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% O2) and hypoxia (5% oxygen, 5% O2). 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% O2 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% O2 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.

中文翻译：

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氧浓度影响体外培养的卵母细胞从头 DNA 甲基化和转录

生殖生物学方法依赖于来自通过激素刺激获得的成熟卵泡的体外卵泡培养物，以产生待受精并发育成健康胚胎的中期 II 卵母细胞。这种技术通常用于啮齿动物和人类物种。DNA甲基化是一个动态过程，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。这种调控对于一类基因，即印记基因尤为重要，其表达模式对下一代至关重要。这项工作的目的是为未成熟小鼠卵母细胞建立一个体外培养系统，该系统将允许操纵特定因素，以便更深入地分析建立转录调控相关甲基化模式的调控机制。体外培养系统由未成熟的小鼠卵母细胞开发而成，这些卵母细胞在两种不同的条件下生长为胚泡 (GV)：常氧（20% 氧气，20% O2）和缺氧（5% 氧气，5% O2）。培养的卵母细胞根据它们的大小进行分类。减少的代表性亚硫酸氢盐测序 (RRBS) 和 RNA-seq 文库是从培养中产生的，并与体内生长的卵母细胞进行比较。在体外培养的卵母细胞中，随着卵母细胞的生长，全局甲基化和 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。在常氧环境中生长的卵母细胞的转录组显示染色质重组和女性生殖基因的丰富表达，而在 5% O2 条件下，转录本偏向于细胞应激反应。为了进一步证实结果，我们基于我们的模型开发了一种功能测定，用于使用减少甲基化和转录的药物来表征卵母细胞甲基化。当组蛋白甲基化和转录过程减少时，来自生长卵母细胞基因体的 CGI 处的 DNA 甲基化呈现出较低的甲基化谱。我们的观察揭示了在体外培养的不同氧浓度的卵母细胞和体内培养的小鼠卵母细胞之间 DNA 甲基化和转录物的变化。在 20% O2 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，这表明较高的氧浓度有利于离体培养条件下的卵母细胞成熟。我们的研究结果揭示了体外培养模型中卵母细胞从未成熟卵母细胞发育为 GV 卵母细胞的表观遗传机制。