PARENT SESSION
Oocyte Development
88
OKADAIC ACID INDUCES THE MITOCHONDRIAL ACTIVITY OF BOVINE COCS DURING IN VITRO MATURATION.
Choi, Sun-Ho1, Seong, Hwan-Hoo1, Oh, Sung-Jong1, Chang, Won-Kyung1, Chung, Il-Jeong1, Yoon, Jong-Taek2, Chung, Yung-Ho3, 1 2 3
ABSTRACT- Okadaic acid (OA), a specific phosphatase inhibitor, can stimulate formation of maturation promoting factor (MPF) and MPF is known to be a key regulator of both mitotic and meiotic cell cycle. Even though high concentration (25 nM) of OA increased plasminogen activator activity in porcine oocytes cumulus cell complex, but inhibited only progression to metaphase II. In this study, experiments was conducted to examine the effects of OA on metaphase of meiosis II and the mitochondrial activity of cytoplasm in bovine COCs during in vitro maturation. Bovine COCs were collected from the slaughterhouse cow ovary and matured in TCM199 supplemented with 0.1% PVA, 25ug/ml cycloheximide (CX), 25ug/ml CX (6hrs culture) plus 2 uM OA or 2 uM OA only at a atmosphere 5% CO2, 95% air 39oC for 0, 6, 12 or 24 hrs. Nuclear types of matured COCs were removed cumulus cells by 0.5% hyaluronidase sol. and oocytes were fixed in 1:3 acetic acid ethyl alcohol for 72 hrs and then stained with 0.1% aceto-orcein sol. For the detection of fluorescent intensity (FI) of matured oocytes, cumulus cells were removed same as performed above and were stained with 20 nM mito tracker for 20 min. at 39oC. Mitochondrial activity with FI of matured oocytes was imaged by laser confocal microscopy (Fluoview, Olympus, Japan) and were measured scanned face on 5 um from median to endpoint of oocytes. Statistical analysis of nuclear types observed the three replicates was carried out with ANOVA and Fisher,s protected least significant difference test using the STATVIEW program. FI of matured oocytes was compared the multiples of the least intensity among the measured oocytes. Maturing in TCM199 supplemented with 0.1% PVA, 25 ug/ml CX, 25 ug/ml CX plus OA or 2 uM OA for 24 hrs, the nuclear stage of metaphase II were 74%, 7%, 45% 51% or 73%, respectively. There were much significant differences among the treatment (P<0.01). When the COCs were matured in 0.1% PVA, CX plus OA and 2 uM OA only, FI was revealed the increasing tendency during the process of maturation. Whereas FI in CX was decreased about 3 times compared to the other treatments of 6hrs maturation. We conclude that OA regulates bovine COCs maturation and induces the mitochondrial activity during the process of maturation.
KEY WORDS: Okadaic acid, mitochondrial activity, bovine COCs, IVM