Laboratory of Endometrium, Endometriosis and Reproductive Medicine, Department of Development and Regeneration, KU Leuven, Herestraat 49 Box 611, 3000, Leuven, Belgium.
Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, VIB Center for Brain & Disease Research, KU Leuven, Herestraat 49 Box 802, 3000, Leuven, Belgium.
Reprod Biol Endocrinol. 2023 Apr 15;21(1):37. doi: 10.1186/s12958-023-01085-7.
Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.
To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.
We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IPR and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells.
These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.
早期胚胎着床是一个复杂的现象，其特征是具有着床能力的囊胚和接受性的子宫内膜。胚胎发育和子宫内膜容受性必须同步，它们之间进行充分的双向对话对于母体识别和着床是必要的。蛋白酶被描述为囊胚分泌的蛋白，参与孵化过程和早期着床事件。这些酶刺激子宫内膜上皮细胞（EEC）中的细胞内钙信号通路。然而，蛋白酶诱导钙信号的确切分子参与者、随后的下游信号通路以及其激活的生物学影响仍不清楚。
为了鉴定人源和鼠源子宫内膜上皮细胞中感兴趣的受体和离子通道的基因表达，进行了 RNA 测序、RT-qPCR 和原位杂交实验。进行钙微荧光实验以研究它们的功能表达。
我们表明，胰蛋白酶在鼠和人源的 EEC 中引发细胞内钙振荡，并鉴定出蛋白酶激活受体 2（PAR2）是在 EEC 中引发蛋白酶诱导的钙反应的分子实体。此外，本研究通过显示细胞内钙储存的耗竭和再填充通过 PLC、IPR 和 STIM1/Orai1 复合物发生，揭示了 PAR2 下游信号中的分子参与者。最后，在存在特异性 PAR2 激动剂的体外实验中，人源子宫内膜上皮细胞中的“着床窗口期”标志物被上调。
这些发现为囊胚衍生的蛋白酶信号提供了新的见解，并将 PAR2 作为母体传感器分配给由发育中的囊胚释放的信号。
