The Function Of DNMTS During The Embryo Implantation Of Mouse

Objective:To investigate the molecular mechanism of DNMTs during the blastocyst implantation, mRNA and protein expression of DNMTs were detected in early pregnancy mouse endometrium in this research.Methods:1. Establishment of mouse model of pregnancy. Pregnant Dl to D7 mice were randomly selected.The endometria of pregnant mice were collected and stored at -80℃immediately or embedded by paraffin.2. FQ-PCR was used to detect the mRNA level of DNMTs in the endometria of pregnant mice.3.Immunohistochemistry was used to analyze the expression of DNMTs protein in the endometria of early pregnant mice.4. Establishment of the induction mouse model by 5-aza-CdR, the pregnant mice on D1 was randomly chosed to inject i.p. (200μl per injection) with the of DNMTs inhibitor, 5-aza-CdR with the concentration of 0, 0.1, or 0.5 mg/kg).5. The mRNA level of DNMTs in the endometria of normal mice and the treated mice by 5-aza-CdR on pregnant D3 and D5 were detected by FQ-PCR.6.Immunohistochemistry was used to analyze the protein expression of DNMTs and Hoxa10 in the endometria of normol and the inducted mice with the treatment of 5-aza-CdR on the pregnant D3 and D5.7. FQ-PCR and Immunohistochemistry were used to analyze the mRNA level and protein expression of Hoxa10 in the endometria of normol and the inducted mice on the pregnant D5.8.The number of implantated blastocysts was evaluated by the way of Trypan blue injection through tail vein on pregnant D5.Result:1. The FQ-PCR data showed that the mRNA levels of DNMT1 in endometria of pregnant mice was gradually elevated,and reached the maximum level on pregnant D3 (P<0.05), then gradually declined to the level of D1 from D4 to D7; The mRNA expression of DNMT3a in endometria of pregnant mice resembling to the DNMT1, gradually increased,and reached a maximum level on D3 (P<0.05), then gradually declined from D4 to D7 in the same. The DNMT3b expression in mRNA gradually increased, and reached a maximum level on D3 ,then remarkably declined from D5 to D7(P<0.01) in the endometria of pregnant mice.2. Immunohistochemistry staining showed that DNMT1 protein was strong stained in the nucleus of luminal epithelia, gland epithelia and stromal cells, weak stained in the cytoplasm; DNMT3a protein was located in the nucleus of luminal epithelia, gland epithelia and stromal cells; DNMT3b protein was mainly located in the cytoplasm of luminal epithelia, gland epithelia and stromal cells, and less in nucleus. The expression patern in protein level in the endometria of pregnant mice was in concordance with mRNA level of of DNMTs, thus the expression of DNMTs protein was increased,and gradually declined when it reached a maximum level on D3 (P<0.05).3. The mRNA levels of DNMTs were down-regulated without significance in endometria of the mice treated for two days (pregnant D3) or four days (pregnant D5) by 5-aza-CdR at concentration of 0.1 mg/kg, comparing with the control group (P>0.05). The expression of DNMT3b in mRNA level of the mice endometria was down-regulated without significance (P>0.05) when treated for two days while with significance (P<0.05)when treated for four days by 5-aza-CdR at concentration of 0.1 mg/kg. The expression of DNMT1, DNMT3a and DNMT3b in mRNA level in the endometria were remarkably down-regulated with significance after two or four days'treatment with 5-aza-CdR at concentration of 0.5 mg/kg, when compared with the control ones.4. After treated with 5-aza-CdR, immunohitochemistry staing showed that DNMT1 was mainly located in the cytoplasm of luminal epithelia, gland epithelia and stromal cells, DNMT3a was located at the nucleus of luminal epithelia, gland epithelia and stromal cells and DNMT3b was located at cytoplasm of luminal epithelia, gland epithelia and stromal cells. The protein level of DNMTs almost the same as the they were in mRNA level.5. No significant difference was observed in the number of implantation sites in the group treated by 5-aza-CdR at concentration of 0.1 mg/kg, when compared with control. No implantation site was observed in the mice when treated by 5-aza-CdR at level of 0.5 mg/kg. The uteri were smaller on the pregnant D5 in the mice treated by 0.5 mg/kg 5-aza-CdR treated mice than the control. No significant difference in size was found between the embryos from 0.1 mg/kg 5-aza-CdR treatment mice and the control.6. The expression of Hoxa10 mRNA and protein were higher than the control group after the four days'treatment of 0.1 mg/kg 5-aza-CdR but had no significant difference(P>0.05). When treated by 0.5 mg/kg 5-aza-CdR, the mRNA and protein level of Hoxa10 in endometria were significant lower than those in the control(P<0.05) .Conclusion:1. mRNA and protein expression of DNMTs in the endometria of pregnant mice were distributed regularly indicated that DNMTs might participated in the implantation process.2. DNMTs might promote expression of some cellular adhesion molecule, glycoproteins, cytokine, making the best reception of endometrium via decreasing methylation of those genes related to reception.