the authors demonstrate that disruptions to Peg3 influences aspects of female behavior that are critical for mediating maternal effects on offspring development, such as postpartum licking/grooming, and that effects of Peg3 are dependent on the maternal genetic background.

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Paternal Influence on Female Behavior: The Role of Peg3 in Exploration,
Olfaction, and Neuroendocrine Regulation of
Maternal Behavior of Female Mice
Frances A. Champagne, James P. Curley,
and William T. Swaney
Columbia University
N. S. Hasen
University of Wisconsin
Eric B. Keverne
University of Cambridge
Genomic imprinting represents a mechanism through which parent-of-origin effects on offspring devel-
opment may be mediated. However, investigation of the influence of imprinted genes on behavior has
been limited. Here the authors investigate the role of the maternally imprinted/paternally expressed gene,
Peg3, in several aspects of behavior using both 129Sv- and B6-Peg3 mutant female mice. Virgin Peg3
females on both genetic backgrounds were less exploratory and had higher rates of defecation with
strain-dependent effects on activity levels and olfactory discrimination. Reproductive success, pup
retrieval, and postnatal maternal care of pups were reduced in these females whereas indices of maternal
aggression were higher among B6 Peg3-KO females. Differences in maternal care were apparent in
females caring for biological or cross-fostered offspring and deficits in pup retrieval apparent beyond the
immediate postpartum period. Oxytocin receptor binding in the MPOA and LS was reduced in Peg3-KO
females. Thus, the authors demonstrate that disruptions to Peg3 influences aspects of female behavior
that are critical for mediating maternal effects on offspring development, such as postpartum licking/
grooming, and that effects of Peg3 are dependent on the maternal genetic background.
Keywords: Peg3, maternal, oxytocin, strain differences, behavior
The discovery that maternal and paternal genomes do not
contribute equally to offspring phenotype reveals a unique
mechanism through which parent-of-origin effects may be me-
diated. The bases of this discovery were the growth and devel-
opmental abnormalities observed among chimeric mouse em-
bryos in which there was either a high maternal (Pg) or paternal
(Ag) genome content (Allen et al., 1995; Barton, Surani, &
Norris, 1984; Surani & Barton, 1983). Pg embryos were found
to be growth restricted relative to normal embryos whereas Ag
embryos were much larger than normal. Conversely, brain size
was reduced in Ag embryos and enhanced in Pg embryos.
Further exploration of the mechanisms that drive these effects
implicated germline epigenetic modification of either the ma-
ternal or paternal genome leading to uni-parental gene expres-
sion (Keverne, Fundele, Narasimha, Barton, & Surani, 1996;
Lees-Murdock & Walsh, 2008). Since the discovery of this
phenomenon, now referred to as genomic imprinting, approxi-
mately 100 genes have been identified that are maternally
expressed/paternally silenced (imprinted) or paternally ex-
pressed/maternally imprinted. To understand the function of
these genes and the evolutionary role of genomic imprinting,
mouse models have been developed in which the effects of
targeted deletion of these genes can be explored.
Though the principal focus of functional studies of imprinted
genes has been physiology and growth (Frontera, Dickins, Plagge,
& Kelsey, 2008), the effects of these genes on brain development
and the expression pattern of these genes within the brain suggest
that genomic imprinting would have a role in parent-of-origin
effects on behavior (Davies, Isles, Humby, & Wilkinson, 2007;
Wilkinson, Davies, & Isles, 2007) that is not limited to the unique
contributions of sex chromosomes. Paternally expressed/
maternally imprinted genes are highly expressed within the hypo-
thalamus whereas maternally expressed/paternally imprinted genes
are highly expressed within the cerebral cortex and striatum (Allen
et al., 1995; Keverne et al., 1996). Corresponding to the function
of these brain regions, targeted deletion of paternally expressed
genes is associated with suckling deficits, longer latencies to pup
retrieval, impairments in male sexual behavior, and aberrant cir-
cadian rhythms (Curley, Barton, Surani, & Keverne, 2004; Kozlov
et al., 2007; Lefebvre et al., 1998; Li et al., 1999; Swaney, Curley,
Champagne, & Keverne, 2007) whereas disruption of maternally
expressed genes is associated with increased cognitive deficits
(Davies et al., 2007; Plagge et al., 2005). Based on the function of
these genes, several hypotheses have emerged regarding the evo-
Frances A. Champagne, James P. Curley, and William T. Swaney,
Department of Psychology, Columbia University; N. S. Hasen, Center for
Women’s Health Research, University of Wisconsin; Eric B. Keverne,
Sub-Department of Animal Behaviour, University of Cambridge, Cam-
bridge, United Kingdom.
