, Volume 24, Issue 9–10, pp 718–729 | Cite as

Fipronil induces apoptosis and cell cycle arrest in porcine oocytes during in vitro maturation

  • Wenjun Zhou
  • Ying-Jie Niu
  • Zheng-Wen Nie
  • Yong-Han Kim
  • Kyung-Tae Shin
  • Jing GuoEmail author
  • Xiang-Shun CuiEmail author


Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 μM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 μM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.


Fipronil Porcine Oocyte maturation Apoptosis MPF 



This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2018R1A2B6001173), Republic of Korea. Also, I would like to thank Doctor Namgoong for providing cyclin B1-GFP and H2B-mCherry expression plasmid.

Compliance with ethical standards

Conflict of interest

We declare that we have no conflict of interest.

Supplementary material

10495_2019_1552_MOESM1_ESM.avi (15.6 mb)
Supplementary material 1 Movie S1. Time-lapse imaging of control group during in vitro maturation. The oocytes were injected with H2B-mcherry (red) and cyclin B1-GFP (green) mRNAs and cultured in vitro for time-lapse imaging (AVI 15978 kb)
10495_2019_1552_MOESM2_ESM.avi (13.5 mb)
Supplementary material 2 Movie S2. Time-lapse imaging of 100 μM fipronil (FPN)-treated group during in vitro maturation. The oocytes were injected with H2B-mcherry (red) and cyclin B1-GFP (green) mRNAs and cultured in vitro for time-lapse imaging with 100 μM FPN treatment (AVI 13788 kb)


