132 INCIDENCE OF OXIDATIVE STRESS-INDUCED APOPTOSIS IN OVINE EMBRYOS IS ALLEVIATED IN VITRO BY TROLOX, A WATER-SOLUBLE VITAMIN E ANALOGUE

Abstract

The ability of ovine embryos to develop in vitro can be curtailed severely by culture conditions that predispose, or medium constituents such as docosahexaenoic acid (DHA; C22: 6n − 3) that are vulnerable to oxidative stress (Reis et al., 2003 Reprod. Abs. Ser. 30, 48). This study examined the consequences of culture in such adverse circumstances in terms of resultant blastocyst cell metabolism and survival, and investigated whether presence of a water-soluble vitamin E analogue (Trolox; Sigma, St. Louis, MO, USA) from the zygote stage onward could safeguard ovine blastocyst integrity. Day 6 blastocysts were produced in Synthetic Oviductal Fluid (10 replicates) supplemented with 0.4% w/v fatty acid-free BSA in the absence (SBSA) or presence of DHA (SBSAD) or in the same media with 200 μM Trolox (SBSAT, SBSADT). For determination of the functional competence of their mitochondria, individual blastocysts (5 replicates) were incubated for 3 h in the presence of [2-¹⁴C]pyruvic acid (American Radiolabeled Chemicals, St. Louis, MO, USA; Sp. act. 5.5 mCi mmol⁻¹) using a ‘hanging drop’ technique, and then fixed for cell counts (Hoechst 33342). In addition, the incidence of apoptosis in corresponding blastocysts (5 replicates) was determined by TUNEL assay (Apoptag Red kit, Serologicals Corporation, Norcross, GA, USA). Cell counts and pyruvate metabolism data (log transformed) were tested using 2-way ANOVA. Blastocyst apoptotic cell indices were analyzed by Generalized Linear Model (binomial distribution; Genstat 6, Version 6.2). Neither DHA nor Trolox significantly affected metabolism of [2-¹⁴C]pyruvic acid on a ¹⁴CO₂ per cell (pmol/3 h) basis (Table 1) in Day 6 blastocysts, but there was an interaction (DHA × Trolox, P < 0.001). Total cell numbers were lowered (P = 0.053) in blastocysts produced in the presence of DHA, and Trolox did not significantly affect this parameter. However, inclusion of Trolox reduced apoptotic indices (mean ± SEM; P < 0.001) which, for blastocysts from SBSA, SBSAD, SBSAT and SBSADT, were 12 ± 0.8 (n = 43), 13 ± 1.1 (20), 9 ± 0.7 (64) and 9 ± 0.8% (36), respectively. The data indicate that Trolox supplementation of DHA-containing media sustained mitochondrial metabolism in Day 6 ovine blastocysts. Moreover, independently of DHA status, Trolox lowered the incidence of apoptosis, presumably by protecting vital but vulnerable cellular constituents. Funded by SEERAD; AR supported by Portuguese Ministry of Science and Technology.

Table 1 
Mean (± SEM) pyruvate metabolism indices (pmol/3 h) and cell numbers for Day 6 blastocysts