The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm
The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm
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DOI：
10.1093/humrep/deaa085
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发表时间：
2020-05-01
影响因子：
6.1
通讯作者：
Hu, Liang
中科院分区：
文献类型：
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作者：
Lan, Yueyun;Zhang, Shuoping;Hu, Liang
研究问题：卵丘颗粒细胞的线粒体DNA(MtDNA)拷贝数是否与卵母细胞胞质成熟有关？摘要回答：与生发泡(GV)颗粒细胞的mtDNA拷贝数相比，中期I(MI)卵母细胞的线粒体DNA拷贝数似乎较低。CGCs和卵母细胞之间的相互作用提供了适当的能量平衡，这是哺乳动物卵母细胞发育所必需的。此外，在卵母细胞-卵丘复合体(OCC)中，具有较高mtDNA拷贝数的成熟卵母细胞往往具有具有较高mtDNA拷贝数的相应CGCs。研究设计、大小、持续时间：这是一项前瞻性研究，来自2018年2月24日至2019年12月21日在中信湘雅生殖遗传医院接受体外受精的70名妇女的302个卵胞浆内单精子注射(ICSI)体外受精。根据其相应卵母细胞的成熟情况，将其分为GV期、MI期和MII期。三个阶段的CGC样本量(n=302)分别为63(CGC(GV))、70(CGC(MI))和169(CGC(MII))。部分样本(n=257)用于线粒体DNA拷贝数的定量，其余(n=45)样本用于线粒体基因表达水平的分析。此外，我们从257个用于线粒体DNA拷贝数的OCC中提取了82个未成熟卵细胞，其中包括36个GV卵母细胞和46个MI卵母细胞，用于分析卵母细胞的线粒体DNA。分别分离每个卵母细胞的CGCs。用实时荧光定量聚合酶链式反应技术检测细胞线粒体DNA拷贝数和基因表达。MAIN结果和Chance的作用：选择MT-ND1、MT-CO1和β-珠蛋白基因来评估mtDNA含量，并检测MT-ND1、MT-CO1、PGC-1α和TFAM的mRNA表达。采用多重置换扩增技术对257个CGC和82个未成熟卵母细胞的基因组进行扩增，并从45个CGC中提取RNA。与CGV相比，CGCMI的mtDNA拷贝数显著减少。MT-ND1测定CGC(GV)：CGC(MI)为[270+/-302]：[134+/-201]，P=0.015。在MT-CO_1测定中，CGC(GV)：CGC(MI)为[205+/-228]：[92+/-112]，P=0.026。CGC(GV)和CGC(MII)的mtDNA差异无统计学意义。MT-ND1检测中，CGC(GV)：CGC(MII)为[270+/-302]：[175+/-223]，P=0.074。MT-CO_1测定中，CGC(GV)：CGC(MII)为[205+/-228]：[119+/-192]，P=0.077。CGC(MI)和CGC(MII)的mtDNA拷贝数差异无统计学意义。MT-ND1检测中，CGC(MI)：CGC(MII)为[134+/-201]：[175+/-223]，P=0.422。MT-CO_1测定中，CGC(MI)：CGC(MII)为[92+/-112]：[119+/-192]，P=0.478。为了验证上述结果的可靠性，我们进一步分析了14例GV、MI和MII卵母细胞的cGCs的mtDNA拷贝数，结果表明CGC(MI)的mtDNA拷贝数可能较低。MT-ND1法CGC(GV)：CGC(MI)为[249+/-173]：[118+/-113]，P=0.016；而MT-CO1法CGC(GV)：CGC(MI)为[208+/-199]：[83+/-98]，P=0.109。CGC(GV)和CGC(MII)的mtDNA差异无统计学意义。MT-ND1检测中，CGC(GV)：CGC(MII)为[249+/-173]：[185+/-200]，P=0.096。MT-CO_1测定中，CGC(GV)：CGC(MII)为[208+/-199]：[114+/-139]，P=0.096。CGC(MI)和CGC(MII)的mtDNA差异也无统计学意义。MT-ND1测定中，CGC(MI)：CGC(MII)为[118+/-113]：[185+/-200]，P=0.198。MT-CO_1测定中，CGC(MI)：CGC(MII)为[83+/-98]：[114+/-139]，P=0.470。此外，MT-ND1、MT-CO1、PGC-1α和TFAM在CGC(GV)、CGC(MI)和CGC(MII)之间的表达水平没有统计学差异(P>；0.05)。因此，cGCs中mtDNA拷贝数的变化是否与卵母细胞的不同发育阶段有关尚未得到进一步证实。此外，样本量相对较小。研究结果的广泛意义：从GV期到MI期，CGCs的mtDNA拷贝数减少，从MI到MII期，CGCs的mtDNA拷贝数保持稳定。在卵母细胞成熟的不同阶段，CGCs的线粒体DNA可能会发生自我降解和复制，以满足相应卵母细胞的能量需求和卵母细胞胞质的成熟。
STUDY QUESTION: Is the mitochondrial DNA (mtDNA) copy number of cumulus granulosa cells (CGCs) related to the maturation of oocyte cytoplasm?SUMMARY ANSWER: Compared with the mtDNA copy number of CGCs from germinal vesicles (GV), CGCs from Metaphase I (MI) oocytes appear to have a lower mtDNA copy number.WHAT IS KNOWN ALREADY: The growth and development of CGCs and oocyte are synchronised. The interaction between CGCs and the oocyte provides the appropriate balance of energy, which is necessary for mammalian oocyte development. Moreover, in the oocyte-cumulus complex (OCC), mature oocytes with higher mtDNA copy numbers tend to have corresponding CGCs with higher mtDNA copy numbers.STUDY DESIGN, SIZE, DURATION: This is a prospective study of 302 OCCs obtained from 70 women undergoing in vitro fertilisation with intracytoplasmic sperm injection (ICSI) at the Reproductive and Genetic Hospital of CITIC-Xiangya, between 24 February 2018 and 21 December 2019. The CGCs were divided into three groups (GV, MI and MII stages) based on the maturation status of their corresponding oocyte. The sample sizes (n = 302) of CGCs in the three stages were 63 (CGC(GV)), 70 (CGC(MI)) and 169 (CGC(MII)), respectively. Some of the samples (n = 257) was used to quantify the mtDNA copy number, while the rest (n = 45) were used to analyse the expression