342 DISTINCT EFFECTS OF FOLLICULAR FLUID ON CUMULUS CELL APOPTOSIS AND PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Abstract

Supplementing medium with follicular fluid (FF) during in vitro maturation (IVM) enhances the developmental competence of porcine oocytes, indicating that factors present in FF are beneficial to cytoplasmic maturation. Previous findings suggest that porcine FF contains high levels of superoxide dismutase activity and exerts a beneficial effect on cytoplasmic maturation by protecting oocytes from oxidative stress (Tatemoto et al. 2004 Biol. Reprod. 71, 1150–1157). Since oxidative stress is a potent inducer of apoptosis, the aim of the present study was to examine the temporal effects of FF during IVM on cumulus cell apoptosis and oocyte developmental competence. Ovaries of prepubertal pigs were collected from a local abattoir and antral follicles, 3 to 7 mm in diameter, were aspirated. Cumulus–oocyte complexes (COCs) with at least 3 uniform layers of compact cumulus cells (CCs) were recovered, washed, and transferred to maturation medium (MM) with or without 25% FF. At 22 h of IVM, COCs from each group were washed and transferred to fresh MM with or without 25% FF, forming 4 groups: -FF/-FF, -FF/+FF, +FF/-FF, and +FF/+FF. Cohorts of COCs were TUNEL stained at 22 and 44 h of IVM using the In Situ Cell Death Detection kit (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer's instructions, and apoptotic CCs were visualized using confocal microscopy. Oocytes denuded at 44 h, that had a polar body, were treated with ionomycin and 6-dimethylaminopurine to induce parthenogenetic development, and were cultured for 7 days in NCSU-23 medium at 38.5°C in 5% O₂, 5% CO₂, and 90% N₂. Data were subjected to ANOVA and Tukey's post-hoc test. At 22 h of IVM, the presence of FF reduced the proportion of apoptotic CCs in COCs (2.1% vs. 4.6%). COCs matured with FF from 22 to 44 h of IVM had much lower proportions of apoptotic CCs (+FF/+FF: 0.9%; −FF/+FF: 2.6%) compared with those matured without FF (+FF/−FF: 10.3%; −FF/−FF: 17.8%). The rate of maturation to the metaphase-II stage was greater when oocytes were matured with FF from 0 to 22 h of IVM (−FF/−FF: 68.6%; −FF/+FF: 72.8%; +FF/−FF: 89.2%; +FF/+FF: 86.2%). Maturation without FF for the entire IVM interval reduced the proportion of activated oocytes that formed blastocysts compared with the other groups (−FF/−FF: 25.1%; −FF/+FF: 44.6%; +FF/−FF: 46.6%; +FF/+FF: 47.3%). Despite a 4-fold difference in the proportion of apoptotic CCs between COCs of the +FF/−FF and −FF/+FF groups, exposure to FF for the first or second half of IVM was as beneficial to oocyte developmental competence as exposure to FF for the entire IVM interval. This suggests that the protective effect of FF in reducing oxidative stress on oocytes during IVM is distinct from the effect on oocyte developmental competence.