  1. 1.
    Konwick BJ, Fisk AT, Garrison AW, Avants JK, Black MC (2005) Acute enantioselective toxicity of fipronil and its desulfinyl photoproduct to Ceriodaphnia dubia. Environ Toxicol Chem 24:2350–2355CrossRefPubMedGoogle Scholar
  2. 2.
    Varró P, Gyori J, Világi I (2009) In vitro effects of fipronil on neuronal excitability in mammalian and molluscan nervous systems. Ann Agric Environ Med 16:71–77PubMedGoogle Scholar
  3. 3.
    Vidau C, Brunet J-L, Badiou A, Belzunces LP (2009) Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2. Toxicol In Vitro 23:589–597CrossRefPubMedGoogle Scholar
  4. 4.
    Das PC, Cao Y, Cherrington N, Hodgson E, Rose RL (2006) Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes. Chem Biol Interact 164:200–214CrossRefPubMedGoogle Scholar
  5. 5.
    Ki Y-W, Lee JE, Park JH, Shin IC, Koh HC (2012) Reactive oxygen species and mitogen-activated protein kinase induce apoptotic death of SH-SY5Y cells in response to fipronil. Toxicol Lett 211:18–28CrossRefPubMedGoogle Scholar
  6. 6.
    Leghait J, Gayrard V, Picard-Hagen N et al (2009) Fipronil-induced disruption of thyroid function in rats is mediated by increased total and free thyroxine clearances concomitantly to increased activity of hepatic enzymes. Toxicology 255:38–44CrossRefPubMedGoogle Scholar
  7. 7.
    Khan S, Jan M, Kumar D, Telang A (2015) Firpronil induced spermotoxicity is associated with oxidative stress, DNA damage and apoptosis in male rats. Pestic Biochem Physiol 124:8–14CrossRefPubMedGoogle Scholar
  8. 8.
    Stehr CM, Linbo TL, Incardona JP, Scholz NL (2006) The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae. Toxicol Sci 92:270–278CrossRefPubMedGoogle Scholar
  9. 9.
    Qureshi IZ, Bibi A, Shahid S, Ghazanfar M (2016) Exposure to sub-acute doses of fipronil and buprofezin in combination or alone induces biochemical, hematological, histopathological and genotoxic damage in common carp (Cyprinus carpio L.). Aquat Toxicol 179:103CrossRefPubMedGoogle Scholar
  10. 10.
    Banerjee BD, Seth V, Ahmed RS (2001) Pesticide-induced oxidative stress: perspective and trends. Rev Environ Health 16:1–40CrossRefPubMedGoogle Scholar
  11. 11.
    Miranda MD, de Bruin VM, Vale MR, Viana GS (2000) Lipid peroxidation and nitrite plus nitrate levels in brain tissue from patients with Alzheimer’s disease. Gerontology 46:179–184CrossRefGoogle Scholar
  12. 12.
    Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K (2009) Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 20:346–353CrossRefPubMedGoogle Scholar
  13. 13.
    Gupta S, Sekhon L, Kim Y, Agarwal A (2010) The role of oxidative stress and antioxidants in assisted reproduction. Curr Womens Health Rev 6:227–238CrossRefGoogle Scholar
  14. 14.
    Lee JE, Kang JS, Ki Y-W et al (2011) Akt/GSK3β signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells. Toxicol Lett 202:133–141CrossRefPubMedGoogle Scholar
  15. 15.
    de Castilhos Ghisi N, Ramsdorf WA, Ferraro MVM, de Almeida MIM, de Oliveira Ribeiro CA, Cestari MM (2011) Evaluation of genotoxicity in Rhamdia quelen (Pisces, Siluriformes) after sub-chronic contamination with Fipronil. Environ Monit Assess 180:589–599CrossRefGoogle Scholar
  16. 16.
    Girgis SM, Yassa VF (2013) Evaluation of the potential genotoxic and mutagenic effects of fipronil in rats. J Mediterr Ecol 12:5–11Google Scholar
  17. 17.
    Çelik A, Ekinci SY, Güler G, Yildirim S (2014) In vitro genotoxicity of fipronil sister chromatid exchange, cytokinesis block micronucleus test, and comet assay. DNA Cell Biol 33:148–154CrossRefPubMedGoogle Scholar
  18. 18.
    Balaban RS, Nemoto S, Finkel T (2005) Mitochondria, oxidants, and aging. Cell 120:483–495CrossRefGoogle Scholar
  19. 19.
    Chen D, Li X, Liu X et al (2017) NQO2 inhibition relieves ROS effects on mouse oocyte meiotic maturation and embryo development. Biol Reprod 97:598–611CrossRefPubMedGoogle Scholar
  20. 20.
    Luberda Z (2005) The role of glutathione in mammalian gametes. Reprod Biol 5:5–17PubMedGoogle Scholar
  21. 21.
    Bedaiwy MA, Elnashar SA, Goldberg JM et al (2012) Effect of follicular fluid oxidative stress parameters on intracytoplasmic sperm injection outcome. Gynecol Endocrinol 28:51–55CrossRefPubMedGoogle Scholar
  22. 22.
    Palini S, Benedetti S, Tagliamonte MC et al (2014) Influence of ovarian stimulation for IVF/ICSI on the antioxidant defence system and relationship to outcome. Reprod Biomed Online 29:65–71CrossRefPubMedGoogle Scholar
  23. 23.
    Orrenius S, Gogvadze V, Zhivotovsky B (2007) Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47:143–183CrossRefPubMedGoogle Scholar
  24. 24.
    Clarke PG, Clarke S (1995) Historic apoptosis. Nature 378:230CrossRefPubMedGoogle Scholar
  25. 25.
    Elledge SJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274:1664–1672CrossRefPubMedGoogle Scholar
  26. 26.
    Nebreda AR, Ferby I (2000) Regulation of the meiotic cell cycle in oocytes. Curr Opin Cell Biol 12:666–675CrossRefPubMedGoogle Scholar
  27. 27.
    Choi W-J, Banerjee J, Falcone T, Bena J, Agarwal A, Sharma RK (2007) Oxidative stress and tumor necrosis factor–α–induced alterations in metaphase II mouse oocyte spindle structure. Fertil Steril 88:1220–1231CrossRefPubMedGoogle Scholar
