Environmental Contaminants and Infertility in Women Ulrike Luderer, M.D., Ph.D. Division of...

Environmental Contaminants and Environmental Contaminants and Infertility in Women Infertility in Women

Ulrike Luderer, M.D., Ph.D.Ulrike Luderer, M.D., Ph.D.

Division of Occupational and Environmental Division of Occupational and Environmental Medicine, Department of MedicineMedicine, Department of Medicine

Department of Developmental and Cell BiologyDepartment of Developmental and Cell Biology

Infertility and premature ovarian Infertility and premature ovarian failurefailure

12-13% of all couples are infertile12-13% of all couples are infertile Premature ovarian failure is responsible for 20-Premature ovarian failure is responsible for 20-

30% of infertility in women30% of infertility in women Defined as menopause before age 40Defined as menopause before age 40 Affects 1-2% of women Affects 1-2% of women

Ovarian follicular developmentOvarian follicular development

primaryPrimordial

Secondary AntralMature/Preovulatory

Ovulation Ovulated OocyteCorpora Lutea

Hypothalamus

Anterior Pituitary

GnRH

LH, FSH

Ovary

Inhibin

E, P

E

Hormonal regulation of ovarian Hormonal regulation of ovarian functionfunction

The human The human menstrual cyclemenstrual cycle

FSHmIU/ml

Primordial

Primary

Secondary/Preantral

Antral

Large antral/Preovulatory

Not responsive togonadotropins

Responsive togonadotropins Gonadotropin-dependent

Photos of sheep follicles(Juengel and McNatty, 2005Human Reprod Update11:144-61)

Stages of Follicular DevelopmentStages of Follicular Development

Infertility and AgeInfertility and Age

Age of Wife 1965 1982 1995 2002

15 to 19 0.6 2.1

20 to 24 3.6 10.6 4.7

25 to 29 7.2 8.7 7.1

30 to 34 14.0 13.6 9.1 10.9

35 to 39 18.4 24.6 10.5

40 to 44 27.7 27.2 19.6 20.2

Total, 15 to44

13.3 13.9 12.0 11.3

a Percent of married couples excluding those surgically sterilized.

Age: 5 mos gest birth puberty menopause# oocytes: 7 million 2 million 400,000 0

Age-Related Decline in Human Oocytes

- Only 350 oocytes progress to ovulation between puberty and menopause

Causes of premature ovarian failureCauses of premature ovarian failure

Cytogenetic abnormalities of the X chromosomeCytogenetic abnormalities of the X chromosome Fragile X syndromeFragile X syndrome

Other genetic mutationsOther genetic mutations 1717hydroxylase, aromatase, FOXL2, BMP15, LH-R, FSH-Rhydroxylase, aromatase, FOXL2, BMP15, LH-R, FSH-R

Immune disturbancesImmune disturbances Viral infectionViral infection Chemical or physical agentsChemical or physical agents Cause is unknown in many casesCause is unknown in many cases

Environmental exposures that cause Environmental exposures that cause ovarian failureovarian failure

SmokingSmoking decreased fecundity, earlier menopausedecreased fecundity, earlier menopause

Polycyclic aromatic hydrocarbonsPolycyclic aromatic hydrocarbons Dimethylbenz(a)anthracene, benzo(a)pyrene, 3-Dimethylbenz(a)anthracene, benzo(a)pyrene, 3-

methylcholanthrenemethylcholanthrene Anticancer drugsAnticancer drugs

Alkylating agents (e.g. cyclophosphamide)Alkylating agents (e.g. cyclophosphamide) DoxorubicinDoxorubicin ProcarbazineProcarbazine

Ionizing radiationIonizing radiation 2-bromopropane2-bromopropane VinylcyclohexeneVinylcyclohexene

Types of cell deathTypes of cell death

NecrosisNecrosis Passive, catastrophicPassive, catastrophic Morphological features:Morphological features:

SwellingSwelling No chromatin remodelingNo chromatin remodeling Plasma membrane Plasma membrane

blebbingblebbing

ApoptosisApoptosis Active, regulatedActive, regulated Morphological features:Morphological features:

