Environmental Contaminants and Infertility in Women

1
Environmental Contaminants and Infertility in
Women

• Ulrike Luderer, M.D., Ph.D.
• Division of Occupational and Environmental
  Medicine, Department of Medicine
• Department of Developmental and Cell Biology

2
Infertility and premature ovarian failure

• 12-13 of all couples are infertile
• Premature ovarian failure is responsible for
  20-30 of infertility in women
• Defined as menopause before age 40
• Affects 1-2 of women

3
Ovarian follicular development
Antral
Secondary
primary
Mature/ Preovulatory
Primordial
Ovulation
Ovulated Oocyte
Corpora Lutea
4
Hormonal regulation of ovarian function
Hypothalamus
E, P
GnRH
Inhibin
E
Anterior Pituitary
LH, FSH
Ovary
5
The human menstrual cycle
FSH mIU/ml
6
Stages of Follicular Development
Not responsive to gonadotropins
Responsive to gonadotropins
Gonadotropin-dependent
Primordial
Large antral/Preovulatory
Photos of sheep follicles (Juengel and McNatty,
2005 Human Reprod Update 11144-61)
Primary
7
Infertility and Age
8
Age-Related Decline in Human Oocytes
Age 5 mos gest birth puberty menopause
oocytes 7 million 2 million 400,000 0
- Only 350 oocytes progress to ovulation between
puberty and menopause
9
Causes of premature ovarian failure

• Cytogenetic abnormalities of the X chromosome
• Fragile X syndrome
• Other genetic mutations
• 17a-hydroxylase, aromatase, FOXL2, BMP15, LH-R,
  FSH-R
• Immune disturbances
• Viral infection
• Chemical or physical agents
• Cause is unknown in many cases

10
Environmental exposures that cause ovarian failure

• Smoking
• decreased fecundity, earlier menopause
• Polycyclic aromatic hydrocarbons
• Dimethylbenz(a)anthracene, benzo(a)pyrene,
  3-methylcholanthrene
• Anticancer drugs
• Alkylating agents (e.g. cyclophosphamide)
• Doxorubicin
• Procarbazine
• Ionizing radiation
• 2-bromopropane
• Vinylcyclohexene

11
Types of cell death

• Necrosis
• Passive, catastrophic
• Morphological features
• Swelling
• No chromatin remodeling
• Plasma membrane blebbing

• Apoptosis
• Active, regulated
• Morphological features
• Shrinkage
• Chromatin condensation
• Plasma membrane budding
• Proteolysis
• Inhibitory regulators, Bcl-2
• Nuclear lamins, cytoskeletal proteins
• Inhibitor of caspase activated DNAse
• DNA fragmentation

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Apoptosis in Ovarian Follicles
Pro-apoptotic Bcl-2 proteins
Anti-apoptotic Bcl-2 proteins
-

damage- induced pathway
cytochrome c
-
caspase 9
Apoptosome

Apaf-1 activation
bid
caspase 3
death receptor pathway
Death domain adaptor proteins
Death Inducing Signaling Complex
caspase 8
13
Reactive Oxygen Species Generation and
Detoxification
NO
O2 e-
O2-
ONOO-
2H O2-
SOD
GSHPx
H2O2 O2
GSSG 2H2O
2GSH
H2O2
CAT
Fe
O2-
2H2O O2
O2 OH- OH
Oxidative damage to DNA, protein, lipids
14
Oxidation and Reduction of Glutathione
Glucose-6- Phosphate
Lipid-OOH or H2O2
GSH
NADP
GSH Peroxidase
GSH Reductase
G-6-P Dehydrogenase
Lipid-OH or H2O
GSSG
NADPH
6-Phospho- gluconate
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Preovulatory follicle culture

• 25-26 day old pre-pubertal rats were injected
  with 10 I.U. eCG
• 48 h later preovulatory follicles were dissected
  by hand
• Immediately processed for assays (0h, negative
  control)
• OR
• cultured for 2 to 48h with
• MEM medium alone (positive control)
• OR
• Ovine FSH, 75 ng/mL (negative control)
• OR
• Various treatments

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FSH stimulates follicular GSH synthesis
17
FSH suppresses ROS formation DHR