Correspondence concerning this article should be addressed to F. A.
Champagne, Department of Psychology, Columbia University, Schermer-
horn Hall, 1190 Amsterdam Avenue, New York, NY 10027. E-mail:
fac2105@columbia.edu
Behavioral Neuroscience
2009, Vol. 123, No. 3, 469–480
© 2009 American Psychological Association
0735-7044/09/$12.00DOI: 10.1037/a0015060
469

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lution of genomic imprinting, including conflict hypothesis and
mother-infant coadaptation (Curley et al., 2004; Hager & John-
stone, 2003; Haig, 1993).
In the current study, we explore the role of the paternally
expressed gene Peg3 in the behavior of adult female mice. Peg3 is
strongly expressed in the placenta and the developing embryo,
particularly in the hypothalamus (Li et al., 1999). The Peg3 gene
is located on mouse proximal chromosome 7 and encodes a large
zinc finger protein (Kuroiwa et al., 1996; Relaix et al., 1996) that
has been implicated in p53 mediated apoptosis (Deng & Wu, 2000;
Relaix et al., 2000). Because of the transmission pattern of pater-
nally expressed/maternally imprinted genes, the biological off-
spring of females with the Peg3 mutation do not possess the
mutation if the male used to generate the litter is genetically wild
type. As such, we can differentiate the effects of the mutation
within the mother and offspring. Mutant offspring exhibit impair-
ments in sucking and rate of growth with associated developmental
delays (Curley et al., 2004). As adults, male Peg3 mutants have
deficits in sexual behavior and olfactory recognition of sexually
receptive mates (Swaney et al., 2007; Swaney et al., 2008) and
Peg3 mutant females show longer latencies to retrieve pups after
hormonal priming and have reduced hypothalamic oxytocin neu-
rons (Curley et al., 2004; Li et al., 1999). Interestingly, genetically
wild type offspring reared by these Peg3 females also display
impairments in growth and behavior suggesting that there is a
transmission of the effects of the mutation in the absence of the
transmission of the mutant Peg3 gene (Curley, Champagne, Bate-
son, & Keverne, 2008).
Previous studies of Peg3 function in female mice have been
limited to the analysis of pup retrieval in females generated on a
129Sv strain. However, our previous studies have demonstrated
that this behavior does not predict postpartum maternal care of
pups and thus may not be critical for shaping offspring develop-
ment (Champagne, Curley, Keverne, & Bateson, 2007). In the
present study, we describe the behavioral effects of the Peg3 gene
on both the original 129Sv lineage and on a C57Bl/6J (B6) genetic
background. This design allows us to address two important ques-
tions: (1) the stability of the effects of the Peg3 mutation over
multiple generations (32 generations in the case of 129Sv females)
and (2) the effect of Peg3 in the context of two phenotypically
divergent genetic backgrounds, allowing for the analysis of the
interaction between background genotype, phenotype and the ef-
fects of Peg3. Conclusions regarding the functional role of genes
often come from knockout models in which the gene is studied in
one genetic context (i.e., background strain). However, to fully
understand gene function, converging evidence from multiple
models should be used to account for gene-environment and gene-
gene interactions. The more active and exploratory patterns of
behavior exhibited by B6 mice also permits a more thorough
examination of the behavioral consequences of the Peg3 mutation.
In addition to comparison of pup retrieval across these strains, here
we present the first investigation of the effects of Peg3 on loco-
motor activity, response to novelty, olfaction, postpartum maternal
care of pups and aggression toward intruders. Given the impor-
tance of specific forms of maternal care in shaping offspring
development (Champagne & Curley, 2005; Meaney, 2001), we
have further examined postpartum maternal behavior in response
to own and cross-fostered pups and the effects of the Peg3 muta-
tion on the density of oxytocin receptors (OTR) in those brain
regions previously shown to mediate individual differences in
postpartum care.
