The onset of gene expression from the new embryonic genome requires the conversion of chromatin from a transcriptionally repressive to permissive state. One component of this conversion is the extensive global acetylation of nuclear histones, including acetylation of lysine 9 within histone 3 (H3K9ace). H3K9ace is not detected in the newly fertilized embryo, with acetylation starting around the time of first DNA replication 1. This reprogramming coincides with the period in which embryos are exposed to the culture environment during assisted reproduction technologies. We showed that embryo culture caused a marked perturbation in the onset of H3K9ace which in turn altered the earliest round of gene expression 1. In this study we explored which aspects of culture are associated with this perturbation.
Quantitative indirect immunofluorescence was used to assay changes in global H3K9ace levels. We compared the H3K9ace levels in zygotes fertilized in vitro and then culture in vitro (IVF) with zygotes fertilized and matured within the reproductive tract (Fresh) and those fertilized in situ and then cultured in vitro (ISF). H3K9ace was significantly greater in IVF zygotes compared to either ISF or Fresh, while ISF was higher than Fresh. The effect of IVF was independent of media formulation. Superovulation had no impact compared to spontaneous ovulation.
Our results confirm that manipulation of the embryo in vitro causes a global disturbance in epigenetic reprogramming and shows that this effect is compounded by IVF. This appears to be a stress response to the conditions of culture per se, since another major putative stressor, superovulation, had no impact. These results confirm the utility of H3K9ace measurements as a biomarker of the effects of stress on the early embryo.