Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, Vic 3168, Australia.
Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Vic 3168, Australia.
Hum Reprod. 2018 May 1;33(5):942-953. doi: 10.1093/humrep/dey052.
What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these?
Changes to DNA methylation in a 5' cytosine-phosphate-guanine 3' (CpG) island in the mtDNA-specific replication factor (DNA polymerase gamma (POLG)) of mtDNA-deficient oocytes mediates an increase in mtDNA copy number by the 2-cell stage that positively modulates the expression of nuclear genes, which affect cellular and metabolic processes, following autologous mitochondrial supplementation.
Too few copies of mtDNA in mature oocytes can lead to fertilisation failure or preimplantation embryo arrest. mtDNA-deficient oocytes that progress to blastocyst express genes associated with poor cellular and metabolic processes, transcriptional activation and mitochondrial biogenesis.
STUDY DESIGN, SIZE, DURATION: Using a pig oocyte model, we assessed mtDNA-deficient and mtDNA-normal oocytes during in vitro maturation for mtDNA variants and levels of DNA methylation in POLG. We supplemented mtDNA-deficient oocytes with autologous populations of mitochondria to determine if there were changes to DNA methylation in POLG that coincided with increases in mtDNA copy number. We assessed metaphase II mtDNA-deficient and mtDNA-normal oocytes by RNA sequencing to identify differentially expressed genes and compared their profiles to blastocysts derived from mtDNA-normal, mtDNA-deficient and supplemented mtDNA-deficient oocytes.
PARTICIPANTS/MATERIALS, SETTING, METHODS: mtDNA variant analysis (n = 24), mtDNA copy number (n = 60), POLG gene expression (n = 24), and RNA sequencing (n = 32 single; and 12 pooled cohorts of n = 5) were performed on oocytes and embryos. DNA methylation of a CpG island in POLG was determined quantitatively by pyrosequencing on oocytes to 2-cell embryos (n = 408). Bioinformatics tools were used to assess differences between mtDNA-normal and mtDNA-deficient oocytes and between mtDNA-normal and mtDNA-deficient oocytes and supplemented oocytes and their blastocyst stage equivalents.
Whilst mtDNA-deficient oocytes regulated variants less stringently during maturation (P < 0.05), there were no differences in the ratio of variants in mature-stage oocytes. However, mtDNA-normal mature oocytes had significantly more molecules affected due to their higher copy number (P < 0.0001). Normal mature oocytes differently DNA methylated a CpG island in POLG compared with mtDNA-deficient oocytes (P < 0.01). Supplementation of mtDNA-deficient oocytes modulated DNA methylation at this CpG island leading to a mtDNA replication event prior to embryonic genome activation inducing significant increases in mtDNA copy number. RNA-Seq identified 57 differentially expressed genes (false discovery rate (FDR) < 0.05) between the two cohorts of oocytes with blastocyst stage gene expression altered by supplementation of mtDNA-deficient oocytes (P < 0.05) including genes associated with metabolic disorders. One key factor was branched chain amino acid transaminase 2 (BCAT2), a regulator of amino acid metabolism and associated with diabetes.
Sequence data are available on the NCBI Sequence Read Archive under the project number PRJNA422295. RNA sequencing data were deposited into NCBI Gene Expression Omnibus, under the accession number GSE108900.
LIMITATIONS, REASONS FOR CAUTION: Whilst this work was conducted in a species that is highly relevant to human reproduction, the outcomes need to be tested in human oocytes and blastocysts prior to clinical application.
The outcomes demonstrate a mechanism of action following mtDNA supplementation of mtDNA-deficient oocytes that results in improved gene expression at the blastocyst stage of development.
STUDY FUNDING/COMPETING INTERESTS: This work was funded by OvaScience Inc. OvaScience did not influence the study design, analysis of results or interpretation of the data.
