Estrogen Receptor Expression in the Hybrid Peromyscus
I.
Introduction
The Peromyscus Polionotis (PO) is a generally socially monogamous species, and is the
most common peromyscus in the United States. The Peromyscus Maniculatus, (BW)
common to the southeastern region of the United States, is generally a polygynous
species. In this study, the social interactions and behavior of the offspring are observed in
three different crosses. POxPO generally yields socially monogamous species, and
BWxBW yields socially polygynous species. In both, non-hybrid crosses, the males do
not necessarily participate in parental care and child rearing, and there is a visible
increase in aggression. The focus of the male in these crosses is as much reproduction as
possible to spread their genes. The male is at battle with the female to exceed the
maternal expenditure of resources to the offspring, while the maternal genome tries to
limit it. In the hybrid cross, the female BW must be crossed with the male PO or the
offspring becomes enlarged and inviable. In the hybrids, the males seem to lose the gene
that contributes to the desire to solely use the female for reproduction, and actually
control the maternal energy expense to the offspring. Biparental care of the offspring then
also occurs. The phenomena that occurs when the female BW mates with the male PO is
that DNA methylation occurs, therefore transcription is silenced, and the gene will not be
expressed. The count of estrogen receptor α containing cells can inform whether or not
these hypothesies are correct. Estrogen α can increase maternal traits such as licking and
grooming. In general, estrogen α in females allows the peromyscus to demonstrate a
higher degree of prosocial behavior. However highly social males are predicted to
1
express significantly lower levels of ER than females. Prosocial behavior can include
positive social interactions, the formation of pair bonds, as well as parental care.
II.
Purpose
To determine if crossing the Peromyscus Polionotis with the Peromyscus Maniculatus
increases monogamy and prosocial behavior.
III.
Materials and Methods
The brains of Peromyscus Polionotis and Peromyscus Maniculatus are collected and fixed
via transcardial perfusion with 4% buffered paraformaldehyde and 2.5% acrolein. Fixed brains
were stored in 25% sucrose at 4°C until they were sectioned at 30 µm on a freezing sliding
microtome. Sections were stored at –20°C in cryoprotectant (Watson et al., 1986) Prior to
preparing the slides, the tissue was processed by ABC Immunochemistry. It is stained with a
1:10K titration, and incubated with Santa Cruz Antibody. Antibody marks the estrogen receptors
so they are visible later. The tissue is then mounted onto frosted microslides using 0.9% saline.
The tissue is mounted from the anterior of the brain to the posterior. Following mounting, the
tissue is then dehydrated and cleared in Xylene. The slides are coverslipped using xylene based
mounting media, and scraped for excess medium. The slides are now ready to be scored. Using a
40x microscope, and Image Pro Plus software, the number of estrogen receptor containing cells
can be counted. Four areas of the brain are looked at; medial preoptic area, (MPOA)
ventromedial nucleus of the hypothalamus, (VMH) Medial Amygdala, (MeA) and the bed
nucleus of the stria terminalis. (BST) These areas were chosen because they are involved in a
variety of aspects of reproduction and social behavior. (Cushing, Wynne-Edwards, 2006) In the
2
statistical analysis, differences between sexes to determine sexual dimorphism were measured by
using one-way ANOVA. species. Statistical tests were conducted separately for each sex and
brain region. Results were considered significant at P < 0.05. The medial amygdala is the only
region displaying sexual dimorphism. (P=0.0311)
IV.
Results
Expression of estrogen α is sexually dimorphic in the medial amygdala, [F(1,11) 6.28,
P<0.05]; Figure 2. The MeA in females had a mean of 81.9. The males had a mean of 12. 3. The
other areas of the brain tested did not show statistical significance for sexual dimorphism.
MPOA[F(1,11) 0.0226, P>0.05]; Figure 1. VMH[F(1,11)0.7814, P>0.05]; Figure 3.
BST[F(1,11)1.02, P>0.05]; Figure 4.
Mean Number of Cells Containing
ERα in MPOA
800
600
Figure 1
400
200
0
Female
Male
3
Mean Number of Cells Containing
ERα in MeA*
780
580
Figure 2
380
180
-20
Female
Male
Mean Number of Cells Containing
ERα in VMH
800
Figure 3
600
400
200
0
Female
Male
Mean Number of Cells Containing
ERα in BST
800
600
Figure 4
400
200
0
Female
Male
4
Among males and females, there were no results that were statistically significant. For
MPOA in females [F(1,11) 0.0116, P>0.05]; Figure 5. For MeA in females [F(1,11)
0.041, P>0.05]; Figure 6. For VMH in females [F(1,11) 0.0554, P>0.05]; Figure 7. For
BST in females [F(1,11) 2.154, P>0.05]; Figure 8. For MPOA in males [F(1,11) 1.58,
P>0.05]; Figure 9. For MeA in males [F(1,11) 0.692, P>0.05]; Figure 10. For VMH in
males [F(1,11) 0.0899, P>0.05]; Figure 11. For BST in males [F(1,11) 0.789, P>0.05];
Figure 12.
Mean Number of Cells Containing
ERα in MPOA in Females
800
Figure 5
600
400
200
0
BW
Hybrid
Mean Number of Cells Containing
ERα in MeA in Females
800
Figure 6
600
400
200
0
BW
Hybrid
5
Mean Number of Cells Containing
ERα in VMH in Females
Figure 7
800
600
400
200
0
BW
Hybrid
Mean Number of Cells Containing
ERα in BST in Females
800
700
600
Figure 8
500
400
300
200
100
0
BW
Hybrid
Mean Number of Cells Containing
ERα in MPOA in Males
800
Figure 9
600
400
200
0
PO
Hybrid
6
Mean Number of Cells Containing
ERα in MeA in Males
780
Figure 10
580
380
180
-20
PO
Hybrid
Mean Number of Cells Containing
ERα in VMH in Males
Figure 11
800
600
400
200
0
PO
Hybrid
Mean Number of Cells Containing
ERα in BST
in Males
Figure 12
800
600
400
200
0
PO
Hybrid
7
V.
Discussion
The data did not support a difference between the parental sex and the offspring. The females
comparison, and males comparison does not show statistical significance, therefore it is difficult
to make the generalization that the hybrids have more receptors or not. But there are some
incidences where the crosses have a greater average mean than the BW females. But in the social
species, PO, male offspring generally have more receptors than the crossed males. In the oneway ANOVA to test sexual dimorphism, the results for the medial amygdala show there is a
statistical significance. This means this portion of the brain is sexually dimorphic, with crossed
females exhibiting more estrogen α receptors than crossed males. In order to further test these
results, perhaps a larger sample size could be used. It would also be worthwhile to test the
offspring crosses we are missing, such as BW males, and PO females.
8
References
Cushing BS, Kramer KM. 2004. Mechanisms underlying epigenetic effects of early social
experience: the role of neuropeptides and steroids. Neuroscience Biobehavior
Cushing BS, Wynne-Edwards KE. 2006. Estrogen Receptor- Distribution in Male Rodents Is
Associated with Social Organization. Journal of Comparative Neurology 494:595– 605
Kramer KM, Carr M, Schmidt JV, Cushing BS. 2006. Parental regulation of central patterns of
estrogen receptor α. Neuroscience 142:165-173.
9