Method
Subjects
Adult Day 70 female wild type (WT) and Peg3-KO mice tested
in these studies were of the inbred 129Sv and C57BL/6J (B6)
strain and were housed on a reversed 12H dark-light cycle at the
Sub-Department of Animal Behavior at the University of Cam-
bridge, with lights on at 8 p.m. and off at 8 a.m. Peg3-KO mice
were originally generated on the 129Sv inbred strain at the Well-
come CRC Institute at the University of Cambridge by insertion of
a 4.8kb ?geo cassette into the 5? coding exon of the Peg3 gene (a
detailed description of the methods can be found in Li et al., 1999).
To generate Peg3-KO mice on the B6 strain, homozygous
Peg3-KO 129sv males were mated with WT B6 females to pro-
duce heterozygous Peg3-KO hybrid offspring. Males from these
litters were backcrossed with WT B6 females and the subsequent
offspring genotyped by tail biopsy to identify the mutant male
offspring. Males carrying the Peg3 mutation were then bred with
WT B6 females. This backcross was repeated over a total of 20
generations to produce B6 Peg3-KO females. Expression of the
Peg3 gene in the B6 adult female brain was found to be consistent
with the pattern previously described in 129Sv mice (Li et al.,
1999; Figure 1). Females were weaned at postnatal Day 28 (PND
28) at which time genotype was confirmed using cartilage from a
2 mm tail biopsy. Tissue was incubated in a 1 ml volume Eppen-
dorf tube, in PBS containing X-gal (1 mg/ml), 5 mM K4Fe(CN)6-
3H2O, 5 mM K3Fe(CN)6, 1 mM MgCl2, and 0.02% NP-40 at room
temperature on an orbital shaker overnight until color developed.
Female pups were weaned into (16” ? 5” ? 5”) cages of up to five
Figure 1.
mouse indicating the expression pattern of Peg3.
LacZ staining of coronal brain sections of an adult female B6
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individuals with same-sex, same-age, same-strain, and same-
genotype cage-mates (typically 2–3 littermates and 2–3 nonlitter-
mates) and provided with ad libitum access to water and the
RM1(E) chow diet (Lillico). All behavioral observations took
place during the dark period of the light cycle, with the onset of
testing occurring at least 1 hour after the onset of the dark cycle.
No behavioral trial took place on the day of cage cleaning, and all
trials were carried out under dim red light illumination. All exper-
imental procedures were conducted in accordance with the terms
of a project license issued by the U.K. Home Office to E.B.
Keverne under the Animals (Scientific Procedures) Act, 1986.
Procedure
Behavioral assessment of WT and Peg3-KO mice commenced
when these females were 70 days of age. Fourteen WT and 14
Peg3-KO females of each strain were first tested for exploratory
behavior in the open-field apparatus. After a 4-day interval, fe-
males were tested for olfactory discrimination and habituation and
then mated. After testing, females were housed 2 to 3 per cage with
an adult male (purchased from Harlan, U.K.) for a period of 14
days. Males were then removed and females monitored daily.
Within 1 to 2 days before parturition females were singly housed.
On the day of birth (PND 0), the dam was weighed, pups were
counted, and weighed and retrieval behavior was tested. Only
litters containing four or more pups were included in subsequent
analyses. From PND 1 to PND 6 mother-pup interactions in the
home-cage were observed. Litters were left undisturbed until PND
28 at which time pups were weaned. Two weeks postweaning, five
females per strain and genotype were sacrificed by cervical dislo-
cation and whole brains extracted for oxytocin receptor binding
analysis.
A second cohort of 8 WT and 11 Peg3-KO B6 adult females
was also mated to determine group differences in maternal behav-
ior toward cross-fostered pups and to measure levels of maternal
aggression. Females were housed and mated as in the previous
cohort. On the day of birth (PND 0), pups were removed and
replaced with a litter of 129Sv-B6 hybrid pups provided by a donor
female. From PND 1 to PND 6, mother-pup interactions in the
home-cage were observed. On PND 7, maternal aggression testing
was performed followed by a test of pup retrieval.
