The impact of ART on genome‐wide oxidation of 5‐methylcytosine and the transcriptome during early mouse development
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Abstract
The use of assisted reproductive technologies (ART) has been increasing over the past three decades due to the elevated frequency of infertility problems. Other factors such as easier access to medical aid than in the past and its coverage by health insurance companies in many developed countries also contributed to this growing interest. Nevertheless, a negative impact of ART on transcriptome and methylation reprogramming is heavily discussed. Methylation reprogramming directly after fertilization manifests itself as genome-wide DNA demethylation associated with the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the pronuclei of mouse zygotes. To investigate the possible impact of ART particularly on this process and the transcriptome in general, pronuclear stage mouse embryos obtained upon spontaneous ovulation or superovulation through hormone stimulation representing ART were subjected to various epigenetic analyses. A whole-transcriptome RNA-Seq analysis of pronuclear stage embryos from spontaneous and superovulated matings demonstrated altered expression of the Bbs12 gene known to be linked to Bardet-Biedl syndrome (BBS) as well as the Dhx16 gene whose zebrafish ortholog was reported to be a maternal effect gene. Immunofluorescence staining with antibodies against 5mC and 5hmC showed that pronuclear stage embryos obtained by superovulation have an increased incidence of abnormal methylation and hydroxymethylation patterns in both maternal and paternal pronuclear DNA compared to their spontaneously ovulated counterparts. Single-cell RT-qPCR analyses of the Tet1, Tet2 and Tet3 genes encoding the enzymes that convert 5mC to 5hmC revealed no significant expression differences between pronuclear stage embryos from spontaneously and superovulated matings that may contribute to the observed superovulation-induced abnormalities of methylation reprogramming. To analyze the possible contribution of TET-independent demethylation mechanisms such as replication dependent passive processes, 5mC and 5hmC levels of pronuclear stage mouse embryos were determined by immunofluorescence analyses after inhibition of DNA replication with aphidicolin. Inhibition of DNA replication had not effect on abnormal methylation and hydroxymethylation patterns that still persisted in the superovulated group. However, the onset of DNA replication which was also analyzed in these experiments was remarkably delayed in the superovulated group. Cumulatively, these results imply that superovulation influences both replication-dependent and -independent or yet unknown demethylation mechanisms in pronuclear stage mouse embryos.
Overall, the data of my thesis further support a negative impact of ovarian stimulation on the transcriptome and epigenetic reprogramming during gametogenesis and early embryogenesis. These findings may pave the way for optimization of ART techniques in order to minimize the related problems.