241 STRATEGIES TO IMPROVE GLUTATHIONE CONTENT OF IN VITRO-MATURED BOVINE OOCYTES

Abstract

Glutathione is the main non-enzymatic defense against oxidative stress and a critical part of oocyte maturation and normal fertilization. Our aim was to test different strategies to manipulate cellular glutathione (GSH) content of bovine in-vitro-matured (IVM) oocytes and study the development of embryos produced from such oocytes. The reducing agents lipoic acid (LA, intracellular) and dihydrolipoic acid (DHLA, extracellular) were compared to the cell-permeable reduced glutathione (GSH) donor glutathione ethyl ester (OET) for their effect on oocyte GSH content, oocyte maturation, and blastocyst development (OET only). Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured for 24 h in a humidified atmosphere of 6% CO2 at 38.5�C in modified tissue culture medium (mTCM199) supplemented with 2% (LA, DHLA) or 10% (OET) fetal calf serum (FCS; Gibco, Grand Island, NY, USA), 0.1 IU bLH and 0.1 IU bFSH (Sioux Biochemicals, Sioux City, IA, USA). COCs were matured in the presence of either LA (100 µm) or DHLA (100 µm) alone or in combination with L-cystine (CYS; 0.6 mm), CYS alone, or OET at 1, 3, and 5 mm. COCs matured under control and experimental conditions were denuded of cumulus cells (40 IU hyaluronidase) and scored for maturity. GSH content of MII oocytes was determined by colorimetric assay (Northwest Life Science Specialties, LLC, Vancouver, WA, USA). Oocytes matured in OET were inseminated with frozen/thawed bull sperm (2 � 106 mL-1), cultured to the blastocyst stage (COOK bovine medium, COOK Australia, Brisbane, Queensland, Australia), and subjected to differential cell count (propidium iodide/Hoechst). GSH levels (mean � SEM) and developmental data (percentage) are expressed for n = 18–73 oocytes or embryos and were analyzed by ANOVA or chi-square test (significance, P ≤ 0.05). LA alone failed to increase oocyte GSH content over 2% FCS control levels (6.98 � 0.22 pmol/oocyte v. 5.26 � 0.4 pmol/oocyte). DHLA alone significantly increased oocyte GSH content (9.64 � 0.8 pmol/oocyte) compared to both LA and controls (10% FCS; 4.78 � 0.36 pmol/oocyte). CYS alone (10.18 � 0.58 pmol/oocyte) or in combination with LA (10.84 � 0.37 pmol/oocyte) or DHLA (9.75 � 0.66 pmol/oocyte) significantly increased GSH compared to controls. GSH content of MII oocytes matured in 5 mm OET (8.35 � 0.35 pmol/oocyte) was significantly higher compared to control (5.07 � 0.32 pmol/oocyte), 1 mm (4.21 � 0.18 pmol/oocyte), and 3 mm (7.12 � 0.35 pmol/oocyte) OET treatments. Maturation rates of oocytes were significantly reduced in 2% FCS (51.1–72%) compared to 10% FCS (90.5%). OET treatment (1–5 mm) did not significantly alter maturation rate compared to control (75–89.8%). Blastocyst development of IVM oocytes treated with 1 mm OET (22.5%) was significantly lower compared to 3 mm (42.3%) and 5 mm (41.1%) OET but not to control (33.6%). Blastocysts from IVM oocytes treated with 5 mm OET had significantly higher cell counts compared to controls (126 � 6.4 cells v. 100.8 � 5.2 cells). Bovine IVM is a valuable model for testing the efficacy of various strategies to increase oocyte cellular GSH. Both strategies improve oocyte GSH levels, and an increase in blastocyst cell number occurred with GSH donor treatment (5 mm OET).