| Menstruation is a physiological phenomenon of endometrium periodic shedding in human, some primates, and a few placental mammals. Studying the mechanism on the endometrium periodic breakdown, bleeding and repair is the key to understand and find new treatment strategy for abnormal bleeding.The early research on menstruation may date back to the1940s. Markee transplanted pieces of human endometrium into the anterior chamber of the eyes in rhesus monkeys, and observed the process of menstrual cycle. Based on their observation, the spiral artery in the endometrium underwent a vasoconstriction period for about24hours before menstruation. Therefore, Markee deducted that the endometrium break-down was due to the consequence of tissue hypoxia-ischemia. However, the study on menstruation moved slowly because of the lack of animal model. In the1980’s, mice, the animal model of mammal, were brought into the field on studying menstruation. Then, the model was optimized. In2007, our research group had successfully established the mouse menstrual-like model by pharmacological progesterone withdrawal and carried on some fundamental researches. The genes and pathways in uterus after progesterone withdrawal in mouse menstrual-like model was assayed for Pathway Finder Chip assay. Based on the analysis of the Chip data, we found that the mRNA of genes concerning hypoxia response and angiogenesis were obviously up-regulated. Then, the genes in uterus at12h and16h after progesterone withdrawal were also assayed by us by Genome-wide expression profile Chip. Further, GO analysis suggested a fair amount of genes concerning hypoxia response, including Hifla and Vegf whose mRNA was both significantly up-regulated.In this study, based on the mouse menstrual-like model by physiological progesterone withdrawal in vivo, and decidual endometrium stroma cells relevance to menstruation in vitro, we focused on and investigated the functions of progesterone withdrawal, hypoxia, HIF1A and VEGF and their regulation during endometrium breakdown. The main results of this study were:1. Progesterone withdrawal up-regulated both Hifla and Vegf mRNA expression in both mouse menstrual-like model and mouse/human decidual endometrium stroma cells. Furthermore, progesterone withdrawal activated HIF1A by inducing nuclear translocation during endometrium breakdown in mouse menstrual-like model. These results confirmed the activation of HIF1A and VEGF2. During the breakdown phase of mouse menstrual-like model (0-24h), HIF1A and VEGF proteins were co-located in uterus. ChIP assay revealed that HIF1A directly bound on Vegf promoter region during endometrium breakdown. The binding peak appeared at12h after progesterone withdrawal which was the exact time point when Vegf mRNA reached to its highest level. HIF1A inhibitor administration suppressed both VEGF protein expression and endometrium breakdown, which indicated that HIF1A activation was important to VEGF expression and endometrium breakdown.3. During the reparative phase of mouse menstrual-like model (32-48h), HIF1A and VEGF proteins were co-located in uterus. ChIP assay revealed that HIF1A directly bound on Vegf promoter region was significantaly higher at32h than48h. HIF1A inhibitor administration suppressed both VEGF protein expression and endometrium repair. This result indicated that HIF1A activation was important to VEGF expression and endometrium repair.4. In mouse/human decidual endometrium stroma cells, progesterone withdrawal up-regulated the expression of Vegf mRNA, but this up-regulation was suppressed by Hifla knock-down. These results confirmed at the cellular level that progesterone withdrawal up-regulated Vegf expression via HIF1A.5. Hypoxia detection using pimonidazole revealed its existence in mouse menstrual-like model during endometrium breakdown. While stimulation of hypoxia in mouse menstrual-like model was not able to induce endometrium breakdown unless progesterone withdrawal. These results, for the first time, confirmed hypoxia existence during endometrium breakdown, and indicated that hypoxia is not the initial factor of endometrium breakdown.6. Hypoxic area showed abundant nuclear localization of HIF1A, and hypoxia, HIF1A and VEGF were co-located in mouse menstrual-like model during endometrium breakdown. Stimulation of hypoxia increased HIF1A nuclear translocation and VEGF expression in mouse menstrual-like model as well as Mouse/human decidual endometrium stroma cells. And VEGF expressions were suppressed by Hifla knockdown using specific siRNA in Mouse/human decidual endometrium stroma cells. These results indicated that hypoxia may up-regulate VEGF expression via HIF1A.We also applied the mouse menstrual like model to study the involment of NF-κB and COX-2. We found that COX-2inhibitor significantaly inhibited endometrium breakdown, and also decreased COX-2activity and the PGF2a content in body. The location of NF-κB (both P50and P65) and COX-2protein were correlated. The inhibition of NF-κB also inhibited endometrium breakdown, meanwhile decreased the expression of COX-2mRNA, and suggested that COX-2was regulated by NF-κB. The inhibition of NF-κB and COX-2both decreased the white blood cells invasion during endometrium breakdown. This result suggested that NF-κB increased PGF2a via COX-2to promote white blood cells invasion during endometrium breakdown. |
