340 EFFECT OF GLUCOSE ON THE MORPHOLOGY OF GERMINAL VESICLE AND MEIOTIC PROGRESSION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

Abstract

Glucose metabolism through the pentose phosphate pathway (PPP) seems to play a critical role in meiotic resumption in mouse oocytes (Downs et al. 1998 Biol. Reprod. 58, 1084–1094). However, the role is not clear in porcine oocytes. In the present study, we examined whether glucose affects morphological change of germinal vesicles and the resumption of meiosis in porcine oocytes in a chemically defined medium. In the first experiment, porcine cumulus–oocyte complexes (COCs) were collected from 3–6-mm follicles of slaughterhouse ovaries and cultured in a chemically defined medium, mNCSU37-PVA with/without 5.55 mM glucose in the presence of eCG, hCG, and dibutyryl cAMP for 20–22 h and then in the absence of eCG, hCG, and dibutyryl cAMP for 24 h. In the second experiment, 5.55 mM glucose in the maturation medium was replaced with the same concentration of Na pyruvate. In the third experiment, the PPP inhibitor 6-aminonicotinamide (6-AN) was added to the maturation medium at various concentrations (0, 10, 50, and 100 µM). To determine the activity of glucose-6-phosphate dehydrogenase (G6PD), OCCs were fixed, blocked, and treated with anti-G6PD polyclonal antibody and the secondary antibody labeling a fluorescent material. Results from 3–5 replicates were analyzed by ANOVA and Duncan's multiple range test. When OCCs were cultured in glucose-free chemically defined maturation media, regardless of the presence of hormones and dibutyryl cAMP, germinal vesicle breakdown (GVBD) of oocytes was inhibited (10.0–21.3%), as compared with OCCs cultured in the presence of glucose and hormones (91.4–92.0%). In a majority of oocytes in which GVBD was inhibited, the arrest occurred at the GV-I stage. When OCCs were cultured in maturation media in which glucose was replaced with Na pyruvate, GVBD was not inhibited any more than in control samples that were cultured in the presence of glucose (97.4% vs. 97.1%). However, the incidence of oocytes developing to the metaphase II stage was significantly lower in this condition than in controls (4.8% vs. 49.9%, respectively). A majority of the oocytes were at the metaphase I stage (86.0% vs. 45.5% in controls). The presence of 6-AN in maturation media significantly inhibited GVBD of oocytes (77.3, 29.0, 7.4, and 8.4% at 0, 10, 50, and 100 µM, respectively) and arrested the oocytes at the GV-I stage. Immunocytochemistry with anti-G6PD demonstrated the activity of G6PD in cumulus cells of OCCs. In conclusion, these results demonstrate that glucose plays a critical role in the release of porcine oocytes arrested at the GV-I stage, probably through PPP of cumulus cells. The current results also suggest the possibility of gluconeogenesis in porcine OCCs when glucose in maturation media was replaced with Na pyruvate.