Notch1 mediates cell fate decisions in the mouse uterus and is critical for complete decidualization

Uterine receptivity implies a dialogue between the hormonally primed maternal endometrium and the free-floating blastocyst. Endometrial stromal cells proliferate, avert apoptosis, and undergo decidualization in preparation for implantation; however, the molecular mechanisms that underlie differentiation into the decidual phenotype remain largely undefined. The Notch family of transmembrane receptors transduce extracellular signals responsible for cell survival, cell-to-cell communication, and trans-differentiation, all fundamental processes for decidualization and pregnancy. Using a murine artificial decidualization model, pharmacological inhibition of Notch signaling by gamma-secretase inhibition resulted in significantly decreased deciduoma. Furthermore, a progesterone receptor (PR)-Cre Notch1 bigenic (Notch1d/d) confirmed a Notch1-dependant hypomorphic decidual phenotype. Microarray and pathway analysis, following Notch1 ablation, demonstrated significantly altered signaling repertoire. Concomitantly, hierarchical clustering demonstrated Notch1-dependent differences in gene expression. Uteri deprived of Notch1 signaling demonstrated decreased cellular proliferation; namely, reduced proliferation-specific antigen, Ki67, altered p21, cdk6, and cyclinD activity, and increased apoptotic-profile, augmented cleaved caspase-3, Bad, and attenuated Bcl2. Demonstrated here, the pre-implantation uterus relies on Notch signaling to inhibit apoptosis of stromal fibroblasts and regulate cell cycle progression, which together promotes successful decidualization. In summary, Notch1 signaling modulates multiple signaling mechanisms crucial for decidualization and we provide greater perspective to the coordination of multiple signaling modalities required during decidualization RNA samples from 3-5 separate mice were extracted. All mRNA quantities were normalized against 18S gene expression. Gene expression levels were measured by real-time RT-PCR SYBR Green analysis using the ABI Prism 7700 Sequence Detector System according to manufacturer’s instructions (Applied Biosystems).子宫容受性（Uterine receptivity）指激素致敏的母体子宫内膜（maternal endometrium）与游离胚泡（free-floating blastocyst）之间的对话交流。子宫内膜基质细胞会发生增殖、规避凋亡并经历蜕膜化（decidualization）过程，为胚胎着床做好准备；然而，驱动细胞分化为蜕膜表型的分子机制仍尚未完全阐明。Notch家族跨膜受体（transmembrane receptors）可转导与细胞存活、细胞间通讯及转分化相关的胞外信号，而这些均为蜕膜化与妊娠过程的核心基础事件。本研究利用小鼠人工蜕膜化模型，通过γ分泌酶抑制剂（gamma-secretase inhibition）对Notch信号进行药理学阻断，结果导致蜕膜瘤（deciduoma）的生成量显著降低。进一步通过孕激素受体（progesterone receptor, PR）-Cre Notch1双转基因（bigenic）小鼠（Notch1d/d）验证，证实了Notch1依赖的功能减退型蜕膜表型（hypomorphic decidual phenotype）。在Notch1基因敲除后开展的基因芯片（microarray）与通路分析显示，细胞信号传导谱发生显著改变。同时，分层聚类（hierarchical clustering）结果表明，基因表达存在Notch1依赖性差异。缺失Notch1信号的子宫组织，其细胞增殖能力下降——具体表现为增殖特异性抗原Ki67（proliferation-specific antigen Ki67）表达量降低、p21、细胞周期蛋白依赖性激酶6（cdk6）与细胞周期蛋白D（cyclinD）活性异常，且凋亡特征谱增强：裂解型半胱天冬酶-3（cleaved caspase-3）与Bcl-2相关死亡蛋白（Bad）的表达水平上调，而B细胞淋巴瘤因子2（Bcl2）的表达则受到抑制。本研究证实，植入前子宫依赖Notch信号来抑制基质成纤维细胞的凋亡并调控细胞周期进程，二者共同促进蜕膜化的顺利进行。总而言之，Notch1信号调控了蜕膜化过程中至关重要的多种信号通路机制，本研究为蜕膜化过程中多种信号模式的协同调控提供了全新视角。本研究提取了3-5只独立小鼠的RNA样本，所有mRNA的定量均以18S基因的表达作为内参进行标准化。基因表达水平通过实时逆转录聚合酶链反应（real-time RT-PCR）SYBR Green法进行检测，所用仪器为ABI Prism 7700序列检测系统（ABI Prism 7700 Sequence Detector System），实验操作严格按照应用生物系统公司（Applied Biosystems）的产品说明书进行。