Gene pathways targeted during Gene pathways targeted during phthalatephthalate--induced testicular induced testicular

dysgenesisdysgenesis

Kevin W. Kevin W. GaidoGaido, Ph.D., Ph.D.

HamnerHamner Institutes for Health SciencesInstitutes for Health Sciences

Research Triangle Park, NC USAResearch Triangle Park, NC USA

Presentation OutlinePresentation Outline

•• Phthalate profile studyPhthalate profile study

•• Time courseTime course

•• Promoter analysis Promoter analysis

•• Species comparisonSpecies comparison

Public Health IssuePublic Health Issue

•• Reported increases in the incidence of Reported increases in the incidence of cryptorchidism, cryptorchidism, hypospadiashypospadias, prostate and , prostate and testicular cancer, and reduced fertility. testicular cancer, and reduced fertility.

•• Testicular Testicular dysgenesisdysgenesis syndromesyndrome (TDS)(TDS)

–– Disruption of male reproductive tract development during Disruption of male reproductive tract development during fetal life by genetic or environmental factors.fetal life by genetic or environmental factors.

Laboratory ObservationsLaboratory Observations

•• Exposure of fetal male ratsExposure of fetal male rats toto antiandrogensantiandrogens leads to reproductive disorders that are similar to those to reproductive disorders that are similar to those of concern in the human male population.of concern in the human male population.

–– Cryptorchidism, reproductive tract defects, Cryptorchidism, reproductive tract defects, reduced fertility, cancerreduced fertility, cancer

Environmental Environmental AntiandrogensAntiandrogens

•• Insecticide metabolites (Insecticide metabolites (p,pp,p’’--DDE, HPTE)DDE, HPTE)

•• Fungicides (Fungicides (VinclozolinVinclozolin, , ProcymidoneProcymidone))

•• Herbicide (Herbicide (LinuronLinuron))

•• Some Some ImidazoleImidazole and and TriazoleTriazole antifungalsantifungals

•• Phthalate esters (DBP; DEHP)Phthalate esters (DBP; DEHP)

•• Organophosphate pesticides (Organophosphate pesticides (FenitrothionFenitrothion))

TestisTestis

SRYSRYSertoli Sertoli CellCell

BipotentialBipotentialGonadGonad

Supporting cell Supporting cell precursorprecursor

Germ cell Germ cell PrecursorPrecursor

SteroidogenicSteroidogenic cell cell precursorprecursor

MISMIS

Male duct Male duct and organsand organs

MullerianMullerian Duct Duct RegressionRegression

TT

DHTDHT

WolffianWolffian ductsductsepididymisepididymisvas deferensvas deferensseminal vesiclesseminal vesicles

testicular descenttesticular descent

External genitaliaExternal genitaliapenispenisscrotumscrotumprostateprostate

Development of the Male Development of the Male Reproductive TractReproductive Tract

gonocytegonocyte

LeydigLeydig CellCell

Development of the Male Reproductive TractDevelopment of the Male Reproductive Tract

44

Gestation (weeks)Gestation (weeks)

55 66 77 88 99 1010 1111 1212 1313 1414 1515 4040

TestosteroneTestosterone

MMüüllerianllerian ductductregressionregression

WolffianWolffian duct duct differentiationdifferentiation

Male external Male external genital genital

differentiationdifferentiation

External External genital growthgenital growth

Testis descentTestis descent

00 2020 40401212 1414 1616 1818 2020

TestosteroneTestosterone

Gestation dayGestation day Postnatal Postnatal dayday

Growth of Growth of external external genitaliagenitalia

Testicular Testicular descentdescent

MMüüllerianllerian ductductregressionregression

Differentiation of Differentiation of external genitalia external genitalia

and prostateand prostate

WolffianWolffian duct duct differentiationdifferentiation

RatRat HumanHuman

Testicular Testicular descentdescent

Fetal TestisFetal Testis

• Widely used plasticizers that impart flexibility and strength to a variety of consumer goods and plastics

• Found in food packaging, clothing, paints, toys and personal care products

• Anti-androgenic

• Does not bind to androgen receptor

PhthalatesPhthalates

•• CryptorchidismCryptorchidism

•• Male reproductive tract malformationsMale reproductive tract malformations–– Epididymal malformationsEpididymal malformations

–– hypospadiashypospadias

•• Testicular Testicular dysgenesisdysgenesis–– Regions of Regions of LeydigLeydig cell hyperplasiacell hyperplasia

