Kyle Ireton Dr. Fredrick Stormshak

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 content/uploads/2008/02/doctor-with-patient.jpg

Two Regulatory Mechanisms of Steroids: Genomic (Slow) Vs. Non-Genomic (Fast)

Genomic actions of steroids: “Slow”

Non-genomic actions of steroids: “Fast”

Possible Non-Genomic Actions of Estrogen Biological Basis for Investigation Estradiol (E 2 ) 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 E 2 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 E 2, in a live domestic animal model? er_horm_rec/dbd/er-ere-system-big.gif

Working Hypothesis A strong, quantifiable correlation exists between nER and the membrane binding protein for E 2, in the ovine endometrium. ogyPages/M/MembraneProteins.gif

Methods Two groups ovariectomized ewes E 2 upregulates nER production P 4 suppresses nER production Koligian and Stormshak (1977)

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 DayGroup 1 EwesGroup 2 Ewes 1Estradiol (E 2 ) 25 μgE2E2 2E2E2 E2E2 3 Progesterone (P 4 ) 10 mg P4P4 4 P4P4 P4P4 5 P4P4 P4P4 6 P4P4 P4P4 7 P4P4 P4P4 8E2E2 P4P4 9E2E2 P4P4 10(none) 11Recover Tissue

Methods Inter-caruncular tissue of endometrium collected Tissue processed and E 2 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 [ 3 H]- estradiol- 17β) quantifies specific binding activity of E 2, per mg Protein Nuclear Tissue Assays: DNA assay quantifies DNA in nucleus Radioreceptor assay quantifies nuclear binding of E 2, per femtomole DNA

Results: Nuclear Binding Activity of E 2 In units of fmol E 2 bound/μg DNA P-value <.07

Results: Membrane Binding Activity of E 2 In units of fmol E 2 bound/ mg membrane protein P-value <.05

Conclusions and Future Investigations Conclusions: Results support hypothesis that nER shares quantifiable correlation to E 2 membrane binding protein, in live domestic animal models Future Investigations: Investigate blocking action of P 4 on E 2 nuclear/membrane binding (this Fall) Further elucidate intracellular mechanisms of non- genomic 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