ShrinkageShrinkage Chromatin condensationChromatin condensation Plasma membrane Plasma membrane

buddingbudding ProteolysisProteolysis

Inhibitory regulators, Bcl-2Inhibitory regulators, Bcl-2 Nuclear lamins, Nuclear lamins,

cytoskeletal proteinscytoskeletal proteins Inhibitor of caspase Inhibitor of caspase

activated DNAseactivated DNAse DNA fragmentationDNA fragmentation

Apoptosis in Ovarian FolliclesApoptosis in Ovarian Follicles

damage-inducedpathway

Apaf-1activation

Apoptosomecaspase 9

Anti-apoptotic Bcl-2 proteins

-

caspase 3

Pro-apoptotic Bcl-2 proteins

deathreceptorpathway Death

InducingSignalingComplex

caspase 8

-

+

cytochrome c

bid

+

Death domainadaptor proteins

Reactive Oxygen Species Reactive Oxygen Species Generation and DetoxificationGeneration and Detoxification

O2 + e- O2-•

SOD2H+

O2-•

H2O2 + O2

H2O2CAT

2H2O + O2O2

-•

Fe++

O2 + OH- + OH•

GSHPx

2GSHGSSG + 2H2O

Oxidative damage to DNA,protein, lipids

ONOO-NO•

Oxidation and Reduction of Oxidation and Reduction of GlutathioneGlutathione

Lipid-OOHor H2O2

Lipid-OHor H2O

GSH Peroxidase GSH Reductase G-6-P Dehydrogenase

GSH

GSSG

NADP

NADPH

Glucose-6-Phosphate

6-Phospho-gluconate

Preovulatory follicle culturePreovulatory follicle culture

25-26 day old pre-pubertal rats were injected with 10 I.U. 25-26 day old pre-pubertal rats were injected with 10 I.U. eCGeCG

48 h later preovulatory follicles were dissected by hand48 h later preovulatory follicles were dissected by hand Immediately processed for assays (0h, negative control) Immediately processed for assays (0h, negative control)

OROR cultured for 2 to 48h with :cultured for 2 to 48h with :

MEM medium alone (positive control)MEM medium alone (positive control)

OROR Ovine FSH, 75 ng/mL (negative control)Ovine FSH, 75 ng/mL (negative control)

OROR Various treatmentsVarious treatments

FSH stimulates follicular GSH synthesisFSH stimulates follicular GSH synthesis

0.0

1.0

2.0

3.0

0h MEM FSH FSH+BSO

Treatment

GSH (nmol/follicle)

4 h 12 h

24 h 48 h*

* *

** * *

‡‡‡

†

† †

† †

†

FSH suppresses ROS formation: DHRFSH suppresses ROS formation: DHR

0

1

2

3

0 2 4 6 8 10 12 14

Duration of incubation (hours)

DHR fluorescence (fold 0h) MEM FSH FSH+BSO

†

††

‡*

*

*

0h H2O2 12h, MEM 12h, FSH 12h, FSH+BSO

FSH suppresses ROS formation: DCFFSH suppresses ROS formation: DCF

0

2

4

6

8

10

12

14

16

18

20

0 4 8 12 16 20 24 28 32 36 40 44 48 52

Duration of incubation (hours)

DCF fluorescence (fold 0h)

MEM

FSH

FSH+BSO

†

*

*

*

*

*

*

†

** *

* * *

*

*

†

0h

MEM, 24h

FSH,48h

FSH+BSO, 48h

0

100

200

300

400

500

0h MEM FSH FSH+BSO

Treatment

TUNEL positive GC per field

4 h experiments12 h experiments24 h experiments48 h experiments

***

† † *

*

*

*

*

†† †

*

†‡

‡

0

20

40

60

80

100

0h MEM FSH FSH+BSO

Treatment

TUNEL positive TC per field

*

*

*

*

*†

*

*

*

*

†

†‡

GSH depletion induces apoptosis



Apaf-1activation

Apoptosomecaspase 9


-

caspase 3




caspase 8

-

+

cytochrome c

bid

+


0

20

40

60

80

100

120

0h MEM FSH FSH+BSO

Treatment

Positive GC per 400x field

4h

12h

24h

48h

0

5

10

15

20

25

30

0h MEM FSH FSH+BSO

Treatment

Positive TC per 400x field

GSH depletion and apoptosis: Caspase 3

*

*

*

*

*

*

‡

‡

†

†

† †

†

††

††

* *

*

*

* ** *

FSH+BSO48h

FSH, 48h

MEM48h

†**

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 0 1 5 10

GSH (nmol/follicle)

