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Non-Genomic Actions of Steroids in 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