Sakaloglou Prodromos, Lazaros Leandros, Bouba Ioanna, Markoula Sofia, Zikopoulos Athanasios, Drakaki Eirini, Anagnostaki Ismini, Potiris Anastasios, Stavros Sofoklis, Gerede Angeliki, Domali Ekaterini, Drakakis Peter, Tzavaras Theodoros, Georgiou Ioannis
Laboratory of Medical Genetics and Human Reproduction, School of Health Sciences, Faculty of Medicine, University of Ioannina, 451 10 Ioannina, Greece.
Medical Genetics and Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Ioannina University Hospital, 451 10 Ioannina, Greece.
Int J Mol Sci. 2024 Nov 27;25(23):12722. doi: 10.3390/ijms252312722.
Retrotransposable elements are implicated in genome rearrangements and gene expression alterations that result in various human disorders. In the current study, we sought to investigate the potential effects of long interspersed elements-1 (LINE-1) overexpression on the integrity and methylation of DNA and on the expression of three major pluripotency factors (OCT4, SOX2, NANOG) during the preimplantation stages of human embryo development. Human MI oocytes were matured in vitro to MII and transfected through intracytoplasmic sperm injection (ICSI) either with an EGFP vector carrying a cloned active human LINE-1 retroelement or with the same EGFP vector without insert as control. The occurrence of retrotransposition events was screened by fluorescent microscopy. The in vitro preimplantation development as well as the methylation, pluripotency, and DNA double-strand breaks (DSBs) of the transfected embryos were examined. LINE-1 retrotransposons gave rise to new retrotransposition events in the transfected embryos. LINE-1 injected embryos were characterized by accelerated asymmetrical cell division, multiple cellular fragments, cleavage arrest, and degeneration. Early OCT4 expression remained unaltered, but cleavage arrest and a high fragmentation rate hindered the expression of SOX2/NANOG at the morula stage. Increased DNA DSBs were observed in cleavage-stage blastomeres, while no methylation changes were detected before the cleavage arrest. Our data provide evidence that LINE-1 retrotransposition in human preimplantation embryos may induce DNA DSBs, while at the same time, it appears to interfere with the expression patterns of pluripotency factors. The morphological, structural, and cleavage abnormalities of the transfected embryos show that aberrant retroelement expression may negatively affect human embryo development.
逆转录转座元件与基因组重排和基因表达改变有关，这些改变会导致各种人类疾病。在本研究中，我们试图研究长散在核元件1（LINE-1）过表达对人类胚胎发育植入前阶段DNA完整性和甲基化以及三种主要多能性因子（OCT4、SOX2、NANOG）表达的潜在影响。将人类MI期卵母细胞体外成熟至MII期，并通过胞浆内单精子注射（ICSI），用携带克隆的活性人类LINE-1逆转录元件的EGFP载体或不含插入片段的相同EGFP载体作为对照进行转染。通过荧光显微镜筛选逆转录转座事件的发生。检查转染胚胎的体外植入前发育以及甲基化、多能性和DNA双链断裂（DSB）情况。LINE-1逆转录转座子在转染胚胎中引发了新的逆转录转座事件。注射LINE-1的胚胎表现为不对称细胞分裂加速、多个细胞碎片、卵裂停滞和退化。早期OCT4表达保持不变，但卵裂停滞和高碎片化率阻碍了桑椹胚期SOX2/NANOG的表达。在卵裂期的卵裂球中观察到DNA DSB增加，而在卵裂停滞前未检测到甲基化变化。我们的数据提供了证据，表明人类植入前胚胎中的LINE-1逆转录转座可能诱导DNA DSB，同时，它似乎会干扰多能性因子的表达模式。转染胚胎的形态、结构和卵裂异常表明，异常的逆转录元件表达可能对人类胚胎发育产生负面影响。
