胰岛素对犬卵母细胞体外成熟的影响

研究不同浓度胰岛素及不同培养时间对犬卵母细胞体外成熟率的影响，为改善犬卵母细胞体外培养体系提供参考，采用切割法收集卵巢表面卵丘—卵母细胞复合体(cumulus oocyte complexes, COCs)，在含有0.6%葡萄糖的TCM199中添加不同浓度的胰岛素（0、3、6、9 IU/mL），38.5 ℃、5% CO₂培养箱内成熟培养，观察卵丘扩散程度，剥离卵丘细胞获得裸卵后，室温下固定15 min；Hoechst 33342染色，压片，荧光显微镜下观察核形态，用SPSS 14.0软件统计试验数据。不同浓度胰岛素培养48 h后，各组卵丘细胞扩散效果都不明显；卵母细胞核成熟期没有达到减数分裂中期（MⅡ），但是6 IU/mL胰岛素组生发泡破裂期（GVBD）比率（35.88%±14.63%）显著高于对照组（11.25%±9.75%）；6 IU/mL胰岛素组延长培养时间至72和96 h后，MⅠ-MⅡ期卵母细胞成熟率分别为13.33%±1.5%、20.8%±1.9%。以上结果表明，犬体外成熟培养基中添加胰岛素既没有提高犬卵母细胞核成熟到MⅡ期，也没有改善犬卵母细胞卵丘扩散效果。但是，在6 IU/mL浓度下延长培养时间，相对增加了成熟率。

The Effect of Insulin on Maturation of Canine Oocytes in vitro

In the present study we investigated the effects of supplementing the in vitro maturation (IVM) medium with insulin on the incidence of maturation to MⅡ. In order to provide parameters for canine oocyte IVM system, cumulus oocyte complexes (COCs) were released by slicing; oocytes were cultured in TCM199 supplemented with 0.6% gulocse and different concentrations of insulin (0, 3, 6, or 9 IU/mL) at 38.5 ℃, 5% CO₂ in air. Observed cumulus cells which were removed from around oocytes using a small glass pipette, denuded oocytes were fixed in then Hoechst 33342 at room temperature for 15 min. Nuclear status was observed under UV light using a fluorescence microscope. Longer culture time to 72 and 96 hours, then statistical analysis by SPSS 14.0. There is no significant different on cumulus expansion. No oocyte development to MⅡbut the percentage of oocytes at GVBD stage was different among the groups 6 IU/mL, and the control. The percentage of oocytes get to M Ⅱin the 72 and 96 h treatment groups with 6 IU/mL insulin groups 20.8%±1.9% vs 13.33%±1.5%, respectively. These results indicated that the addition of insulin to the in vitro maturation medium of canine oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect cumulus expansion, however, the maturation rate of oocyte increased by delayed culture period.