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Exploring the Role of nER in Non-Genomic Estrogen Regulation of the Ovine Endometrium

Exploring the Role of nER in Non-Genomic Estrogen Regulation of the Ovine Endometrium. Kyle Ireton Dr. Fredrick Stormshak. Relevance. Biological Relevance Steroids regulate reproductive organs/processes and pituitary gland Health Related Issues

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Exploring the Role of nER in Non-Genomic Estrogen Regulation of the Ovine Endometrium

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  1. Exploring the Role of nER in Non-Genomic Estrogen Regulation of the Ovine Endometrium Kyle Ireton Dr. Fredrick Stormshak http://jeremy29.smugmug.com/photos/317349190_8xiJ8-X3.jpg

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

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

  4. Genomic actions of steroids: “Slow” http://scienceblogs.com/clock/upload/2006/11/a2%20steroid-receptor.jpg

  5. Non-genomic actions of steroids: “Fast” http://www.scq.ubc.ca/wp-content/uploads/2006/07/transduction.gif

  6. 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 Razandiet al. (1999) in transfected CHO cells

  7. 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/ster_horm_rec/dbd/er-ere-system-big.gif

  8. 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/BiologyPages/M/MembraneProteins.gif

  9. Methods • Two groups ovariectomized ewes • E2upregulatesnER production • P4 suppresses nERproduction • Koligian and Stormshak (1977) http://www.salmonellablog.com/43_flock_of_sheep.jpg

  10. 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

  11. 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

  12. 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

  13. Results:Nuclear Binding Activity of E2 In units of fmol E2 bound/μg DNA P-value <.07

  14. Results: Membrane Binding Activity of E2 In units of fmol E2 bound/ mg membrane protein P-value <.05

  15. 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 non-genomic activity (ERK 1/2, PI3K, PIP2 pathways)

  16. 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

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