Pup Retrieval Test
On the day of birth, the lactating female and pups were removed
from the home cage briefly (approximately 10 seconds) and bed-
ding was disturbed throughout the cage. Three pups from the litter
were then randomly placed away from the nest end of the home
cage, and the mother was then reintroduced to the cage. The
latency (in seconds) to sniff a pup, retrieve each of three pups,
nestbuild, and crouch over pups was recorded. If a female had not
completed this response within 15 minutes the test was terminated,
resulting in a latency of 900 seconds for any behaviors not yet
observed. After testing, all pups were returned to the home cage.
All testing occurred within the colony room.
Maternal Observations
The procedure for assessing maternal behavior in mice was
adapted from previous work examining natural variations in ma-
ternal care in rats (Champagne, Francis, Mar, & Meaney, 2003).
Maternal behavior was scored from PND 1 to PND 6. Observers
were trained to a high level of interrater reliability (i.e., ?0.90).
Dams were observed in their home cage during the dark-phase of
the light cycle under dim red light (?5 lux) and not disturbed for
the duration of the 6-day observation period. Each day consisted of
four observation periods, two within the first 5 hours after the
onset of the dark cycle (0800 to 1300) and two within 7 hours of
the end of the dark cycle (1300 to 2000). Each observation was 60
minutes in duration and no observation sessions took place within
the 1-hr period before or after the transition from the light to dark
cycle. Within each observation period, the behavior of each mother
was scored every 3 minutes (20 observations/period ? 4 periods
per day ? 80 observations/mother/day ? 480 observations per
mother over the 6 days). The following behaviors were scored:
mother off pups, mother licking and grooming any pup (both body
and anogenital licking were included), mother in nursing posture
over pups, nest-building (while in contact with pups, nursing pups,
or not in contact with pups), self-grooming, eating, and drinking.
Variations in nursing posture previously observed in lactating rat
dams were not evident in the 6 days postpartum and nursing thus
describes a crouched arch posture over pups. We have used this
method in previous studies of natural variations in maternal care in
129Sv and B6 mice (Champagne et al., 2007).
Open-Field Test
The open field test is a standard tool for measuring exploratory
and anxiety-like behavior in rodents (for reviews see Archer, 1973;
Belzung & Griebel, 2001; Crawley, 1985). There is also evidence
from several rodent models that illustrate the relationship between
open-field measures and the indices of maternal care to be ex-
plored in these females (Clinton et al., 2007; Neumann, Kromer, &
Bosch, 2005). The open-field used was a 24” ? 24” ? 16” plastic
box. The sides of this box were black and the floor consisted of
white tiles. The behavior of the animal in this field was recorded
with a video camera mounted on a tripod adjacent to the field.
Coding of these video recordings was completed using a DOS-
based program designed to give summaries of the amount of time
spent in the inner and outer area of the field, as well as the overall
activity of the animal. Females were tested in the open-field at
approximately 70 days of age and confirmed to be in diestrus on
the day of testing after analysis of the cytology of a vaginal smear.
On the day of testing, the mouse was removed from its home cage
and placed directly into one corner of the open field. After a
10-min session, the mouse was removed and returned to its home
cage. Counts of fecal boli were assessed at this time. All testing
was conducted under red (dark phase) lighting conditions. During
analysis of the recordings, the field was divided into a grid of 10 ?
10 squares. For the purposes of analysis, inner field exploration
was defined as the time spent in the inner 9 ? 9 squares, activity
was defined as the number of square crossings and pauses in
movement within the field were defined as the duration of time
spent immobile. The length of time spent immobile is a measure
created from a log starting 5 seconds after the rodent stops engag-
ing in grid crossings during the test and indicates the duration of
time during which there are no grid crossings. Though there may
be some aspects of “freezing” included in this measure, it is a more
general indicator of immobility.
