Redox-Induced Apoptosis of Human Oocytes in Resting Follicles In Vitro

Abstract

Background: The age-related depletion of the resting follicle (RF) stock occurs as a result of two processes: atresia and entry in growth phase. Due to difficulties in obtaining sufficient numbers of RFs for study, little is known about the apoptotic mechanisms for RF atresia. The present study was designed to investigate the effects of oxidative stress on the apoptosis of RF oocytes.

Methods: RFs isolated from human adult ovaries were cultured in vitro, treated with H₂O₂ at various concentrations (50 μM, 100 μM, 1.0 mM) for 1 hour, and observed for up to 48 hours. The oxidant-induced apoptosis of oocytes were observed by detection of DNA fragments, mitochondria membrane potantial (MMP) and cytochrome c release.

Results: Based on nuclear morphology and TUNEL (terminal deoxynucleotidyl transferase-mediated dDTP nick end-labeling), oocyte apoptosis was observed in the RFs treated with 50 μM H₂O₂ with rates of 35% and 43% at 24 and 48 h after treatment, respectively. But intensive oxidative stress (1 mM H₂O₂) caused mainly necrosis as measured by quantifying propidium iodide (PI)-positive oocytes (44% within 12 hours), with lower level of apoptosis (17%) being observed at 24 hours after treatment. RFs treated with 100 μM H₂O₂ showed both apoptosis with the similar rate observed at 50μM and necrosis (13% PI-positive oocytes). Although pre-incubation with cyclosporine A (CsA) could effectively prevent oxidant-induced MMP collapse, but failed to suppress apoptosis of oocytes in RFs.

Conclusions: Oocytes in RFs in adult ovaries retain their ability to undergo apoptosis under oxidative stress, which is both dose- and time-dependent.

Keywords

Ovary oocyte oxidative stress apoptosis resting follicle

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References

1. Gougeon A . Regulation of ovarian follicular development in primates: Facts and hypotheses. Endocr Rev 1996;17:121-155.

2. Vaskivuo TE , Tapanainen JS . Apoptosis in the human ovary. Reprod Biomed Online 2003;6:24-35.

3. Gougeon A , Ecochard R , Thalabard JC . Age-related changes of the population of human ovarian follicles: Increase in the disappearance rate of non-growing and early-growing follicles in aging women. Biol Reprod 1994;50:653-663.

4. Faddy MJ , Gosden RG , Gougeon A , Richardson SJ , Nelson JF . Accelerated disappearance of ovarian follicles in mid-life: Implications for forecasting menopause. Hum Reprod 1992;7:1342-1346.

5. Pesce M , De Felici M . Apoptosis in mouse primordial germ cells: A study by transmission and scanning electron microscope. Anat Embryol (Berl) 1994;189:435-440.

6. Ratts VS , Flaws JA , Kolp R , Sorenson CM , Tilly JL . Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 1995;136:3665-3668.

7. Morita Y , Tilly JL . Oocyte apoptosis: Like sand through an hourglass. Dev Biol 1999;213:1-17.

8. Vaskivuo TE , Anttonen M , Herva R , et al. Survival of human ovarian follicles from fetal to adult life: Apoptosis, apoptosis-related proteins, and transcription factor GATA-4. J Clin Endocrinol Metab 2001;86:3421-3429.

9. Chun SY , Eisenhauer KM , Minami S , Billig H , Perlas E , Hsueh AJ . Hormonal regulation of apoptosis in early antral follicles: Follicle-stimulating hormone as a major survival factor. Endocrinology 1996;137:1447-1456.

10.

10. Perez GI , Robles R , Knudson CM , Flaws JA , Korsmeyer SJ , Tilly JL . Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nat Genet 1999;21:200-203.

11.

11. Depalo R , Nappi L , Loverro G , et al. Evidence of apoptosis in human primordial and primary follicles. Hum Reprod 2003;18:2678-2682.

12.

12. Johnson FB , Sinclair DA , Guarente L . Molecular biology of aging. Cell. 1999;96:291-302.

13.

13. Papa S , Skulachev VP . Reactive oxygen species, mitochondria, apoptosis and aging. Mol Cell Biochem 1997;174:305-319.

14.

14. Shigenaga MK , Hagen TM , Ames BN . Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A 1994;91:10771-10778.

15.

15. Ozawa T . Genetic and functional changes in mitochondria associated with aging. Physiol Rev 1997;77:425-464.

16.

16. Sohal RS , Weindruch R . Oxidative stress, caloric restriction, and aging. Science 1996;273:59-63.

17.

17. Pasqualotto EB , Agarwal A , Sharma RK , et al. Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures. Fertil Steril 2004;81:973-976.

18.

18. Kane DJ , Sarafian TA , Anton R , et al. Bcl-2 inhibition of neural death: Decreased generation of reactive oxygen species. Science 1993;262:1274-1277.

19.

19. Quillet-Mary A . Jaffrezou JP , Mansat V , Bordier C , Naval J , Laurent G . Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem 1997;272:21388-21395.

20.

20. Liu L , Trimarchi JR , Keefe DL . Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes. Biol Reprod 2000;62:1745-1753.

21.

21. Tatemoto H , Sakurai N , Muto N . Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during in vitro maturation: Role of cumulus cells. Biol Reprod 2000;63:805-810.

22.

