Endometrial Hoxall, Meis1, Cdh1 And Ctnnb1 Expression With Controlled Ovarian Hyperstimulation During Peri-implantation Period In Mice

Part I Endometrial Hoxall, Meisl, Cdh1 and Ctnnb1 expression with Controlled ovarian stimulation in miceObject:To evaluate the endometrial receptivity with controlled ovarian hyperstimulation (COH) protocols by investigating the expression of Hoxal1, Meisl, Cdh1 and Ctnnb1 in endometrium during peri-implantation period in mice.Method:human mimic IVF mouse model was used. The mimick COH protocols, included GnRH agonist plus human menopausal gonadotropin HMG (GnRH agonist group), GnRH antagonist plus HMG (GnRH antagonist group) and HMG alone (HMG group). Uterus samples were dissected from the mice with the COH or naturally ovulated mice at 48 h after the COH or ovulation (the control group). Uterus sample was divided into two parts, which were stored at-80℃for extraction of protein and total RNA. The molecular mRNA levels were examined by real-time PCR, and protein levels were detected by Western blot.Result:The mRNA and protein expressions of Hoxal1, Meisl, Cdh1 and Ctnnb1 were decreased in all the COH groups. Among the COH groups, Hoxal1 and Ctnnbl were the lowest in the GnRH agonist group, and Meisl and Cdhl were lower in the GnRH analogue groups than the HMG group. There were positive correlations between the expressions of Hoxal 1 and Ctnnbl as well as the expressions of Meisl and Cdhl among the groups.Conclusion:The COH protocols particularly with GnRH analogues suppressed Hoxal1, Meisl, Ctnnb1 and Cdhl expressions in mouse endometrium during peri-implantation period. PartⅡConstruction of pEGFP-Hoxa11 and pDsRed-Meis1 Eukaryotic Expression Vector and Expression in CHO Cell LineObject:constructed the recombinanit plasmid pEGFP-Hoxa11 and pDsRed-Meis1 to investigate the interaction between Hoxa11 and Ctnnb1, as well as Meis1 and Cdh1.Method:The full sequence of Hoxa11 and Meis1 was got by PCR. After enzyme digestion by EcoRI and Kpn1, ligation overnight by T4 DNA ligase and transformation by DH5a, we got the recombinant plasmid of pEGFP-Hoxa11 and pDsRed-Meis1, which were identified by PCR, enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into CHO cell lines, and detected by fluorescence microscope and Western blotting analysis.Result:The recombinant plasmid pEGFP-Hoxa11 and pDsRed-Meis1 were successfully constructed. The green and red fluorescence was visible in transfected CHO cells under fluorescent microscope and Hoxa11 and Meis1 expressions were significantly increased in pEGFP-Hoxa11 transfection compared with empty vetor transfection.Conclusion:The expression vector pEGFP-Hoxa11 and pDsRed-Meis1 are successfully constructed, which can co-express Hoxa11-EGFP protein and Meis1-DsRed protein in CHO cell lines. PartⅢHoxall and Meisl transfection in vivo promoted endometrial expressions of Ctnnbl and Cdhl respectivelyObject:Investigated the interaction between Hoxa11 and Ctnnb1, as well as Meis1 and Cdh1.Method:Untreated female mice (10 in total) were mated and examined every 8 h until detection of vaginal plugs, which was designated as day 0 of the pregnancy (pd 0). The mice were anesthetized 30-60 h following plug detection with 25μl of the expressive plasmid (pEGFP-Hoxa11 or pDsRed-Meisl)/liposome complex and its vector control (pEGFP-N1 or pDsRed-N1)/liposome complex were injected into the bases of the right or left horn of the uterine respectively.48h after the surgery,10 mice were executed by cervical vertebrae dislocation under anesthesia, and then the uteri were excised. Uterus samples were divided into three parts. One part was used for making cryosections to detect the exogenous gene expression by fluorescence microscope screening; one part was stored at-80℃for protein extraction to examine the expressions of CDH1 and CTNNB1, and the third part was fixed in formalin, embedded in paraffin, sectioned into 5μm sections, and mounted onto slides.Result:1、Hoxa11-EGFP and Meis1-Red fusion proteins were expressed in luminal epithelial cells, glandular cells and stromal cells.2、Western blot identified the expression of Hoxa11 and Meis1 after in vivo transfection. Overexpression of Hoxa11 enhanced Ctnnb1 expression, and Meis1 overexpression promoted Cdh1 expression.3、Immunohistochemical data showed that CDH1 and CTNNB1 were expressed in luminal cells, glandular cells and in stromal cells of endometrium transfected with pDsRed-N1 and pEGFP-N1 during the implantation window, particularly on the membrane of luminal cells and glandular cells. The expression of CDH1 in endometrium were significantly increased with pDsRed-Meis1 transfection (HSCORE:1.4±0.046) compared with the pDsRed-N1 transfection (HSCORE:0.58±0.04, p<0.001). The expression of CTNNB1 in endometrium transfected with pEGFP-Hoxa11 (HSCORE:1.85±0.06) was significantly increased compared with those in the uterus transfected with pEGFP-N1 (HSCORE:0.973±0.064, p <0.001).Conclusion:Hoxal1 and Meis1 over-expression significantly promoted the expression of endogenous Ctnnbl and Cdhl respectively, which suggested that they would be associated with regulation of expression of Ctnnbl and Cdhl in murine endometrium.