Study of the embryo-derived factors in endometrial stromal cells

胚胎与子宫内膜基质细胞（ESCs）之间复杂的细胞相互作用阻碍了对着床机制的理解。尽管体外系统植入有助于理解这些复杂的体内生物现象，但在伦理上不可能在这些实验中使用人类胚胎。因此，建立小鼠体外植入模型是了解植入机制的必要条件。作为第一步，我们尝试建立脱个体化的体外模型。收集小鼠ESCs，在添加10% FCS、雌二醇（E2, 0.1nM）和黄体酮（P, 100nM）的DMEM中孵育。EP培养的ESCs转化为大的蜕膜样细胞，并产生蜕膜蛋白。在超微结构上，这些细胞在转化过程中具有丰富的粗面内质网。这些结果表明，体外培养的小鼠胚胎干细胞对卵巢类固醇有反应，并表现出类似于体内去人格化的形态和功能变化。因此，该培养体系可作为小鼠脱个体化的体外模型。作为第二步，我们在体外研究了胚胎的发育和滋养细胞对ESCs的侵袭。去个性化模型。4周龄小鼠ESCs在添加E2 （0.1 nM）和P （100 nM）的DMEM中培养9天。将孕4 d小鼠胚泡与含或不含E2/P的ESCs共培养，不含ESCs的胚泡作为对照（单培养）。培养12 d后，在光镜和透射电镜下观察囊胚发育情况及其与ESCs的相互作用。结果表明：1)无论是否添加E2/P，单培养囊胚在培养第12天均发生变性，而与ESCs共培养囊胚无变性；2)在共培养中，与不含E2/P的培养基相比，添加E2/P的培养基中生长的胚胎面积显著增加；3)在不含E2/P的培养基中，滋养细胞对ESCs的侵袭明显，而在有E2/P的培养基中，滋养细胞对ESCs的侵袭受到抑制。;4)ESCs与囊胚共培养时，在不加E2/P的情况下也能转化为蜕膜样细胞。这些结果表明，蜕细胞促进胚胎发育并调节滋养层侵袭，而胚胎可能产生一些蜕细胞刺激因子，从而表明蜕细胞与胚胎之间存在相互作用。少

Progress in understanding the mechanism of implantation has been hampered by the complexity of cellular interactions between embryos and endometrial stromal cells (ESCs). Although in vitro system of implantation may contribute to understanding such complex biological phenomena in vivo, it is ethically impossible to use human embryos in these experiments. Therefore, the establishment of in vitro model of mouse implantation is mandatory to understand the mechanism of implantation. As a first step, we tried to establish in vitro model of decidualization. Mouse ESCs were collected and incubated in DMEM supplemented with 10% FCS, estradiol (E2, 0.1nM) and progesterone (P, 100nM).ESCs cultured with EP transformed into large and decidua-like cells and produced decidual protein. Ultrastructurally, these cells became to have abundant rough endoplasmic reticulum during the transformation. These findings show that mouse ESCs in culture respond to ovarian steroids and showed morphological and func … More tional changes similar to decidualization in vivo. Thus, this culture system may serve as an in vitro model of mouse decidualization.As a second step, development of embryos and invasion of the trophoblast into ESCs were studied using this in vitro. model of decidualization. ESCs isolated from 4 week old mice were cultured for 9 days in DMEM supplemented with E2 (0.1 nM) and P (100 nM). The blastocysts isolated from 4 day pregnant mice were co-cultured with ESCs with or without E2/P, and the blastocysts were also cultured without ESCs as control (single culture). After 12-day culture, the blastocysts development and their interaction with ESCs were examined under light microscopy and transmission electron microscopy. The results were as follows : 1) blastocysts in the single culture were degenerated by the 12th day of culture regardless of E2/P addition, while the blastocysts co-cultured with ESCs survived without degeneration ; 2) in the co-culture, the area of growing embryos significantly increased in E2/P containing medium, compared with those without E2/P ; 3) the invasion of trophoblasts into ESCs was apparent in medium without E2/P, while it was inhibited in medium with E2/P. ; 4)ESCs were transformed into decidua-like cells in spite of culture without E2/P when they were co-cultured with blastocysts. These results suggest that decidual cells enhance the embryonic development and regulate the trophoblastic invasion, while embryos may produce some decidualization-stimulating factor(s), thus indicating the presence of a mutual interaction between decidualized ESCs and embryos. Less