小鼠不同阶段卵泡同步分离方法的初步建立及评估

Preliminary establishment and evaluation of a method for synchronized isolation of mouse follicles across different developmental stages

目的:建立并评估同步分离卵巢内不同发育阶段卵泡的方法——酶消化后滤网过滤法。方法:运用混合有胶原酶Ⅰ和脱氧核糖核酸酶Ⅰ的消化酶来消化小鼠卵巢预切组织,然后依次通过不同孔径(100 μm、40 μm、20 μm)的滤网以实现不同发育阶段卵泡同步分离的目的,将翻转冲洗100 μm滤网得到的卵泡记为A组(>100 μm),翻转冲洗40 μm滤网得到的卵泡记为B组(40~100 μm),翻转冲洗20 μm滤网得到的卵泡记为C组(20~40 μm)。评估并比较经不同孔径滤网筛选获得的卵泡群的数量、大小、形态、活力和发育潜能。结果:数量上,C组获得的卵泡数目最多A组次之,B组最少;形态上,C组卵泡基底膜完整率高于A组和B组( P<0.001, P<0.001)。活力上,A组卵泡的存活率70.59%(120/170)高于B组51.79%(58/112)和C组35.90%(28/78)( P=0.001 6, P<0.001)。发育潜能上,经过96 h体外培养,A组卵泡直径由(113.64±9.57) μm增长至(150.95±45.90) μm( P=0.002 4),B组卵泡直径由(88.12±9.12) μm增长至(120.61±18.00) μm ( P<0.001),C 组卵泡在培养第24 h内出现单层颗粒细胞贴壁,卵母细胞与颗粒细胞之间失去连接结构,卵母细胞完全裸露,未能培养成功。 结论:酶消化后多重滤网过滤法能快速有效获得大量形态完整、有活力和发育潜能良好的卵泡,且能达到不同发育阶段卵泡同步分离的目的。

Objective:To establish and evaluate a follicle isolation method, combination of enzymatically digestion with strainer filtration, for synchronously collecting mouse follicles across different developmental stages.Methods:Pre-cutted ovarian tissue blocks were digested with enzymes mixed by Collagenase I and DNase I, and then filtered through three different pore size cell strainers to synchronously separate the follicles at developmental stages. The follicles obtained by turning over and washing the 100 μm strainer were recorded as group A (>100 μm), as same as the follicles obtained by turning over and washing the 40 μm strainer were recorded as group B (40-100 μm), and the follicles obtained by turning over and washing the 20 μm strainer were recorded as group C (20-40 μm). The quantity, morphology, viability and developmental potency were examined among these harvested follicles.Results:In terms of quantity, follicles in group C accounted for most, followed by follicles in group A, and the follicles in group B were the least. Morphologically, the basal membrane integrity rate of follicles in group C was higher than that of groups B and A ( P<0.001 and P<0.001, respectively). As for viability, the survival rate of follicles in group A was 70.59% (120/170), which was higher than that in group B (51.79%, 58/112) and group C (35.90%, 28/78) ( P=0.001 6 and P<0.001, respectively). In terms of developmental potency, after 96 h of in vitro culture, the diameter of follicles in group A increased from (113.64±9.57) μm to (150.95±45.90) μm ( P=0.002 4), and the diameter of follicles in group B increased from (88.12±9.12) μm to (120.61±18.00) μm ( P<0.001). In group C, monolayer-layer granulosa cells were attached to the follicles within 24 h of culture, and the connection structure between oocytes and granulosa cells was lost. The oocytes were completely exposed and failed to be cultured successfully. Conclusion:Combining enzymatically digestion with multiple strainers filtration is a rapid and effective method for follicle collecting, which is capable to isolate different developmental stage mouse follicles synchronously with morphological integrity, favorable viability and good developmental potency.