Expression Of BrdU,Musashi-1 And P63 In Mouse Endometrium And Their Significance

Objective:The endometrium, lining of the uterus, is a highly active organ that is periodically remodeled during the lifespan. Many literatures have demonstrated that the presence of an adult stem cell (ASC) population in this tissue because of its cyclic regenerative capacity. In this study, we aim at (1) identifying and localizing the putative ASC population in the murine uterus using the 5-bromo-2-deoxyuridine (BrdU) labeling method to detect label-retaining cells (LRCs) that carry on cell cycle slowly, (2) analyzing their relation to endometrial growth and differentiation with immunohistochemistry via some well-known un-differentiation markers such as P63 and Musashi-1's expression in mice endometrium, (3) then ascertaining whether P63 or Musashi-1 could be used as specific endometrial stem cell marker using double immunohistochemistry of P63/ BrdU and Musashi-1/BrdU.Methods:3-day-old Kunming white mice had been subcutaneously injected with BrdU twice daily for 3 consecutive days. Mice were then allowed to grow without further labeling. Thereafter, five mice were sacrificed to harvest their uteri at week 1,2,3,4,6,8 and 10. Immunohistobiochemistry was then used to analyze BrdU and stem cell marker P63 and Musashi-1 expression in the mice endometrium. And then select some P63 or Musashi-1 positive samples for double-immunohistochemical staining of P63/BrdU or Musashi-1/ BrdU. Enumeration of BrdU, musashi-1 and P63-labeled cells was performed in a high power microscope. Eventually, the results were statistically analyzed by SPSS statistical software.Result:1. BrdU-labeling cells in the mouse uterine endometrium:at postnatal 1 st week, the majority of epithelial and stromal cells were stained for BrdU when glandular development commences. Nuclear staining was apparent in the stroma and luminal epithelium. The percentage of BrdU-positive cells began to decrease gradually thereafter. At postnatal 3rd week, there were still many BrdU-positive cells in stroma, mainly located near stroma glands, meanwhile, there was also a small amount of BrdU-positive cells in luminal epithelium and glandular epithelium. After the 6th week, only scattered BrdU-positive cells were observed in the surface of epithelium. After the 8th week, almost no BrdU-positive cells were found in endometrial epithelium, but a few of stromal cells showed BrdU-positive in this phase, and most of them located at the interface of stromal and myometrial junction.Statistical analysis showed that BrdU-positive cell number in epithelium was significantly different (P<0.05) from in stroma.2.The expression of P63 in the mouse uterine endometrium:P63 was widely expressed in endometrial surface epithelium and stroma at the postnatal 1 st week. With the growth and development of the endometrial glands, P63 positive cells were discovered in the glandular epithelium. At postnatal 3rd week, some P63 positive cells were seen in the basal parts of endometrial stroma, and a small amount of P63-positive cells also occurred in the epithelium. At postnatal 8th week, P63-positive cells were mainly found adjacent to the stromal glands.At postnatal 10th week, only a few P63-positive cells were found a in the basal parts of the stroma.3.The expression of Musashi-1 in the mouse uterine endometrium:At postnatal 1st week, a lot of Musashi-1-Positive cells were observed in endometrial epithelium and stroma. Thereafter, the number of Musashi-1-Positive cells decreased gradually. During postnatal 6-week phase, the Musashi-1-Positive cells mainly located in the stroma, while a few of positive cells also occurred in the glandular epithelium. After 10th week, the expression of Musashi-1-positive cells was only occasional, and these Musashi-1-Positive cells mainly located adjacent to endometrial-myometrial junction, nevertheless epithelial Musashi-1-positive cells were not detected. 4.The result of immunohistochemical double staining:very few P63/BrdU double positive cells were detected in mice endometrium after 8 weeks phase, which mainly located at the endometrial-myometrial junction and underneath the luminal epithelium; Musashi-1/BrdU double positive cells were discoved in subepithelial basement. At the 8th week, occasional Musashi-1/ BrdU double positive cells were found around perivascular stroma and adjacent to the endometrial-myometrial junction.Conclusion:1. Using the LRC technique we demonstrated the presence of adult endometrial stem cells in mouse uteri. These cells appear likely to be located mainly at the stroma around the blood vessel and in the interface of endometrial-myometrial junction.2. Along with the growth and maturity of the endometrium, P63 and Musashi-1-positive cells decreased gradually in mouse endometrium, suggesting that P63 and Musashi-1 participate in the normal endometrial proliferation, and P63 and Musashi-1 are inevitable in the growth and maturation of mouse uterine endometrium, as well as their cyclical regeneration and differentiation.3. By means of double staining of P63/BrdU and Musashi-1/BrdU we affirmed that P63 and Musashi-1 might play a essential role in the development of endometrium, and the both could be used as assistant endometrial stem cell markers.