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Non-Genomic Actions of Steroids in Ovine Endometrium
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Non-Genomic Actions of Steroids in Ovine Endometrium

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  • 1. 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 Razandi et 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/st er_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/Biol ogyPages/M/MembraneProteins.gif
  • 9. Methods  Two groups ovariectomized ewes  E2 upregulates nER production  P4 suppresses nER production  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 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 E2 P4 9 E2 P4 10 (none) (none) 11 Recover Tissue Recover Tissue
  • 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 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 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
  • 14. 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
  • 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