Laboratory of Germ Cell Physiology and Engineering, Faculty of Agriculture, Shinshu University, Nagano, Japan.
Laboratory of Applied Reproductive Science, Faculty of Agriculture, Shinshu University, Nagano, Japan.
Biol Reprod. 2024 Nov 11;111(5):1156-1167. doi: 10.1093/biolre/ioae125.
In vitro culture of ungrown oocytes in preantral follicles is one of the intriguing subjects being pursued to produce viable eggs in assisted reproductive technology. Previous studies have succeeded in obtaining mature eggs after in vitro culture of preantral follicles, while denuded undeveloped oocytes, which are obtained occasionally when collecting preantral follicles, seem to be almost useless. Moreover, methods to culture them efficiently to produce viable eggs have not been established yet. The present study was conducted to demonstrate in vitro culture of mouse denuded undeveloped oocytes by reconstructing granulosa cell-oocyte complexes, and to analyze cellular communication in reconstructed granulosa cell-oocyte complexes. Single denuded undeveloped oocytes were aggregated with 1 × 104 granulosa cells in wells with U-shaped bottoms in a low-binding cell culture plate for 8 days under either 20% or 5% O2, and then the reconstructed granulosa cell-oocyte complexes formed were cultured on a collagen-coated culture membrane insert for 4 days under 5% O2. At day 8 of culture, the rates of reconstructed granulosa cell-oocyte complexes formation were significantly higher in the culture group under 5% O2 (64.9%) than that under 20% O2 (42.3%; P < 0.001); furthermore, the formation of transzonal projections was observed. After maturation and fertilization, we produced matured eggs and blastocysts at higher rates (>90% and 61.9%, respectively) in the group cultured under 5% O2. After transferring 126 two- to four-cell stage embryos, six live pups were obtained. This is the first report that demonstrates production of viable eggs after in vitro culture of denuded undeveloped oocytes from preantral follicles by reconstruction of granulosa cell-oocyte complexes.
在体外培养未成熟的卵母细胞于原始卵泡中是在辅助生殖技术中产生可育卵子的一个令人感兴趣的课题。之前的研究已经成功地从原始卵泡中获得成熟的卵子，而在收集原始卵泡时偶尔获得的裸卵似乎几乎没有用。此外，还没有建立有效地培养它们以产生可育卵子的方法。本研究旨在通过重建颗粒细胞-卵母细胞复合物来展示体外培养小鼠裸未成熟卵母细胞，并分析重建的颗粒细胞-卵母细胞复合物中的细胞通讯。将单个裸未成熟卵母细胞与 1×104 个颗粒细胞在 U 形底的孔中聚集，在低结合细胞培养板中于 20%或 5%O2 下培养 8 天，然后将形成的重建颗粒细胞-卵母细胞复合物在 5%O2 下培养在胶原包被的培养膜插入物上 4 天。在培养的第 8 天，在 5%O2 下培养的重建颗粒细胞-卵母细胞复合物形成率（64.9%）显著高于在 20%O2 下培养的复合物形成率（42.3%；P<0.001）；此外，观察到透明带投射的形成。在成熟和受精后，我们在 5%O2 下培养的组中以更高的比率（分别为>90%和 61.9%）产生成熟的卵子和囊胚。在移植 126 个 2-至 4 细胞期胚胎后，获得了 6 只活幼仔。这是第一个通过重建颗粒细胞-卵母细胞复合物从原始卵泡中体外培养裸未成熟卵母细胞产生可育卵子的报道。
