Membrane-Initiated
Estrogen Signaling in the
Ovine Endometrium
Brian Kitamura
Dr. Fred Stormshak
Cecily Bishop
Relevance

Economic Impact
Implications for reproduction in litter bearing
species
 Conceptus implantation/attachment


Human Health
Breast cancer/uterine cancer
 Hypothesized to cause cells to multiply
 Fast acting estrogen effects for vasodilation

Rationale
Classical Method of Steroid Action
HSP90
SR
S 1
4
3
5
2
SBP
Nucleus
S
6
mRNA
7
Protein
Rationale
Previous Observations

In vitro studies



High affinity Estradiol (E2) binding sites in plasma membrane
of Chinese Hamster Ovary cells (Razandi et al.)
Rapid activation of MAPK pathway by E2 (Song et al.)
In vivo studies



Winter 2004-2005
High affinity E2 binding sites in ovine endometrium
KD = 2.13 x 10-13 M
Objectives of Research



E2 effect on the MAPK Pathway has been
studied in in vitro studies
Is there physiological significance in the animal
model?
Examine the effect of E2 on the MAPK
pathway in vivo
Methods


Ovariectomized subjects
Tissue samples from
ovine endometrium




Endometrium is highly
responsive to E2
Hormone treated to
mimic estrous cycle
General anesthetic
Uterus exposed via
midventral laparotomy
Methods




(interior tissue of the uterus
known as the endometrium)
Figure courtesy of P.L. Senger, Pathways to
Pregnancy and Parturition, First Revised
edition, 1999


Uterus opened to reveal
endometrium
First tissue sample taken
5 micrograms E2 injected via
uterine artery
Tissue collected at 5, 10, 15
and 30 minutes post injection
of E2
Tissue frozen at -80 C
Returned to laboratory
Methods
Time Scheme
Hormone Schedule
Days 1-2
E2 Given
Days 8-9
Days 3-7
P4 Given
E2 Given
Day 10
Day 11
Rest
Surgery
Experiment Timeline
0 min 5 min 10min
First Sample
Taken, E2
Injected
Samples Taken
15 min
30 min
Final Sample Taken
Methods
Our Hypothesis
picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asp
Methods
Testing the Hypothesis
Tissue homogenized and separated into nuclear
and cytosolic portions
 Proteins separated via western blot
 Probed for presence of phosphorylated
ERK1/ERK2
Methods
Testing the Hypothesis
picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asp
Results
Presence of Phosphorylated ERK1/2
Minutes: 0
5
10
15
30
5
7.36
45.5
26.67
8.75
3
9.45
39.67
27.33
10
Relative Intensity:
Conclusion

Exogenous estrogen acts via a membrane
generated signal to stimulate the MAPK
pathway in the ovine endometrium
Acknowledgements




Howard Hughes Medical Intership
Undergraduate Research, Innovation,
Scholarship, and Creativity (URISC)
Department of Animal Sciences, College of
Agriculture
Stormshak Lab
Thank You!