327THE EFFECTS OF GLUCOSE AND GLUCOSAMINE ON CUMULUS EXPANSION AND NUCLEAR MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES

Abstract

Glucose is the primary energy substrate consumed by bovine COCs during in vitro maturation (IVM), with most accounted for by glycolysis (L-lactate production). However, antral follicular fluid (FF) contains less than half the glucose of standard IVM media (TCM199=5.6mM, FF=2.3mM). We have previously demonstrated that from 20 to 24h of IVM, a significant proportion of the glucose utilized is directed into pathways other than lactate production (Sutton et al., 2003 Reproduction 126, 27–34). We hypothesize that glucose is utilized for cumulus matrix synthesis. The aim of this study was to determine the influence of glucosamine (an intermediate for matrix components) on FSH-stimulated glucose uptake and cumulus expansion. The influence of different glucose concentrations and glucosamine on nuclear maturation was also investigated. Bovine COCs were collected from abattoir-derived ovaries. In Exp. 1, individual COCs (n=60, 3 replicates) were cultured in 10-μL drops of TCM199 (plus pyruvate, hCG and BSA, containing 5.6mM glucose), ±FSH (0.1IUmL−1) and ±glucosamine (5mM). After 20h, COCs were transferred to fresh media and cultured a further 4h. Cumulus expansion and glucose/L-lactate levels in spent medium from 0–4-h and 20–24-h culture periods were measured. In Experiment 2, COCs (n=300, 6 replicates) were cultured in groups of 10 in 100μL of Bovine FF medium (a defined medium based on the composition of bovine antral FF, also containing amino acids, FSH, hCG and BSA) ±glucosamine (5mM) in 2.3 or 5.6mM glucose, or in conventional TCM199 IVM media (as above). Nuclear maturation was assessed at 24 and 30h using orcein staining. Treatment differences were determined using two-way ANOVA. The influence of FSH and glucosamine (Exp. 1) on the measured parameters was evident at 20–24h, with FSH increasing diameter, glucose uptake and L-lactate production (P<0.05). Although glucosamine alone did not influence diameter or glucose/L-lactate concentrations, glucosamine plus FSH led to a decrease in glucose uptake compared to FSH-stimulation alone (P<0.05). The proportion of oocytes at MII (Exp. 2) was significantly lower when COCs were cultured in low glucose (main effect, 24h: 2.3mM=38% v. 5.6mM=64%; P<0.005). The presence of glucosamine tended to stimulate meiotic maturation (main effect, 24h: 0mM=45% v. 5mM=59%; P=0.1). MII frequency in TCM199 controls at 24h was 68%. These experiments support the hypothesis that synthesis of cumulus matrix is a major pathway for glucose metabolism, especially in the absence of glucosamine. Furthermore, oocytes matured in media based on a physiological concentration of glucose (2.3mM), have delayed meiosis compared to oocytes cultured in higher glucose (5.6mM). Thus, glucose has multiple functions, involving matrix formation and meiosis regulation during bovine IVM. Supplementation of medium with glucosamine appears to partly reduce the dependency of COCs on glucose. Supported by Australian Research Council and COOK Australia.