Gene expression profiling of mouse uterine epithelial cells isolated on morning of day 4 of pregnancy

Embryo implantation into a receptive endometrium is tightly regulated by a variety of maternal factors, including cytokines, growth factors and transcription factors. Previous studies identified the leukaemia inhibitory factor (LIF), produced in uterine glands, as an essential factor for implantation. It was shown that LIF acts via its cell surface receptor to activate the transcription factor STAT3 in the uterine epithelial cells. However, the mechanisms via which STAT3 promotes uterine receptivity remain unknown. To address the molecular pathways regulated by STAT3 in the uterus, we created mice in which Stat3 gene is conditionally inactivated in uterine epithelium. These mutant mice are infertile due to implantation failure and exhibit a lack of embryo attachment to the luminal epithelium. Gene expression profiling of the epithelial tissue impaired in STAT3 activation revealed dysregulated expression of specific components of junctional complexes, including E-cadherin, β-catenin, and claudins, which critically regulate epithelial cell polarity and embryo attachment. Additionally, mice lacking functional epithelial STAT3 showed markedly reduced stromal proliferation and differentiation, indicating that this transcription factor controls stromal function via a paracrine mechanism. The stromal defect arose from a drastic reduction in the production of several members of the epidermal growth factor (EGF) family in luminal epithelium of mutant uteri and consequent lack of activation of EGF receptor signaling and mitotic activity in the stromal cells. Collectively, our results uncovered intricate signaling networks operating downstream of STAT3 in uterine epithelium that regulate epithelial cell polarity, and stromal proliferation and differentiation, which are critical determinants of successful implantation. To identify the downstream targets of STAT3 in mouse uterine epithelial cells during pregnancy, we performed gene expression profling of mouse uterine epithelial cells on day 4 of pregnancy between Stat3 flox control and SW d/d mice. This led to the identification of several junctional molecules (Claudins and Catenins) that are negatively regulated by STAT3 at the time of implantation. Mouse uterine epithelial cells were isolated from control and knockout mice on the morning of day 4 of pregnancy. (n=3 for each sample), pooled total RNA from these cells was then hybridized to high density affymetrix microarrays according to the Affymetrix protocol (Mouse Genome 430A 2.0 Array) .胚胎植入容受性子宫内膜（receptive endometrium）的过程受到多种母体因子的严格调控，涵盖细胞因子、生长因子与转录因子。既往研究已证实，由子宫腺体分泌的白血病抑制因子（leukaemia inhibitory factor, LIF）是胚胎植入的必需因子。已有研究表明，LIF可通过其细胞表面受体激活子宫上皮细胞中的转录因子STAT3。然而，STAT3促进子宫容受性的具体分子机制仍未阐明。 为探究子宫内STAT3所调控的分子通路，我们构建了在子宫上皮中条件性失活Stat3基因的小鼠模型。该突变小鼠因胚胎植入失败而不育，且表现出胚胎无法黏附于宫腔上皮的表型。对STAT3激活缺陷的上皮组织进行基因表达谱分析后发现，连接复合体（junctional complexes）的特定组分存在表达失调情况，包括E-钙粘蛋白（E-cadherin）、β-连环蛋白（β-catenin）与闭合蛋白（claudins），这些组分对上皮细胞极性及胚胎黏附至关重要。此外，缺乏功能性上皮STAT3的小鼠还出现基质增殖与分化显著受损的表型，提示该转录因子可通过旁分泌机制调控基质功能。 该基质缺陷源于突变小鼠宫腔上皮中多种表皮生长因子（epidermal growth factor, EGF）家族成员的表达水平大幅下调，进而导致基质细胞中表皮生长因子受体信号通路激活不足及有丝分裂活性缺失。 综上，本研究揭示了子宫上皮中STAT3下游的复杂信号网络，该网络调控上皮细胞极性以及基质增殖与分化，而这些正是胚胎成功植入的关键决定因素。为鉴定妊娠期间小鼠子宫上皮细胞中STAT3的下游靶基因，我们对妊娠第4天的Stat3 flox对照小鼠与SW d/d小鼠的子宫上皮细胞开展了基因表达谱分析。我们于妊娠第4天清晨从对照组与敲除组小鼠中分离子宫上皮细胞（每组样本n=3），收集混合后的总RNA，并按照Affymetrix实验流程（Mouse Genome 430A 2.0 Array）将其与高密度Affymetrix基因芯片进行杂交。