247 ADENOSINE TRIPHOSPHATE CONTENT AND SUPEROXIDE DISMUTASE ACTIVITY IN SINGLE OOCYTES BEFORE AND AFTER IN VITRO MATURATION

Abstract

The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Analyses of energy and redox status parameters are emerging technologies useful for further oocyte quality characterisation. Mitochondrial (mt) activity is a necessary feature involved in cytoplasmic maturation, and the primary function of mitochondria is adenosine triphosphate (ATP) production. Mitochondria distribution pattern and ATP content are important parameters in the evaluation of oocyte metabolic activity, particularly activities driving microtubules dynamics leading to chromosomes segregation. Superoxide dismutase (SOD), a first-line antioxidant enzyme, has also been hypothesised as being associated to oocyte quality. The aim of the present study was to analyse ATP content and SOD activity in single equine oocytes examined before and after in vitro maturation. Cumulus–oocyte complexes surrounded by a compact cumulus oophorus were recovered from the ovaries of slaughtered mares and analysed before or after in vitro maturation (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent evaluation of signs of meiotic maturation, and only those oocytes showing cumulus expansion, regular ooplasmic size (>160 μm in diameter) and morphology, and 1st polar body extrusion were selected for analysis. Adenosine triphosphate intracellular levels were analysed by luciferin-luciferase bioluminescent reaction (ATPlite, PerkinElmer, Monza, Italy). Quantification of SOD activity was performed by spectrophotometrical assay with WST1 and by polyacrylamide native gel and nitro blue tetrazolium reduction method. Intracellular ATP levels were influenced by meiotic stage in that oocytes at the germinal vesicle stage (GV, n = 15) showed 1.25 ± 0.8 pmol cell^–1, whereas metaphase II (MII) oocytes (n = 15) showed significantly higher levels (2.29 ± 1.69 pmol cell^–1; P < 0.05). This is in line with our previous observations on mt distribution pattern analysed by Mitotracker Orange CMTM Ros staining and confocal microscopy (Ambruosi et al. 2009). In vitro-matured MII oocytes showed significantly higher rates of perinuclear mt distribution pattern, indicating mt aggregation around meiotic metaphase spindle, compared with GV oocytes (3/12, 25% v. 0/13, 0% in GV oocytes; P < 0.05). Superoxide dismutase spectrophotometrical activity was 0.72 ± 0.55 U mg^–1 prot in GV oocytes (n = 4) and 2.33 ± 0.33 U mg^–1 prot in MII oocytes (n = 2; P < 0.001). In native gel SOD activity was 16 285.05 arbitrary densitometric units (ADU) in a GV oocyte and 22 501.35 ADU in a MII oocyte. To our knowledge, this is the first study reporting intracellular SOD activity in single oocytes in mammals. Moreover, this is the first study reporting ATP content in single equine oocytes. Observed quantitative differences seem to be related to meiotic stage.