小鼠体外发育卵母细胞生长分化因子-9基因表达

Growth differentiation factor-9 gene expression in in vitro cultured oocytes in rats

目的体外培养小鼠窦前卵泡得到MⅡ期卵母细胞,比较发育过程中体外与体内卵母细胞生长分化因子-9(GDF-9)的基因表达量,初步探讨GDF-9的表达对卵母细胞体外发育成熟的影响。方法出生D10雌性昆明小鼠50只,机械方法分离窦前卵泡,体外培养12d。分别于体外培养D2、D4、D6、D8、D10、D12分离卵母细胞作为体外发育组;同窝雌性小鼠出生后D12、D14、D16、D18、D20、D22卵母细胞作为体内发育组;使用半定量逆转录多聚酶链反应技术分别检测两组单个卵母细胞GDF-9基因表达量。应用计算机全自动图像分析仪测量PCR产物电泳条带的几何平均光密度,基因表达量用相对光密度表示:检测基因光密度/管家基因(β-actin)光密度。结果培养第12天观察306个卵泡,274个卵泡成活(89.5%),143个窦腔形成(51.8%);第13天观察,155个卵母细胞成熟(56.6%)。体外发育D2、D4、D6、D8、D10、D12卵母细胞GDF-9基因表达相对光密度分别是0.83±0.08、0.52±0.09、0.45±0.13、0.49±0.09、0.49±0.09、0.68±0.08;体内发育D12、D14、D16、D18、D20、D22卵母细胞GDF-9基因表达相对光密度分别是0.64±0.35、0.48±0.10、0.52±0.10、0.66±0.08、0.72±0.09、0.91±0.11;体外发育D8￣12卵母细胞GDF-9表达量明显低于同期体内发育卵母细胞(P<0.05)。结论小鼠窦前卵泡体外培养后可以生长发育,部分得到成熟的卵母细胞。小鼠卵母细胞GDF-9基因表达量随发育时间的改变发生变化;体外发育D8￣12卵母细胞GDF-9基因表达量低于同期体内发育的卵母细胞可能是其发育潜能较低的原因之一。

Objective To explore the relation between oocyte maturation and growth differentiation factor-9 （GDF-9） gene expression. Methods Ovariectomy was performed in 50 Kunming female mice of 10 days old, and the preantral follicles were isolated from the ovaries and cultured in medium drops for 12 days. Oocytes and somatic cells were mechanically isolated. The oocytes cultured in vitro for 2, 4, 6, 8, 10, and 12 days constituted the in vitro cultured group and the oocytes obtained from female mice of 12, 14, 16, 18, 20, and 22 days old served as the in vivo group. Semi-quantitative RT-PCR and agar gel electrophoresis were performed to quantify GDF-9 gene expression in each oocyte. Results Follicle survival, antrum formation and maturation rate was 89.5%, 51.8% and 56.6% in the in vitro cultured follicles, respectively. GDF-9 gene expression on days 2, 4, 6, 8, 10, and 12 in in vitro cultured oocytes was 0.83±0.08, 0.52±0.09, 0.45±0.13, 0.49±0.09, 0.49±0.09, and 0.68± 0.08, respectively; GDF-9 gene expression in in vivo grown oocytes of 12, 14, 16, 18, 20, and 22 days were 0.64±0.35, 0.48±0.10, 0.52±0.10, 0.66±0.08, 0.72±0.09, and 0.91 ±0.11, respectively. Between days 8 and 12, GDF-9 gene expression in in vitro cultured oocyte was significantly lower than that in in vivo grown oocytes （P〈0.05）. Conclusion MII oocytes can be obtained from in vitro culture of the preantral follicles. GDF-9 gene expression in the oocytes varies with their growth stages. Between days 8 and 12 of in vitro culture, GDF-9 gene expression in the cultured oocytes is different from that in in vivo grown oocytes.