Upload
ashlie-merritt
View
213
Download
0
Tags:
Embed Size (px)
Citation preview
Exploring the Role of nER in Non-Genomic Estrogen Regulation of the Ovine EndometriumKyle Ireton
Dr. Fredrick Stormshak http://jeremy29.smugmug.com/photos/317349190_8xiJ8-X3.jpg
RelevanceBiological Relevance
Steroids regulate reproductive organs/processes and pituitary gland
Health Related IssuesSteroids regulate onset
and development of breast and uterine cancer, and promote cardiovascular health
http://physicians.truthaboutdialysis.com/wp-content/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 InvestigationEstradiol (E2) is essential for rapid development of the
endometrial lining, for reception of a fertilized ovum (egg)
In Vitro Basis for InvestigationSpecific 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 FocusDoes 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/ster_horm_rec/dbd/er-ere-system-big.gif
Working HypothesisA strong, quantifiable correlation exists between nER and
the membrane binding protein for E2, in the ovine endometrium.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/MembraneProteins.gif
MethodsTwo groups
ovariectomized ewesE2 upregulates nER
productionP4 suppresses nER
productionKoligian and Stormshak
(1977)
http://www.salmonellablog.com/43_flock_of_sheep.jpg
Injection Schedule
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
Day Group 1 Ewes Group 2 Ewes
1 Estradiol (E2) 25 μg E2
2 E2 E2
3 Progesterone (P4) 10 mg
P4
4 P4 P4
5 P4 P4
6 P4 P4
7 P4 P4
8 E2P4
9 E2P4
10 (none) (none)
11 Recover Tissue Recover Tissue
MethodsInter-caruncular tissue of
endometrium collectedTissue processed and E2
nuclear and membrane binding activity counted
P.L. Senger, Pathways to Pregnancy and Parturition, First Revised edition, 1999
Tissue Sample AnalysisMembrane Tissue Assays:
BCA protein assay quantifies membrane proteinRadioreceptor assay (utilizing [3H]- estradiol- 17β)
quantifies specific binding activity of E2, per mg Protein
Nuclear Tissue Assays:DNA assay quantifies DNA in nucleusRadioreceptor assay quantifies nuclear binding of E2, per
femtomole DNA
Results:Nuclear Binding Activity of E2
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
Group 1 (E2)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
Group 2 (P4)
In units of fmol E2 bound/μg DNA
P-value <.07
Results: Membrane Binding Activity of E2
0
5
10
15
20
25
30
35
Group 1 (E2)
0
5
10
15
20
25
30
35
Group 2 (P4)
In units of fmol E2 bound/ mg membrane protein
P-value <.05
Conclusions and Future InvestigationsConclusions:
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 non-
genomic activity (ERK 1/2, PI3K, PIP2 pathways)
AcknowledgmentsDr. Stormshak, Professor Emeritus and HHMI MentorMary Meaker, Lab TechnicianBrian Kitamura, HHMI 2005 participantKevin Ahern, HHMI program CoordinatorHHMI and URISC programs