  28. 28.
    Tamura H, Takasaki A, Miwa I et al (2008) Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res 44:280–287CrossRefPubMedGoogle Scholar
  29. 29.
    Sugino N (2005) Reactive oxygen species in ovarian physiology. Reprod Med Biol 4:31–44PubMedPubMedCentralGoogle Scholar
  30. 30.
    Bolcunfilas E, Rinaldi VD, White ME, Schimenti JC (2014) Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway. Science 343:533–536CrossRefGoogle Scholar
  31. 31.
    Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51:6304–6311PubMedGoogle Scholar
  32. 32.
    De Oliveira PR, Bechara GH, Denardi SE, Oliveira RJ, Mathias MIC (2012) Cytotoxicity of fipronil on mice liver cells. Microsc Res Tech 75:28–35CrossRefPubMedGoogle Scholar
  33. 33.
    Han J, Wang Q-C, Zhu C-C et al (2016) Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation. Toxicol Appl Pharmacol 300:70–76CrossRefPubMedGoogle Scholar
  34. 34.
    Zhang Y, Han J, Zhu C-C et al (2016) Exposure to HT-2 toxin causes oxidative stress induced apoptosis/autophagy in porcine oocytes. Sci Rep 6:33904CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Kitajima TS, Ohsugi M, Ellenberg J (2011) Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes. Cell 146:568–581CrossRefPubMedGoogle Scholar
  36. 36.
    Prochazka R, Petlach M, Nagyova E, Němcová L (2011) Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins. Reproduction 141:425–435CrossRefPubMedGoogle Scholar
  37. 37.
    Varani S, Elvin JA, Yan C et al (2002) Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol Endocrinol 16:1154–1167CrossRefPubMedGoogle Scholar
  38. 38.
    Oqani RK, Lin T, Lee JE, Kim SY, Kang JW, Jin DI (2017) Effects of CDK inhibitors on the maturation, transcription, and MPF activity of porcine oocytes. Reprod Biol 17:320–326CrossRefPubMedGoogle Scholar
  39. 39.
    Šefčíková Z, Babeľová J, Čikoš Š et al (2018) Fipronil causes toxicity in mouse preimplantation embryos. Toxicology 410:214–221CrossRefPubMedGoogle Scholar
  40. 40.
    Xu C, Niu L, Liu J et al (2019) Maternal exposure to fipronil results in sulfone metabolite enrichment and transgenerational toxicity in zebrafish offspring: indication for an overlooked risk in maternal transfer? Environ Pollut 246:876–884CrossRefPubMedGoogle Scholar
  41. 41.
    Chaube SK, Prasad PV, Thakur SC, Shrivastav TG (2005) Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 10:863–874CrossRefPubMedGoogle Scholar
  42. 42.
    Han J, Wang T, Fu L et al (2015) Altered oxidative stress, apoptosis/autophagy, and epigenetic modifications in Zearalenone-treated porcine oocytes. Toxicol Res 4:1184–1194CrossRefGoogle Scholar
  43. 43.
    Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157CrossRefPubMedGoogle Scholar
  44. 44.
    Tsujimoto Y, Shimizu S, Eguchi Y, Kamiike W, Matsuda H (1997) Bcl-2 and Bcl-xL block apoptosis as well as necrosis: possible involvement of common mediators in apoptotic and necrotic signal transduction pathways. Leukemia 11(Suppl 3):380–382PubMedGoogle Scholar
  45. 45.
    Lin F, Ma XS, Wang ZB et al (2014) Different fates of oocytes with DNA double-strand breaks in vitro and in vivo. Cell Cycle 13:2674CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    de Oliveira PR, Bechara GH, Denardi SE, Oliveira RJ, Mathias MI (2012) Genotoxic and mutagenic effects of fipronil on mice. Exp Toxicol Pathol 64:569–573CrossRefPubMedGoogle Scholar
  47. 47.
    Valdiglesias V, Giunta S, Fenech M, Neri M, Bonassi S (2013) γH2AX as a marker of DNA double strand breaks and genomic instability in human population studies. Res Rev Mutat Res 753:24–40CrossRefGoogle Scholar
  48. 48.
    Marangos P, Carroll J (2012) Oocytes progress beyond prophase in the presence of DNA damage. Curr Biol 22:989–994CrossRefPubMedGoogle Scholar
  49. 49.
    Kuroda T, Naito K, Sugiura K, Yamashita M, Takakura I, Tojo H (2004) Analysis of the roles of Cyclin B1 and Cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection1. Biol Reprod 70:154–159CrossRefPubMedGoogle Scholar
  50. 50.
    Karlsson-Rosenthal C, Millar J (2006) Cdc25: mechanisms of checkpoint inhibition and recovery. Trends Cell Biol 16:285CrossRefPubMedGoogle Scholar
  51. 51.
    Park JH, Park YS, Lee JB et al (2016) Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells. J Appl Toxicol 36:10–23CrossRefPubMedGoogle Scholar
  52. 52.
    Han B, Mura M, Andrade CF et al (2005) TNFα-induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. J Immunol 175:8303–8311CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wenjun Zhou
    • 1
  • Ying-Jie Niu
    • 1
  • Zheng-Wen Nie
    • 1
  • Yong-Han Kim
    • 1
  • Kyung-Tae Shin
    • 1
  • Jing Guo
    • 2
    • 3
    Email author
  • Xiang-Shun Cui
    • 1
    Email author
  1. 1.Department of Animal ScienceChungbuk National UniversityCheongjuRepublic of Korea
  2. 2.Chongqing Key Laboratory of Human Embryo EngineeringChongqingChina
  3. 3.Chongqing Reproductive and Genetics InstituteYu Zhong DistrictChongqingChina

Personalised recommendations