Estudio de la replicación del ADN mitocondrial durante el desarrollo preimplantacional en ratones

Abstract

Mitochondria exert essential functions both before and after oocyte fertilization takes place. Currently, it remains unclear whether mitochondrial genome (mtDNA) replication takes place during the preimplantational development of mammalian embryos. Previous studies show that mtDNA copy number remains relatively stable from oocytes until implantation. Nevertheless, it is possible that this is the result of the destruction and replication of mtDNA taking place simultaneously. Polγ is the only enzyme responsible for DNA polymerization during mitochondrial DNA replication. This enzyme, encoded by the gene Polg, allocated on chromosomic DNA, has three different isoforms generated by alternative splicing. In this study, we have analyzed the differences in mtDNA content at the blastocyst stage between control embryos and embryos where two or three of Polg isoforms have been ablated at the zygote stage in order to determine whether mtDNA replication occurs during this developmental period. Gene ablation was achieved by means of CRISPR/Cas9 gene editing technology. A first experiment targeted a genomic locus that generated KO embryos for isoforms 1 and 3, whereas a second experiment targeted another locus that generated KO embryos for all isoforms of Polg. Gene ablation was achieved by microinjection of CRISPR components (Cas9 mRNA and sgRNA) at the zygote stage and control embryos were microinjected with Cas9 mRNA alone. Microinjected embryos were allowed to develop to the blastocyst stage in vitro, subsequently genotyped to identify KO embryos and subjected to mtDNA analysis by quantitative PCR (qPCR). mtDNA relative copy number was significantly lower in embryos lacking isoforms 1 and 3 compared to control, wild type (WT) embryos (1±0.141 vs. 1.87±0.239, for KO vs. WT embryos, respectively, ANOVA p<0,05). Similar results were obtained when mtDNA copy number was compared between embryos lacking all isoforms of Polg and WT embryos (1±0.139 vs. 2.60±0.259, for KO vs. WT embryos, respectively, ANOVA p<0,05). These results uncover that mtDNA replication takes place during preimplantation development being mediated by Polγ.