5-aza-2'-deoxycytidine leads to reduced embryo implantation and reduced expression of DNA methyltransferases and essential endometrial genes

Abstract

Background: The DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and principal findings: Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type-specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5' flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions: The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.

Figure 1. Effects of 5-aza-CdR on embryo implantation, cell proliferation, and decidualization.

(A–C) Embryo implantation sites (blue dots with arrows) in mice treated with or without 5-aza-CdR, stained by intravenous Chicago Blue B injection on day 5 of pregnancy (D5). (D) Graph showing the number of implantation sites in each group (n = 6 for each group). Bars indicate the mean±S.D. **p<0.001. (E–G) Hematoxylin and eosin staining of longitudinal uterine sections on D7 after 4 days treatment with 5-aza-CdR, indicates defected development of uterus during post-implantation period in mice treated by 0.5 mg/kg 5-aza-CdR. de, decidua; em, embryo; S, stroma. (H–J) Endometrial decidualization as indicated by alkaline phosphatase activity (blue) on PD6 in oil-induced decidulized uteruses (red indicates negative staining). (K–M) Endometrial stromal cell proliferation on PD6 in oil-induced decidualized uteruses, indicated by immunostaining with rabbit anti-Ki67 (brown). (N) Graph showing the number of Ki67-positive stromal cells in each group (n = 6 for each group). Bars indicate the mean±S.D. **p<0.001.

Figure 2. Effects of 5-aza-CdR on Dnmt1, Dnmt3a, and Dnmt3b expression in mouse endometrium by western blotting.

Mice were treated for 2 or 4 days with 0 (control), 0.1, or 0.5 mg/kg 5-aza-CdR, and tissues were collected for analysis on PD3 or PD5. β-actin was assessed as a loading control. *p<0.01; **p<0.001.

Figure 3. Effects of 5-aza-CdR on Dnmt1, Dnmt3a, and Dnmt3b expression in mouse endometrium by immunohistochemistry.

Mice were treated for 2 or 4 days with 0 (control), 0.1, or 0.5 mg/kg 5-aza-CdR, and sections were prepared on PD3 or PD5. Immunoreactivity (brown) of Dnmt1 (A–F), Dnmt3a (G–L), and Dnmt3b (M–R) is shown. Sections that were not exposed to the primary antibody were used as negative controls (S–U) and stained with Hematoxylin and eosin. le, luminal epithelium; ge, glandular epithelium; S, stroma.

Figure 4. Effects of 5-aza-CdR on DNA methylation of genes controlling implantation in endometrial tissues.

(A) Esr1 promoter and 5′-UTR (exon 1) containing 19 CpG sites. (B) 5′-UTR (exon 1) of Pgr containing 16 CpG sites. (C) Hoxa10 promoter and 5′-UTR (exon 1) containing 21 CpG sites. Each row of circles represents a single cloned allele (five clones per mouse). Each circle represents a single CpG site. Filled and open circles indicate methylated and unmethylated cytosines, respectively.

Figure 5. Effects of 5-aza-CdR on Esr1, Pgr, and Hoxa10 protein levels in mouse endometrium.

Mice were treated with 0 (control), 0.1, or 0.5 mg/kg 5-aza-CdR for 4 days, and endometrium was analyzed by western blotting on PD5. β-actin was assessed as a loading control. *p<0.01; **p<0.001.

Figure 6. Immunohistochemistry of Esr1 (A, B), Pgr (C, D), and Hoxa10 (E, F) in mouse endometrium on PD5.

Mice were treated daily with or without 0.5 mg/kg 5-aza-CdR. The inset boxes in the upper right corners are enlarged region of each figure. le, luminal epithelium; ge, glandular epithelium; s, stroma.