–– Multinucleated Multinucleated gonocytesgonocytes

–– Abnormal seminiferous cord formationAbnormal seminiferous cord formation

Phthalates Disrupt Male Rat Phthalates Disrupt Male Rat Reproductive DevelopmentReproductive Development

Decrease in Anogenital Distance Decrease in Anogenital Distance Among Male Infants with Prenatal Among Male Infants with Prenatal

Phthalate ExposurePhthalate Exposure

Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, CalafatCalafatAM, Mao CS, AM, Mao CS, RedmonRedmon JB, JB, TernandTernand CL, Sullivan S, CL, Sullivan S,

Teague JL, and the Study for Future Families Research Teague JL, and the Study for Future Families Research Team. Team.

Environ Health Environ Health PerspectPerspect

Phthalate Ester Phthalate Ester Reproductive Reproductive effectseffects

DimethylDimethyl --DiethylDiethyl --DioctylDioctyl--teretere --DiDi--nn-- butylbutyl ++benzobutylbenzobutyl ++DiDi--nn-- pentylpentyl ++DiDi--(2(2--ethylhexyl)ethylhexyl) ++

Phthalate Profile StudyPhthalate Profile Study

Phthalate estersPhthalate estersDEHPDEHP DBPDBP

DMPDMP DEPDEP

Study DesignStudy Design

Gestation day: 16 19 2112

Isolate and snap freeze testis for RNA isolationIsolate and snap freeze testis for RNA isolation

AffymetrixAffymetrix microarraymicroarrayresultsresults

•• Investigated approximately 30,000 genes Investigated approximately 30,000 genes

•• 391 genes significantly altered (1.3%)391 genes significantly altered (1.3%)

–– 225 unknown transcribed sequences225 unknown transcribed sequences

•• DMP, DEP, DOTPDMP, DEP, DOTP

–– Not significantly different from control or each other.Not significantly different from control or each other.

•• DBP, BBP, DPP, DEHPDBP, BBP, DPP, DEHP

–– All significantly different from control and DMP, DEP, All significantly different from control and DMP, DEP, DOTPDOTP

–– But not each otherBut not each other

Con

trol

Con

trol

DE

PD

EP

DM

PD

MP

DO

TPD

OTP

DB

PD

BP

DE

HP

DE

HP

DP

PD

PP

BB

PB

BP

391 unique genes391 unique genes

-- Universal mean determined for Universal mean determined for each geneeach gene

--Red = increased relative to meanRed = increased relative to mean--Green = reducedGreen = reduced

Gene OntologyGene Ontology167 genes 167 genes

•• Lipid, cholesterol, sterol homeostasis Lipid, cholesterol, sterol homeostasis -- -- -- --

•• SteroidogenesisSteroidogenesis -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

•• Lipid, cholesterol, sterol transport Lipid, cholesterol, sterol transport -- -- -- -- -- -- --

•• Transcription Factors Transcription Factors -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

•• Signal Transduction Signal Transduction -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

•• Oxidative Stress Oxidative Stress -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

•• Cytoskeleton Cytoskeleton -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

•• UnclassifiedUnclassified -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

31 (19 %) 31 (19 %) ↓↓

12 (7 %) 12 (7 %) ↓↓

10 (6 %) 10 (6 %) ↓↓

9 (5 %)9 (5 %)

22 (13 %)22 (13 %)

11 (7 %) 11 (7 %) ↓↓

13 (8 %)13 (8 %)

59 (35 %)59 (35 %)

SteroidogenesisSteroidogenesis

HDLHDL

SRSR--B1B1

CellCellMembraneMembrane

ARAR

CholesterolCholesterolSynthesisSynthesis

CholesterolCholesterol P450sccP450scc

MitochondrialMitochondrialMembraneMembraneStARStAR

DBPDBP

DBPDBP

pregnenolonepregnenolone

Testosterone Testosterone 1717ßß--HSDHSD

androstenedioneandrostenedione

1717--OH progesteroneOH progesterone1717αα--hydroxylasehydroxylase

33ßß--HSDHSDprogesteroneprogesterone

17, 20 17, 20 -- lyaselyaseCYP17CYP17

LdlrLdlrVldlrVldlrcd36cd36

PBRPBRDBIDBIP450 ORP450 ORferredoxinferredoxin

Insulin signalingInsulin signalingLipogenesisLipogenesisSterol transportSterol transportTranscription FactorsTranscription Factors

Tes

tost

ero

ne

Tes

tost

ero

ne

(( ngng /

test

is)

/tes

tis)

