Armant D R, Kameda S
Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Exp Zool. 1994 Jun 1;269(2):146-56. doi: 10.1002/jez.1402690208.
We have investigated the invasive activity of mouse trophoblast cells during embryo implantation in vitro by culturing blastocysts with extracellular matrix (ECM) purified from mouse endometrium obtained on day 4 of pregnancy. Endometrium was dissected from lyophilized mouse uteri, and intact ECM was isolated by sequential precipitation in nonionic detergent and high salt. Electron microscopic examination of the ECM revealed typical collagen fibers plus an amorphous material resembling basement membrane. Electrophoretic analysis of the ECM revealed an enrichment of high molecular weight proteins, and immunoblotting indicated the presence of fibronectin, laminin, entactin, and type IV collagen, but not the intracellular proteins 2',3'-cyclic nucleotide-3'-phosphodiesterase or vimentin. Mouse blastocysts cultured with this ECM attached to it within 3 days, and the trophoblast cells began to migrate through the matrix in a manner resembling trophoblast invasion in utero. Unlike blastocysts cultured on plastic surfaces, the trophoblast did not flatten and become disorganized, but retained a polarized, spherical structure. Fluorescent microscopy with fluorescein isothiocyanate-labeled phalloidin revealed a high degree of microfilament organization and established that actin was absent from the ECM preparation. In the presence of a serum substitute, differentiation continued through yolk sac formation. Without serum components, yolk sac did not form; however, light and electron microscopic examination indicated that the invasive behavior of trophoblast cells persisted and was comparable to that of trophoblasts cultured in the presence of the serum substitute. A three-dimensional model for investigating trophoblast behavior in ECM from the endometrium should be of great value in elucidating the cellular and molecular events surrounding the process of blastocyst implantation.
我们通过将囊胚与从妊娠第4天获得的小鼠子宫内膜纯化的细胞外基质（ECM）一起培养，在体外研究了胚胎植入过程中小鼠滋养层细胞的侵袭活性。从冻干的小鼠子宫中解剖出子宫内膜，并通过在非离子去污剂和高盐中顺序沉淀分离出完整的ECM。对ECM的电子显微镜检查显示有典型的胶原纤维以及一种类似基底膜的无定形物质。对ECM的电泳分析显示高分子量蛋白质富集，免疫印迹表明存在纤连蛋白、层粘连蛋白、巢蛋白和IV型胶原，但不存在细胞内蛋白2',3'-环核苷酸-3'-磷酸二酯酶或波形蛋白。用这种ECM培养的小鼠囊胚在3天内附着其上，滋养层细胞开始以类似于子宫内滋养层侵袭的方式穿过基质迁移。与在塑料表面培养的囊胚不同，滋养层没有变平并变得无序，而是保留了极化的球形结构。用异硫氰酸荧光素标记的鬼笔环肽进行荧光显微镜检查显示微丝高度有序排列，并确定ECM制剂中不存在肌动蛋白。在存在血清替代物的情况下，分化持续到卵黄囊形成。没有血清成分时，卵黄囊不形成；然而，光镜和电镜检查表明滋养层细胞的侵袭行为持续存在，并且与在血清替代物存在下培养的滋养层细胞的侵袭行为相当。一个用于研究来自子宫内膜的ECM中滋养层行为的三维模型对于阐明围绕囊胚植入过程的细胞和分子事件应该具有很大价值。
