effects of butyrolactoneⅠ on maturation and development competence of porcine oocytes In vitro | 丁内酯-Ⅰ影响猪卵母细胞的体外成熟和发育能力

中国人. 体外成熟的卵母细胞存在核成熟与胞质成熟不一致的情况，从而使得体外成熟的卵母细胞质量不高。本实验采用cdc2激酶抑制剂丁内酯-Ⅰ(butyrolactone Ⅰ, BL-Ⅰ)抑制猪卵母细胞核体外成熟，期望解决卵母细胞体外培养过程中细胞核与细胞质发育的不同步问题。结果表明，分别用0、10、20、40和80 μmol/L BL-Ⅰ处理猪卵母细胞44 h后，对卵母细胞成熟的抑制程度与BL-Ⅰ浓度的升高成正比。用20、40和80 μmol/L处理时，处于生发泡(GV)期的卵母细胞分别为25%、47%和67%，与对照组(5%)相比差异极显著(P＜0.01)；而到达MⅡ期的卵母细胞分别为38%、9%和0，与对照组(67%)相比差异极显著(P＜0.01)。将20、40和80 μmol/L处理44 h后的卵母细胞，再在不含BL-Ⅰ的卵母细胞体外成熟培养液中培养44 h后，到达MⅡ期的卵母细胞为57%、41%和5%，与抑制44 h时相比均有显著差异，表明BL-Ⅰ对卵母细胞成熟的抑制作用是可逆的。20 μmol/L BL-Ⅰ抑制培养20 h再正常培养24 h(20 h＋+24 h-处理组)后卵母细胞的成熟率显著低于对照组(正常培养44 h)(65.6 % vs 70.1%， P＜0.05)。对20 h＋+24 h-处理组成熟的卵母细胞进行孤雌激活，其卵裂率、囊胚率和囊胚总细胞数均好于对照组，但无显著差异；对20 h＋+24 h-处理组成熟的卵母细胞进行核移植(NT)，获得重构胚的卵裂率、囊胚率和囊胚总细胞数均优于对照组，并且卵裂率有显著差异(68.9 % vs 62.4%， P＜0.05)。结果提示BL-I处理对猪卵母细胞的核成熟有明显抑制作用，这种抑制作用是可逆的，对猪卵母细胞孤雌胚胎发育能力和核移植胚胎的发育能力有一定的提高。

英语. There is not synchronized between nuclear maturation and cytoplasmic maturation of in vitro maturated oocytes. In order to solve this problem, we use butyrolactone I (BL-Ⅰ), cdc2 kinase inhibitor, to prevent porcine oocyte nuclear maturation in vitro. Porcine oocytes were cultured for 44 h in the medium containing different concentrations of BL-Ⅰ(0, 10, 20, 40 and 80 mol/L, respectively). With the increase of BL-Ⅰ concentration, the inhibition efficiency on maturation of porcine oocytes was increased. When treated with BL-Ⅰ in 20, 40 and 80 mol/L, respectively, the oocytes reached 25%, 47% and 67%, respectively, at germinal vesicle(GV) stage, and 38%, 9% and 0% at MII stage, which were both significant difference with control group. In addition, After removed BL-Ⅰand incubated for additional 44 h in ooycte maturation media, these oocytes at MⅡ stage were significant increased and reached 51%, 47% and 5%, respective, which showed the BL-Ⅰinhibition to maturation of oocytes is reversible. Additionally, oocytes incubated with 20 μmol/L BL-Ⅰ for 20 and 24 h without BL-Ⅰ (20 h＋+24 h-treatment group), showed low percentage at MⅡ stage compared with the control group (culture for 44 h without BL-Ⅰ) (P 0.05, 65.6% vs 70.1%). When the matured oocytes from 20 h＋+24 h- treatment group were parthenogenetic activated, the cleavage rate, blastocyst rate and blastocyst total cell number had increased, but no significant difference compared with the control group. When the matured oocytes were used for nuclear transfer, the cleavage rate, blastocyst rate and blastocyst total cell number were better than that of the control group, but only significant difference in cleavage rate (P 0.05, 69% vs 62%). These results indicate that BL-Ⅰ can significantly inhibit the porcine oocytes maturation, and that the inhibition can be reversed by removing BL-Ⅰ. These results also show that matured oocytes treated with the proper concentration of BL-Ⅰ can slightly improved the development ability of parthenogenetic and nuclear transfer embryos.