In mammals, the endometrium undergoes dynamic changes in response to estrogen and progesterone to prepare for blastocyst implantation. Two distinct types of endometrial epithelial cells, the luminal (LE) and glandular (GE) epithelial cells play different functional roles during this physiological process. Previously, we have reported that Notch signaling plays multiple roles in embryo implantation, decidualization, and postpartum repair. Here, using the uterine epithelial‐specific Ltf‐iCre, we showed that Notch1 signaling over‐activation in the endometrial epithelium caused dysfunction of the epithelium during the estrous cycle, resulting in hyper‐proliferation. During pregnancy, it further led to dysregulation of estrogen and progesterone signaling, resulting in infertility in these animals. Using 3D organoids, we showed that over‐activation of Notch1 signaling increased the proliferative potential of both LE and GE cells and reduced the difference in transcription profiles between them, suggesting disrupted differentiation of the uterine epithelium. In addition, we demonstrated that both canonical and non‐canonical Notch signaling contributed to the hyper‐proliferation of GE cells, but only the non‐canonical pathway was involved with estrogen sensitivity in the GE cells. These findings provided insights into the effects of Notch1 signaling on the proliferation, differentiation, and function of the uterine epithelium. This study demonstrated the important roles of Notch1 signaling in regulating hormone response and differentiation of endometrial epithelial cells and provides an opportunity for future studies in estrogen‐dependent diseases, such as endometriosis.
The uterine luminal epithelium, glandular epithelium, and stromal cells are vital for the establishment of pregnancy. Previously studies have shown various methods to isolate mouse uterine epithelium and stromal cells, including Laser Capture Microdissection (LCM), enzyme digestion, and Immunomagnetic beads. Despite the importance of the endometrial epithelium as the site of implantation and nutritional support for the conceptus, there is no isolated method to separate the luminal epithelium and glandular epithelium. Here, we establish a novel enzyme-based way to separate two types of epithelium and keep their viability. In this article, we analyzed their purity by mRNA level, immunostaining, and transcriptome analysis. Our isolation method revealed several unstudied luminal and glandular epithelial markers in transcriptome analysis. We further demonstrated the viability of the isolated epithelium by 2D and 3D cultures. The results showed that we successfully separated the endometrial luminal epithelium and glandular epithelium. We also provided an experimental model for the following study of the physiological function of the different parts of the uterus and related diseases.
Fibrosis is a pathological process characterized by abnormal activation of fibroblasts with increased synthesis of extracellular matrix components, including collagens. It may lead to loss of proper tissue architecture and organ function in clinical diseases such as systemic sclerosis and liver fibrosis. Excess accumulation of collagens is considered the primary indicator of fibrosis. Notch signaling has been reported to be involved in the fibrosis of many different organs, including the liver. Our previous study showed that the uterine-specific over-activation of canonical Notch1 signaling in the mouse uterus (Pgr cre/+ Rosa26 N1ICD/+ , OEx) results in complete infertility as a consequence of multiple developmental and physiological defects, together with increased collagen accumulation evidenced by Masson's staining. In this study, we further detected expressions of all 44 collagen genes in these Notch1 gain-offunction transgenic mice and found that 18 collagens have been largely affected. In another aspect, using an intrauterine adhesion model (IUA), we mimicked fibrosis in the mouse uterine. The results suggested that Notch receptors were upregulated only 3 days after induction, and most of the fibril-forming collagen began to upregulate 6 days after the surgery. Furthermore, when induced IUA in the N1ICD-OEx mice, the expression of collagens and fibrosis levels were significantly enhanced. At last, as a Notch signaling inhibitor, the γ-secretase inhibitor N-[N-(3,-5-difl uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) pretreatment could alleviate the expression of collagens and the symptoms of fibrosis. These results demonstrate that Notch signaling may play a role in upregulating collagens expression in endometrial fibrosis and might be a potential target of fibrosis therapy in the endometrium.
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