Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.
Reproduction. 2021 Sep 9;162(4):307-318. doi: 10.1530/REP-21-0209.
In vitro generation of fertile oocytes has been reported in several mammalian species. However, oocyte integrity is compromised by in vitro culture. Here, we aimed to understand the factors affecting oocyte competency by evaluating mitochondrial function and transcriptome as well as lipid metabolism in in vivo-derived oocytes and in vitro grown and matured (IVGM) oocytes under atmospheric (20%) and physiological (7%) O2 concentration. We used single-cell RNA-sequencing as well as Gene Ontology and KEGG analyses to identify the molecular pathways affecting the developmental competence of oocytes. Oocytes grown under 20% O2 conditions showed a significant decrease in mitochondrial membrane potential, upregulation of ceramide synthesis pathway-associated genes, and high ceramide accumulation compared with oocytes grown under 7% O2 conditions and in vivo-grown oocytes. This suggests that excess ceramide level causes mitochondrial dysfunction and poor developmental ability of the oocytes. Mitochondrial DNA copy number was lower in IVGM oocytes irrespective of O2 concentration in culture, although there was no common abnormality in the expression of genes related to mitochondrial biosynthesis. In contrast, some oocytes produced under 7% O2 conditions showed gene expression profiles similar to those of in vivo-grown oocytes. In these oocytes, the expression of transcription factors, including Nobox, was restored. Nobox expression correlated with the expression of genes essential for oocyte development. Thus, Nobox may contribute to the establishment of oocyte competency before and after the growth phase. The comprehensive analysis of IVGM oocytes presented here provides a platform for elucidating the mechanism underlying functional oocyte production in vivo.
在几种哺乳动物中已经报道了体外生成可育卵母细胞的方法。然而，卵母细胞的完整性会受到体外培养的影响。在这里，我们旨在通过评估线粒体功能和转录组以及在大气（20%）和生理（7%）氧气浓度下体内衍生卵母细胞和体外生长和成熟（IVGM）卵母细胞中的脂质代谢，来了解影响卵母细胞能力的因素。我们使用单细胞 RNA 测序以及基因本体论和 KEGG 分析来鉴定影响卵母细胞发育能力的分子途径。与在 7%氧气条件下生长的卵母细胞和体内生长的卵母细胞相比，在 20%氧气条件下生长的卵母细胞的线粒体膜电位显著降低，神经酰胺合成途径相关基因上调，神经酰胺积累增加。这表明过多的神经酰胺水平导致线粒体功能障碍和卵母细胞发育能力差。无论培养中氧气浓度如何，IVGM 卵母细胞中的线粒体 DNA 拷贝数都较低，尽管与线粒体生物合成相关的基因表达没有共同异常。相比之下，一些在 7%氧气条件下产生的卵母细胞表现出与体内生长的卵母细胞相似的基因表达谱。在这些卵母细胞中，转录因子，包括 Nobox 的表达得到了恢复。Nobox 的表达与卵母细胞发育所必需的基因的表达相关。因此，Nobox 可能有助于在生长阶段前后建立卵母细胞能力。这里提出的对 IVGM 卵母细胞的综合分析为阐明体内功能性卵母细胞产生的机制提供了一个平台。
