Abstract
Purpose
To determine the level of apoptosis, and alteration of FoxO3 (forkhead box O3 transcription factor) expression and phosphorylation in human granulosa cells amongst polycystic ovary syndrome (PCOS) patients and control group.
Methods
We recruited infertile women with PCOS (n = 14) and compared them with infertile women due to tubal blockage or male factor infertility (n = 14, controls). GnRH agonist and gonadotropins were used for ovarian stimulation. Follicular fluids from large follicles (>16 mm) were pooled and granulosa cells (GCs) were isolated using cell strainer methodology. Apoptosis of purified GCs was measured by flow cytometry using Annexin V and propidium iodide. Quantitative real-time PCR and western blotting were performed to assess alteration of FoxO3 expression and phosphorylation in GCs.
Results
There were higher percentages of early and late apoptosis in GCs of PCOS patients than in the control group. FoxO3 mRNA level and total FoxO3 protein were significantly higher in PCOS group than in the control group. The ratio of p-FoxO3/total FoxO3 decreased significantly in PCOS than in the control group. It was inferred that unphosphorylated (active form) FoxO3 was higher in GCs of PCOS patients. Apoptosis was significantly and positively correlated with the total FoxO3 and negatively correlated with the p-FoxO3 protein levels in PCOS patients.
Conclusions
Activation and overexpression of FoxO3 in granulosa cells of PCOS women correlated with higher apoptosis levels in these cells suggesting that FoxO3 may be a candidate for the higher apoptosis in granulosa cells from women with PCOS.
Similar content being viewed by others
References
Ehrmann DA (2005) Polycystic ovary syndrome. New Engl J Med 352:1223–1236
Moran L, Teede H (2009) Metabolic features of the reproductive phenotypes of polycystic ovary syndrome. Hum Reprod Update 15:477–488
Köninger A, Koch L, Edimiris P, Enekwe A, Nagarajah J, Kasimir-Bauer S, Kimmig R, Strowitzki T, Schmidt B (2014) Anti-Mullerian Hormone: an indicator for the severity of polycystic ovarian syndrome. Arch Gynecol Obstet 290:1023–1030
Honnma H, Endo T, Henmi H, Nagasawa K, Baba T, Yamazaki K, Kitajima Y, Hayashi T, Manase K, Saito T (2006) Altered expression of Fas/Fas ligand/caspase 8 and membrane type 1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats. Apoptosis 11:1525–1533
Yu YS, Sui HS, Han ZB, Wei L, Luo MJ, Tan JH (2004) Apoptosis in granulosa cells during follicular atresia: relationship with steroids and insulin-like growth factors. Cell Res 14:341–346
Maeda A, Inoue N, Matsuda-Minehata F, Goto Y, Cheng Y, Manabe N (2007) The role of interleukin-6 in the regulation of granulosa cell apoptosis during follicular atresia in pig ovaries. J Reprod Dev 53:481–490
Dijkers PF, Birkenkamp KU, Lam EW-F, Thomas NSB, Lammers J-WJ, Koenderman L, Coffer PJ (2002) FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal protein kinase B—enhanced cell survival through maintenance of mitochondrial integrity. J Cell Biol 156:531–542
Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, Burgering BM, Medema RH (2002) The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J Immunol 168:5024–5031
Accili D, Arden KC (2004) FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117:421–426
Obexer P, Geiger K, Ambros P, Meister B, Ausserlechner M (2007) FKHRL1-mediated expression of Noxa and Bim induces apoptosis via the mitochondria in neuroblastoma cells. Cell Death Differ 14:534–547
Suh CS, Jee BC, Choi YM, Kim JG, Lee JY, Moon SY, Kim SH (2002) Prognostic implication of apoptosis in human luteinized granulosa cells during IVF–ET. J Assist Reprod Gen 19:209–214
Urman B, Tiras B, Yakin K (2004) Assisted reproduction in the treatment of polycystic ovarian syndrome. Reprod Biomed Online 8:419–430
Iavazzo C, Vitoratos N (2010) Polycystic ovarian syndrome and pregnancy outcome. Arch Gynecol Obstet 282:235–239
Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA (2003) Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301:215–218
Rotterdam ESHRE/ASRM sponsored consensus workshop group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 19:41–47
Nikolettos N, Kupker W, Al-Hasani S, Demirel L, Schöpper B, Sturm R, Diedrich K (2000) ICSI outcome in patients of 40 years age and over: a retrospective analysis. Eur J Obstet Gyn R B 91:177–182
