Membrane-Initiated Membrane-Initiated Estrogen Signaling in Estrogen Signaling in

the Ovine the Ovine EndometriumEndometrium

Brian KitamuraBrian Kitamura

Dr. Fred StormshakDr. Fred Stormshak

Cecily BishopCecily Bishop

RelevanceRelevance

Economic ImpactEconomic Impact Implications for reproduction in litter Implications for reproduction in litter

bearing speciesbearing species Conceptus implantation/attachmentConceptus implantation/attachment

Human HealthHuman Health Breast cancer/uterine cancerBreast cancer/uterine cancer Hypothesized to cause cells to multiplyHypothesized to cause cells to multiply Fast acting estrogen effects for Fast acting estrogen effects for

vasodilationvasodilation

RationaleRationaleClassical Method of Steroid ActionClassical Method of Steroid Action

Protein

SR

HSP90

S 1

2

3

7

4

5

mRNA

Nucleus

6

SBP S

RationaleRationalePrevious ObservationsPrevious Observations

In vitroIn vitro studies studies High affinity Estradiol (EHigh affinity Estradiol (E22)) binding sites in plasma binding sites in plasma

membrane of Chinese Hamster Ovary cells membrane of Chinese Hamster Ovary cells (Razandi (Razandi et alet al.).)

Rapid activation of MAPK pathway by ERapid activation of MAPK pathway by E2 2 (Song (Song et et alal.).)

In vivoIn vivo studies studies Winter 2004-2005Winter 2004-2005 High affinity EHigh affinity E22 binding sites in ovine binding sites in ovine

endometriumendometrium KKDD = 2.13 x 10 = 2.13 x 10-13 -13 MM

Objectives of ResearchObjectives of Research

EE22 effect on the MAPK Pathway has effect on the MAPK Pathway has been studied in been studied in in vitroin vitro studies studies

Is there physiological significance in Is there physiological significance in the animal model?the animal model?

Examine the effect of EExamine the effect of E22 on the on the MAPK pathway MAPK pathway in vivoin vivo

MethodsMethods Ovariectomized Ovariectomized

subjectssubjects Tissue samples from Tissue samples from

ovine endometriumovine endometrium Endometrium is highly Endometrium is highly

responsive to Eresponsive to E22

Hormone treated to Hormone treated to mimic estrous cyclemimic estrous cycle

General anesthetic General anesthetic Uterus exposed via Uterus exposed via

midventral midventral laparotomylaparotomy

MethodsMethods Uterus opened to Uterus opened to

reveal endometriumreveal endometrium First tissue sample First tissue sample

takentaken 5 micrograms E5 micrograms E2 2

injected via uterine injected via uterine arteryartery

Tissue collected at 5, Tissue collected at 5, 10, 15 and 30 minutes 10, 15 and 30 minutes post injection of Epost injection of E22

Tissue frozen at -80 CTissue frozen at -80 C Returned to laboratoryReturned 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

MethodsMethodsTime SchemeTime Scheme

P4 Given

Days 3-7 Days 8-9

E2 Given

Days 1-2 Day 11Day 10

E2 Given Rest Surgery

Hormone Schedule

Samples Taken

10min 30 min

First Sample Taken, E2 Injected

5 min0 min 15 min

Experiment Timeline

Final Sample Taken

MethodsMethodsOur HypothesisOur Hypothesis

picture courtesy of picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asphttp://www.biocarta.com/pathfiles/h_erkPathway.asp

MethodsMethods Testing the HypothesisTesting the Hypothesis

Tissue homogenized and separated Tissue homogenized and separated into nuclear and cytosolic portionsinto nuclear and cytosolic portions

Proteins separated via western blotProteins separated via western blot Probed for presence of Probed for presence of

phosphorylated ERK1/ERK2phosphorylated ERK1/ERK2

MethodsMethodsTesting the HypothesisTesting the Hypothesis

picture courtesy of picture courtesy of http://www.biocarta.com/pathfiles/h_erkPathway.asphttp://www.biocarta.com/pathfiles/h_erkPathway.asp

ResultsResultsPresence of Phosphorylated Presence of Phosphorylated

ERK1/2ERK1/2

Minutes: 0 5 10 Minutes: 0 5 10 15 3015 30Relative Intensity:

5 7.36 45.5 26.67 8.75

3 9.45 39.67 27.33 10

ConclusionConclusion

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

AcknowledgementsAcknowledgements

Howard Hughes Medical IntershipHoward Hughes Medical Intership Undergraduate Research, Undergraduate Research,

Innovation, Scholarship, and Innovation, Scholarship, and Creativity (URISC) Creativity (URISC)

Department of Animal Sciences, Department of Animal Sciences, College of AgricultureCollege of Agriculture

Stormshak LabStormshak Lab

Thank You!Thank You!