Study of the embryo-derived factors in endometrial stromal cells

胚胎和子宫内膜间质细胞（ESCs）之间复杂的细胞相互作用阻碍了对着床机制的理解。尽管体外植入系统可能有助于理解体内这种复杂的生物学现象，但在这些实验中使用人类胚胎在伦理上是不可能的。因此，建立小鼠胚胎着床的体外模型对于了解胚胎着床机制具有重要意义。作为第一步，我们尝试建立体外蜕膜化模型。收集小鼠胚胎干细胞，在添加10%FCS、雌二醇（E2，0.1nM）和孕酮（P，100 nM）的DMEM中培养，用EP培养的胚胎干细胞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变得具有丰富的粗面内质网。这些发现表明，培养的小鼠胚胎干细胞对卵巢类固醇激素有反应，并表现出形态和功能上的差异。 ...更多信息 与体内蜕膜化相似的典型变化。因此，该培养体系可以作为小鼠蜕膜化的体外模型。作为第二步，胚胎发育和滋养层细胞侵入ESCs的体外研究。蜕膜化模型。将从4周龄小鼠分离的ESC在补充有E2（0.1 nM）和P（100 nM）的DMEM中培养9天。将从妊娠4天的小鼠分离的囊胚与具有或不具有E2/P的ESCs共培养，并且囊胚也在不具有ESCs的情况下培养作为对照（单一培养）。培养12 d后，在光镜和透射电镜下观察囊胚发育及其与胚胎干细胞的相互作用。结果表明：1）单独培养的囊胚在培养第12天时，无论是否添加E2/P，囊胚均发生退化，而与ESCs共培养的囊胚存活，未发生退化; 2）在共培养中，添加E2/P的培养液中，生长胚胎的面积显著增加; 3）E2/P对ESCs的侵袭作用明显，而E2/P对ESCs的侵袭作用受到抑制; 4）ESCs与囊胚共培养时，即使在E2/P不存在的情况下，也能转化为蜕膜样细胞。这些结果表明，蜕膜细胞促进胚胎发育并调节滋养层细胞的侵袭，而胚胎可能产生某些促蜕膜化因子，从而表明蜕膜化ESCs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