22. Abir R , Roizman P , Fisch B , et al. Pilot study of isolated early human follicles cultured in collagen gels for 24 hours. Hum Reprod 1999;14:1299-1301.

23.

23. Abir R , Fisch B , Nitke S , Okon E , Raz A , Ben Rafael Z . Morphological study of fully and partially isolated early human follicles. Fertil Steril 2001;75:141-146.

24.

24. Okuno S , Shimizu S , Ito T , et al. Bcl-2 prevents caspase-independent cell death. J Biol Chem 1998;273:34272-34277.

25.

25. Reers M , Smiley ST , Mottola-Hartshorn C , Chen A , Lin M , Chen LB . Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 1995;260:406-417.

26.

26. Reers M , Smith TW , Chen LB . J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991;30:4480-4486.

27.

27. Chen Q , Ames BN . Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci U S A 1994;91:4130-4134.

28.

28. Bladier C , Wolvetang EJ , Hutchinson P , de Haan JB , Kola I . Response of a primary human fibroblast cell line to H2O2: Senescence-like growth arrest or apoptosis? Cell Growth Differ 1997;8:589-598.

29.

29. Bachvarova R , Baran MM , Tejblum A . Development of naked growing mouse oocytes in vitro. J Exp Zool 1980;211:159-169.

30.

30. Hovatta O , Wright C , Krausz T , Hardy K , Winston RM . Human primordial, primary and secondary ovarian follicles in long-term culture: Effect of partial isolation. Hum Reprod 1999;14:2519-2524.

31.

31. Salustri A , Camaioni A , Di Giacomo M , Fulop C , Hascall VC . Hyaluronan and proteoglycans in ovarian follicles. Hum Reprod Update 1999;5:293-301.

32.

32. Stojkovic M , Kolle S , Peinl S , et al. Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro. Reproduction 2002;124:141-153.

33.

33. Webber LJ , Stubbs S , Stark J , et al. Formation and early development of follicles in the polycystic ovary. Lancet 2003;362:1017-1021.

34.

34. Cataldo NA , Dumesic DA , Goldsmith PC , Jaffe RB . Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: Relationship to apoptosis. Hum Reprod 2000;15:1889-1897.

35.

35. Tilly JL , Kowalski KI , Johnson AL , Hsueh AJ . Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991;129:2799-2801.

36.

36. Mikkelsen AL , Host E , Lindenberg S . Incidence of apoptosis in granulosa cells from immature human follicles. Reproduction 2001;122:481-486.

37.

37. Wu J , Zhang L , Wang X . Maturation and apoptosis of human oocytes in vitro are age-related. Fertil Steril 2000;74:1137-1141.

38.

38. Negoescu A , Lorimier P , Labat-Moleur F , et al. In situ apoptotic cell labeling by the TUNEL method: Improvement and evaluation on cell preparation. J Histochem Cytochem 1996;44:959-968.

39.

39. Grasl-Kraupp B , Ruttkay-Nedecky B , Koudelka H , Bukowska K . Bursch W , Shulte-Hermann R . In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology 1995;21:1465-1468.

40.

40. Wyllie AH , Kerr JF , Currie AR . Cell death: The significance of apoptosis. Int Rev Cytol 1980;68:251-306.

41.

41. Majno G , Joris I . Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995;146:3-15.

42.

42. Billig H , Chun SY , Eisenhauer K , Hsueh AJ . Gonadal cell apoptosis: Hormone-regulated cell demise. Hum Reprod Update 1996;2:103-117.

43.

43. Armstrong DG , Webb R . Ovarian follicular dominance: The role of intraovarian growth factors and novel proteins. Rev Reprod 1997;2:139-146.

44.

44. Morita Y , Perez GI , Paris F , et al. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat Med 2000;6:1109-1114.

45.

45. Chaube SK , Prasad PV , Thakur SC , Shrivastav TG . Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 2005;10:863-874.

46.

46. Liu L , Trimarchi JR , Keefe DL . Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol. Biol Reprod 1999;61:1162-1169.

47.

47. Wiese AG , Pacifici RE , Davies KJ . Transient adaptation of oxidative stress in mammalian cells. Arch Biochem Biophys 1995;318:231-240.

48.

48. Castilho RF , Kowaltowski AJ , Meinicke AR , Vercesi AE . Oxidative damage of mitochondria induced by Fe(II)citrate or tbutyl hydroperoxide in the presence of Ca2+: Effect of coenzyme Q redox state. Free Radic Biol Med 1995;18:55-59.

49.

49. Kroemer G , Zamzami N , Susin SA . Mitochondrial control of apoptosis. Immunol Today 1997;18:44-51.

50.

50. Marchetti P , Decaudin D , Macho A , et al. Redox regulation of apoptosis: Impact of thiol oxidation status on mitochondrial function. Eur J Immunol 1997;27:289-296.

51.

51. Crompton M . The mitochondrial permeability transition pore and its role in cell death. Biochem J 1999;341:233-249.

52.

52. Liu X , Kim CN , Yang J . Jemmerson R , Wang X . Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 1996;86:147-157.

53.

53. Ikemoto H , Tani E , Ozaki I , Kitagawa H , Arita N . Calphostin C-mediated translocation and integration of Bax into mitochondria induces cytochrome c release before mitochondrial dysfunction. Cell Death Differ 2003;7:511-520.