线粒体 DNA（mtDNA）缺陷和 mtDNA 正常卵母细胞之间的分子差异是什么，以及线粒体补充如何改变这些差异？
mtDNA 缺陷卵母细胞中 mtDNA 特异性复制因子（DNA 聚合酶γ（POLG））中 5' 胞嘧啶-磷酸-鸟嘌呤 3'（CpG）岛的 DNA 甲基化变化介导 mtDNA 拷贝数在 2-细胞阶段增加，这种增加通过自体线粒体补充正向调节核基因的表达，核基因影响细胞和代谢过程。
成熟卵母细胞中 mtDNA 拷贝数过少可导致受精失败或植入前胚胎停滞。进展到囊胚的 mtDNA 缺陷卵母细胞表达与细胞和代谢过程、转录激活和线粒体生物发生相关的基因。
研究设计、大小、持续时间：使用猪卵母细胞模型，我们在体外成熟过程中评估 mtDNA 缺陷和 mtDNA 正常卵母细胞的 mtDNA 变体和 POLG 中的 DNA 甲基化水平。我们用自体线粒体群体补充 mtDNA 缺陷卵母细胞，以确定 POLG 中的 DNA 甲基化是否与 mtDNA 拷贝数的增加同时发生。我们通过 RNA 测序评估中期 II mtDNA 缺陷和 mtDNA 正常卵母细胞，以鉴定差异表达基因，并将其与来自 mtDNA 正常、mtDNA 缺陷和补充 mtDNA 缺陷卵母细胞的囊胚进行比较。
参与者/材料、设置、方法：mtDNA 变体分析（n = 24）、mtDNA 拷贝数（n = 60）、POLG 基因表达（n = 24）和 RNA 测序（n = 32 个单样本；和 12 个 n = 5 的混合样本）在卵母细胞和胚胎上进行。定量焦磷酸测序法检测卵母细胞到 2-细胞胚胎的 POLG 中 CpG 岛的 DNA 甲基化（n = 408）。使用生物信息学工具评估 mtDNA 正常和 mtDNA 缺陷卵母细胞之间以及 mtDNA 正常和 mtDNA 缺陷卵母细胞和补充卵母细胞及其囊胚阶段等效物之间的差异。
虽然 mtDNA 缺陷卵母细胞在成熟过程中对变体的调控不那么严格（P < 0.05），但成熟卵母细胞中的变体比例没有差异。然而，由于其更高的拷贝数，mtDNA 正常成熟卵母细胞受影响的分子数量明显更多（P < 0.0001）。正常成熟卵母细胞与 mtDNA 缺陷卵母细胞相比，POLG 中的 CpG 岛的 DNA 甲基化方式明显不同（P < 0.01）。mtDNA 缺陷卵母细胞的补充调节了这个 CpG 岛的 DNA 甲基化，导致 mtDNA 复制事件发生在胚胎基因组激活之前，从而显著增加 mtDNA 拷贝数。RNA-Seq 确定了两个卵母细胞队列之间 57 个差异表达基因（错误发现率（FDR）< 0.05），其中 mtDNA 缺陷卵母细胞补充后的囊胚阶段基因表达发生改变（P < 0.05），包括与代谢紊乱相关的基因。一个关键因素是支链氨基酸转氨酶 2（BCAT2），它是氨基酸代谢的调节剂，与糖尿病有关。
序列数据可在 NCBI Sequence Read Archive 上获得，项目编号为 PRJNA422295。RNA 测序数据已被存入 NCBI Gene Expression Omnibus，注册号为 GSE108900。
局限性、谨慎的原因：虽然这项工作是在与人类生殖高度相关的物种中进行的，但在将其应用于临床之前，需要在人类卵母细胞和囊胚中进行测试。
研究结果表明了 mtDNA 缺陷卵母细胞补充 mtDNA 后的作用机制，该机制导致囊胚阶段发育的基因表达改善。
研究资金/利益冲突：这项工作由 OvaScience Inc. 资助。OvaScience 并未影响研究设计、结果分析或数据解释。
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