Sodium fluoride disrupts DNA methylation of H19 and Peg3 imprinted genes during the early development of mouse embryo

Abstract

Sodium fluoride (NaF) is associated with embryonic and fetal development abnormalities, but the mechanism by which this occurs is unclear. DNA methylation, an important epigenetic reprogramming mechanism, is essential for normal embryonic development. Thus, we investigated the effect of NaF on DNA methylation in early mouse embryos, as well as mouse sperm and liver using bisulfite sequencing and ELISA. Data indicate that H19, a paternally imprinted gene, compared to control embryos, was less methylated in 8-cell embryos from pregnant mice treated with NaF (100 mg/l) in drinking water for 48 h. Peg3, a maternally imprinted gene, and the Line1 repeated sequence were similarly methylated in NaF-treated and control embryos. Oral ingestion of NaF for 35 days did not significantly change Line1 and genomic global DNA methylation in the liver. H19, Rasgrf1, Line1, and genomic global DNA methylation were also similar in NaF-treated and control sperm. Female mice mated with NaF-treated male mice (35 days) had less methylated H19, but Peg3 was significantly more methylated. Line1 was similarly methylated in treated 8-cell embryos, compared to control embryos. NaF treatment of male mice before copulation significantly increased the expression of H19 in blastocysts, whereas H19 expression was not detected in 8-cell embryos. Data suggest that NaF may interact directly with the embryo to disrupt the maintenance of normal gene imprinting during pregnancy. Long-term NaF exposure of males may not directly affect DNA methylation of the sperm and liver, but the sperm may signal to early embryos with abnormal gene imprinting.

Sodium Fluoride Affects DNA Methylation of Imprinted Genes in Mouse Early Embryos

“…According to an earlier report [Zhu et al, 2014], genomic DNA samples were dissolved in 0.3 M NaOH at 37 ° C for 20 min, mixed with 2 volumes of 2% low melting point agarose, and dropped into chilled mineral oil to form agarose beads. Then they were treated with freshly made bisulfite solution (2.5 M sodium metabisulfite, 125 m M hydroquinone, pH 5.0) at 55 ° C for 12 h in the dark followed by equilibrating the beads 3× with 1 ml of TE buffer.…”

“…Then they were treated with freshly made bisulfite solution (2.5 M sodium metabisulfite, 125 m M hydroquinone, pH 5.0) at 55 ° C for 12 h in the dark followed by equilibrating the beads 3× with 1 ml of TE buffer. After desulfonation in 0.5 ml 0.3 M NaOH for 0.5 h, the beads were washed with TE buffer and H 2 O, respectively, and stored at -20 ° C [Zhu et al, , 2014.…”

“…Spermatogenesis is a critical process for H19 methylation, but H19 also undergoes methylation reprogramming after fertilization. And sodium fluoride is reported to decrease the methylation degree of H19 in 8-cell embryos of mice [Zhu et al, 2014]. In contrast, Peg3 is a paternally expressed gene and imprinting occurs only in oocytes [Lucifero et al, 2002].…”

“…Over-intake of fluorine causes pathological changes such as dental fluorosis and liver damage although it is important for the metabolism of calcium and phosphor [Gupta et al, 2007]. Moreover, fluorine was recently reported to cross the placental barrier, affecting early embryonic development as well as testicular spermatogenesis in which DNA methylation is erased and re-established [Zhu et al, 2014].DNA methylation, especially methylation of imprinted genes, is an important epigenetic mechanism during early embryonic development and is susceptible to a series of environmental factors such as alcohol, fluorine, and radiation [Ge et al, 2013[Ge et al, , 2014Fu et al, Key Words DNA methylation · Embryonic development · H19 · Peg3 · Sodium fluoride …”

“…Peg3 has been reported to inhibit Wnt signaling, which is very important for embryo development [Jiang et al, 2010]. Its methylation level in 8-cell embryos increased when the mated male mice were treated with sodium fluoride [Zhu et al, 2014].…”