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Habituation-Dishabituation Olfactory Testing
As is the case for open-field exploration, there is strong
empirical support for the role of olfactory ability in maternal
care, with gross disruptions of olfaction leading to enhanced
maternal responsivity in virgins and decreased maternal respon-
sivity in lactating females (Fleming & Rosenblatt, 1974). The
habituation-dishabituation test examines olfactory discrimina-
tory ability by measuring the frequency and duration of ap-
proach to repeated presentations of two olfactory stimuli (Baum
& Keverne, 2002). This test determines the ability to detect
differences between two distinct olfactory stimuli by examining
habituation (reduced olfactory investigation) to repeated pre-
sentations of the same stimuli and dishabituation (increased
olfactory investigation) when a new stimuli is introduced. Fe-
males were tested 4 days after completion of the open-field test
and confirmed to be in diestrus. Females were singly housed 30
minutes before testing. At the start of testing, the cage lid
(containing food and water bottle) was removed and replaced
with a clean cage lid. The first three stimuli presentations
determined baseline levels of approach to the stimuli and ha-
bituated the animals to the testing procedure. There was 30 ?l
of water placed on a piece of filter paper that was then posi-
tioned on the cage lid. Number of approaches toward the stimuli
and the duration of these approaches were recorded using a
stopwatch. After a 2-min exposure and a 1-min interval, the
procedure was repeated twice, such that there were a total of
3 ? 2-min presentations of the water. The olfactory testing
phase was then initiated using two stimuli: urine pooled from
unfamiliar group-housed dietrus 129Sv females or urine pooled
from unfamiliar group-housed diestrus B6 females. The ability
to distinguish these volatile urinary cues likely requires main
rather that accessory olfactory function. Though the ability to
distinguish between 129Sv and B6 urine may not be relevant to
reproductive success of females in an ecological context, the
inability to distinguish these two urine types would indicate a
gross rather than subtle impairment in olfactory processes,
which would have reproductive consequences (Fleming &
Rosenblatt, 1974). The first stimulus was presented for 3 ?
2-min exposure periods, each separated by a 1-min interval. In
each case, 30 ?l of urine was placed on the filter paper and
placed on the lid of the cage. After a 1-min interval, the second
urine stimulus was presented for 3 ? 2-min exposure periods
each separated by a 1-min interval. The order of presentation of
the two urine types (B6 then 129Sv or 129SV then B6) was
counterbalanced. After testing females were returned to the
home cage.
In the analysis of the data obtained from this testing, overall
investigation (frequency and duration) of the stimuli was com-
pared with a repeated measures ANOVA. In addition, the habitu-
ation to the first urine type was determined as the frequency/
duration of approach to the first presentation subtracting the
frequency/duration of approach to the third presentation, with
higher values indicating increased habituation. Dishabituation was
determined to be the frequency/duration of approach to the first
presentation of the second urine type subtracting the frequency/
duration of approach to the third presentation of the first urine
type. These values were calculated for each animal and compared
as a function of genotype using a t test.
Maternal Aggression
On postpartum Day 7, each B6 female was exposed to an
intruder male for 7 minutes in her home-cage. All tests took place
between 4 and 7 hours after the onset of the dark light cycle.
Immediately before the behavioral test, pups were removed from
the home-cage. Previous work has demonstrated that removing
pups does not diminish the expression of maternal aggression in
mice (Svare, Betteridge, Katz, & Samuels, 1981). Postpartum Day
7 was chosen as this is within the period of high levels of maternal
aggression in mice that occurs between postpartum Days 4 and 10
(Svare, 1990). At the commencement of the test, an adult, group-
housed, virgin B6 intruder male was placed in the dam’s home
cage at the opposite end from the nest-site. Each test was recorded
on videotape and reviewed posttest to analyze maternal aggression.
No male was used more than twice, with WT and Peg3 mutant
females receiving males with equal levels of previous test experi-
ence. Males were given at least 1 day of rest between tests. The
latency, frequency and total duration of the following behaviors
were quantified by individuals blind to the genotype of females—
sniff the male, lunge at the male without contact, chase the male,
or bite the male (aggressive behaviors); freeze when approached
by the male, run away from the male, or establish a subordinate
posture—the female stands on her hind legs with her paws upright
and nape displayed (subordinate behaviors); self-groom, climb on
the cage, or bury themselves in bedding (distractive behaviors).