NOAELNOAEL

LOAELLOAEL

Substantial Reductions in Testosterone Substantial Reductions in Testosterone Occur Before Developmental EndpointsOccur Before Developmental Endpoints

0.1 1 10 100 1000

0.0

0.5

1.0

1.5

*

**

Dose DBP (mg/kg/day)

Cholesterol transportCholesterol transport

Relative Relative ExpressionExpression

Relative Relative ExpressionExpression

mRNAmRNA

StARStAR

Scavenger ReceptorScavenger ReceptorProteinProtein

00..0011 00..11 11 1100 110000 1100000000..00

00..55

11..00

11..55

****

**

DDoossee DDBBPP ((mmgg//kkgg//ddaayy))

00..0011 00..11 11 1100 110000 1100000000..00

00..55

11..00

11..55

******

**

DDoossee DDBBPP ((mmgg//kkgg))0.10.1 11 1010 100100 10001000

0.00.0

0.50.5

1.01.0

1.51.5

****

**

Dose DBP (mg/kg/day)Dose DBP (mg/kg/day)

0.10.1 11 1010 100100 100010000.00.0

0.50.5

1.01.0

1.51.5

****

**

Dose DBP (mg/kg/day)Dose DBP (mg/kg/day)

Multinucleated Multinucleated GonocytesGonocytes

DBP-induced cryptorchidism

Role of insl3 in Testicular Descent

scrotumscrotum

normalnormalpositionposition

IntraIntra--abdominalabdominalpositionposition

InguinalInguinalcanalcanal

TransTrans--abdominal abdominal descent (Insl3descent (Insl3--mediated)mediated)

Scrotal descentScrotal descent(androgen(androgen--mediated)mediated)

Location of androgen Location of androgen receptor antagonistreceptor antagonistexposed testesexposed testes

--

Role of insl3 in Testicular DescentRole of insl3 in Testicular Descent

scrotumscrotum

normalnormalpositionposition

IntraIntra--abdominalabdominalpositionposition

InguinalInguinalcanalcanal

TransTrans--abdominal abdominal descent (Insl3descent (Insl3--mediated)mediated)

Scrotal descentScrotal descent(androgen(androgen--mediated)mediated)

Location of DBPLocation of DBP--exposed testesexposed testes

PhthalatePhthalate--Induced Testicular Induced Testicular DysgenesisDysgenesis

Fetal Fetal LeydigLeydig CellCell

GonocyteGonocyte

Sertoli CellSertoli Cell

SRB1SRB1LDLRLDLRVLDLRVLDLRCD36CD36

CholesterolCholesterol

CholesterogenesisCholesterogenesis

SteroidogenesisSteroidogenesis

Oxidative Oxidative StressStress

Insl3Insl3

αα inhibininhibin

Reproductive tract developmentReproductive tract development

Testicular DescentTesticular Descent

TestosteroneTestosterone

Sertoli Cell Sertoli Cell developmentdevelopment

LHRLHRCNPCNP

cc--KITKIT

FGF signalingFGF signaling

PhthalatePhthalate--Induced Testicular Induced Testicular DysgenesisDysgenesis

Fetal Fetal LeydigLeydig CellCell

GonocyteGonocyte

Sertoli CellSertoli Cell

↓↓ SRB1SRB1↓↓ LDLRLDLR↓↓ VLDLRVLDLR↓↓ CD36CD36

CholesterolCholesterol

↓↓ CholesterogenesisCholesterogenesis↓↓ LipogenesisLipogenesis↓↓ Insulin SignalingInsulin Signaling

SteroidogenesisSteroidogenesis

↓↓Oxidative Oxidative StressStress

↓↓ Insl3Insl3

↓↓ αα inhibininhibin

Malformed Reproductive tractMalformed Reproductive tract

CryptorchidismCryptorchidism

↓↓ TestosteroneTestosterone

Altered Sertoli Cell Altered Sertoli Cell developmentdevelopment

↓↓ LHRLHR↓↓ CNPCNP↑↑ AVR (AVR (--))

XXX

X X

X XX

X

Disrupted Cell JunctionsDisrupted Cell Junctions↓↓ GJA1GJA1↑↑ TestinTestin

↓↓ cc--KITKIT↓↓Dax1Dax1

Multinucleated Multinucleated GonocyteGonocyte

↓↓FGF signalingFGF signaling

Early gene changes in the fetal Early gene changes in the fetal testis after DBP exposuretestis after DBP exposure

