Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, 34134, Vezneciler, Istanbul, Turkey.
Argenit Technology, ITU ARI Technocity, ITU Ayazaga Campus, 34467, Sariyer, Istanbul, Turkey.
Reprod Sci. 2022 Aug;29(8):2208-2222. doi: 10.1007/s43032-022-00953-8. Epub 2022 Apr 27.
In humans and most animals, maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is considered as an universal assumption. Recently, several lines of evidence suggest that different species seem to employ distinct mechanisms to prevent the inheritance of paternal mtDNA. There are few studies in the literature on the molecular basis of sperm mtDNA elimination in mammals and paternal mtDNA transmission in humans. Endonuclease G (ENDOG) is a mitochondrial nuclease encoded by nuclear ENDOG gene. The critical importance of ENDOG gene on paternal mitochondrial elimination (PME) has been previously demonstrated in model organisms such as C. elegans and D. melanogaster. However, its mechanism in human is still unclear. Therefore, we aimed to evaluate whether nuclear ENDOG gene copy number could be a potential marker of paternal mtDNA transmission or not.Male factor infertility patients diagnosed with different infertility subgroups such as azoospermia, oligoteratozoospermia, astheno-teratozoospermia were included in this study: 13 infertile men and 25 healthy men as control group. Quantitative real-time polymerase chain reaction (qPCR) analysis and dual-color Fluorescence in situ hybridization (FISH) method were used to compare the groups. FISH method was applied to verify qPCR results and two signals were observed in nearly all patients. ENDOG gene copy number data were evaluated by comparing them with entire human mtDNA next-generation sequencing (NGS) analysis results obtained through bioinformatics and proteomics tools. Mitochondrial whole genome sequencing (WGS) data allowed determination of novel and reported variations such as single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphism (MNP), insertion/deletion (INDEL). Missense variants causing amino acid substitution were filtered out from patients' mtDNA WGS data.Relative copy number of target ENDOG gene in male infertility patients [0.49 (0.31 - 0.77)] was lower than healthy controls [1.00 (0.66 - 1.51)], and statistical results showed significant differences between the groups (p < 0.01). A total of 38 missense variants were detected in the genes encoding the proteins involved in the respiratory chain complex. Moreover, we detected paternal mtDNA transmissions in the children of these patients who applied to assisted reproductive techniques.In conclusion, this study reveals that ENDOG gene may be an important factor for the PME mechanism in humans. To the best of our knowledge, this is the first study in humans about this topic and assessment of ENDOG gene sequencing and gene expression studies in a larger sample size including patients with male factor infertility would be our future project.
在人类和大多数动物中，线粒体和线粒体 DNA（mtDNA）的母系遗传被认为是普遍存在的假设。最近，有几条证据表明，不同的物种似乎采用不同的机制来防止父系 mtDNA 的遗传。关于哺乳动物精子 mtDNA 消除和人类父系 mtDNA 传递的分子基础，文献中研究甚少。内切核酸酶 G（ENDOG）是一种由核 ENDOG 基因编码的线粒体核酸内切酶。在模式生物如秀丽隐杆线虫和黑腹果蝇中，已经证明了 ENDOG 基因对父系线粒体消除（PME）的重要性。然而，其在人类中的机制尚不清楚。因此，我们旨在评估核 ENDOG 基因拷贝数是否可以作为父系 mtDNA 传递的潜在标志物。
这项研究纳入了患有不同不育亚组的男性因素不育患者，如无精子症、少精子症、弱精子症-畸形精子症：13 名不育男性和 25 名健康男性作为对照组。使用实时定量聚合酶链反应（qPCR）分析和双色荧光原位杂交（FISH）方法进行比较。应用 FISH 方法验证 qPCR 结果，在几乎所有患者中均观察到两个信号。通过比较它们与通过生物信息学和蛋白质组学工具获得的下一代测序（NGS）分析结果，评估 ENDOG 基因拷贝数数据。线粒体全基因组测序（WGS）数据允许确定新的和报道的变异，如单核苷酸多态性（SNP）、多位点多态性（MNP）、插入/缺失（INDEL）。从患者的 mtDNA WGS 数据中过滤出导致氨基酸替换的错义变体。
男性不育患者的目标 ENDOG 基因相对拷贝数[0.49（0.31-0.77）]低于健康对照组[1.00（0.66-1.51）]，组间差异有统计学意义（p<0.01）。在参与辅助生殖技术的这些患者的子女中，我们检测到了编码呼吸链复合物蛋白的基因中的 38 个错义变体。
总之，这项研究表明，ENDOG 基因可能是人类 PME 机制的重要因素。据我们所知，这是关于该主题的首例人类研究，评估 ENDOG 基因测序和包括男性因素不育患者在内的更大样本量的基因表达研究将是我们未来的项目。