0 100 100 100 100

GEE

BSO

* *

††

0

50

100

150

200

0 0 1 5TUNEL positive cells per 400x field

Granulosa cells

Theca cells

*

*

†

†

0 100 100 100 BSO

GEE

†

GSH replacement reverses effect of BSO GSH replacement reverses effect of BSO on GC apoptosison GC apoptosis

Intraovarian benz(a)pyrene destroys Intraovarian benz(a)pyrene destroys primordial follicles in miceprimordial follicles in mice

Adapted from Takizawa et al, 1984, Cancer Research 44:2571

0

20

40

60

80

100

120

0.01 0.1 1 10 100

Dose (microgram per ovary)

Primordial follicles

(%treated versus untreated)

0

100

200

300

400

0 0.1 1 10 100

DMBA dose (μ )M

400 TUNEL positive GC per x field

12h 24h 48h

DMBA induces granulosa cell DMBA induces granulosa cell apoptosisapoptosis

*

*

*

0 μM

0.1 μM 10 μM

100 μM



Apaf-1activation

Apoptosomecaspase 9


-

caspase 3




caspase 8

-

+

cytochrome c

bid

+


DMBA induces granulosa and theca DMBA induces granulosa and theca cell apoptosiscell apoptosis

0

5

10

15

20

25

30

35

0 0.1 1 10 100

DMBA dose (μ )M

# 3 / 400 caspase positive cells x field

GC TC

0 μM 10 μM

**

**

**



Apaf-1activation

Apoptosomecaspase 9


-

caspase 3




caspase 8

-

+

cytochrome c

bid

+


DMBA-induced apoptosis is DMBA-induced apoptosis is preceded by increased Baxpreceded by increased Bax

0

5

10

15

20

25

0 0.1 1 10 100

DMBA (μ )M

# /400 Bax positive GC x field

12h 24h 48h

0

5

10

15

20

0 0.1 1 10 100

DMBA (μ )M

# /400 Bax positive TC x field

12h 24h 48h

0 μM

10 μM0.1 μM

* *

*

*** *

* **

DMBA-induced apoptosis is DMBA-induced apoptosis is preceded by increased ROSpreceded by increased ROS

0.0

1.0

2.0

3.0

4.0

5.0

0 8 16 24 32 40 48

Hours

DCF Intensity (fold 0h)

FSH FSH+0.5 DMBA FSH+1.0 DMBA FSH+10 DMBA

*

**

* *

**

***

Effect of treatment, p=0.003, FSH differs from 1.0 and 10 μM DMBA at p<0.01Effect of time p<0.001; * significantly different from 0h

0

10

20

30

40

50

60

70

80

90

100

FSH FSH+GEE FSH+DMBA FSH+DMBA+GEE

Treatment

#TUNEL positive cells/400x field

Granulosa cells

Theca cells

GSH supplementation prevents GSH supplementation prevents DMBA-induced GC apoptosisDMBA-induced GC apoptosis

*

*

*

†

†

0

50

100

150

200

250

FSH FSH+ DMBA FSH+BSO FSH+ DMBA+ BSO

Treatment

TUNEL positive GC per 400x field

Granulosa cells Theca cells

GSH depletion enhances DMBA-GSH depletion enhances DMBA-induced GC apoptosisinduced GC apoptosis

*

*†

FSH

FSH+DMBA

FSH+BSO

FSH+DMBA+BSO

GST-Mediated Glutathione GST-Mediated Glutathione ConjugationConjugation

O || CH2CH2ClH2N-P-N | CH2CH2Cl O -

GSH

GSTA1

RX + GSH RSG + HXGST

1.

2.

Phosphoramide mustard

O || CH2CH2ClH2N-P-N | CH2CH2SG O - Monoglutathionyl-

Phosphoramide mustard

0

20

40

60

80

100

120

sal/sal sal/50 sal/300 BSO/50

Treatment

Percent TUNEL positive or atretic

TUNEL positive

Atretic * ***

* *

Sal/ 300 mg/kg Sal/ 50 mg/kg

Cyclophosphamide induces apoptosis in granulosa cells

0

20

40

60

80

100

120

sal/sal sal/50 sal/300 BSO/50

Treatment

Percent TUNEL positive or atretic

TUNEL positive

Atretic

**

* * * ** *

* *

4HC induces apoptosis in COV434 4HC induces apoptosis in COV434 cells: TUNELcells: TUNEL

