Kyle Ireton
Dr. Fredrick Stormshak
http://jeremy29.smugmug.com/photos/317349190_8xiJ8-X3.jpg
Relevance
Biological Relevance
 Steroids regulate
reproductive
organs/processes and
pituitary gland
Health Related Issues
 Steroids regulate onset
and development of
breast and uterine cancer,
and promote
cardiovascular health
http://physicians.truthaboutdialysis.com/wpcontent/uploads/2008/02/doctor-with-patient.jpg
Two Regulatory Mechanisms of
Steroids:
Genomic (Slow) Vs.
Non-Genomic (Fast)
Genomic actions of steroids:
“Slow”
http://scienceblogs.com/clock/upload/2006/11/a2%20steroid-receptor.jpg
Non-genomic actions of steroids:
“Fast”
http://www.scq.ubc.ca/wp-content/uploads/2006/07/transduction.gif
Possible Non-Genomic
Actions of Estrogen
Biological Basis for Investigation
 Estradiol (E2) is essential for rapid development of the
endometrial lining, for reception of a fertilized ovum (egg)
In Vitro Basis for Investigation
 Specific plasma membrane binding site for E2 in
endometrium first observed by Pietras and Szego (1977)
 Translocation of up to 3% nuclear estrogen receptor (nER)
protein from nucleus to plasma membrane demonstrated
by Razandi et al. (1999) in transfected CHO cells
Research Focus
 Does a quantifiable correlation exist between nER and the
membrane binding protein for E2, in a live domestic
animal model?
http://www.ks.uiuc.edu/Research/pro_DNA/st
er_horm_rec/dbd/er-ere-system-big.gif
Working Hypothesis
 A strong, quantifiable correlation exists between nER and
the membrane binding protein for E2, in the ovine
endometrium.
http://users.rcn.com/jkimball.ma.ultranet/Biol
ogyPages/M/MembraneProteins.gif
Methods
 Two groups
ovariectomized ewes
 E2 upregulates nER
production
 P4 suppresses nER
production
 Koligian and Stormshak
(1977)
http://www.salmonellablog.com/43_flock_of_sheep.jpg
Injection Schedule
Day Group 1 Ewes
Group 2 Ewes
1
Estradiol (E2) 25 μg E2
2
E2
3
4
Progesterone (P4) 10 P4
mg
P4
P4
5
P4
P4
6
P4
P4
7
P4
P4
8
E2
P4
9
E2
P4
10
(none)
(none)
11
Recover Tissue
Recover Tissue
E2
 Alternating injections
simulate natural estrous
cycle of ewes
 Greater levels on nER
predicted in Group 1
 Hence, greater binding
activity predicted for
Group 1 ewes
Methods
 Inter-caruncular tissue of
endometrium collected
 Tissue processed and E2
nuclear and membrane
binding activity counted
P.L. Senger, Pathways to Pregnancy and Parturition,
First Revised edition, 1999
Tissue Sample Analysis
Membrane Tissue Assays:
 BCA protein assay quantifies membrane protein
 Radioreceptor assay (utilizing [3H]- estradiol- 17β)
quantifies specific binding activity of E2, per mg Protein
Nuclear Tissue Assays:
 DNA assay quantifies DNA in nucleus
 Radioreceptor assay quantifies nuclear binding of E2, per
femtomole DNA
Results:
Nuclear Binding Activity of E2
In units of fmol E2 bound/μg DNA
Group 1 (E2)
Group 2 (P4)
0.2
0.2
0.18
0.18
0.16
0.16
0.14
0.14
0.12
0.12
0.1
0.1
0.08
0.08
0.06
0.06
0.04
0.04
0.02
0.02
0
0
P-value <.07
Results:
Membrane Binding Activity of E2
In units of fmol E2 bound/ mg membrane protein
Group 1 (E2)
Group 2 (P4)
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
0
P-value <.05
Conclusions and Future
Investigations
Conclusions:
 Results support hypothesis that nER shares quantifiable
correlation to E2 membrane binding protein, in live
domestic animal models
Future Investigations:
 Investigate blocking action of P4 on E2 nuclear/membrane
binding (this Fall)
 Further elucidate intracellular mechanisms of nongenomic activity (ERK 1/2, PI3K, PIP2 pathways)
Acknowledgments
Dr. Stormshak, Professor Emeritus and HHMI Mentor
Mary Meaker, Lab Technician
Brian Kitamura, HHMI 2005 participant
Kevin Ahern, HHMI program Coordinator
HHMI and URISC programs