18
FSH suppresses ROS formation DCF
0h
H2O2
12h, MEM
12h, FSH
12h, FSHBSO

















19
GSH depletion induces apoptosis


0h















FSHBSO, 48h
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Apoptosis in Ovarian Follicles
Pro-apoptotic Bcl-2 proteins
Anti-apoptotic Bcl-2 proteins
-

damage- induced pathway
cytochrome c
-
caspase 9
Apoptosome

Apaf-1 activation
bid
caspase 3
death receptor pathway
Death domain adaptor proteins
Death Inducing Signaling Complex
caspase 8
21
GSH depletion and apoptosis Caspase 3
MEM 48h







FSH, 48h



















FSHBSO 48h


22
GSH replacement reverses effect of BSO on GC
apoptosis
23
(No Transcript)
24
Intraovarian benz(a)pyrene destroys primordial
follicles in mice
Adapted from Takizawa et al, 1984, Cancer
Research 442571
25
DMBA induces granulosa cell apoptosis
0 mM
100 mM
0.1 mM
10 mM



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Apoptosis in Ovarian Follicles
Pro-apoptotic Bcl-2 proteins
Anti-apoptotic Bcl-2 proteins
-

damage- induced pathway
cytochrome c
-
caspase 9
Apoptosome

Apaf-1 activation
bid
caspase 3
death receptor pathway
Death domain adaptor proteins
Death Inducing Signaling Complex
caspase 8
27
DMBA induces granulosa and theca cell apoptosis
0 mM
10 mM






28
Apoptosis in Ovarian Follicles
Pro-apoptotic Bcl-2 proteins
Anti-apoptotic Bcl-2 proteins
-

damage- induced pathway
cytochrome c
-
caspase 9
Apoptosome

Apaf-1 activation
bid
caspase 3
death receptor pathway
Death domain adaptor proteins
Death Inducing Signaling Complex
caspase 8
29
DMBA-induced apoptosis is preceded by increased
Bax
0 mM
10 mM
0.1 mM










30
(No Transcript)
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DMBA-induced apoptosis is preceded by increased
ROS










Effect of treatment, p0.003, FSH differs from
1.0 and 10 mM DMBA at plt0.01 Effect of time
plt0.001 significantly different from 0h
32
GSH supplementation prevents DMBA-induced GC
apoptosis





33
GSH depletion enhances DMBA-induced GC apoptosis


FSH

FSHDMBA
FSHBSO
FSH DMBA BSO
34
GST-Mediated Glutathione Conjugation
RX GSH
1.
RSG HX
GST
GSH
2.
O CH2CH2Cl H2N-P-N
CH2CH2Cl O -
GSTA1
Phosphoramide mustard
Monoglutathionyl- Phosphoramide mustard
35
Cyclophosphamide induces apoptosis in granulosa
cells
Sal/ 300 mg/kg
Sal/ 50 mg/kg










36
4HC induces apoptosis in COV434 cells TUNEL
0 mM
1 mM
10 mM
Effect of 4HC dose, plt0.001 Effect of time,
plt0.001
50 mM
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4HC causes oxidative stress

0
50



38
GSH depletion enhances 4HC-induced apoptosis
4HC (µM)
0
BSO (µM)
100
100
100
0
0
plt0.001, effects of 4HC, BSO and time p0.012,
4HC x BSO interaction
39
Radiation increases ROS in COV434 granulosa cells



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Glutathione Synthesis
?-glutamyl- cysteinyl glycine (GSH)
glutamate
41
Stable transfection of Gclc and Gclm in COV434
cells
42
Gcl subunit overexpression increases GSH
concentrations
43
Gclc overexpression protects against ionizing
radiation
44
ROS and apoptosis in granulosa cells

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

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Acknowledgments

• Luderer Lab Current Members
• Mabel Cortés-Wanstreet
• Yvonne Hoang
• Brooke Nakamura
• Laura Ortiz
• Victoria Flores
• Past Lab Members
• Miyun Tsai-Turton
• Youming Tan
• Brian Luong
• Pedro Morgado
• Sarah Lopez
• Matilde Gonzalez
• Jennifer Lavorin

• UC Irvine
• Charles Limoli
• University of Washington
• Terry Kavanagh
• University of Leiden
• Peter Schrier
• UCI Developmental Biology Center and Chao Family
  Comprehensive Cancer Center
• Funding
• NIEHS ES10963
• Center for Occupational and Environmental Health

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Pedro
Brian
Azuka
Yvonne
Mabel
Brooke
Miyun