title: 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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Reproductive Biology, Chongqing Medical University, Chongqing, People's Republic of China.
authors.forename: Yu-Bin
authors.initials: YB
authors.lastname: Ding
authors.forename: Chun-Lan
authors.initials: CL
authors.lastname: Long
authors.forename: Xue-Qing
authors.initials: XQ
authors.lastname: Liu
authors.forename: Xue-Mei
authors.initials: XM
authors.lastname: Chen
authors.forename: Liang-Rui
authors.initials: LR
authors.lastname: Guo
authors.forename: Yin-Yin
authors.initials: YY
authors.lastname: Xia
authors.forename: Jun-Lin
authors.initials: JL
authors.lastname: He
authors.forename: Ying-Xiong
authors.initials: YX
authors.lastname: Wang
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000090205:DNA Methyltransferase 3A; D000077209:Decitabine; D010064:Embryo Implantation / Q000187:drug effects*; D004717:Endometrium / Q000187:drug effects* / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D005260:Female; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D011247:Pregnancy; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism; D000097584:DNA Methyltransferase 3B
chemical_list: D001374:Azacitidine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000077209:Decitabine; D047628:Estrogen Receptor alpha; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins; D011980:Receptors, Progesterone; D000097584:DNA Methyltransferase 3B; C000715535:Dnmt3a protein, mouse; C092119:Hoxa10 protein, mouse; C000619381:Dnmt1 protein, mouse