DIFFERENCES IN METABOLIC PATTERNS IN DOG OOCYTES MAY EXPLAIN VARIATIONS IN IN VITRO MATURATION SUCCESS

Ovarian follicular size influences developmental competence of domestic dog oocytes. Specifically, oocytes from follicles > 2 mm in diameter are more successful at completing nuclear maturation than those from follicles 2 mm or less. The objectives were to determine the influence of follicular size, maturation time and meiotic status on patterns of glycolysis, glucose oxidation, pyruvate uptake and glutamine oxidation in the dog oocyte. Our hypothesis was that metabolism differed between oocytes in large versus small follicles. Oocytes (n = 531) from adult ovaries were classified into three groups based on follicular size (small, < 1 mm, n = 252 oocytes; medium, 1 to 2 mm, n = 231; and large, > 2 mm, n = 48). Oocytes were cultured (38.5°C in 5% CO2) for 0, 24 or 48 h in TCM 199 + 25 mM βmercaptoethanol + 10 ng/ml epidermal growth factor + 0.25 mM pyruvate + 2.0 mM glutamine + 0.1% polyvinyl alcohol (IVM medium). After in vitro culture, oocytes were denuded, assessed for metabolic activity and evaluated for nuclear status. For metabolic assessments, oocytes were incubated for 3 h (38.5°C) in 3 ml of IVM medium containing (1) 0.005 mM [0.064 mCi/ml] D-53H-glucose (glycolysis) + 1 mM D-614C [0.053 mCi/ml] glucose (glucose oxidation) or (2) 0.001 mM [0.041 mCi/ml] L-G-3H-glutamine + 1 mM [0.027 mCi/ml] 1-14C pyruvate, placed on the lid of a centrifuge tube containing 25 mM NaHCO3 and trapped radioactivity measured. Only oocytes at an appropriate stage of nuclear maturation for each culture period (n = 380) were included in data analysis (e.g., oocytes with a germinal vesicle at 24 and 48 h culture were excluded). Differences in oocyte metabolism among groups were analyzed by ANOVA (main effects being follicular class, culture interval and meiotic status). Oocytes from large follicles metabolized all substrates at a higher rate than those from smaller ones (P < 0.05). Glucose metabolism also was influenced by duration of culture and meiotic status (P < 0.05). Among all follicular groups, glycolytic rate was lowest in oocytes cultured for 24 h (P < 0.05) compared to 0 and 48 h. However, there was no difference (P > 0.05) in glycolytic activity between oocytes immediately after release from the follicle and after culture for 48 h. At 48 h after in vitro culture, glycolytic rate of metaphase II oocytes was significantly higher than that of metaphase I (P < 0.05). Glucose oxidation of oocytes from large follicles decreased (P < 0.05) over the culture interval compared to constant (P > 0.05) values for oocytes from small and medium follicles. At culture onset (0 h), oocytes from small follicles predominantly metabolized pyruvate, while those from medium and large follicles primarily metabolized glucose (P < 0.05). Although oocytes from large follicles metabolized glutamine at a higher rate at 24 h than other energy substrates (P < 0.05), there was no change (P > 0.05) in substrate utilization among oocytes obtained from small and medium follicles. In conclusion, dog oocytes from different size ovarian follicles express different metabolic activities when cultured in vitro. The elevated metabolic activity of oocytes from large follicles may be responsible for their higher developmental competence during in vitro maturation. (Funded by NIH-5-KO1RR020564-02). (poster)