Abstract
The present study was conducted to examine the effect of ascorbic acid (AsA) on the cytoplasmic maturation of cumulus-denuded porcine oocytes during in vitro maturation (IVM) culture. After mechanical removal of cumulus cells, cumulus-denuded oocytes (DOs) were cultured for 44 h in BSA-free NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and 0 to 750 μM ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative, and then assessed for their developmental changes, including the progression of meiosis, in vitro fertilization (IVF) parameters and subsequent embryo development. Treatment with 250 μM AA-2G during IVM culture in DOs showed no influence on the rates of maturation (60-61%), sperm penetration (86%), and polyspermy (69-71%) following IVF, but enhanced the ability (43%) to form male pronucleus (MPN) compared to that of DOs treated without AA-2G (20%) (P<0.05). Although this rate of MPN formation of DOs was still lower than that of COCs (66%) (P<0.05), the addition of 250 μM AA-2G to maturation medium in DOs apparently enabled IVM-IVF-derived zygotes to develop to the blastocyst stage (3%). The levels of intracellular AsA and glutathione in DOs matured with AA-2G were significantly higher than those in DOs matured without AA-2G (P<0.05). Moreover, the length of DNA migration, analyzed using the comet assay, was increased by oxidative damage in DOs treated without AA-2G during IVM culture, but treatment with AA-2G abated DNA damage, indicating that intracellular AsA supplied by AA-2G can potentiate the cellular protection of oocytes against oxidative stress through its scavenging activity during IVM culture. Thus, the present findings suggest that the preventing oxidative stress in oocytes during meiotic maturation supports the cytoplasmic maturation responsible for the subsequent developmental competence post-fertilization.
