Dynamic regulation of DNA methyltransferases in human oocytes and preimplantation embryos after assisted reproductive technologies

Abstract

DNA methylation is a key epigenetic modification which is essential for normal embryonic development. Major epigenetic reprogramming takes place during gametogenesis and in the early embryo; the complex DNA methylation patterns are established and maintained by DNA methyltransferases (DNMTs). However, the influence of assisted reproductive technologies (ART) on DNA methylation reprogramming enzymes has predominantly been studied in mice and less so in human oocytes and embryos. The expression and localization patterns of the four known DNMTs were analysed in human oocytes and IVF/ICSI embryos by immunocytochemistry and compared between a reference group of good quality fresh embryos and groups of abnormally developing embryos or embryo groups after cryopreservation. In humans, DNMT1o rather than DNMT1s seems to be the key player for maintaining methylation in early embryos. DNMT3b, rather than DNMT3a and DNMT3L, appears to ensure global DNA remethylation in the blastocysts before implantation. DNMT3L, an important regulator of maternal imprint methylation in mouse, was not detected in human oocytes (GV, MI and MII stage). Our study confirms the existence of species differences for mammalian DNA methylation enzymes. In poor quality fresh embryos, the switch towards nuclear DNMT3b expression was delayed and nuclear DNMT1, DNMT1s and DNMT3b expression was less common. Compared with the reference embryos, a smaller number of cryopreserved embryos showed nuclear DNMT1, while a delayed switch to nuclear DNMT3b and an extended DNMT1s temporal expression pattern were also observed. The spatial and temporal expression patterns of DNMTs seem to be disturbed in abnormally developing embryos and in embryos that have been cryopreserved. Further research must be performed in order to understand whether the potentially disturbed embryonic DNMT expression after cryopreservation has any long-term developmental consequences.

Sperm DNA Methylation, Infertility and Transgenerational Epigenetics

“…Ovarian stimulation has been linked to altered imprinted gene methylation in embryos [127,128]. Vitrified mouse oocytes have reduced methylation at imprinted genes in blastocysts, due to reduced DNA methyltransferase expression [129], and this was also found in a human oocyte cryopreservation study [130]. However, a study using human sperm cryopreservation noted no changes to methylation at imprinted genes, spermatogenesis-related genes or MTHFR in the sperm DNA [131].…”

NLRPs, the subcortical maternal complex and genomic imprinting

“…It is currently unknown if the mechanism of methylation establishment is the same in humans as several differences have been observed. For example, despite DNMT3L having no catalytic activity, this co-factor is essential for the establishment of all germline-derived maternally methylated imprinted regions in mice, but DNMT3L is not expressed in human oocytes (Petrussa et al 2014) (Table 2). It is therefore assumed that methylated loci in human oocytes recruit DNMT3A directly via their PWWP domain that recognizes H3K36me3 (Dhayalan et al 2010).…”

“…Alternatively, NLRP7-SCMC may ensure the correct cellular localization and nuclear translocation of epigenetic factors during oocyte development. Immunostaining for the two mammalian de novo methyltransferases DNMT3A and DNMT3B has revealed that, like DNMT1, they also have a cytoplasmic localization in human oocytes (Petrussa et al 2014). This suggests that low abundant protein complexes containing the DNMTs may associate to specific DNA sequences possibly by direct interaction with known NLRP7-interacting chromatin regulators YY1 (Mahadevan et al 2014) or ZBTB16 (Singer et al 2015).…”

Vitrification Affects Nuclear Maturation and Gene Expression of Immature Human Oocytes

“…Reduction of DNMT1 gene expression may cause alteration in the patterns of DNA methylation, resulting in disruption of gene expression, genomic imprinting, and genome stabilization, which can lead to cell death. The expression of DNMT1, seems to be more disturbed in poor quality (fresh and cryopreserved) embryos, in comparison to good quality cryopreserved embryos and delayed fresh embryos (9). It should be noted that, apart from limitations in sample collection, the gene expression analysis on limited number of oocytes was a challenge, due to the small amount of RNA which was extracted.…”

“…DNMT1 is responsible for the sustainability of the methylation patterns during replication (8). Recently, Petrussa et al showed that DNMT1 was constitutively present in the nuclei of human oocytes and embryos at all stages of preimplantation development until Day 7 after fertilization (9). In mouse, a desirable correlation has been found between aberrant genome-wide DNA methylation patterns, abnormal embryonic development, and preimplantation embryonic loss (10).…”