Time CourseTime Course

•• 500 mg/kg/day500 mg/kg/day

•• 0, 0.5, 1, 3, 6, 12, 18, & 24 h0, 0.5, 1, 3, 6, 12, 18, & 24 h

•• Between Between gdgd 18 and 1918 and 19

•• Testosterone RIATestosterone RIA

•• RTRT--PCRPCR

•• MicroarraysMicroarrays at 1, 3, 6, 18 hat 1, 3, 6, 18 h

DBP causes a biphasic reduction in DBP causes a biphasic reduction in fetal testicular testosterone and fetal testicular testosterone and

gene expressiongene expression

0.50.500 55 1010 1515 2020 252500

100100

200200

300300

400400

Time (h)Time (h)

Tes

tost

ero

ne

Co

nce

ntr

atio

n

Tes

tost

ero

ne

Co

nce

ntr

atio

n

pg

/ T

esti

sp

g /

Tes

tis

Rel

ativ

e E

xpre

ssio

nR

elat

ive

Exp

ress

ion

00 11 22 33 66 1212 1818 24240.00.0

0.50.5

1.01.0

1.51.5StARStARSRSR--B1B1SCCSCCCYP17CYP17

TimeTime

Time course heat mapTime course heat map

11 33 66 1818 11 33 66 1818ControlControl DBPDBP

Time (h):Time (h):

DBP induces immediate early DBP induces immediate early gene expressiongene expression

00 55 1010 1515 2020 252500

55

1010

1515

btg2btg2

cc--fosfos

EGREGR--11

EGREGR--22Gro1Gro1JunDJunD

Time (h)Time (h)

Res

po

nse

Res

po

nse

Promoter AnalysisPromoter Analysis

DnaseDnase footprintingfootprintingChromatin Chromatin ImmunoprecipitationImmunoprecipitationEMSAEMSAWestern analysisWestern analysis

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

Control DEP 100 DBP 500 DBPTreatment (mg/kg)

Test

oste

rone

Con

cent

ratio

n (n

g/m

g to

tal p

rote

in)

*

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Control DEP 100 DBP 500 DBP

Tretment (mg/kg)

Rela

tive

Gen

e E

xpre

ssio

n

StARSR-B1Cyp11a1cyp17

* **

*

*

*

DBP inhibition of Testosterone DBP inhibition of Testosterone biosynthesis pathwaybiosynthesis pathway

Automated Automated DNaseDNase I I FootprintingFootprinting

DNA subjected to DNasedigestion

DNA regions bound by protein are protected from digestion

DNA fragments are amplified with LigationMediated-PCR using promoter specific primers

Changes in Changes in StARStAR promoter architecture promoter architecture using using in vivoin vivo DNaseDNase footprintingfootprinting

Promoter Location and Sequence (bp upstream)

10 20 30 40 50 60 70 80 90 100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300R

elat

ive

Flou

rese

nce

0

10000

20000

30000

40000

TTCGTTCACGAGACGGAATTTACGAAGGTCGCCTTCCGACACGTAGTAGTGAACTCTATTTTTTCGGTTGTCAGTGACACGTCTCCCAGTTCCTACCTTACTAACGGAGTATGACGTCACGACTGGTTCCACCCTCCCCCGTCTGGTCTGAGCCGTCCGTCCCCTTTGGTCACCAGTGGCTCTACCCCGTTTCCCGTATTGAAGAGGTTTCAAAATTTACCTCTGAATTTCGGGACAGGAGTTCGAGGTCCAAGTCGTAAGTGAATTTACGGGTCATTGACCCCACT

500 DBP100 DBP500 DEPCorn Oil

Changes in Changes in StARStAR promoter architecture promoter architecture using using in vivoin vivo DNaseDNase footprintingfootprinting

Promoter Location and Sequence (bp upstream)

10 20 30 40 50 60 70 80 90 100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300R

elat

ive

Flou

rese

nce

0

10000

20000

30000

40000

TTCGTTCACGAGACGGAATTTACGAAGGTCGCCTTCCGACACGTAGTAGTGAACTCTATTTTTTCGGTTGTCAGTGACACGTCTCCCAGTTCCTACCTTACTAACGGAGTATGACGTCACGACTGGTTCCACCCTCCCCCGTCTGGTCTGAGCCGTCCGTCCCCTTTGGTCACCAGTGGCTCTACCCCGTTTCCCGTATTGAAGAGGTTTCAAAATTTACCTCTGAATTTCGGGACAGGAGTTCGAGGTCCAAGTCGTAAGTGAATTTACGGGTCATTGACCCCACT

