Post on 13-Jan-2016
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
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
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
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
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
**
**
**
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
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**
*
Glutathione SynthesisGlutathione Synthesis
glutamate
COOH|(CH2)2
|CHNH2
|COOH
CH2SH |CONHCHCOOH|(CH2)2
|CHNH2
|COOH
CH2SH |CONHCHCO-NHCH2COOH|(CH2)2
|CHNH2
|COOH
CH2SH |H2NCHCOOH
1 2
H2NCH2COOH
1: glutamate cysteine ligase (glutamyl-cysteine synthetase)2: glutathione synthetase
cysteine
glycine
glutamyl-cysteinyl glycine(GSH)
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
Treatment Time (Hours)
Fold Control Absorbance (A.U.)
Gclc Overexpression (C6a)
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.)
Gclm Overexpression (M5a)
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.)
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