192 EXPRESSION OF SELECTED ANTIOXIDANT ENZYMES IN BOVINE OVIDUCT EPITHELIAL CELL (BOEC) IN RESPONSE TO ELEVATED TEMPERATURES IN VITRO

Abstract

Elevated temperatures have a negative impact on bovine reproduction. One of its effects is an increased concentration of reactive oxygen species (ROS) which may lead to female infertility. Oxidative stress impairs oocyte maturation, fertilization, and embryo development, and it also influences the reproductive tract. One of the defence mechanisms against the increase of ROS is the synthesis of antioxidants. Thus, the aim of this study was to analyse the expression of antioxidant enzymes (superoxide dismutase 1, SOD1; catalase, CAT; and glutathione peroxidase 1, GPX1) in bovine oviduct epithelial cells (BOEC) cultured with or without embryos at elevated temperatures. Ovaries and oviducts were collected from a slaughterhouse. BOECs were mechanically isolated from the oviducts. The oocytes were isolated from ovaries and then maturated and fertilized in vitro. BOEC, after formation of aggregates, were cultured (variant I) in 40-µL droplets of cultured medium (TCM199 25 mM HEPES medium supplemented with 10% FBS, 10 µg mL^–1 gentamicin, and 50 µg mL^–1 streptomycin) overlaid with mineral oil. Twenty aggregates per droplet were cultured at control (38.5°C) and elevated (41°C) temperatures for 168 h in 5% CO₂ in air. Analogously, in variant II, BOEC aggregates were co-cultured with 15 bovine embryos per droplet. Subsequently, the SOD1, CAT, and GPX1 mRNA levels were analysed in BOEC by real-time RT–PCR (Light Cycler, Roche Diagnostics, Warsaw, Poland) and normalized to S18/H2A gene expression. Relative quantification was determined with LightCycler software version 3.5 (Roche Diagnostics) by the second derivative maximum method. Statistical analyses were performed by Portable Statgraphics 5.0 Centurion (Statpoint Technologies Inc., Warrenton, VA). Mean values of SOD1, CAT, and GPX1 expression in BOEC in RT-qPCR analysis were compared using Tukey's HSD test (a = 0.01). Elevated temperature leads to an up-regulation of SOD1 in BOEC cultured (38°C: 0.76 ± 0.12 a.u., n = 44; 41°C: 1.07 ± 0.21 a.u., n = 48) and co-cultured with bovine embryos (38°C: 0.71 ± 0.11 a.u., n = 36; 41°C: 1.04 ± 0.2 a.u., n = 36) and the difference was statistically significant (P < 0.01). The CAT gene expression in BOEC was constant in variant I (38°C: 0.56 ± 0.22 a.u., n = 56; 41°C: 0.58 ± 0.27 a.u., n = 56) and variant II (38°C: 0.48 ± 0.27 a.u., n = 32; 41°C: 0.59 ± 0.29 a.u., n = 24). Also, GPX1 gene expression in BOEC was constant in variant I (38°C: 0.66 ± 0.23 a.u., n = 60; 41°C: 0.61 ± 0.19 a.u., n = 56) and in variant II (38°C: 0.59 ± 0.19 a.u., n = 36; 41°C: 0.64 ± 0.22 a.u., n = 36). In conclusion, elevated temperature leads to an activation of the BOEC's defence mechanisms which are based on SOD1 expression, and which may protect cells against oxidative stress. Elevated temperature doesn't affect the cat and GPX1 expression in BOEC. The presence of embryos does not affect the expression of antioxidant enzymes in BOEC.

Research was supported by COST DPN/DWM/MZ/5670/08/09.