Altered DNA methylation and Dnmt expression in obese uterus may cause implantation failure.

Obesity is defined by increased adipose tissue volume and has become a major risk factor for reproduction. Recent studies have revealed a substantial link between obesity and epigenetics. The epigenome is dynamically regulated mainly by DNA methylation. DNA methylation, which is controlled by DNA methyltransferases (Dnmts), has been widely studied because it is essential for imprinting and regulation of gene expression. In our previous study, we showed that the levels of Dnmt1, Dnmt3a and global DNA methylation was dramatically altered in the testis and ovary of high-fat diet (HFD)-induced obese mice. However, the effect of HFD on Dnmts and global DNA methylation in mouse uterus has not yet been demonstrated. Therefore, in the present study, we aimed to evaluate the effect of HFD on the level of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l and global DNA methylation in uterus. Our results showed that HFD significantly altered the levels of Dnmts and global DNA methylation in the uterus. The total expression of Dnmt1, Dnmt3a and Dnmt3b was significantly upregulated, while level of Dnmt3l and global DNA methylation were dramatically decreased (p < 0.05). Furthermore, we observed that the expression of Dnmt3b and Dnmt3l was significantly increased in endometrium including gland and epithelium (p < 0.05). Although Dnmt3b was the only protein whose expression significantly increased, the level of global DNA methylation and Dnmt3l significantly decreased in stroma and myometrium (p < 0.05). In conclusion, our results show for the first time that obesity dramatically alters global DNA methylation and expression of Dnmts, and decreased DNA methylation and Dnmt expression may cause abnormal gene expression, especially in the endometrium.

Antioxidant supplementation may effect DNA methylation patterns, apoptosis, and ROS levels in developing mouse embryos.

This study was designed to address the question: does antioxidant-containing embryo culture media affect DNA methyltransferases, global DNA methylation, inner cell mass/trophoblast differentiation, intracellular reactive oxygen species (ROS) levels, and apoptosis? Mouse zygotes were cultured in embryo culture media containing MitoQ, N-acetyl-L-cysteine (NAC), acetyl-L-carnitine (ALC), α-lipoic acid (ALA), or the mixture of NAC + ALC + ALA (AO) until the blastocyst stage, whereas in vivo-developed blastocysts were used as control. Protein expression levels of Dnmt1, 3a, 3b, and 3l enzymes were analyzed by immunofluorescence and western blot, while global DNA methylation, apoptosis, and ROS levels were evaluated by immunofluorescence. NAC, ALC, and MitoQ significantly increased the levels of all Dnmts and global methylation. ALA significantly induced all Dnmts, whereas global methylation did not show any difference. NAC and mixture AO applications significantly induced Nanog levels, ALA and MitoQ increased Cdx2 levels, while the other groups were similar. ALA and MitoQ decreased while ALC increased the levels of intracellular ROS. This study illustrates that antioxidants, operating through distinct pathways, have varying impacts on DNA methylation levels and cell differentiation in mouse embryos. Further investigations are warranted to assess the implications of these alterations on the subsequent offspring.

Unveiling the impact of DNA methylation machinery: Dnmt1 and Dnmt3a in orchestrating oocyte development and cellular homeostasis.

DNA methylation can be considered the most prominent in controlling the gene expression responsible for the balance between cell proliferation and cell death. In this study, we aimed to analyze the distinct contributions of Dnmt1 and Dnmt3a enzymes in oocyte maturation, survival, autophagy, reactive oxygen species (ROS) production, and compensation capacity of Dnmt3b and Dnmt3l enzymes in mouse oocytes. Following confirming the suppression of Dnmt1or Dnmt3a through siRNA application, the assessment involved immunofluorescence staining for Dnmts, 5mC, p62, and ROS levels. Cell death rates showed a noticeable increase while oocyte maturation rates exhibited significant reduction. Global DNA methylation showed a decline, concomitant with elevated p62 and ROS levels upon Dnmt1 or Dnmt3a knockdown. Remarkably, silencing of Dnmt1 led to an upsurge in Dnmt3a expression, whereas Dnmt3a knockdown triggered an increase in Dnmt1 levels. Furthermore, Dnmt3l expression exhibited a notable decrease after silencing of either Dnmt1 or Dnmt3a, while Dnmt3b levels remained comparable between control and siRNA-treated groups. Collectively, this study underscores the pivotal roles of Dnmt1 and Dnmt3a in orchestrating various facets of oocyte development, encompassing maturation, survival, autophagy, and ROS production. These findings offer valuable insights into the intricate regulatory network governed by DNA methylation machinery within the context of oocyte physiology.

DNA methyltransferase (Dnmt) silencing causes increased Cdx2 and Nanog levels in surviving embryos.

Epigenetic mechanisms are one of the essential regulators of gene expression which do not involve altering the primary nucleotide sequence. DNA methylation is considered among the most prominent epigenetic mechanisms in controlling the functions of genes related to cell differentiation, cell cycle, cell survival, autophagy, and embryo development. DNA methyl transferases (Dnmts) control DNA methylation, the levels of which are differentially altered during embryonic development, and may determine cell differentiation fate as in the case of pluripotent inner cell mass (ICM) or trophectoderm (TE). In this study, we aimed to analyze the role of Dnmt1 and Dnmt3a enzymes in ICM (using the Nanog marker) and TE (using the Cdx2 marker) differentiation, autophagy (using p62 marker), reactive oxygen species (ROS) production, and apoptosis (using TUNEL) during mouse preimplantation embryo development. Following knockdown of Dnmt1 and Dnmt3a in zygotes, expression levels of Cdx2 in the trophectoderm and Nanog in the inner cell mass were measured, as well as p62 levels, reactive oxygen species (ROS) production, and apoptosis levels after 96 hours in embryo culture. We found that knockdown of Dnmt1 or Dnmt3a significantly induced Cdx2 and Nanog expression. Similarly, p62 expression, ROS levels and apoptosis significantly increased after silencing. This study shows that Dnmt genes are highly crucial for embryonic fate determination and survival. Further studies are required to reveal the specific targets of these methylation processes related to cell differentiation, survival, autophagy, and ROS production in mouse and human preimplantation embryos.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.