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Apoptosis

, Volume 22, Issue 1, pp 98–107 | Cite as

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro

  • Meenakshi Tiwari
  • Anima Tripathi
  • Shail K. ChaubeEmail author
Article

Abstract

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H2O2)-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H2O2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H2O2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H2O2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H2O2-induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H2O2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.

Keywords

Rat egg Granulosa cells Hydrogen peroxide Bax/Bcl-2 DNA fragmentation Apoptosis 

Notes

Acknowledgements

This study was financially supported by Department of Science and Technology, Ministry of Science and Technology, Government of India (Grant No. EMR/2014/000702).

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of interest exist.

Supplementary material

10495_2016_1324_MOESM1_ESM.tif (2.6 mb)
Fig. 1 Representative photograph showing protective effects of encircling granulosa cells on H2O2-induced changes in Bax, Bcl-2 and cytochrome c expression levels in COCs cultured for 3 h in vitro. Encircling granulosa cells prevented H2O2-mediated increased expression of Bax (A2, white arrow), reduced expression of Bcl-2 (B2, white arrow) and increased expression of cytochrome c (C2, white arrow) as compare to their respective controls (A1, B1 and C1). Lower panel shows β-actin expression level as a control for upper panel photographs (D1 and D2). Three independent experiments using 36-42 COCs were conducted to confirm the observations. Bar 80 µm (TIF 2628 KB)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Meenakshi Tiwari
    • 1
  • Anima Tripathi
    • 1
  • Shail K. Chaube
    • 1
    Email author
  1. 1.Cell Physiology Laboratory, Department of Zoology, Institute of ScienceBanaras Hindu UniversityVaranasiIndia

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