The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm

Autor: Liang Hu, Fei Gong, Shuoping Zhang, Ge Lin, Changfu Lu, Yueyun Lan
Rok vydání: 2020
Předmět:
Zdroj: Human Reproduction. 35:1120-1129
ISSN: 1460-2350
0268-1161
DOI: 10.1093/humrep/deaa085
Popis: 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 (CGCGV), 70 (CGCMI) and 169 (CGCMII), 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 β-globin genes were chosen for the assessment of mtDNA content, and mRNA expressions of MT-ND1, MT-CO1, PGC-1α 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 CGCGV: CGCMI was [270 ± 302]: [134 ± 201], P = 0.015. In the MT-CO1 assay, CGCGV: CGCMI was [205 ± 228]: [92 ± 112], P = 0.026. There was no statistical difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [270 ± 302]: [175 ± 223], P = 0.074. In the MT-CO1 assay, CGCGV: CGCMII was [205 ± 228]: [119 ± 192], P = 0.077. No statistical difference of mtDNA copy number was observed between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [134 ± 201]: [175 ± 223], P = 0.422. In the MT-CO1 assay, CGCMI: CGCMII 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 CGCMI may be lower. The mtDNA copy number of CGCGV and CGCMI was statistically different in the MT-ND1 assay where CGCGV: CGCMI was [249 ± 173]: [118 ± 113], P = 0.016, but in the MT-CO1 assay, CGCGV: CGCMI was [208 ± 199]: [83 ± 98], P = 0.109. There was no significant difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [249 ± 173]: [185 ± 200], P = 0.096. In the MT-CO1 assay, CGCGV: CGCMII was [208 ± 199]: [114 ± 139], P = 0.096. There was also no significant difference in mtDNA between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [118 ± 113]: [185 ± 200], P = 0.198. In the MT-CO1 assay, CGCMI: CGCMII was [83 ± 98]: [114 ± 139], P = 0.470. Moreover, there were no statistical differences in the expression levels of MT-ND1, MT-CO1, PGC-1α and TFAM between CGCGV, CGCMI and CGCMII (P > 0.05). LARGE SCALE DATA N/A LIMITATIONS, 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. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the National Key R&D Program of China (Grant 2018YFC1003100, to L.H.), the science and technology major project of the Ministry of Science and Technology of Hunan Province, China (grant 2017SK1030, to G.L.), the National Natural Science Foundation of China (grant 81873478, to L.H.), and Merck Serono China Research Fund for Fertility Experts (to L.H.). There is no conflict of interest.
Databáze: OpenAIRE
Externí odkaz: