Estrogen receptor alpha expression in Peromyscus hybrids related to social behavior of the
parent of origin
Lauren Chiec
Introduction:
The intention of this research project was to examine the relationship between estrogen
receptor alpha (ERα) expression and the social behavior of the parent of origin. To do this I
compared ERα in two species of Peromyscus, Peromyscus maniculatus and Peromyscus
polionotus, as well as the hybrids resulting from cross-breeding between the two species. P.
polionotus male mice (―beach mice‖) are classified as socially monogamous, maintaining an
exclusive territory with a single female and helping to raise the offspring (Foltz, 1981).
However, Peromyscus maniculatus male mice (―deer mice‖) are considered polygynous, moving
about within a large home range and mating with multiple females (Birdsall and Nash, 1973). It
has been shown through empirical studies comparing microtines (voles) that ERα expression is
higher in males of polygynous populations than monogamous populations, specifically in brain
regions such as the medial amygdala (MeA), the bed nucleus of the stria terminalis (BST), the
medial preoptic area (MPOA), and the ventromedial hypothalamus (VMH) (Cushing and
Wynne-Edwards, 2006; Wu et al., 2011). Here, the relative ERα expression of P. maniculatus, P.
polionotus, and the hybrids resulting from the cross-breeding of the two species were examined
to determine if there is any significant difference between male and female mice of both species
or between the hybrids in the MeA, BST, MPOA, and the VMH.
Previous studies have supported the hypothesis that ERα expression is higher in
polygynous species when contrasted with monogamous species. One such study performed by
Cushing and Wynne-Edwards compared ERα expression along with reproductive strategies and
behavior in several different species of voles (2006). Male pine voles (Microtus pinetorum), a
monogamous species, showed the lowest levels of ERα expression in the medial amygdala
(MeA) when compared to male montane voles (M. montanus) that have a polygynous
reproductive strategy and meadow voles (M. pennsylvanicus) that are generally considered
polygynous, but show signs of prosocial behavior under certain conditions. In addition, male
pine voles showed lower levels of ERα in the bed nucleus of the stria terminalis (BST) when
compared with male montane voles. In the highly social species, ERα expression was sexually
dimorphic in the MeA and BST, with females expressing higher levels of ERα than males.
The above pattern of ERα expression and reproductive behavior has also held true in
other experiments with different vole species. For example, one study looked at two distinct
populations of mandarin voles (Microtus mandarinus) (Wu et al., 2011). Males from the
Chengcun population are known for possessing monogamous reproductive behaviors, less
aggression, and more prosocial behavior when compared to males from the Xinzheng population
that are known for possessing polygynous traits. Consistent with previous results, the less
prosocial Xinzheng population showed significantly higher ERα immunoreactivity (IR) than the
Chengcun population in the MeA, MPOA, and VMH (Wu et al., 2011).
Although oxytocin (OT) and vasopressin (V1a) have both been shown to play a role in
establishing reproductive behavior, they do not explain the behavior completely (Cushing and
Wynne-Edwards, 2006). Neither oxytocin nor vasopressin receptor expression are sexually
dimorphic (Insel and Shapiro, 1992; Insel et al., 1994). However, oxytocin seems to play a larger
role in female reproduction and vasopressin is more important in regulating male reproductive
behavior (Winslow et al., 1993; Williams et al., 1994; Insel and Hulihan, 1995; Insel et al., 1998;
Cushing and Carter, 2000; Cushing et al., 2001). Therefore, estrogen has been chosen as the
focus of the study because it could play a role in the establishment of the response to
neuropeptides such as oxytocin and vasopressin.
To look at the regulation of ERα and patterns of expression, another study was performed
that looked at hybrid offspring from phenotypically distinct populations of prairie voles
(Microtus ochrogaster) (Kramer et al., 2006). Illinois prairie voles are monogamous and show
low levels of ERα expression and Kansas prairie voles are less prosocial, display more
characteristics associated with polygyny, and exhibit higher levels of ERα expression. The
results showed that hybrid males display ERα expression patterns similar to males from the
mother’s population, while hybrid females showed ERα expression patterns similar to females
from the father’s population. These results suggest that parentage has a significant effect on ERα
expression. Kansas-Illinois (KI) males with Kansas (KN) mothers showed more ERα expression
than IL, IK, and KN males (Kramer et al., 2006).
