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 Corpora Lutea Ovulated Oocyte

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. Secondary/Preantral Antral 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 11:144-61) 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. 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 Types of cell death

12. 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

15. 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

16. † † * * † * ‡ ‡ ‡ * † † † * * * FSH stimulates follicular GSH synthesis

17. FSH suppresses ROS formation: DHR * * ‡ * † † †

18. FSH suppresses ROS formation: DCF H2O2 0h 12h, MEM 12h, FSH 12h, FSH+BSO * * † * † * * * * * * * * * † * *

19. FSH,48h ‡ † * * * † * * MEM, 24h * † * * * GSH depletion induces apoptosis * ‡ 0h † * ‡ * † † † † * † * * * * * FSH+BSO, 48h

20. 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 † † † * † * * * † * * * † † * † * † * * FSH+BSO 48h * *

22. † † * * * GEE 0 100 100 100 100 BSO † † † * GEE 0 100 100 100 BSO GSH replacement reverses effect of BSO on GC apoptosis

24. Intraovarian benz(a)pyrene destroys primordial follicles in mice Adapted from Takizawa et al, 1984, Cancer Research 44:2571

25. DMBA induces granulosa cell apoptosis 0 mM 100 mM 0.1 mM 10 mM * * *

26. 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 0.1 mM 10 mM * * * * * * * * * *

31. DMBA-induced apoptosis is preceded by increased ROS * * * * * * * * * * Effect of treatment, p=0.003, FSH differs from 1.0 and 10 mM DMBA at p<0.01 Effect of time p<0.001; * significantly different from 0h

32. GSH supplementation prevents DMBA-induced GC apoptosis * † * † *

33. GSH depletion enhances DMBA-induced GC apoptosis † * FSH * FSH+DMBA FSH+BSO FSH+ DMBA +BSO

34. O || CH2CH2Cl H2N-P-N | CH2CH2SG O - GST-Mediated Glutathione Conjugation 1. RX + GSH 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, p<0.001 Effect of time, p<0.001 50 mM

37. 4HC causes oxidative stress * 0 50 * * *

38. GSH depletion enhances 4HC-induced apoptosis 4HC (µM) 0 0 100 100 100 BSO (µM) 0 p<0.001, effects of 4HC, BSO and time; p=0.012, 4HC x BSO interaction

39. Radiation increases ROS in COV434 granulosa cells * * *

40. CH2SH | CONHCHCO-NHCH2COOH | (CH2)2 | CHNH2 | COOH CH2SH | CONHCHCOOH | (CH2)2 | CHNH2 | COOH COOH | (CH2)2 | CHNH2 | COOH 1 2 CH2SH | H2NCHCOOH H2NCH2COOH glycine cysteine 1: glutamate cysteine ligase (-glutamyl-cysteine synthetase) 2: glutathione synthetase Glutathione Synthesis -glutamyl- cysteinyl glycine (GSH) glutamate

41. CMV9b C6b CM3a COV434 CM2a M8c C6a CM4b M5a M7a MWM CMV-GCLc CMV-GCLm 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.

45. 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 Acknowledgments • 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

46. Pedro Brian Azuka Yvonne Mabel Brooke Miyun