500 DBP100 DBP500 DEPCorn Oil

SF-1 GATA4 c/ebpβ c/ebpβ SF-1 sp1 AP-1 c/ebp GATA / PR

EMSAEMSA

Promoter Location and Sequence (bp upstream)

10 20 30 40 50 60 70 80 90 100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300R

elat

ive

Flou

rese

nce

0

10000

20000

30000

40000

TTCGTTCACGAGACGGAATTTACGAAGGTCGCCTTCCGACACGTAGTAGTGAACTCTATTTTTTCGGTTGTCAGTGACACGTCTCCCAGTTCCTACCTTACTAACGGAGTATGACGTCACGACTGGTTCCACCCTCCCCCGTCTGGTCTGAGCCGTCCGTCCCCTTTGGTCACCAGTGGCTCTACCCCGTTTCCCGTATTGAAGAGGTTTCAAAATTTACCTCTGAATTTCGGGACAGGAGTTCGAGGTCCAAGTCGTAAGTGAATTTACGGGTCATTGACCCCACT

500 DBP100 DBP500 DEPCorn Oil

SF-1 GATA4 c/ebpβ c/ebpβ SF-1 sp1 AP-1 c/ebp GATA / PR

Olig

oon

lyα−

c/eb

pβ

α−SF

-1

∗

α−G

ATA4

EMSAEMSA

Promoter Location and Sequence (bp upstream)

10 20 30 40 50 60 70 80 90 100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300R

elat

ive

Flou

rese

nce

0

10000

20000

30000

40000

TTCGTTCACGAGACGGAATTTACGAAGGTCGCCTTCCGACACGTAGTAGTGAACTCTATTTTTTCGGTTGTCAGTGACACGTCTCCCAGTTCCTACCTTACTAACGGAGTATGACGTCACGACTGGTTCCACCCTCCCCCGTCTGGTCTGAGCCGTCCGTCCCCTTTGGTCACCAGTGGCTCTACCCCGTTTCCCGTATTGAAGAGGTTTCAAAATTTACCTCTGAATTTCGGGACAGGAGTTCGAGGTCCAAGTCGTAAGTGAATTTACGGGTCATTGACCCCACT

500 DBP100 DBP500 DEPCorn Oil

SF-1 GATA4 c/ebpβ c/ebpβ SF-1 sp1 AP-1 c/ebp GATA / PR

Olig

oon

lyα−

c/eb

pβα−

CREB

α−CR

EM

∗

0

5

10

15

20

25

Corn Oil 500 DEP 100 DBP 500 DBP

Treatment

% In

put

StARcyp17cyp11a1SR-B1

***

*

*

*

SF1SF1

GATA4GATA4

c/ebpc/ebp ββ

0

1

2

3

4

5

6

7

8

9

10

Corn Oil 500 DEP 100 DBP 500 DBP

Treatment

% In

put

StAR cyp17

cyp11a1 SR-B1

0

2

4

6

8

10

12

14

Corn Oil 500 DEP 100 DBP 500 DBP

Treatment

% In

put

StARcyp17cyp11a1SR-B1

*

*

p=0.

016

p=0.

043

p=0.

19

p=0.

32

*

p=0.

23

p=0.

29

p=0.

43

p=0.

006

*

0

2

4

6

8

10

12

Corn Oil 500 DEP 100 DBP 500 DBP

Treatment

% In

put

StARcyp17cyp11a1SR-B1

p=0.

12

p=0.

13

p=0.

19

p=0.

37

p=0.

083

sp1sp1

Chromatin Chromatin ImmunoprecipitationImmunoprecipitationquantifies changes in specific TF bindingquantifies changes in specific TF binding

TATADEP

Proposed MechanismProposed Mechanism

TATADEP

Proposed MechanismProposed Mechanism

Proposed MechanismProposed Mechanism

TATADBPDBP

Effect of DBP on Mouse Fetal Effect of DBP on Mouse Fetal

TestisTestis

MBP PharmacokineticsMBP Pharmacokinetics Maternal Plasma- Single 500 mg/kg

0.1 1 10 1000

100000

200000

300000

400000

500000

600000MBP-RAT-GD19MBP-MOUSE- GD 18

Time (hr)

Fetal Plasma- Single 500 mg/kg

0.1 1 10 1000

100000

200000

300000

400000

500000 MBP- Rat- GD 19MBP- Mouse GD 18

Time (hr)

