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”

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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)

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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