Tests were to be immediately terminated if overt aggression was
observed, although this was not the case with any females tested
here. Females who did not display a given behavior during the
7-min test were assigned a latency of 420 seconds for that behavior.
Oxytocin Receptor Binding Assay
Mice were sacrificed through rapid decapitation 2 weeks post-
weaning and brains were extracted, placed briefly in isopentane,
and kept at ?80 °C until processed. Females were confirmed to be
in diestrus at the time of sacrifice through cytological analysis of
vaginal swabs. Brains were sectioned in the coronal plane at 16
?m, and sections thaw mounted onto poly-L-lysine coated slides
that were stored at ?80 °C until the assay was performed. Slide-
mounted coronal brain sections were processed for receptor auto-
radiography using
England Nuclear, Boston) as previously described (Francis, Cham-
pagne, & Meaney, 2000).125I-OVT has a very high affinity for OT
receptors (Kd? 0.048 ? 0.008 nM) and a 10-fold greater affinity
for oxytocin receptors than OT (Elands et al., 1988). The affinity
of this compound for OT receptors is approximately 300-fold
higher than for V1 and V2 vasopressin receptors (Elands et al.,
1988). After a prewash in Tris-HC1 (50 mM, pH 7.4), slides were
exposed to a 75-min incubation (at room temperature) of 60 pM
125I-OVT in Tris with MgCl (10 mM), BSA (0.1%), and bacitracin
(0.05%). Nonspecific binding was defined in adjacent sections by
adding 50 nM Thr4Gly7-oxytocin (a concentration previously
found to completely displace 60 pM125I-OVT; Elands et al., 1988)
to the incubation buffer. The final 35-min wash was performed at
room temperature in 50 mM Tris (pH 7.4), 10 mM MgCl to reduce
background radiation. After air-drying, the slides were exposed to
BioMax MR film (Kodak) for 48 hours.
standards (Amersham) were included in the cassette for quantifi-
125I-d(CH2)5[Tyr-Me)2,Tyr-NH2
9] OVT (New
125I autoradiographic
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CHAMPAGNE, CURLEY, SWANEY, HASEN, AND KEVERNE

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cation. All autoradiograms were analyzed using an image-analysis
system (MC1D-4, Interfocus Imaging, Cambridge, U.K.). Three
sections were analyzed bilaterally for each brain region. For each
animal, total and nonspecific binding was measured for each
region and the difference taken to yield specific binding. Specific
binding was greater than 90% of total binding. The statistical
analysis was performed on the mean of these values for each
animal by brain region according to the mouse brain atlas (Paxinos
& Franklin, 2003). Receptor density was measured in the medial
preoptic area (MPOA), lateral septum (LS), bed nucleus of the
stria terminalis (BNST), the basolateral (BL), medial (MD), and
central nuclei (CN) of the amygdala and the ventral medial hypo-
thalamus (VMH).
Results
Open-Field Exploration
Among 129Sv mice, the Peg3 mutation significantly increased
the time spent immobile [t(26) ? 4.16, p ? .001] and decreased
the number of grid crossings [t(26) ? 4.34, p ? .001] as well as the
amount of time spent in the inner area of the field [t(26) ? 2.86,
p ? .01; Figure 2A]. The number of grid crossings in both the
outer [t(26) ? 4.14, p ? .001] and inner [t(26) ? 2.96, p ? .01]
area of the field were lower among the Peg3-KO females (Figure
2A). In contrast, the Peg3 mutation on the B6 genetic background
had the effect of increasing levels of activity as evidenced by the
elevated number of total squares crossed by B6 Peg3-KO females
[t(26) ? 2.86, p ? .05]. Though there was a trend toward WT
females spending more time immobile than Peg3-KO females
during the test [t(26) ? 1.82, p ? .08] this effect was not statis-
tically significant. Consistent with the effect found in 129Sv fe-
males, B6 Peg3-KO females spent significantly less time in the
inner area of the open-field [t(26) ? 2.86, p ? .05; Figure 2B].
