173 EFFECTS OF GLUCOSE METABOLISM DURING IN VITRO MATURATION ON CYTOPLASMIC MATURATION OF GOAT OOCYTES

Abstract

It is well known that oocyte maturation consists of 2 processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to resumption of the first meiosis and extrusion of the first polar body (PB1), and cytoplasmic maturation is manifested as acquisition of the ability to complete pre-implantation development. Although it is recognised that energy supply is essential for oocyte maturation and there have been many reports on the effect of glucose metabolism on oocyte nuclear maturation, studies on the effect of glucose metabolism on ooplasmic maturation are limited. In the present study, goat oocytes recovered from slaughterhouse ovaries were cultured for 24 h in a simplified CR1 (sCR1) medium (NaCl, KCl, NaHCO₃, CaCl₂, BSA, and eCG) supplemented with glucose (10 mM) and/or lactate (3.5 mM) in the presence or absence of pentose phosphate pathway (PPP) inhibitor dehydroepiandrosterone (DHEA, 100 μM) or glycolysis inhibitor iodoacetate (1 μM). At the end of maturation culture, oocytes with PB1 were either activated by treatment with ionomycin plus 6-DMAP to observe embryo development, or assayed for total glutathione concentrations (GSX) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios. Embryos were cultured for 9 days in CR1aa medium (NaCl, KCl, NaHCO₃, calcium lactate, sodium pyruvate, glutamine, EAA, NEAA, and FCS) at 38.5°C under 5% CO₂ in humidified air. In the absence of inhibitors, oocyte maturation rates of 82, 65, and 76%, and blastocyst rates of 7, 0, and 7%, were obtained, respectively, after oocytes were matured in sCR1 supplemented with glucose, lactate, or both. When oocytes were matured in sCR1 containing glucose and lactate in the presence of DHEA or iodoacetate, oocyte maturation rates were 69 and 67%, respectively, with no blastocyst produced in either case. However, whereas the presence of DHEA produced 12% morulae, no morulae were observed in the presence of iodoacetate. Furthermore, GSX concentrations (pmol/oocyte) were 8.5, 6.5, and 7.2, whereas GSH/GSSG ratios were 1.8, 0.3, and 0.5, respectively, after oocyte maturation without inhibitors or with 300 μM DHEA or 3 μM iodoacetate. The difference in GSX concentration was statistically significant (P < 0.05; one-way ANOVA) between DHEA and iodoacetate. In conclusion, using a culture system (sCR1 containing 3.5 mM lactate) that sustained oocyte nuclear maturation but did not support blastocyst development, we have studied the effect of PPP and glycolysis of glucose metabolism on the cytoplasmic maturation of goat oocytes. The results suggest that both PPP and glycolysis are essential for ooplasmic maturation of goat oocytes, and that both promote oocyte cytoplasmic maturation by increasing glutathione synthesis and reduction.

This study was supported by grants from the National Basic Research Program of China (Nos. 2012CB944403 and 2014CB138503) and the China National Natural Science Foundation (Nos. 31272444 and 30972096).