O-215 Copper oxide nanoparticles (CuONPs) impairs mitophagy and promotes DNA methylation to suppress mouse embryonic pluripotency
Authors: Luo Y , Zeng X , Dai X , Li J , Huang G 
Published: July 2024

Abstract

Study question
Does exposure to copper oxide nanoparticle (CuONPs) have adverse effects on early embryonic development? What is the potential mechanism?

Summary answer
CuONPs exposure can damage mitophagy and lead to α-KG reduction, resulting DNA hypermethylation, thereby reducing the pluripotency of pre-implantation embryos in mice.

What is known already
Environmental nanomaterials contamination is a great concern for organisms including human. Exposure to nanomaterials have been associated with fecundity and fertility in couples conceiving via assisted reproduction technology. Among these bio-effects, nanomaterials induced epigenetic changes are of particular interest. Copper oxide nanoparticles (CuONPs) are widely used in a huge range of applications which might pose potential risk to organisms. Recent studies reported that CuONPs exposure resulted in transgenerational toxicity in Caenorhabditis elegans associated with possible epigenetic regulation. However, whether CuONPs exposure affect mammalian embryonic development, and if so the underlying mechanisms, are unclear.

Study design, size, duration
For the human experiments, we measured the copper ion content in women’s urine, and assessed the expression of pluripotent genes in 3PN embryos treated with CuONPs. For the embryo experiments, we collected zygotes from ICR mice and cultured them in KSOM medium with CuONPs (10 μg/mL) until blastocyst stage. For the cell experiments, we treated mouse embryonic stem cells (mESCs) with CuONPs (2 μg/mL) for four days to detect cell proliferation and pluripotency.

Participants/materials, setting, methods
The ICP-MS method was used to detect copper ion content in women’s urine. Time–lapse incubator was used to document the developmental potential of embryos. To quantify the metabolites of embryo, we employed metabolomics. Single-cell multiomics sequencing that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression were performed to detect genes expression and epigenetic modification. We used real-time PCR, immunofluorescence, and western blot to measure the expression of pluripotency related genes.

Main results and the role of chance
In human, we discovered a significant negative relationship between the urine’s copper ion content and clinical pregnancy rate. In embryo experiments, CuONPs exposure has been shown to have adverse effects on embryo developmental potential, resulting morula and blastocyst transformation arrest, as well as reduction in blastocyst rate and live birth rate. Furthermore, CuONPs treatment led to a reduction in blastocyst quality, indicating a decrease in blastocyst cell count, particularly in ICM cells. CuONPs also inhibited the pluripotency of blastocysts and mESCs, showing down-regulated expression of pluripotent genes. Mechanistically, CuONPs aggregated in lysosomes and decreased the mitophagic flux of embryos, which caused damaged mitochondria to accumulate and ultimately caused mitochondrial dysfunction. Notably, in embryos treated with CuONPs, there was a decrease in α-ketoglutarate (α-KG) due to the disruption of the tricarboxylic acid (TCA) cycle. α-KG is an essential cofactor for α-KG dependent dioxygenases such as the ten-eleven translocation (TET) family of DNA demethylase. The lack of α-KG in CuONPs-treated embryos led to DNA hypermethylation, which reduced chromatin accessibility, and suppressed the expression of pluripotent genes like sox2, klf2, and klf4. The poor pluripotency ultimately leads to abnormal embryonic development in CuONPs-treated embryos.

Limitations, reasons for caution
The molecular mechanism by which CuONPs arrest the development of pre-implantation embryos remains to be determined. In addition, future work should be extended to human embryos to determine whether this mechanism is conserved between mice and humans.

Wider implications of the findings
The findings of this work indicated the relationship between CuONPs and epigenetic modification. The results will support a more thorough understanding into the biosafety of CuONPs, and offer fresh perspectives on the potential uses of nanomaterials in the field of reproductive medicine in the future.

Trial registration number
Not Applicable