Number of squares crossed in the inner area versus outer area of
the field was not significantly different as a function of genotype
among B6 mice though there was a trend toward Peg3-KO females
exhibiting higher numbers of crossings in the inner area of the field
[t(26) ? 1.88, p ? .07]. These behavioral differences were appar-
ent throughout the 10-min test. Interestingly, despite significant
strain differences in behavior, Peg3-KO females on both back-
grounds had higher rates of defecation during testing [WT-
129Sv ? 1.0 ? .23, 129Sv Peg3-KO ? 2.5 ? .44, t(26) ? 3.90,
p ? .01; WT-B6 ? 1.14 ? .25, B6 Peg3-KO ? 2.29 ? .43,
t(26) ? 4.26, p ? .05].
Olfactory Discrimination
Repeated measures analysis of the behavior of 129Sv WT and
Peg3 mutant females indicated no significant effect of genotype on
the number of approaches or the time spent sniffing the stimuli,
though there was a main effect of exposure time/type on these
behaviors [approaches: F(8, 26) ? 4.49, p ? .001; time spent
sniffing: F(8, 26) ? 4.29, p ? .001; Figure 3A] indicating the
change in behavior occurring during successive presentations of
the stimuli. More relevant to the question of habituation and
dishabituation is the comparison between the behavior toward the
first urine presentation compared to the third urine presentation
(habituation; a1 vs. a3 on Figure 3) and the change in behavior
from the third presentation of the first urine type and the first
presentation of the second urine type (dishabituation; a3 vs. b1 on
Figure 3). However, there was no significant effect of genotype on
either of these behavioral dimensions among 129Sv mice. Re-
peated measures analysis of the olfactory behavior of B6 females
indicated a main effect of genotype on the number of approaches
to the stimulus [F(1, 26) ? 10.98, p ? .01] and an interaction
between genotype and exposure time/type on the number of ap-
proaches [F(8, 26) ? 1.98, p ? .05]. Similar effects were found for
duration of time spent sniffing the stimulus [genotype: F(1, 26) ?
6.5, p ? .05; genotype X exposure F(8, 26) ? 2.1, p ? .05;
Figure 3B]. Though B6 females with the Peg3 mutation did not
differ significantly in the number of approaches and time spent
sniffing the water or the first urine stimulus, there was a significant
effect of genotype on these variables when the second urine
stimulus was presented (p ? .01). Peg3 females were significantly
less exploratory of the second urine type regardless of whether it
was B6 or 129Sv urine. Both WT and Peg3-KO females exhibited
habituation to the urine stimulus, showing reduced exploration of
this stimulus over successive presentations. Behavioral dishabitu-
ation in response to the second urine type differed significantly as
a function of genotype, with WT females showing increased ap-
proach (p ? .01) and time spent sniffing (p ? .05) the novel
stimulus and Peg3-KO females showing no significant change in
behavior in response to the new olfactory stimulus.
Reproductive Success
After olfactory testing all females were mated with a same strain
male for a 2-week period. Females were monitored daily to deter-
mine the date of birth of the litter and to assess litter size and
weight. Among the 129Sv females, 12 of the 14 WT females gave
birth to litters; 2 of those females were excluded from further
testing because of a litter size of fewer than 4 pups. Ten of the 14
Peg3-KO females gave birth; 2 of those females were excluded
500
A. 129Sv Females
e (s)
me spent immobile
ti
ed
total squares crosse
***
300
250
ea (s)
ent in the inner are
time sp
1000
100 00
200
300
400
**
50
0
100
50
150
200
20000
400
600
800
***
1000 1000 300
250
s)
e spent immobile (s
time
a (s)
ent in the inner area
time spe
0
WTPeg3
B. B6 Females
500
**
**
WT Peg3
0
WT Peg3
200200
400
600
800
100
50
150
200
300
tal squares crossed
tot
100100
200
300
400
0
WT Peg3
0
WT Peg3
0
WTPeg3
Figure 2.
spent immobile, time spent in the inner area, and total activity observed
among (A) 129Sv females as a function of genotype and (B) B6 females as
a function of genotype.?p ? .05.??p ? .01.???p ? .001.
Open-field behavior of WT and Peg3 mutant females. Time
473
PATERNAL INFLUENCE ON FEMALE BEHAVIOR