For this current experiment, two different species of mice and the hybrids resulting from
the cross-breeding of these mice were studied to examine their ERα expression patterns. The
hybrids studied from the cross between these two species are from P. maniculatus mothers and
P. polionotus fathers. This cross results in offspring that are smaller than normal but otherwise
healthy. The cross between P. polionotus mothers and P. maniculatus fathers results in
abnormally large offspring that generally die soon after birth. The first cross most likely works
because the father comes from a population that exhibits a monogamous reproductive strategy in
which an autosomal locus from the fathers’ genes undergoes epigenetic silencing (Vrana et al.,
2000). In the cross that results in progeny that are abnormally large and die soon after birth,
Vrana hypothesizes that there is a loss or disruption of imprinting. The alleles from the mother
and father compete, the father’s allele does not get silenced, and an irregular quantity of
resources is allocated to the offspring (2000).
As earlier stated, it has previously been discovered that ERα expression is sexually
dimorphic in the MeA and BST in highly social species, with females expressing higher levels of
ERα (Cushing and Wynne-Edwards, 2006). This study looked at the relative ERα expression in
the MeA, BST, MPOA, and the VMH to determine if ERα expression is sexually dimorphic in
the parents and/or hybrid offspring mice. These areas of the brain were chosen because they are
known to be involved in several elements of reproductive and social behavior. In addition to
comparing only the P. maniculatus mothers, P. polionotus fathers, and the hybrid offspring, this
study intended to include data from P. maniculatus males and P. polionotus females, to allow for
comparison of the offspring ERα expression not only with their parents, but between and within
species as well. However, due to difficulties with the immunocytochemistry staining procedure
and time constraints, this data has not yet been obtained.
Behaviors of the mice were not studied because the project began with the receipt of the
brains from another lab. The brains were cross-sectioned and the process of
immunocytochemistry was used on the sectioned tissue to determine the ERα expression of the
mice. The stained tissue was mounted onto slides, cover-slipped, and then scored to determine
ERα expression.
Materials and Methods:
The Peromyscus brains were collected outside of the lab before the start of this project.
Fixed brains were stored in 25% sucrose at 4⁰C until sectioned at 30μm using a freezing sliding
microtome. Sliced tissue was stored in cryoprotectant until processed for ERα immunoreactivity
(IR) using ABC immunocytochemistry (ICC).
Tissue was first rinsed six times (every ten minutes for one hour) in 0.05M KPBS and
was then incubated for 20 minutes in 1% sodium borohydride to reduce background staining.
After again rinsing with KPBS, tissue was incubated with rabbit ERα polyclonal antibody at a
dilution of 1:100,000 in 0.05M KPBS—0.4% Triton X-100 at room temperature for one hour
and then at 4⁰C for 48 hours. This polyclonal antibody binds both free and bound receptors,
removing variation that may otherwise arise due to differences in circulating hormone levels.
Tissue was then rinsed ten times (every six minutes for one hour) in KPBS and then incubated
for one hour at room temperature in biotinylated goat anti-rabbit IgG (1:600 dilution in 0.4%
Triton X-100). Tissue was again rinsed and then incubated in an avidin-biotin peroxidase
complex for one hour. The tissue was rinsed again in KPBS followed by rinses in 0.175M
sodium acetate. ERα was visualized using a nickel-sulfate-diaminobenzidine chromagen
solution. Tissue was mounted onto subbed glass slides and air dried overnight. Slides were
counterstained with Neutral Red to aid in identifying brain regions, before being dehydrated in
ascending ethanol solutions, cleared in Histoclear, and coverslipped using Histomount (Cushing,
2004).
Prior to the experiment, controls were run to ensure the specificity of the primary
antibody. Tissue was also incubated in secondary antibody without being first incubated in
primary antibody and staining was not observed. In addition, the tissue was incubated in the
primary antibody along with the peptide it was generated against, and again staining did not
occur (Kramer et al., 2005).
Images of the slides were captured at 40X magnification and scored using image analysis
software. Sexes were analyzed separately, and counts were averaged for statistical differences
between sexes within each brain region, comparing the parents to one another as well as
comparing the hybrid offspring to one another. Differences were determined using a hypothesis
tests (t-tests) for the mean and differences were considered significant if P<0.05. In addition, the
sample size is limiting because only one mouse from each category (male and female parents,
hybrid offspring) was studied. Therefore, all four brain regions of all four mice were observed
and compared, with this small sample size possibly allowing for more error. Because of only one
sample from each group was observed, I am therefore unable to include variation in the results,
which would be the next step should this study be continued.
