Epigenetics Program, Babraham Institute, Cambridge, CB22 3AT, UK.
Diseases Network Research Unit, Faculty of Biochemistry and Molecular Medicine, Oulu University, Oulu, Finland.
Clin Epigenetics. 2021 Jun 28;13(1):132. doi: 10.1186/s13148-021-01116-3.
Reproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.
An in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O) and hypoxia (5% oxygen, 5% O). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.
Our observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.
生殖生物学方法依赖于通过激素刺激从成熟卵泡中体外培养卵泡，以产生中期 II 期卵母细胞进行受精，并发育成健康的胚胎。此类技术在啮齿动物和人类中常规使用。DNA 甲基化是一种动态过程，在配子发生和发育的表观遗传调控中发挥作用。在哺乳动物卵母细胞中，DNA 甲基化的建立调节胚胎中的基因表达。这种调节对于一类基因（印迹基因）尤为重要，其表达模式对于下一代至关重要。本工作的目的是建立一个用于不成熟小鼠卵母细胞的体外培养系统，该系统将允许操作特定因素，以更深入地分析建立与转录调控相关的甲基化模式的调控机制。
建立了一种从处于生发泡（GV）阶段的不成熟小鼠卵母细胞进行体外培养的系统，该系统在两种不同条件下进行培养：常氧（20%氧气，20% O）和低氧（5%氧气，5% O）。培养的卵母细胞根据其大小进行分选。从培养的卵母细胞中生成并与体内生长的卵母细胞进行比较的代表性亚硫酸氢盐测序（RRBS）和 RNA-seq 文库。在体外培养的卵母细胞中，随着卵母细胞的生长，全局和 CpG 岛（CGI）甲基化逐渐增加，印迹基因的甲基化与体内生长的卵母细胞相似。在常氧条件下生长的卵母细胞的转录组显示染色质重排和富含雌性生殖基因的表达，而在 5% O 条件下，转录本偏向于细胞应激反应。为了进一步证实这些结果，我们开发了一种基于我们的模型的功能测定法，用于使用降低甲基化和转录的药物来鉴定卵母细胞的甲基化。当组蛋白甲基化和转录过程减少时，生长卵母细胞的基因体 CGIs 的 DNA 甲基化呈现出较低的甲基化谱。
我们的观察结果揭示了在不同氧浓度下体外培养的卵母细胞与体内生长的小鼠卵母细胞之间的 DNA 甲基化和转录的变化。在 20% O 下生长的卵母细胞与体内卵母细胞的 DNA 甲基化和转录相关性更高，表明在体外培养条件下较高的氧气浓度有利于卵母细胞的成熟。我们的结果揭示了在体外培养模型中从不成熟卵母细胞到 GV 卵母细胞的发育过程中的表观遗传机制。