Effect of 4HC dose, p<0.001Effect of time, p<0.001

0

10

20

30

40

50

60

70

80

90

100

110

0 1 10 50

4HC dose (μ )M

% TUNEL positive cells

12h 24h

**

**

*0 μM

1 μM

10 μM

50 μM

0

10

20

30

40

50

60

70

80

0 1 10 50

4HC dose (μ )M

# 8- /200 OHdG positive x field

4HC causes oxidative stress4HC causes oxidative stress

*

0 50

*0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 1 10 50 H2O2

4HC (μ )M

( 0 DCF slope fold

μ )M control *

*

0

20

40

60

80

100

120

140

160

0 1 10 0 1 10

# Hoechst positive cells/200x field

12h 24h

GSH depletion enhances 4HC-GSH depletion enhances 4HC-induced apoptosisinduced apoptosis

BSO (µM)

4HC (µM)

100100100000

p<0.001, effects of 4HC, BSO and time; p=0.012, 4HC x BSO interaction

Radiation increases ROS in COV434 Radiation increases ROS in COV434 granulosa cellsgranulosa cells

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 2 4 6 8 10

Radiation dose (Gray)

Absorbance

48h72h96h

0.0

0.5

1.0

1.5

2.0

2.5

6 24 48

Time since irradiation (h)

DCF fluorescence (fold 0 Gy)

0 Gy1 Gy5 Gy**

*

Stable transfection of Gclc and Stable transfection of Gclc and Gclm in COV434 cellsGclm in COV434 cells

MW

M

CO

V43

4

CM

V9b

CM

4b

CM

3a

CM

2a

C6a

C6b

M5a

M7a

M8c

CMV-GCLc

CMV-GCLm

Gcl subunit overexpression Gcl subunit overexpression increases GSH concentrationsincreases GSH concentrations

*

*

*

**

0

20

40

60

80

100

120

140

COV434 CMV9b CM2a CM3a CM4b C6a C6b M5a M7a M8c

Cell Line

GSH (nmol/mg protein)

Gclc overexpression protects Gclc overexpression protects against ionizing radiation against ionizing radiation

Empty Vector (CMV9b)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 72 96

Treatment Time (Hours)

Fold Control Absorbance (A.U.)

O Gy

1 Gy

5 Gy

Parental Cell Line (COV434)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 72 96



Gclc Overexpression (C6a)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 72 96



Gclm Overexpression (M5a)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 72 96



ROS and apoptosis in granulosa ROS and apoptosis in granulosa cellscellsSpontSpont DMBADMBA 4HC4HC RadRad

Early increase in ROSEarly increase in ROS YesYes YesYes YesYes YesYes

Apoptosis prevented by Apoptosis prevented by GSH supplementationGSH supplementation

YesYes YesYes YesYes YesYes

Apoptosis potentiated Apoptosis potentiated by GSH depletionby GSH depletion

YesYes YesYes YesYes --

Variations in ovarian GSH synthesizing capacity in human Variations in ovarian GSH synthesizing capacity in human populations may be responsible for interindividual differences populations may be responsible for interindividual differences in susceptibility to ovarian toxicants and to premature ovarian in susceptibility to ovarian toxicants and to premature ovarian failure.failure.

AcknowledgmentsAcknowledgments

Luderer Lab Current MembersLuderer Lab Current Members Mabel Cortés-WanstreetMabel Cortés-Wanstreet Yvonne HoangYvonne Hoang Brooke NakamuraBrooke Nakamura Laura OrtizLaura Ortiz Victoria FloresVictoria Flores

Past Lab MembersPast Lab Members Miyun Tsai-TurtonMiyun Tsai-Turton Youming TanYouming Tan Brian LuongBrian Luong Pedro MorgadoPedro Morgado Sarah LopezSarah Lopez Matilde GonzalezMatilde Gonzalez Jennifer LavorinJennifer Lavorin

UC IrvineUC Irvine Charles LimoliCharles Limoli

University of WashingtonUniversity of Washington Terry KavanaghTerry Kavanagh

University of LeidenUniversity of Leiden Peter SchrierPeter Schrier

UCI Developmental Biology UCI Developmental Biology Center and Chao Family Center and Chao Family Comprehensive Cancer CenterComprehensive Cancer Center

FundingFunding NIEHS ES10963NIEHS ES10963 Center for Occupational and Center for Occupational and

Environmental HealthEnvironmental Health

Yvonne MabelBrooke Miyun

Brian

Pedro

Azuka

Environmental Contaminants and Infertility in Women Ulrike Luderer, M.D., Ph.D. Division of...

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