Development in vitro of mouse oocytes from primordial follicles
Development in vitro of mouse oocytes from primordial follicles
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DOI：
10.1095/biolreprod54.1.197
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发表时间：
1996-01-01
影响因子：
3.6
通讯作者：
OBrien, MJ
中科院分区：
文献类型：
--
作者：
Eppig, JJ;OBrien, MJ
这些研究的目的是从新生小鼠卵巢原始卵泡中的卵母细胞开始，在体外实现完整的卵母细胞发育。制定了两步策略：首先将新生小鼠的卵巢在器官培养物中培养8天，然后从器官培养的卵巢中分离发育中的卵母细胞-颗粒细胞复合物，并再培养14天。原始卵泡的卵母细胞体积约为4190μm（3）（直径20μm），在器官培养8天期间，该体积增加了约53810μm（3），最终大小为58000μm3，增加了13.8倍。在第一个实验中，卵母细胞-颗粒细胞复合物在对照培养基或补充有 FSH (0.5 ng/ml)、表皮生长因子 (EGF；1.0 ng/ml) 或 EGF 加 FSH 的培养基中生长。在 14 天培养期结束时，仅回收了在对照培养基或补充有 FSH 的培养基中培养的复合物的 50-60%。相比之下，在补充有EGF的培养基中培养的复合物中超过90%被回收。在对照培养基中生长的卵母细胞的中值尺寸为176 800μm(3)（直径69μm），而在补充EGF的培养基中生长的卵母细胞的中值尺寸稍小（136400μm（3）体积；63μm直径），因为更多较小尺寸的卵母细胞在含有EGF的培养基中存活。在含有 FSH 的培养基中发育后恢复的卵母细胞中有 30% 能够经历生发囊泡破裂 (GVB)。在第二组实验中，从器官培养的卵巢中分离出的卵母细胞-颗粒细胞复合物在补充有0.5或5.0ng/ml FSH或补充有相同浓度的FSH加1.0ng/ml EGF的培养基中培养。再次，在用 EGF 培养的组中观察到卵母细胞回收率增加。各组之间获得 GVB 能力的卵母细胞百分比（32%）或产生极体的 GVB 卵母细胞百分比没有差异，因此表明减数分裂进展到中期 II（22%）。当成熟卵母细胞受精时，在卵母细胞发育过程中，没有EGF的组中，有21％的受精和卵裂到2细胞阶段，而在有EGF培养的组中，42％的受精和卵裂到2细胞阶段。在任何一组中，只有不到 2% 的 2 细胞阶段胚胎发育至囊胚阶段。将一百九十个2细胞期胚胎移植到假孕雌性的输卵管中；两只雌性各产下一只幼崽；一个还活着，另一个显然最近去世了。这里报告的结果清楚地表明，卵母细胞从原始卵泡阶段在体外完全发育是可能的，并为使用实验室动物的卵母细胞作为人类以及具有农业和动物学重要性的动物的卵母细胞发育的模型系统建立了进一步研究的框架。
The objective of these studies was to achieve complete oocyte development in vitro beginning with the oocytes in the primordial follicles of newborn mouse ovaries. A two-step strategy was developed: first the ovaries of newborn mice were grown in organ culture for 8 days, and then the developing oocyte-granulosa cell complexes were isolated from the organ-cultured ovaries and cultured for an additional 14 days. The oocytes of primordial follicles are approximately 4190 mu m(3) in volume (20 mu m in diameter), and this volume increased by approximately 53 810 mu m(3) to a final size of 58 000 mu m3-a 13.8-fold increase-during the 8 days of organ culture. In the first experiment the oocyte-granulosa cell complexes were grown in control medium or in medium supplemented with FSH (0.5 ng/ml), epidermal growth factor (EGF; 1.0 ng/ml), or EGF plus FSH. Only 50-60% of the complexes cultured in control medium or in medium supplemented with FSH were recovered at the end of the 14-day culture period. In contrast, more than 90% of the complexes cultured in medium supplemented with EGF were recovered. The median size of the oocytes grown in control medium was 176 800 mu m(3) (69-mu m diameter), while the median size of those grown in medium supplemented with EGF was slightly smaller (136 400 mu m(3) volume; 63-mu m diameter), due to the survival of more smaller-size oocytes in EGF-containing medium. Thirty percent of the oocytes recovered after development in FSH-containing medium were competent to undergo germinal vesicle breakdown (GVB). In the second set of experiments, oocyte-granulosa cell complexes isolated from organ-cultured ovaries were cultured in medium supplemented with either 0.5 or 5.0 ng/ml FSH or with these same concentrations of FSH plus 1.0 ng/ml EGF. Again, increased oocyte recovery was observed in the groups cultured with EGF. There was no difference among the groups in the percentage of the oocytes that acquired competence to undergo GVB (32%) or in the percentage of GVB oocytes that produced a polar body, thus indicating progression of meiosis to metaphase II (22%). When the mature oocytes were inseminated, 21% underwent fertilization and cleavage to the 2-cell stage in the groups without EGF during oocyte development, while 42% underwent fertilization and cleavage to the 2-cell stage in the groups cultured with EGF. Less than 2% of the 2-cell-stage embryos developed to the blastocyst stage in any of the groups. One hundred and ninety 2-cell-stage embryos were transferred to the oviducts of pseudopregnant females; two females produced one pup each; one was living and the other had apparently died recently. The results reported here clearly show that complete development of oocytes in vitro from the primordial follicle stage is possible and establish the framework for further studies using oocytes from laboratory animals as model systems for the development of oocytes from humans as well as from animals of agricultural and zoological importance.