Gene Expression Following Gene Expression Following DBP ExposureDBP Exposure

Lipid, Sterol, and Cholesterol homeostasisLipid, Sterol, and Cholesterol homeostasis

GeneGene MouseMouse RatRat Gene TitleGene TitleDhcr7Dhcr7 77--dehydrocholesterol dehydrocholesterol reductasereductaseAacsAacs acetoacetylacetoacetyl--CoACoA synthetasesynthetaseClpsClps NSNS colipasecolipase, pancreatic , pancreatic -- lipid catabolismlipid catabolismCyp51Cyp51 cytochromecytochrome P450, family 51P450, family 51Hsd17b7Hsd17b7 hydroxysteroidhydroxysteroid (17(17--beta) beta) dehydrogenasedehydrogenase 77Idi1Idi1 isopentenylisopentenyl--diphosphatediphosphate delta delta isomeraseisomeraseLssLss lanosterollanosterol synthasesynthaseStard4Stard4 StARStAR--related lipid transfer domain containing 4related lipid transfer domain containing 4Scd1Scd1 stearoylstearoyl--Coenzyme A Coenzyme A desaturasedesaturase 11Scd3Scd3 ------ stearoylstearoyl--coenzyme A coenzyme A desaturasedesaturase 33Sc5dSc5d sterolsterol--C5C5--desaturasedesaturase

Multinucleated Multinucleated gonocytesgonocytes in mouse in mouse fetal testis following DBP exposurefetal testis following DBP exposure

Seminiferous Seminiferous cord diametercord diameter

MNG / CordMNG / Cord Nuclei / MNGNuclei / MNG

DBP targets common and divergent DBP targets common and divergent pathways in the Mouse and Ratpathways in the Mouse and Rat

DBPDBP

RatRat

MouseMouse

IEGIEG

SteroidogenesisSteroidogenesis

Sertoli / germ cellTestinTestinFGFFGFTSP1TSP1CTGFCTGFIntegrinIntegrin signallingsignalling

Reproductive tractReproductive tractDevelopmentDevelopment

Cord FormationCord Formation&&

MNGMNGIEGIEG

Insl3Insl3 Testicular DescentTesticular Descent

Leydig Cell

??

Testis transplant modelTestis transplant model

Testosterone

0.00

0.50

1.00

1.50

2.00

2.50

Corn Oil DBP

ng T

esto

ster

one/

mg

Prot

ein

Application of Genomics to Application of Genomics to Reproductive Health RisksReproductive Health Risks

HighHigh--throughput,throughput,MicroarrayMicroarray analysisanalysis

Animal StudiesAnimal StudiesReproductive TractReproductive Tract

Additional StudiesAdditional Studies••DoseDose--ResponseResponse••Time CourseTime Course••ProfileProfile••PharmacokineticsPharmacokinetics••Species comparisonsSpecies comparisons

Testicular Testicular DysgenesisDysgenesisSyndromeSyndrome

Systems Systems Biology & Biology &

Computational Computational BBBB--DR ModelsDR Models

Decreased AR activity at target tissue

AR mediated reproductive tract malformationsAR mediated reproductive tract malformations

Fetal Androgen InsufficiencyFetal Androgen InsufficiencyDecreased Decreased

TestosteroneTestosteroneDecreased Decreased

DHTDHTBlock Androgen Block Androgen

ReceptorReceptorMutated Mutated ReceptorReceptor

AcknowledgmentsAcknowledgments

•• KaminKamin JohnsonJohnson

•• Adam Adam KuhlKuhl

•• Elena Elena KleymenovaKleymenova -- GSKGSK

•• Jack Liu Jack Liu -- GSKGSK

•• Susan RossSusan Ross

•• Kim Kim LehmannLehmann

•• JananJanan HensleyHensley

•• Delong LiuDelong Liu

•• Kim Kim BoekelheideBoekelheide -- BrownBrown

•• Susan Susan BorghoffBorghoff -- ILSILS

•• Paul Foster Paul Foster -- NIEHSNIEHS

•• Chris Thompson Chris Thompson -- SanofiSanofi--AventisAventis

•• Valerie Shultz Valerie Shultz -- SanofiSanofi--AventisAventis

•• Stan Young Stan Young -- NISSNISS

•• Russ Russ WolfingerWolfinger -- SASSAS

•• ACCACC--LRILRI

•• NIH R01 ES011803NIH R01 ES011803--01A101A1

•• NIH R21 ES011754NIH R21 ES011754--0101

•• EPA STAR R830766EPA STAR R830766