Results:
ERα expression was determined by the mean number of cells expressing ERα
immunoreactivity. P. maniculatus female ERα IR was compared to male P. polionotus
expression in the MPOA, MeA, BST, and VMH. In addition, ERα IR was compared between the
male and female offspring-hybrids. Results can be found in Figure 1.
In all four brain regions studied, there was no significant difference in ERα IR between
P. maniculatus and P. polionotus parents. In addition, in the MPOA, BST, and VMH, no
difference in ERα IR was seen between male and female offspring. One significant difference
was found however in the MeA between the male and female offspring, with the female hybrids
showing an average of 81.9 cells expressing ERα IR and the male hybrids showing an average of
12.3 cells expressing ERα IR. Therefore, sexually dimorphic expression of ERα IR was found
only between the male and female hybrid offspring and only in the MeA. Results are
summarized in Figure 1 below.
Figure 1. Mean number of cells expressing ERα immunoreactivity in the medial preoptic area (MPOA),
medial amygdala (MeA), bed nucleus of the stria terminalis (BST), and ventromedial hypothalamus (VMH)
in P. maniculatus females, P. polionotus males, and the male and female hybrid offspring. Columns grouped
under bars are significantly different. Results show that no sexual dimorphism was found between the parent
species. Sexual dimorphism was found to be significant between the male and female hybrid offspring in the
MeA, but not in the MPOA, BST, or VMH. These results are based on statistical hypothesis testing of the
means (t-testing), with P<0.05 considered significant. Results are limited by sample size of 1 in each group,
therefore variation is not accounted for.
Discussion:
Previous studies have found that sexual dimorphism in ERα IR exists in the MeA and
BST for prairie voles, with female prairie voles exhibiting significantly higher levels (Cushing
and Wynne-Edwards, 2006). This study shows that sexual dimorphism is upheld in hybrids
resulting from the cross-breeding of two different species of mice. Although no significant
difference was seen between the male and female parents, it is most likely because the parents
are from two separate species that exhibit completely different reproductive and social behavior.
P. maniculatus mothers come from a polygynous population with relatively low levels of
prosocial behavior. P. polionotus fathers come from a population of mice that exhibit
monogamous reproductive relationships and relatively high levels of prosocial behavior. The
differences between species, gender, and behavior make it difficult to accurately compare the
parents to each other. As stated earlier, this study had planned to include data from P.
maniculatus males and P. polionotus females in order to study the relationship of ERα
expression based on gender within species and to compare the hybrid offspring to the two
species of mice.
In the hybrid offspring, the sexual dimorphism between males and females is seen in the
significant difference in ERα IR expression in the MeA. This result is consistent with previous
findings of sexual dimorphism, with females exhibiting greater levels of ERα IR than males. In a
study published by Cushing and Wynne-Edwards, sexual dimorphism was seen more in Illinois
prairie voles, the more social species, when compared with Kansas prairie voles (2006). In
addition, the males from the more social Illinois population exhibited less ERα IR expression in
the MeA and BST. So, the sexual dimorphism and reduction of ERα in the male hybrids seen in
this study may be associated with increased prosocial behavior.
The reason that sexual dimorphism was only seen in the MeA may be because it plays an
important role in affiliative and aggressive behaviors. In addition, changes in the MeA most
likely do not adversely impact other aspects of sociosexual behavior, whereas changes in other
areas of the brain studied may have serious negative outcomes on reproductive behavior and
reproduction in general.
Without behavioral studies from the hybrid offspring or data from the missing sexes from
both species, it is difficult to make conclusions about the impact of this significant difference in
ERα IR expression. In addition, the low sample size of this study limits the conclusions that can
be made from the results. However, this study does show the existence of sexual dimorphism of
ERα IR expression in hybrid offspring, which calls for further study into the purpose and
mechanism behind this sexual dimorphism. If this study were to continue, the next step would be
to produce a more significant sample size and to include the missing sexes from both species. In
addition, it would be pertinent to begin with behavioral studies of the mice, which would
therefore allow for conclusions to be made about the correlation and possible causes of specific
reproductive behavior in relation to ERαIR expression.
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