Expression, Regulation And Function Of Glutathione Peroxidase 3 In Mouse Uterus During Peri-implantation Process

Implantation and decidualization are crucial steps of the whole pregnancy process. The abnormality during implantation and decidualization is a major limiting factor in assisted reproductive therapies. Reactive oxygen species have traditionally been regarded as by-products of aerobic metabolism. It affects nomal function of reproductive system and involves in the pathogenesis of infertility. Antioxidant enzymes reduce reactive oxygen species and maintain the oxygen balance. Glutathione peroxidase is a very important class of antioxidant enzymes.?Based on our microarray analysis results, Gpx3 was significantly up-regulated in implantation sites compared with inter-implantation sites in mouse uterus, suggesting that Gpx3 may play a role in the process of mouse embryo implantation. So far, the expression, regulating and function of Gpx3 during implantation process were still unclear. This study was going to examine the expression, regulation and function of Gpx3 in mouse endometrium by in situ hybridization, Real-time RT-PCR, Western blot and luciferase assay.The signals of Gpx3 mRNA were not detected by in situ hybridization in mouse preimplantation uterus and the inter-implantation sites on day 5 of pregnancy. Gpx3 mRNA expressed in stromal cells at the implantation sites on day 5 of pregnancy, and the signal then spreaded to the whole decidua from days 6 to 8. There was no Gpx3 expression in mouse?pseudopregnancy uterus from days 1 to 5. Gpx3 was induced in implantation site when delayed implantation was activated. The artificial deciduoma also strongly expressed Gpx3. The expression of Gpx3 mRNA during early pregnancy was further confirmed by Real-time RT-PCR. Compared with the other members of GPX family, the expression of Gpx3 changed more obviously. It may play a major role in pregnant mouse uterus.? In hormone-treatment model of ovariectomized mouse, Gpx3 mRNA was significantly up-regulated by estrogen treatment for 72 h and by progesterone treatment for 12 h. RU486, a progesterone receptor antagonist, could block the up-regulation of Gpx3 by progesterone. Progesterone stimulated GPX3 expression through PR/HIF1αin mouse endometrial stromal cells. The up-regulation could be inhibited by RU486 or Chrysin, a HIF1αinhibitor. Besides, 8Br-cAMP also increased the expression of Gpx3 mRNA in mouse endometrial stromal cells. Sodium selenite increased GPX3 protein by post-transcriptional regulation. miR-138 could significantly down-regulate the activity of Gpx3 3′-UTR reporter by luciferase assay, but it could not inhibit the activity of mutated Gpx3 3′-UTR reporter.? miR-138 also inhibitd the protein level of GPX3 in mouse endometrial stromal cells. Thus, Gpx3 was a target gene of miR-138.The concentration of H2O2 of the implantation sites was significantly lower than that of the inter-implantation sites on day 5 of pregnancy. The expression of Gpx3 mRNA and protein was significantly increased in implantation sites compared with inter-implantation sites on day 5 pregnancy. The other genes of GPX family were not significantly up-regulated in implantation sites by Real-time RT-PCR, indicating that GPX3 palyed a major role of reducing H2O2 in mouse endometrium.The concentration of H2O2 in the supernatant of stromal cells undergoing in vitro decidualization was significantly down-regulated which could be blocked by GPX inhibitor or siGpx3. The result of Real-time RT-PCR showed that Gpx3 mRNA was significantly up-regulated, Gpx1 mRNA was significantly down-regulated, and Gpx4 mRNA was invariable in decidualized cells compared with control. These data indicated that GPX3 also palyed a major role of reducing H2O2 in decidualized cells in vitro. There were similar results in human endometrial stromal cells undergoing in vitro decidualization, indicating that GPX3 had similar functions in human endometrium.After intraperitoneal injection of GPX inhibitor for three days from day 5 to day 7 of pregnancy, the pregnancy rates were significantly reduced on day 8 of pregnancy, indicating that GPX may play an important role in the process of implantation and decidualization. After intraperitoneal injection of excessive sodium selenite, the mice were selenosis and the pregnancy rates were significantly reduced on day 8 of pregnancy, indicating that selenium was closely related with pregnancy process, and GPX3 may also have certain effect in this process as a?selenoprotein. In conclusion, GPX3 may play a major role during mouse embryo implantation and decidualization.