level of mitochondrial genes. Furthermore, we retrieved 82 immature oocytes from among the 257 OCCs used for mtDNA copy numbers, including 36 GV oocytes and 46 MI oocytes, for analysis of oocyte mtDNA.PARTICIPANTS/MATERIALS, SETTING, METHODS: We selected genes with high consistency of real-time PCR results to accurately measure the mtDNA copy number by testing the efficacy and the reproducibility in whole genome amplification (WGA) samples from a human embryonic stem cell line. The CGCs of each oocyte were individually isolated. The mtDNA copy number and gene expression of the CGCs were assessed using real-time PCR techniques. Mitochondrial DNA copy number of the corresponding immature oocytes was also evaluated.MAIN RESULTS AND THE ROLE OF CHANCE: MT-ND1, MT-CO1 and beta-globin genes were chosen for the assessment of mtDNA content, and mRNA expressions of MT-ND1, MT-CO1, PGC-1 alpha and TFAM were also measured. The genome of 257 CGCs and 82 immature oocytes were amplified according to the multiple displacement amplification (MDA) protocol, and RNA was extracted from 45 CGCs. Compared with CGCGV, CGCMI had a significantly lower mtDNA copy number. In the MT-ND1 assay, the CGC(GV): CGC(MI) was [270 +/- 302]: [134 +/- 201], P = 0.015. In the MT-CO1 assay, CGC(GV): CGC(MI) was [205 +/- 228]: [92 +/- 112], P = 0.026. There was no statistical difference in mtDNA between CGC(GV) and CGC(MII). In the MT-ND1 assay, CGC(GV): CGC(MII) was [270 +/- 302]: [175 +/- 223], P = 0.074. In the MT-CO1 assay, CGC(GV): CGC(MII) was [205 +/- 228]: [119 +/- 192], P = 0.077. No statistical difference of mtDNA copy number was observed between CGC(MI) and CGC(MII). In the MT-ND1 assay, CGC(MI): CGC(MII) was [134 +/- 201]: [175 +/- 223], P = 0.422. In the MT-CO1 assay, CGC(MI): CGC(MII) was [92 +/- 112]: [119 +/- 192], P = 0.478. To verify the reliability of the above results, we further analysed the mtDNA copy number of CGCs of 14 patients with GV, MI and MII oocytes, and the results showed that the mtDNA copy number of CGC(MI) may be lower. The mtDNA copy number of CGC(GV) and CGC(MI) was statistically different in the MT-ND1 assay where CGC(GV): CGC(MI) was [249 +/- 173]: [118 +/- 113], P = 0.016, but in the MT-CO1 assay, CGC(GV): CGC(MI) was [208 +/- 199]: [83 +/- 98], P = 0.109. There was no significant difference in mtDNA between CGC(GV) and CGC(MII). In the MT-ND1 assay, CGC(GV): CGC(MII) was [249 +/- 173]: [185 +/- 200], P = 0.096. In the MT-CO1 assay, CGC(GV): CGC(MII) was [208 +/- 199]: [114 +/- 139], P = 0.096. There was also no significant difference in mtDNA between CGC(MI) and CGC(MII). In the MT-ND1 assay, CGC(MI): CGC(MII) was [118 +/- 113]: [185 +/- 200], P = 0.198. In the MT-CO1 assay, CGC(MI): CGC(MII) was [83 +/- 98]: [114 +/- 139], P = 0.470. Moreover, there were no statistical differences in the expression levels of MT-ND1, MT-CO1, PGC-1 alpha and TFAM between CGC(GV), CGC(MI) and CGC(MII) (P > 0.05).LARGE SCALE DATA: N/ALIMITATIONS, REASONS FOR CAUTION: Due to the ethical issues, the study did not quantify the mtDNA content of MII oocytes. Thus, whether the change in mtDNA copy number in CGCs is related to the different developmental stages of oocytes has not been further confirmed. Moreover, the sample size was relatively small.WIDER IMPLICATIONS OF THE FINDINGS: The mtDNA copy number of CGCs decreases from the GV phase to the MI phase and stays steady from the MI to MII stage. At different stages of oocyte maturation, the mtDNA of CGCs may undergo self-degradation and replication to meet the energy requirements of the corresponding oocyte and the maturation of the oocyte cytoplasm.