Optimization of Indirubin-3''-oxime Concentration for In Vitro Development of Porcine oocytes

• Main Authors: Shih-Wen Sha, 沙士文
• Other Authors: Ju Jyh-Cherng
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/29811281260008153629

碩士 === 中興大學 === 動物科學系所 === 95 === Pig oocytes undergo spontaneous germinal vesicle breakdown (GVBD) after being released from follicular environment which potentially hinders GV-related manipulation. It has also been proposed that inhibition of GVBD could enhance development of immature oocytes. The aim of this study was to investigate the effects of specific cdc2 kinase inhibitors - indirubin oxime (IO), on the prevention of GVBD, cytoskeletal changes, and subsequent embryo development in porcine oocytes. In Experiment 1, cumulus-oocyte-complexes (COCs) were treated with low (0, 1.56, 3.13, 6.25 and 12.5 mM) or high (25, 50, 75 and 100 mM) concentrations of IO for 22 h during IVM followed by cytoskeleton examination. Results showed that most of oocytes (more than 90%) remained at the GV stage when IO concentration was higher than 6.25 mM. Some oocytes maintained at GV stage by IO showed fragmented or pulverized ooplasmic microtubule. In Experiment 2, resumption of meiosis after IO removal was evaluated. Twelve to fifty percentage of oocytes underwent GVBD and reached the MII stage. In the high concentration groups, the resumed maturation rate was higher in 25 mM group (60%) than other treatment groups, but all were significantly lower than those in the control groups. In contrast, the maturation rates of all lower concentration groups were 62-77%, and the cytoskeleton of mature oocytes showed normal morphologies. In Experiment 3, the development of IO-treated oocytes was evaluated. With the exception of 12.5 mM group (93%, P < 0.05), the cleavage rates at day 2 had no significant differences among treatments and control (96-97% vs. 99%, P > 0.05). In blastocyst rate, all the treatments groups were significantly lower than the 44 h control group (6-20 vs. 40%, P < 0.05), with no differences among all groups in total cell numbers/blastocysts. Taken together, this study showed that appropriate concentration of IO can effectively prevent GVBD of porcine oocytes without affecting the cytoskeletal confignration and blastocyst rate. Those abnormal cytoskeletal morphologies were proposed being due to prolonged maturation time. Further investigations at molecule level are required to uncover the effects of CDK inhibitors on porcine oocyte maturation in the future.