Superovulation induces alterations in the epigenome of zygotes, and results in differences in gene expression at the blastocyst stage in mice

Abstract

Gamete and embryo manipulations can result in alterations to the epigenome, and are associated with altered gene expression. The initial objective of this study was to determine the transcript level of several epigenetic modifiers in embryos that had been cultured from the 2-cell stage until the late-blastocyst stage in four culture conditions. Cultured embryos were compared to control, in vivo-produced late blastocysts to ascertain if differences in gene expression existed among the culture conditions; none were observed. As all of the embryos used were produced in females that had undergone superovulation, we next compared the transcript level of the same epigenetic modifiers between superovulated, in vivo-produced embryos and embryos produced from natural ovulation. Following in vitro culturing, expression of the genes analyzed was increased in all superovulation groups. We therefore hypothesized that the superovulation procedure-used to increase the number of embryos obtained for experimentation-may have caused an inappropriate acquisition of epigenetic modifications in the maternal genome prior to ovulation, which in turn caused misexpression of genes at the blastocyst stage. To test this hypothesis, we compared the level of global DNA methylation and histone 3 lysine-9 or -14 acetylation in zygotes obtained by natural- or superovulation. Indeed, superovulation decreased global DNA methylation on the maternal pronucleus of zygotes, which inversely correlated with H3K9/14 acetylation. In conclusion, superovulation alters the epigenome of the oocyte, resulting in the dysregulation of gene expression at the blastocyst stage.

Superovulation Influences Methylation Reprogramming and Delays Onset of DNA Replication in Both Pronuclei of Mouse Zygotes

“…In addition, their results indicated a higher failure (14%) to develop to the blastocyst stage for embryos obtained from superovulated matings compared to those from spontaneously ovulated matings (5%). Also similar to our findings, the recently published study of Huffman et al [2015] indicated a hypomethylation of the maternal pronucleus of zygotes from superovulated females. However, this study only analyzed maternal pronuclei of PN3 and PN4 embryos and thus lacks a detailed analysis of paternal pronuclei as well as all PN stages.…”

“…Previous studies also used immunofluorescence staining with antibodies against 5mC to document abnormal genome-wide methylation reprogramming in superovu- lated mouse zygotes and 2-cell embryos [Shi and Haaf, 2002;Huffman et al, 2015]. Shi and Haaf [2002] used mouse 2-cell embryos from superovulated females, nonsuperovulated matings, and IVF for 5mC staining and in vitro development analysis up to the blastocyst stage.…”

“…Studies at the mouse zygote stage detected a higher incidence of abnormal global methylation patterns and reduced developmental potential of mouse preimplantation embryos from superovulated matings [Shi and Haaf, 2002]. More recently, Huffman et al [2015] further linked superovulation to a reduction of global DNA methylation as well as increased histone H3 lysine 9 (H3K9) and H3K14 acetylation in maternal pronuclei of mouse zygotes.…”

Association between assisted reproductive technology and the risk of autism spectrum disorders in the offspring: a meta-analysis

“…found abnormal methylation is particularly involved in ART imprinting disorders related to the context of ASD35. Experiments in animals have suggested that the various steps of the ART procedures such as superovulation, in vitro culture of oocytes or embryo, and IVF might be related to epigenetic defects in the embryos and offspring36. However, abnormal DNA methylation could not be consistently identified in IVF children37.…”

High-Speed Mouse Backcrossing Through the Female Germ Line

“…Several studies have described anomalies in imprinted gene expression when comparing blastocyst stage embryos developing from superovulated versus naturally ovulated oocytes [19–21]; however, subsequent reports found that expression alterations were not associated with abnormalities in methylation patterns [20], and imprinted expression in later stage development was largely normal [22]. Studies investigating postnatal endocrine and reproductive health of offspring resulting from superovulated vs naturally ovulated oocytes found no differences [23, 24].…”