体外培养小鼠卵母细胞及其生长分化因子-9基因表达

体外培养小鼠的窦前卵泡以得到第二次减数分裂中期(MⅡ)卵母细胞,比较体外发育卵母细胞与体内生长的卵母细胞生长分化因子-9(GDF-9)的基因表达量,探讨GDF-9的表达对卵母细胞体外发育成熟的影响。选择体外培养第2天(D2)、D4、D6、D8、D10、D12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;半定量逆转录多聚酶链反应技术分别检测两组MⅠ卵母细胞GDF-9基因表达量。结果体外培养小鼠窦前卵泡可以得到MⅡ期卵母细胞,卵泡成活率、窦腔形成率、卵母细胞成熟率分别达到89·5%、51·8%和56·6%。小鼠卵母细胞GDF-9基因表达量随发育时间的改变而发生变化,而体外发育D8—12卵母细胞GDF-9表达量显著低于同期体内发育卵母细胞(P<0·05)。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的原因之一可能是其发育潜能较低。

Growth Differentiation Factor-9 Gene Expression of Mice Oocytes in Vitro and in Vivo

Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ (MⅡ) oocytes. Oocyte growth differentiation factor-9 (GDF-9) gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of 10-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 (D2), D4, D6, D8, D10, D12 cultured in vitro were named the in vitro group and oocytes of day 12 (D12), D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8-12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo (P＜0.05). We conclude that MⅡ oocytes can be obtained from in vitro culture of preantral follicles. The GDF-9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cultured in vitro may be the cause of their low developmental capacity.