| In the late secretory phase of menstrual cycle, luteal regression leads to decline of progesterone levels which also called progesterone withdrawal. Progesterone withdrawal induced apoptosis of endometrial stromal cells, followed by endometrium undergoes bleeding and shedding, the process was defined as menstruation.Menstruation was divided into two different stages:reversible and irreversible stage according to whether being reversible by progesterone addback. The two stages involved different signal pathways. The former stage is controlled by progesterone. The subsequent latter stage trigger a series of cascade of reactions related to tissue breakdown factors. In human and primate, there are a lot of limits in such efforts to illustrate molecular mechanism of those events. Therefore, the key clarifying the mechanism of menstruation is to build a convenient animal model in vivo which is low-costly, easily operative and suitable for molecular dissection, so it is key point that we find the important period of progesterone reversible stage in mice model to further study mechanism of menstruation. Moreover, some interesting exploration was carried out and the results were also showed as follows:1. It is a great significance issue to determine the key period of progesterone reversible stage in menstruation by adding back progesterone to mice. We successfully identified the key period of endometrial shedding and bleeding could be reversed by progesterone addback in mouse by monitoring bleeding and identifying of tissue morphology with vaginal smear, that is,12-16hour after withdrawal.2. In order to acquire information of differentially expressed genes, three groups of gene expression profile of endometrium including at12h after progesterone withdrawal,16h after progesterone withdrawal and adding back progesterone at12h after progesterone withdrawal in mice were compared by microarray. Finding differential gene expression provided guidance for our further study.3. From data of microarray, key members of MMP family, cycloo-xygenase-2(COX-2) and VEGF were selected as our target molecules. We compared mRNA and protein expression in mouse endometrium. The results was showed:(1) In the early stage of menstruation, when progesterone was added back to mice at12h after progesterone withdrawal, mRNA and protein expression of MMP-3, MMP-10, MMP-11MMP-13and COX-2were inhibited suggesting that these molecules may be functional in the early period of the menstruation;(2) In late stage of menstruation, when progesterone was added back to mice at a critical time point (12h), mRNA and protein expression of MMP-2, MMP-3, MMP-9, MMP-10, MMP-11, MMP-13and COX-2were inhibited largely. Moreover, after the critical time, i.e. progesterone was added back to mice at16h after progesterone withdrawal, mRNA and proteins expression of MMP molecules and COX-2were partly inhibited or could not be inhibited completely.(3) It was interesting that mRNA expression of MMP-7can not be inhibited by progesterone suggesting MMP-7may not play role in shedding and bleeding of endometrium in mice and be active in regeneration of endometrium.(4) Progesterone addback or not did not affect VEGF mRNA and protein expression, which was not consistent with VEGF protein in the mouse uterus tissues localization and this inconsistent may be due to transient increase of VEGF expression.4. Apoptosis of endometrial stromal cell was studied when progesterone was add back to mice. We found when progesterone was added back at12h after progesterone withdrawal, it could effectively block apoptosis of stromal cells. However, after the critical time point, apoptosis of endometrial cells was not inhibited by progesterone. We also analyzed mRNA and proteins expression of p53, mdm2, bcl-2, bax, fas, caspase-3and caspase-8which were apoptosis-related genes. It seemed that anti-apoptotic effect by addback of progesterone may be due to increasing the ratio of bcl-2/bax and decreasing caspase-3expression by progesterone. |
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