Catteau-Jonard S, Jamin SP, Leclerc A, Gonzalès J, Dewailly D, di Clemente N (2008) Anti-Mullerian hormone, its receptor, FSH receptor, and androgen receptor genes are overexpressed by granulosa cells from stimulated follicles in women with polycystic ovary syndrome. J Clin Endocrinol Metab 93:4456–4461
Smith LP, Nierstenhoefer M, Yoo SW, Penzias AS, Tobiasch E, Usheva A (2009) The bile acid synthesis pathway is present and functional in the human ovary. PLoS One 4:e7333
Wolffe AP, Tata JR (1984) Primary culture, cellular stress and differentiated function. Febs L 176:8–15
Ferrero H, Delgado-Rosas F, Garcia-Pascual CM, Monterde M, Zimmermann RC, Simón C, Pellicer A, Gómez R (2012) Efficiency and purity provided by the existing methods for the isolation of luteinized granulosa cells: a comparative study. Hum Reprod 27:1781–1789
Fedorcsák P, Ráki M, Storeng R (2007) Characterization and depletion of leukocytes from cells isolated from the pre-ovulatory ovarian follicle. Hum Reprod 22:989–994
Figenschau Y, Sundsfjord J, Yousef M, Fuskevåg O, Sveinbjörnsson B, Bertheussen K (1997) A simplified serum-free method for preparation and cultivation of human granulosa-luteal cells. Hum Reprod 12:523–531
Al-Gubory KH, Fowler PA, Garrel C (2010) The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 42:1634–1650
Billig H, Furuta I, Hsueh A (1993) Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinol 133:2204–2212
Kaneko T, Saito H, Takahashi T, Ohta N, Saito T, Hiroi M (2000) Effects of controlled ovarian hyperstimulation on oocyte quality in terms of the incidence of apoptotic granulosa cells. J Assist Reprod Gen 17:580–585
Karuputhula NB, Chattopadhyay R, Chakravarty B, Chaudhury K (2013) Oxidative status in granulosa cells of infertile women undergoing IVF. Syst Biol Reprod Med 59:91–98
Nisenblat V, Norman RJ (2009) Androgens and polycystic ovary syndrome. Curr Opin Endocrinol Diabetes Obes 16:224–231
Balen AH, Tan SL, Jacobs HS (1993) Hypersecretion of luteinising hormone: a significant cause of infertility and miscarriage. BJOG 100:1082–1089
Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, Hiroi M (1997) The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 68:312–317
Zhang J, Zhu G, Wang X, Xu B, Hu L (2007) Apoptosis and expression of protein TRAIL in granulosa cells of rats with polycystic ovarian syndrome. J Huazhong Univ Sci Technol 27:311–314
Onalan G, Selam B, Baran Y, Cincik M, Onalan R, Gündüz U, Ural AU, Pabuccu R (2005) Serum and follicular fluid levels of soluble Fas, soluble Fas ligand and apoptosis of luteinized granulosa cells in PCOS patients undergoing IVF. Hum Reprod 20:2391–2395
Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, Raveendran M, Storey A (2008) Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. J Clin Endocrinol Metab 93:881–887
Almahbobi G, Anderiesz C, Hutchinson P, McFarlane JR, Wood C, Trounson AO (1996) Functional integrity of granulosa cells from polycystic ovaries. Clin Endocrinol 44:571–580
Wang X-L, Wu Y, Tan L-B, Tian Z, Liu J-H, Zhu D-S, Zeng S-M (2012) Follicle-stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis. J Biol Chem 287:10166–10177
Matsuda F, Inoue N, Maeda A, Cheng Y, Sai T, Gonda H, Goto Y, Sakamaki K, Manabe N (2011) Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries. J Reprod Develop 57:151–158
Inoue N, Maeda A, Matsuda-Minehata F, Fukuta K, Manabe N (2006) Expression and localization of Fas ligand and Fas during atresia in porcine ovarian follicles. J Reprod Develop 52:723–730
Acknowledgments
The present study was funded by Deputy Ministry for Research, Tehran University of Medical Sciences; Grant No. 91-03-30-19007. We would like to thank Dr. Ensieh Tehraninejad (Vali-e-Asr Reproductive Health Research Centre, Tehran University of Medical Sciences, Tehran, Iran) and Parisa Hayat (Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran) for their kind help with the present research.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests associated with this publication.
Rights and permissions
About this article
Cite this article
Mikaeili, S., Rashidi, B.H., Safa, M. et al. Altered FoxO3 expression and apoptosis in granulosa cells of women with polycystic ovary syndrome. Arch Gynecol Obstet 294, 185–192 (2016). https://doi.org/10.1007/s00404-016-4068-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00404-016-4068-z