1. Membrane-Initiated Estrogen Signaling in the Ovine Endometrium Membrane-Initiated Estrogen Signaling in the Ovine Endometrium Brian Kitamura Dr. Fred Stormshak Cecily Bishop

2. Relevance Economic Impact Economic Impact Implications for reproduction in litter bearing species Implications for reproduction in litter bearing species Conceptus implantation/attachment Conceptus implantation/attachment Human Health Human Health Breast cancer/uterine cancer Breast cancer/uterine cancer Hypothesized to cause cells to multiply Hypothesized to cause cells to multiply Fast acting estrogen effects for vasodilation Fast acting estrogen effects for vasodilation

3. Rationale Classical Method of Steroid Action Protein SR HSP90 S 1 2 3 7 4 5 mRNA Nucleus 6 SBP S

4. Rationale Previous Observations In vitro studies In vitro studies High affinity Estradiol (E 2 ) binding sites in plasma membrane of Chinese Hamster Ovary cells (Razandi et al.) High affinity Estradiol (E 2 ) binding sites in plasma membrane of Chinese Hamster Ovary cells (Razandi et al.) Rapid activation of MAPK pathway by E 2 (Song et al.) Rapid activation of MAPK pathway by E 2 (Song et al.) In vivo studies In vivo studies Winter 2004-2005 Winter 2004-2005 High affinity E 2 binding sites in ovine endometrium High affinity E 2 binding sites in ovine endometrium K D = 2.13 x 10 -13 M K D = 2.13 x 10 -13 M

5. Objectives of Research E 2 effect on the MAPK Pathway has been studied in in vitro studies E 2 effect on the MAPK Pathway has been studied in in vitro studies Is there physiological significance in the animal model? Is there physiological significance in the animal model? Examine the effect of E 2 on the MAPK pathway in vivo Examine the effect of E 2 on the MAPK pathway in vivo

6. Methods Ovariectomized subjects Ovariectomized subjects Tissue samples from ovine endometrium Tissue samples from ovine endometrium Endometrium is highly responsive to E 2 Endometrium is highly responsive to E 2 Hormone treated to mimic estrous cycle Hormone treated to mimic estrous cycle General anesthetic General anesthetic Uterus exposed via midventral laparotomy Uterus exposed via midventral laparotomy

7. Methods Uterus opened to reveal endometrium First tissue sample taken 5 micrograms E 2 injected via uterine artery Tissue collected at 5, 10, 15 and 30 minutes post injection of E 2 Tissue frozen at -80 C Returned to laboratory (interior tissue of the uterus known as the endometrium) Figure courtesy of P.L. Senger, Pathways to Pregnancy and Parturition, First Revised edition, 1999

8. Methods Time Scheme P 4 Given Days 3-7 Days 8-9 E 2 Given Days 1-2 Day 11Day 10 E 2 GivenRestSurgery Hormone Schedule Samples Taken 10min30 min First Sample Taken, E 2 Injected 5 min0 min 15 min Experiment Timeline Final Sample Taken

9. Methods Our Hypothesis picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asp

10. Methods Testing the Hypothesis Tissue homogenized and separated into nuclear and cytosolic portions Proteins separated via western blot Proteins separated via western blot Probed for presence of phosphorylated ERK1/ERK2 Probed for presence of phosphorylated ERK1/ERK2

11. Methods Testing the Hypothesis picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asp

12. Results Presence of Phosphorylated ERK1/2 Minutes: 0 5 10 15 30 Relative Intensity: 5 7.36 45.5 26.67 8.75 3 9.45 39.67 27.33 10

13. Conclusion Exogenous estrogen acts via a membrane generated signal to stimulate the MAPK pathway in the ovine endometrium Exogenous estrogen acts via a membrane generated signal to stimulate the MAPK pathway in the ovine endometrium

14. Acknowledgements Howard Hughes Medical Intership Howard Hughes Medical Intership Undergraduate Research, Innovation, Scholarship, and Creativity (URISC) Undergraduate Research, Innovation, Scholarship, and Creativity (URISC) Department of Animal Sciences, College of Agriculture Department of Animal Sciences, College of Agriculture Stormshak Lab Stormshak Lab

15. Thank You!