317 EFFECT OF REACTIVE OXYGEN SPECIES (ROS) ON ACTIVATION OF BOVINE OOCYTES MATURED IN VITRO

Abstract

Vitamin E is an antioxidant that protects embryos from oxidative stress and supports embryonic development in vitro. This study was carried out to assess the effect of reactive oxygen species (ROS) by measuring the effects of hydrogen peroxide (H₂O₂) and vitamin E (VitE) on chemical activation of in vitro-matured oocytes. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/L-cysteinr, 44 mg/L sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 ng/mL epidermal growth factor. Oocytes were cultured at 38.9°C in 5% CO₂ in humidified air. After 22 h of culture, oocytes with polar bodies were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 ¼M for 5 min) followed by incubation with 6-DMAP (2 mM) for 3.5 h in medium supplemented with or without VitE (100 ¼m). Routine in vitro fertilization (IVF) was also performed for 18 h as control treatment with or without VitE. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO₂, 5% O₂ in humidified air for 16-18 h. H₂O₂ content was measured and rate of development was monitored. For detection of H₂O₂, individual oocytes were stained with 22,72-dichlorofluorescein diacetate (DCFDA) and observed under fluorescence microscope (Hashimoto et al. 2000 Mol. Reprod. Dev. 56, 520-526). Levels of H₂O₂ were analyzed by counting the number of black-white pixels of TIFF images (ImageJ ver. 1.32; http://rsb.info.nih.gov/ij) after conversion of fluorescence image. Data were analyzed using Student's t-test and chi-square test. The H₂O₂ contents were significantly higher (P < 0.05) in the activation group without VitE (218 pixels, n = 49) than IVF without VitE (210 pixels, n = 50). Levels of H₂O₂ in oocytes activated with VitE were also higher (P < 0.05) than in the IVF group with VitE (216 pixels, n = 50 vs. 210 pixels, n = 51). Between groups with or without VitE, the H₂O₂ contents were not different in both activation and IVF. The cleavage rate to the 2-cell stage and development rate to blastocysts after activation with VitE (77.8% and 15.1%, n = 72) were significantly higher (P < 0.05) than in those without VitE (63.3% and 10.2%, n = 60). The results of the present study demonstrate that the chemical activation increased accumulation of H₂O₂ in oocytes compared to the IVF condition. Although ROS level did not affect chemical activation, vitamin E added in the activation medium promoted further development of activated embryos.

This work was supported by a grant from the BioGreen 21 Program (20050301-034-443-026-03-00), Rural Development Administration, Korea.